Biodiversitas vol. 16, no. 1, April 2015

ISSN: 1412-033X E-ISSN: 2085-4722

Journal of Biological Diversity Volume 16 – Number 1 – April 2015

ISSN/E-ISSN: 1412-033X (printed edition), 2085-4722 (electronic)

EDITORIAL BOARD (COMMUNICATING EDITORS): Abdel Fattah N.A. Rabou (Palestine), Alan J. Lymbery (Australia), Alireza Ghanadi (Iran), Ankur Patwardhan (India), Bambang H. Saharjo (Indonesia), Daiane H. Nunes (Brazil), Ghulam Hassan Dar (India), Guofan Shao (USA), Faiza Abbasi (India), Hassan Pourbabaei (Iran), Hwan Su Yoon (South Korea), I Made Sudiana (Indonesia), Ivan Zambrana-Flores (United Kingdom), Joko R. Witono (Indonesia), Katsuhiko Kondo (Japan), Krishna Raj (India), Livia Wanntorp (Sweden), M. Jayakara Bhandary (India), Mahdi Reyahi-Khoram (Iran), Mahendra K. Rai (India), Mahesh K. Adhikari (Nepal), María La Torre Cuadros (Peru), Maria Panitsa (Greece), Muhammad Akram (Pakistan), Muhammad Iqbal (Indonesia), Mochamad A. Soendjoto (Indonesia), Mohib Shah (Pakistan), Mohamed M.M. Najim (Srilanka), Pawan K. Bharti (India), Paul K. Mbugua (Kenya), Rasool B. Tareen (Pakistan), Seweta Srivastava (India), Seyed Aliakbar Hedayati (Iran), Shahabuddin (Indonesia), Shahir Shamsir (Malaysia), Shri Kant Tripathi (India), Stavros Lalas (Greece), Subhash Santra (India), Sugiyarto (Indonesia), T.N.Prakash Kammardi (India)

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Society for Indonesia Biodiversity

Sebelas Maret University Surakarta

BIODIVERSITAS Volume 16, Number 1, April 2015 Pages: 1-9

ISSN: 1412-033X E-ISSN: 2085-4722 DOI: 10.13057/biodiv/d160101

Effects of timber harvest on structural diversity and species composition in hardwood forests FARZAM TAVANKAR1,♼, AMIR ESLAM BONYAD2 1

Department of Forestry, Khalkhal Branch, Islamic Azad University, Khalkhal, Iran. P.O. Box 56817-31367, Tel.: +98 4532451220-2, Fax: +98 42549050452, ♼email: farzam_tavankar@yahoo.com 2 Department of Forestry, Faculty of Natural Resources, University of Guilan, Somehsara, Iran.

Manuscript received: 13 August 2014. Revision accepted: 10 September 2014.

Abstract. Tavankar F, Bonyad AE. 2015. Effects of timber harvest on structural diversity and species composition in hardwood forests.

Biodiversitas 16: 1-9. Forest management leads to changes in structure and species composition of stands. In this research vertical and horizontal structure and species composition were compared in two harvested and protected stands in the Caspian forest of Iran. The results indicated the tree and seedling density, total basal area and stand volume was significantly (P < 0.01) higher in the protected stand. The Fagus orientalis L. had the most density and basal area in the both stands. Species importance value (SIV) of Fagus orientalis in the protected stand (92.5) was higher than in the harvested stand (88.5). While, the SIV of shade-intolerant tree species such as Acer insigne, Acer cappadocicum and Alnus subcordata was higher in the harvested stand. The density of trees and seedling of rare tree species, such as Ulmus glabra, Tilia begonifolia, Zelkova carpinifolia and Fraxinus coriarifolia, was also higher in the protected stand. The Shannon-Wiener diversity index in the protected stand (0.84) was significantly higher (P < 0.01) than in the harvested stand (0.72). The highest diversity value in the harvested stand was observed in DBH of 10-40 cm class, while DBH of 40-70 cm had the highest diversity value in the protected stand.

Key words: Beech stands, Shannon-Wiener index, stand structure, uneven aged management.

INTRODUCTION One main principle of biodiversity protection in multiple management of national forest is the protection of stands structure composition (Eyre et al. 2010; Sohrabi et al.

2011). In forest science, stand structure refers to the withinstand distribution of trees and other plants characteristics such as size, age, vertical and horizontal arrangement, or species composition (Powelson and Martin 2001).

Structural diversity is a straightforward indicator of potential biodiversity in forest landscapes because a diverse stand structure provides better habitat for forest-dwelling organisms. Broadly accepted, a structurally diverse stand provides living space for a number of organisms.

Increasing and maintaining structural diversity in forest stands, also has become an important forest management strategy for adapting climate change. Conservation of forests biodiversity is one of important objective in sustainable forest management (Burton et al. 1992; Brockerhoff et al. 2008). It is common opinion in forest ecology that different management practices are a major determinant of forest diversity and that a more complex forest structure is linked to a high diversity of plant and animal species (Pretzsch 1997; Boncina 2000; Shimatani 2001). Forest management leads to changes in horizontal and vertical structure (Kuuluvainen et al. 1996; North et al.

1999) and in the species composition (Nagaike Hayashi 2004; Uuttera et al. 1997). The idea that biodiversity can be

maintained by managing the structural diversity of stands is a common argument among researchers (Buongiorno et al.

1994; Lindenmayer and Franklin 1997; Sullivan et al.

2001; Franklin et al. 2002; Kant 2002; Varga et al. 2005).

Some silvicultural practices can enhance biological diversity in managed forests, such as retaining old trees (Seymour and Hunter 1999), maintaining adequate levels of dead wood (Sturtevant 1997), establishing mixed stands (Palik and Engstrom 1999) or extending rotation lengths (Ferris et al. 2000).

The Caspian natural forests of Iran also called Hyrcanian forests, are located on the southern border of the Caspian Sea and cover an area of 2 million hectares. The stands in this area are the most valuable and economical. The main benefits of these forests are essentially two-fold: on the one hand there is its wood production while on the other hand there are various physical and social effects frequently termed as forest influence. In many instances, the latter transcends is the significance of forests as producers of wood (Bonyad et al. 2012). The current forest harvesting method in these forests is mainly selective cutting. The main goal of selection cutting management is uneven aged and mixed stands that are close to nature. Selection cutting is the silvicultural practice of harvesting a proportion of the trees in a stand (Pourmajidian and Rahmani 2009). In selective cutting, each tree must be individually assessed to decide whether it should be cut or left. In reality, this method is the practice of removing mature timber or

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thinning to improve the timber stand. Selection cutting improves the health of the stand and releases space for young trees to grow. In the selection system, regeneration, tending, and harvesting all take place concurrently (Marvie Mohadjer 2006). Selection cutting may include opening up areas to allow tree species that require greater light intensity to grow but that are not large enough to meet the legal definition of a clear cut (Nyland 1998; Anderson et al.

2000; Webster and Lorimer 2002; Pourmajidian and Rahmani 2009). Selection cutting is appropriate for forests composed of trees of different sizes and ages. Selection cutting does not have a visual impact on landscapes because only some of trees are removed, a factor that is much appreciated by forest users. Uneven-aged management is one alternative that could generate sustainable harvests while maintaining continuous forest cover and protecting stands diversity (Guldin 1996).

A planned program of silvicultural treatments ensures the conservation and maintenance of biological diversity and richness for sustainable forestry (Torras and Saura 2008; Schumann et al. 2003; Battles and Fahey 2000; Simila et al. 2006). The uneven-aged management can be economically viable while preserving forest stand diversity (Buongiorno et al. 1994, Schulte and Buongiorno 1998, Volin and Buongiorno 1996).

Beech (Fagus orientalis Lipsky) is the most industrial commercial tree species among more than 80 broad-leaved trees and shrubs. Many studies have been carried out on plant biodiversity in Beech stands in Iran and around the world (Sohrabi et al. 2011; Pourmajidian et al. 2009; Brunet et al. 2010; Sefidi et al. 2011; Pourbabaei et al.

2013). The study of forest structure especially in virgin forests is very important and gives us comprehensive information about the condition in forest for programming.

The selection cutting, such as other forestry practices, can leads to changes in stand structure and tree compositions.

The stand structural diversity can be characterized horizontally, i.e. the spatial distribution of trees, and vertically in their height differentiation (Zenner and Hibbs 2000). In this research, stand volume and structure, tree and seedling density, and species composition were compared in the harvested and protected Beech dominated stands.

The objective of this study was effects of timber harvesting on structural diversity and species composition in oriental Beech stands in the Iranian Caspian forests.

MATERIALS AND METHODS Study area The study area is Iranian Caspian forests. These forests are suitable habitats for a variety of hardwood species and include various forest types. Approximately 60% of these forests are used for commercial purposes and the rest of them are more or less degraded (Marvie Mohadjer 2006).

This study was conducted in Nav forests (latitude 37° 38' 34" to 37° 42' 21" N, longitude 48° 48' 44" to 48° 52' 30" E) in Guilan province, north of Iran. Two adjacent compartments of 123 (protected) and 112 (harvested) with areas of 43 and 63 ha were selected for collection of data.

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The physiographical characteristics of these compartments are almost similar. The elevation of these compartments ranges from 850 m to 1,100 m asl. The climate is temperate on based Demarton climate classification, with a mean annual temperature of 9.1°C and mean annual precipitation of 950 mm for along with the 1990 to 2008 years.

Vegetation period maintains for 7 months in average. The original vegetation of this area is an uneven-aged mixed forest dominated by Fagus orientalis and Carpinus betulus, with the companion species Alnus subcordata, Acer platanoides, Acer cappadocicum, Ulmus glabra and Tilia rubra. The soil type is forest brown soil and the soil texture varies between sandy clay loam to clay loam. This study was carried out in two areas, harvested and protected compartments in the Nav forest area of Iran (Nav Forest Management Plan 1998).

Data collection Data were collected by circular sample plots with an area of 0.1 hectare. The sample plots were located on the study area through systematic grid (100 m × 100 m) with a random start point. Diameter at breast height (DBH) of all trees (DBH ≥ 7.5 cm) was measured by diameter tape.

Individuals of trees with DBH < 7.5 cm were counted by species as seedling. Height was measured to the nearest m using Suunto clinometer.

Data analysis Species importance value (SIV) for each specious was calculated by (Ganesh et al. 1996; Krebs 1999; Pourbabaei et al. 2013; Rezaei Taleshi 2014): SIV= Relative density (RD) + relative frequency (RF) + relative dominance (RD).

Basal area was considered for dominancy and relative dominance (RD) calculated by: RD = (basal area of a species × 100) / total basal area of all species. The species diversity index was computed using the Shannon-Wiener information function (Krebs 1999; Sharma et al. 2009; Abedi and Pourbabaei 2010; Pourbabaei et al. 2012) as: H'=-Σni/n log2 ni/n, where: ni = denote to the SIV of a species and n= denote to the sum of total SIV of all species.

The species evenness index was computed using the Pielou’s evenness index (J) as: J = H' / ln S, where ln is Natural logarithm, S is the total species number in each plot. Also species richness (S) was number of species per plot. After checking for normality (Kolmogorov-Smirnov test) and homogeneity of variance (Levene’s test), the means of stand characteristics (tree and seedling density, basal area, stand volume) in two compartments (harvested and protected) were compared using independent samples t test. The means of biodiversity indices (diversity, evenness and richness) in two compartments were also compared using independent samples t test. The means of biodiversity indices in DBH classes compared using a one-way ANOVA. Multiple comparisons were made by Tukey’s test (significance at α < 0.05). Regression analysis was applied to test the relations between DBH and stand volume, tree density and tree height. SPSS 19.0 software was used for statistical analysis; also the results of the analysis were presented using descriptive statistics.

TAVANKAR & BONYAD â&#x20AC;&#x201C; Effects of timber harvest on hardwood forests

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A

Harvested Protected

B

C

Figure 3. Study site map of Nav-forest, northern Iran. A. Guilan Province, Iran, B. Nav-forest within study site, near Nav ( ), Asalem, Talesh, Guilan, Iran, C. Detailed site of forest sampling.

RESULTS AND DISCUSSION Results The stand parameters in two studied compartments are shown in table 1. The results indicated the tree and seedling density in the protected stand was significantly higher (P < 0.01) than the harvested stand. The total basal area and stand volume in the protected stand was also significantly higher (P < 0.01) than the harvested stand (Table 1).

A total of 16 tree species from 8 families were observed in the sample plots (Table 2). The Fagus orientalis had the most density and basal area in the both stands. Density of Beech trees in the harvested stand was 66.8 stem.ha-1 (28.7%), while in the protected stand was 89.1 stem.ha-1 (25.9%). Basal area of Beech trees in the harvested stand was 5.1 m2.ha-1 (29.3%), while in the protected stand was 7.9 m2.ha-1 (31.7%). Indeed, the density percentage of Oriental Beech trees was higher in the harvested stand, while the basal area percentage of Oriental Beech trees was higher in the protected stand. After the Oriental Beech trees, Carpinus betulus had the most density in the harvested (32.6 stem.ha-1 or 14%) and in the protected (54.7 stem.ha-1 or 15.9%) stands. In addition, the density percentage of Carpinus betulus was higher in the protected stand, but the basal area percentage of Carpinus betulus was higher in the harvested stand (16.1% vs. 13.6%). The family of Aceraceae had three species (A. insigne, A. cappadocicum and A. platanoides) in these stands. The density of Aceraceae species in harvested stand was 77.3 stem.ha-1 or 33.2%, while in the protected stand was 99

stem.ha-1 or 29%. Also the basal area of Aceraceae species in the harvested stand was 4.8 m2.ha-1 or 27.6%, while in the protected stand was 6.3 m2.ha-1 or 25.3%. However, the family of Rosaceae had the most number of tree species, but these trees had the minimum density and basal area in two stands. The Rosaceae species include Mespilus germanica, Cerasus avium, Pyrus communis, Prunus divaricata and Sorbus torminalis.

Species Importance Value (SIV) of different tree species in the harvested and protected stands is shown in Figure 1. The SIV of Fagus orientalis in the protected stand (92.5) was higher than the harvested stand (88.5).

While, the SIV of Carpinus betulus, Acer insigne, Acer cappadocicum and Alnus subcordata in the harvested stand was higher than in the protected stand. Also, the SIV of Acer platanoides, Quercus castaneifolia, Tilia begonifolia, Ulmus glabra and Zelkova carpinifolia in the protected stand was the higher than the harvested stand. The SIV of other tree species (Fraxinus coriarifolia, Mespilus germanica, Cerasus avium, Pyrus communis, Prunus divaricata and Sorbus torminalis was almost equal in the harvested and protected stands (Figure 1). Volume of different tree species in the harvested and protected stands is shown in Figure 2. The volume of all tree species in the protected stand was higher than in the harvested stand. The volume of Fagus orientalis in harvested and protected stands was 51.5 and 74.5 m3.ha-1. Seedling of different tree species in harvested and protected stands are shown in Figure 3. The seedling density of all tree species, except of Carpinus betulus, in the protected stand was higher than in

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the harvested stand. The multiple regression analyses applied to test the relations between DBH and tree height in the harvested and protected stands that were statistically significant (P < 0.001) and the result are shown in Figure 4.

The regression analysis between DBH and tree density in the harvested and protected stands was also statistically significant (P < 0.001) and the results are shown in Figure 5. The regression analyses applied to test the relations between DBH and stand volume in the harvested and protected stands that were statistically significant (P < 0.001) and the result are shown in Figure 6.

Biodiversity indices in harvested and protected stands are shown in table 3. The value of diversity index in the protected stand (0.84) was significantly higher (P < 0.01) than harvested stand (0.72). The value of evenness and richness indices in the protected stand were also higher (P < 0.01) than in the harvested stand. ANOVA tests showed the DBH classes had significantly affect (P < 0.01) on the means of biodiversity indices in the harvested and protected stands (Table 4). The highest diversity value in

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the harvested stand was observed in DBH of 10-40 cm class, while DBH of 40-70 cm had the highest diversity value in the protected stand. The highest evenness value in the harvested stand was observed in DBH of 70-100 cm, while DBH of > 100 cm had the highest evenness value in the protected stand. The highest richness value was observed in DBH of 10-40 cm in the both of harvested and protected stands.

The results of t test showed were not significant difference between the values of diversity index in two stands in the DBH class of 10-40 cm (Table 5). While, the values of diversity index in the DBH classes of 40-70, 70100 and > 100 cm in the protected stand were significantly higher than harvested stand (Table 5). The value of evenness index was significantly higher in the protected stand than the harvested stand only in the DBH class of > 100 cm (Table 5). The values of richness index in the all of DBH classes in the protected stand were significantly higher than the harvested stand (Table 5).

Table 1. Stand parameters (mean ± standard deviation) in the study sites.

Parameter

Harvested

Protected

T-Value

Tree density (stem.ha-1) Basal area (m2.ha-1) Volume (m3.ha-1) Seedling density (stem.ha-1) Note: **: P < 0.01.

232.7 ± 57.7 17.4 ± 2.3 154.3 ± 14.2 350.4 ± 18.1

344.1 ± 41.3 24.9 ± 5.2 257.3 ± 17.3 486.8 ± 68.5

11.14** 8.59** 31.05** 10.87**

Table 2. Frequency and basal area of tree species in the study sites.

Tree species Fagus orientalis Lipsky Carpinus betulus L. Acer insigne Boiss.

Acer cappadocicum Gled.

Alnus subcordata C.A.M. Acer platanoides L. Quercus castaneifolia Gled.

Tilia begonifolia Stev.

Ulmus glabra Huds.

Zelkova carpinifolia Diopp Fraxinus coriarifolia Scheel Mespilus germanica L. Cerasus avium L. Pyrus communis L. Prunus divaricata Ledeb.

Sorbus torminalis L.

Family Fagaceae Corylaceae Aceraceae Aceraceae Betulaceae Aceraceae Fagaceae Tiliaceae Ulmaceae Ulmaceae Oleaceae Rosaceae Rosaceae Rosaceae Rosaceae Rosaceae

Density (stem.ha-1) Harvested Protected 66.8 89.1 32.6 54.7 28.1 38.0 26.5 32.6 25.0 30.1 23.3 28.4 9.6 20.5 5.2 20.3 3.3 10.8 2.8 8.5 2.2 4.1 2.0 2.4 1.7 2.0 1.2 1.8 1.0 1.5 0.5 0.8

Basal area (m2.ha-1) Harvested Protected 5.1 7.9 2.8 3.4 2.3 2.6 1.8 2.3 1.1 1.7 0.7 1.4 0.9 1.6 0.7 1.8 0.6 1.4 0.5 1.0 0.3 0.6 0.1 0.2 0.1 0.2 0.1 0.1 0.1 0.1 0.1 0.1

Table 3. Biodiversity indices (mean ± standard deviation) in DBH classes.

Diversity* Evenness Harvested Protected Harvested Protected 10-40 0.78 ± 0.18a 0.70 ± 0.13b 0.51 ± 0.14b 0.46 ± 0.18b 40-70 0.55 ± 0.16b 0.91 ± 0.19a 0.60 ± 0.17a 0.53 ± 0.12b 70-100 0.44 ± 0.15c 0.81 ± 0.20a 0.66 ± 0.15a 0.71 ± 0.16a > 100 0.40 ± 0.10c 0.55 ± 0.13c 0.53 ± 0.14b 0.76 ± 0.19a All trees 0.72 ± 0.15 0.84 ± 0.09 0.61 ± 0.08 0.70 ± 0.07 Note: *: Different letters in each column indicated significant difference at α = 0.05.

DBH (cm)

Harvested 4.96 ± 1.50a 4.51 ± 1.45ab 3.88 ± 1.38b 3.20 ± 1.25c 3.77 ± 1.09

Richness Protected 6.80 ± 1.54b 6.21 ± 1.53ab 5.65 ± 1.58a 4.31 ± 1.62a 4.79 ± 1.08

TAVANKAR & BONYAD â&#x20AC;&#x201C; Effects of timber harvest on hardwood forests

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Table 4. ANOVA results for means of biodiversity indices in DBH class.

Indices

Sites

SS

df

MS

F

P-Value

Diversity

Harvested Protected

3.307 3.839

3 3

1.102 1.279

182.05 201.38

0.000 0.000

Evenness

Harvested Protected

2.736 3.263

3 3

0.912 1.088

112.45 131.52

0.000 0.000

Richness

Harvested Protected

49.293 64.72

3 3

16.431 21.573

28.760 30.536

0.000 0.000

Table 5. Results of t test for comparing means of biodiversity indices in harvested and protected stands according DBH class.

DBH (cm)

Diversity N.S

10-40 1.678 40-70 2.543* 70-100 4.659** > 100 2.324* All trees 5.032** Note: N.S: Not significance, *: P < 0.05, **: P < 0.01

Evenness N.S

1.982 2.104N.S 2.001N.S 3.064** 6.058**

Figure 1. SIV of tree species in the harvested and protected stands.

Figure 2. Volume of tree species in the harvested and protected stands.

Richness 5.528** 3.371** 4.580** 5.051** 4.584**

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Figure 3. Seedling density of tree species in the harvested and protected stands.

Figure 4. Relation between DBH and tree height in the harvested and protected stands.

Figure 5. Relation between DBH and tree density in the harvested and protected stands.

TAVANKAR & BONYAD â&#x20AC;&#x201C; Effects of timber harvest on hardwood forests

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Figure 6. Relation between DBH and stand volume in the harvested and protected stands.

Discussion Understanding the effects of forest management practices on plant species diversity is important for achieving ecologically sustainable forest management (Banda et al. 2006; Nagaike et al. 2006; Liang et al. 2007; Sefidi et al. 2011). The results of this study indicated the tree and seedling density, total basal area and stand volume in the protected stand was higher than in the harvested stand. Managing the forest for periodic income from the sale of trees as raw material for forest products depends on being able to regenerate the forest successfully. Forests are the most species rich of all terrestrial ecosystems and provide essential benefits to society. Forest management plan should describe both short and long term management goals and how to maintain forest productivity. Qiu et al.

(2006) investigated effects of selection cutting on the forest structure and species diversity of evergreen broad-leaved forest in northern Fujian, China. They reported selection cutting of low and medium intensities caused to little variation in the stand structure, while high intensity of selection cutting caused to significantly changing in the stand structure. Sohrabi et al. (2011) studied structural diversity of Beech stands in northern Iran and reported the most diversity of trees is in low height and diameter classes.

The results of this study indicated the density of trees and seedling of rare tree species, for example, Ulmus glabra, Tilia begonifolia, Zelkova carpinifolia and Fraxinus coriarifolia, in the protected stand was higher than in the harvested stand. It is widely demonstrated that more species contribute to greater ecosystem stability.

Nowadays, forest management practices increasingly promote conservation and enhancement of biodiversity.

Forest management typically has a marked affect on plant species diversity, which is an important ecological indicator (Lindenmayer et al. 2000). Poor forest management practices contribute to decline or loss of biodiversity. The conservation of biodiversity has become a major concern for resource managers and conservationists worldwide and it is one of the foundation principles of ecologically sustainable forestry (Carey and Curtis 1996; Hunter 1999).

Our results indicated the species importance value (SIV) of shade-intolerant species such as Acer insigne, Acer cappadocicum and Alnus subcordata in the harvested stand were higher than protected stand. The diversity of a forest stand may not be sufficiently described by tree species diversity alone. Forest ecologically management include forest ecosystem, wood production and non timber values (Lindenmayer et al. 2000; Pourbabaei and Pourrahmati 2009). The forest biodiversity guidelines focus on how best to conserve and enhance biodiversity in forests, through appropriate planning, conservation and management. Tavankar et al. (2011) investigated effects of selection cutting on species diversity of trees and regeneration at a 10 years period in the Caspian forests.

Their results indicated species diversity of tree and regeneration were slightly increased after 10 years from cutting since. Also the researchers reported the species importance value (SIV) of Beech and Hornbeam trees were decreased, but SIV of Maple and Alder trees were increased at the end of period.

The structural attributes of forest stands are increasingly recognized as being of theoretical and practical importance in the understanding and management of forest ecosystems (Franklin et al. 2002). The structural diversity can be characterized by diameter variation of trees in a forest stand. The regression analysis of relation between DBH and tree height showed the height of trees with DBH of > 30 cm in protected stand were higher than in the harvested stand. Pourmajidian and Rahmani (2009) compared stand structure after 12 years in a Beech stand. They reported the stand volume was not significantly changed, but density and basal area of trees significantly increased after 12 years. Structural diversity is an important property of forest stands. Diameter diversity is the most straightforward way for quantifying vertical structure (canopy layering) of a forest stand because diameter is strongly associated with tree height and crown width (Neumann and Starlinger 2001). The regression analysis of relation between DBH and tree density showed the density of trees in the protected stand were higher than in the harvested stand in the all DBH classes. Villela et al. (2006) studied effect of

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selective logging on stand structure in Brazil forests and reported did not differ in stem density and total basal area in logged and unlogged stands, but unlogged stand had more density of large diameter trees and greater mean of canopy height.

Forest managers have been seeking a feasible way to integrate biodiversity issues into management plans. To control forest stand structure may be the most practical way to manage biodiversity in forest ecosystems. The regression analysis of relation between DBH and stand volume showed the trees with DBH of almost 80 cm have the most stand volume in the both harvested and protected stands.

Kia-Daliri et al. (2011) investigated how to marking of trees that will be harvested during selection cutting and its impact on stand structure in a mixed Beech stand in Caspian forest. They reported the most marked and harvested trees were large diameter (DBH > 60 cm), high quality and Beech specimen.

It is now widely accepted that forests should be managed in an ecologically sustainable fashion (Kohm and Franklin 1997; Lindenmayer et al. 2000). Biodiversity is an essential case for life continuance, economical affairs and ecosystems function and resistance (Singh 2002).

Biodiversity measurement is recognized as guidance for conservation plans in local scale. The knowledge of the floristic composition of an area is a perquisite for any ecological and phyto-geographical studies and conservation management activities (Jafari and Akhani 2008).

Forests are among the most diverse and complex ecosystems in the world, providing a habitat for a multitude of flora and fauna. The results of this study indicated the value of biodiversity indices (diversity, evenness and richness) in the protected stand were significantly higher than in the harvested stand. It has been well documented that species composition and diversity can be used as indicators of past management practices in forested areas (Hunter 1999; Kneeshaw et al. 2000). Species richness and diversity are useful indicators of the effects of forest management practices (Nagaike et al. 2006). Species diversity is an important index in community ecology (Myers and Harms 2009). Ecologically sustainable forestry is the practice of land stewardship that integrates growing and harvesting of trees while protecting soil, water, biodiversity and landscape.

In this research effects of timber harvesting on structural diversity and species composition in mixed Beech (Fagus orientalis L.) stands were studied in the Caspian forests of Iran. They are suitable habitats for a variety of hardwood species such as Beech, Hornbeam, oak, maple and Alder. The silvicultural method is single selection cutting and commercial logging is accomplished within the legal framework of forestry management plan in the Caspian forests of Iran. These forests are the most valuable forests in Iran. These forests are known as one of the most basic resources for wood production and have a big share in supplying wood to the related industries. Our suggestion for biodiversity conservation is to leave the tree species that are less dense in these stands, such as Ulmus glabra, Zelkova carpinifolia, Fraxinus coriarifolia and Cerasus avium and logging operation focus on the tree

16 (1): 1-9, April 2015

species that are high density. Diversity of species is correlated to the diversity of their habitats. Marking for trees selection should not be only for harvesting of the wood, but also it should consider the uneven aged structure, keeping the seed trees and their regeneration and the diversity of wood species. The conservation of biological diversity is one of the goals of ecologically sustainable forestry (Lindenmayer et al. 2000). Fully protected areas are often assumed to be the best way to conserve plant diversity and maintain intact forest composition and structure (Banda et al. 2006). Forest protection should aim at ensuring that forests continue to perform all their productive, socio-economic and environmental functions in the future. Forest structure is the important feature in management of forest ecosystems (Zenner and Hibbs 2000; Tavankar 2013).

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B I O D I V E R S IT A S Volume 16, Number 1, April 2015 Pages: 10-15

ISSN: 1412-033X E-ISSN: 2085-4722 DOI: 10.13057/biodiv/d160102

Effect of Alnus subcordata, Acer insigne and Sequoia sempervirens plantations on plant diversity in Hyrcanian forest of Iran ♥

FATEMEH GHEIBI, MOSLEM AKBARINIA , YAHYA KOOCH Faculty of Natural Resources and Marine Sciences, Tarbiat Modares University, 46417-76489, Noor, Mazandaran, Iran.

Tel: + 98-122-6253101 (-3), Fax: + 98-122-6253499, ♥email: akbarim@modares.ac.ir.

Manuscript received: 19 June 2014. Revision accepted: 27 September 2014.

Abstract. Gheibi F, Akbarinia M, Kooch Y. 2015. Effect of Alnus subcordata, Acer insigne and Sequoia sempervirens plantations on plant diversity in Hyrcanian forest of Iran. Biodiversitas 16: 10-15. Forest plantation is a common action in order to restore the degraded forests in Hyrcanian forests of Iran. This study compares the plant biodiversity in four 25-year-old stands of plantation, adjacent understorey of alder (Alnus subcordata C. A. Mey.), maple (Acer insigne Boiss.), sequoia or red wood (Sequoia sempervirens (D. Don) Endl.) and mixed stand (maple and sequoia), located in Salmanshahr of Mazandaran Province, northern Iran. Research carried out in, 10 sample plots with 20m × 20m area which taken by systematic-random in each plantation. All understorey species were identified, recorded and then the biodiversity indices (diversity, richness and evenness) were calculated. Our findings show that the planted species had significant effects on understorey diversity. Statistical comparisons revealed that the highest and lowest diversity (Simpson and Shanon-Winer) and richness (Margalef and Menhinic) indices occurred in sequoia and alder stands, respectively. The evenness indices (Camargo and Smith-Wilson) were significantly greater in maple, sequoia and mixed stands compared with the alder type. As a conclusion, floristic change trends were different according to the planted tree species. A good understanding of the complexity of vegetation processes requires long-term monitoring of vegetation change.

Key words: diversity, evenness, richness, sequoia, understorey.

INTRODUCTION Biodiversity is necessary for mankind life duration, economical issues and for ecosystem stability and function (Singh 2002). Biodiversity is declining at an unprecedented rate and on a global scale. Indeed, loss of ecosystem functions and services associated with such declines has generated international debate (Zhou et al. 2006). Several causes have been identified to explain such loss, including increased land use by an expanding human population (Lambin and Geist 2006) and global climate change (Thuiller 2007). Biodiversity is often used to compare the forest ecosystems the ecological status of forest ecosystems and evaluate the forest communities and ecosystems (Esmailzadeh and Hosseini 2008). Forests support about 65% of the world’s terrestrial taxa (Lindenmayer et al.

2006) and have the highest species diversity for many taxonomic groups including birds, invertebrates and microbes (Lindenmayer et al. 2006). High species diversity in ecosystems led to high food chain and more complex network environment (Lindenmayer et al. 2003). The layers of vegetation in a forest ecosystem support desirable habitats for these taxonomic groups. So forests in the world have the most contribution to biodiversity in terrestrial ecosystems. Loss of native species or alteration and introduction of invasive species through habitat destruction is considerable because of vicinity of forest ecosystems to human population centers (Pilehvar et al. 2010).

Caspian forests of Iran are located in the north of Iran and south coast of Caspian Sea, also known as the

Hyrcanian forests (Takhtajan 1974; Kooch et al. 2014a,b).

These forests cover 1.8 million hectares of land area.

Approximately 60 percent of these forests are used for commercial purposes and the rest of them are degraded.

They are suitable habitats for a variety of hardwood species such as beech, hornbeam, oak, maple, alder, and encompass various forest types including 80woody species (Marvie Mohadjer 2005). Today, the Caspian forests of Iran are depleting rapidly due to population growth, and associated socio-economic problems, industrial development and urbanism (Poorzady and Bakhtiari 2009).

Forest plantation is a common action in order to restore the degraded forests in the Caspian region (Kooch et al. 2012; Mohammadnezhad Kiasari et al. 2013).

Forest plantations are being established at an increasing rate throughout much of the world, and now account for 5% of global forest cover (FAO 2001). Plantations can buffer edges between natural forests and non-forest lands, and improve connectivity among forest patches, which might be important for some populations (Cullen et al.

2004). The primary aim of almost all plantations is the production of large quantities of woodland fiber (e.g. for timber and pulp production). However, there are often important opportunities for biodiversity conservation within plantations (Hartley 2002). Various studies have found that plantations of native or exotic timber species can increase biodiversity by promoting woody understorey regeneration (Carnevale and Montagnini 2002). Plantations promote understorey regeneration by shading out grasses, increasing nutrient status of topsoil (through litter fall), and

GHEIBI et al. – Effect of commercial trees plantations on plant diversity

facilitating the influx of site-sensitive tree species (Cusack and Montagnini 2006).

Numerous studies have shown that the establishment of plantations or restoration plantings on degraded lands can ameliorate unfavorable microclimatic and soil conditions, and provide habitat for seed-dispersing wildlife, there by greatly accelerating natural forest regeneration (Carnus et al. 2006). Previous studies investigated the effect of different land use and also cover on plant biodiversity with different condition (Nagaike 2002; Esmailzadeh and Hosseini 2008; Pilehvar et al. 2010, Taleshi and Akbarinia 2011; Mohammadnejad Kiasari et al. 2013). Here we designed to investigate and compare the plant diversity in the stands of 25-year-old plantation (sequoia, maple, alder and sequoia-maple mixed). The results of this study can be useful for forest plantation and conservation of biodiversity in degraded lands located in northern forests of Iran and same situation. This information also can be used as the database for further research.

MATERIALS AND METHODS Site characteristics The study area is located at the Tilekenar district of Salmanshahr in Mazandaran Province, in the north of Iran, between 36°39'36″N-36°40'01″N and 51°09'55″ E51°10'18″ E at the coast of Caspian sea (Figure 1). Study

stands were located at an altitude of 250 m above sea level and with gentle slope (0-5%). Annual rainfall averages 1300 mm, with wetter months occurring between September and February. In the dry season from April to August, monthly rainfall usually averages less than 40 mm for four months. The soils have textures of loam and clay loam with an acidic pH in the top layers; in the deep layers, soil textures were clay and silty clay and soil pH was less acidic. Previously this area was dominated by degraded natural forests containing native tree species such as Quercus castaneifolia, Zelkova carpinifolia, Parrotia persica, Carpinus betulus, Diospyros lotus and Buxus hyrcana. While 25 years ago after clear cutting (in small areas in degraded natural forests), reforestations have been established (within 3×3 m spaces) in this area with some native species including alder (Alnus subcordata C. A. Mey.), maple (Acer insigne Boiss.), as well as exotic species of sequoia or red wood (Sequoia sempervirens (D. Don) Endl.) and mixed stand (maple and sequoia).

Data collection and diversity measures Research done in, 10 sample plots with 400 m2 (20m×20m) areas taken by systematic-random in each plantation. The entire understorey species were identified, recorded and then the values of diversity (Simpson and Shanon-Wiener indices), richness (Margalef and Menhinic indices) and evenness indices (Camargo and Smith-Wilson indices) were calculated by using PAST and Ecological Methodology software's as follow (Mesdaghi 2001, 2005):

50o 0’0”

Sequoia sempervirens

Caspian Sea

11

Mixed stand Alnus subcordata Acer insigne

Figure 1. Site locations of study area in Mazandaran Province, north of Iran.

12

BIODIVERSITAS

16 (1): 10-15, April 2015

RESULTS AND DISCUSSION ..............................................(1) Where, S is Simpson index; s is the number of species; ni is the number of ith species in sample; N is the number of all species.

..............................................(2) Where, H is Shannon-Wiener index; s is the number of species; PI is the proportion of individuals found in the ith species.

.........................................................(3) Where, R is Margalef index; s is the number of species; N is the number of all species. ................................................................(4) Where, R is Menhinic index; s is the number of species; N is the number of all species.

..................... (5) Where, E is Camargo species evenness indexes; Pi is the ratio of ith species to all species; Pj is the ratio of jth species to all species; S is the number of species.

..(6) Where, Evar is Smith and Wilson index; ni is the number of ith species in sample; nj is the number of jth species in sample; S is the number of all species.

Statistical analysis The normality of the variables was checked by the Kolmogorov-Smirnov test, while Leveneâ&#x20AC;&#x2122;s test was used to examine the equality of the variances. Differences in biodiversity indices (diversity, richness and evenness) among afforested stands were tested with ANOVA Oneway analysis. Duncanâ&#x20AC;&#x2122;s test was used to separate the averages of the dependent variables which were significantly affected by treatment. Significant differences among treatment averages for different parameters were tested at P â&#x2030;¤ 0.05.

A total number of 47 plant species were identified in the studied stands (Table 1).Our findings show that the planted species had significant effects on understorey diversity (Table 2). Statistical comparisons revealed that the highest and lowest diversity (Simpson and ShanonWiner) and richness (Margalef and Menhinic) indices occurred in sequoia and alder stands, respectively (Figure 2a, b , c, d). The evenness indices (Camargo and SmithWilson) were significantly greater in pure maple and sequoia as well as mixed stands compared with the alder type (Figure 2 e,f).

In the early stages after clear cutting due to high intensity light herbaceous plant diversity rapidly increased and sometimes invasive species are dominant (Humphery et al. 2003). Diversity index is the combination of species richness and evenness that have both the species richness and evenness in a quantity collects (Brockway et al. 1998).

Biodiversity in a plantation area increase when trees are cut down to grow seedlings during planting seedlings in a change of fluctuate.

In the present study, the most dominant species in all stands belongs to those after the destruction of the natural area expand sand shows the breakdown of natural ecosystem of the destroyed area (Marvie Mohadjer 2005).

Initially, study area was in the natural forest and slowly become dilapidated due to human influences, and to preventing the process of destruction and human poaching into forest plantation of exotic and native species has been suggested. The destruction of the ecosystem stops and with time recover and return to the natural ecosystem would be require a lot of time finally what is visible the plantation was able to stop the destruction. Various species richness shows that the numbers of plant species in an area are achieved. So far, a large number of species richness, which was invented by the index counts the total number of species (Maguran 1988), as is most celebrated for species richness (Kent and Coker 1992). Our findings showed that the number of species in the stands of sequoia and mixed are more than others, as shown in Figure 3, by the Margalef index. The simple stand most common criterion for assessing species richness of habitats and plant communities is the number of species (Humphrey et al.

1996).

The broken branches in sequoias stand were more detected than other stands that cause more light to penetrate into the stand and may cause a higher diversity in sequoia stand. Dense canopy of alder and maple perhaps is one reason for the low number of species on the forest cover plantation compared to sequoia stand. The result of Fallahchai and Hashemi (2012) research showed that Shanon-Winer diversity index had greater amounts in the Pinus taeda stand than to the other broad-leaved stands. As shown indifferent researches that planting of tree species in a plantation canopy over time, that larger trees are also wider and it would reduce the variation in stand plantation.

Plant diversity will be reduced with closing of canopy cover gradually (Kuksina and Ulanova 2000).Since the sequoia stand that is a species of conifers, its soils are more

GHEIBI et al. â&#x20AC;&#x201C; Effect of commercial trees plantations on plant diversity

acidic than other sand presence of higher percentage of ferns can be a reason for the higher diversity and richness of the stand.

Barbier et al. (2008) in their review study on the effect of tree on herbaceous species diversity and the mechanisms affecting boreal forests also concluded that presence of acidic friendly (Acidophilus) under a canopy of conifers species diversity in these populations will increase. Also, the effect of these have on the soil and encourage more herbaceous plants that are more oriented toward acidic soils to increase some parameters in this stand (Humphery et al.

2002). As alder species belong to those that leaves earlier and shed it after other therefore over the years a massive canopy will be emerge which with the high humidity of the

13

stand can also reduce biodiversity. The numerical value of the indices was not too different, because after 25 years since plantation the plantation covers of different stands become similar to each other.

Table 2. ANOVA for biodiversity indices in the studied stands Biodiversity indices Diversity

F-value Simpson 7.161 Shannon-Wiener 5.426 Richness Margalef 3.374 Menhinic 9.812 Evenness Camargo 11.011 Smith and Wilson 9.331 Note: **Different is significant at the 0.01 level.

Sig. .000** .001** .019** .000** .000** .000**

Table 1. Average percentage of floor coverings in the studied stands.

Scientific name

Sequoia

Maple

Alder

Mixed

Brachypodium pinnatum (L.) P.Beauv.

Carex sylvatica L. Conyza bonariensis (L.) Cronq.

Oxalis corniculata L. Oplismenus undulatifolius (Ard.) P. Beauv.

Calystegia sepium (L.) R.Br. Cyclamen coum Miller.

Primula heterochroma Stapf.

Parietaria officinalis L. Pteris cretica L. Urtica dioica L. Scutellaria tournefortii Benth.

Viola alba L. Fragaria vesca L. Geum urbanum L. Prunella vulgaris L. Hypericum androsaemum L. Polystichum aculeatum (L.) Roth Clinopodium vulgare L. Solanum nigrum L. Stellaria media (L.) Cyr. Cardamine impatiens L. Phytolacca aquatica L. Plantago major L. Hedera pastuchovii Woron.

Danae racemosa (L.) Moench Phyllitis scolopendrium (L.) Newm.

Lamium album L. Sanicula europaea L. Smilax excelsa L. Pteris dentate Forssk Mentha aquatica L. Microstegium vimineum (Trin.) A. Camus.

Carpesium cernuum L. Pimpinella affinis Ledeb Ajuga reptans L. Potentilla reptans L. Tamus communis L. Athyrium filix-femina (L.) Roth Setaria viridis (L.) P. Beauv.

Mercurialis perennis L. Ruscus hyrcanus Woron.

Sambucus nigra L. Rubus persicus Bioss.

Melissa officinalis L. Ilex spinigera (Loes) Loes Unknown

0.1 1.53 0 0 14.94 0 0 0 0 5.26 0 0 1.78 0.12 0 0 0.02 1.9 0 0 0 0 0 0.02 0.14 0 0.04 0 0.38 0.38 0.1 0.2 0.62 0.24 0.22 0.26 0.16 0.04 3.66 0.1 0.06 0.76 0.06 0.08 0 0 0

0.14 1.76 0.18 0.08 15.68 0.2 0.1 0.12 0.54 0.44 0.06 0.04 1.26 0.04 0 0 0 0 0 0 0 0 0 0 0 0 0.04 0 0 0.24 0 0 0.39 0 0 0 0 0 0 0 0 0.78 0 0.04 0 0 0

0 2.57 0 0 51.7 0.64 0 0.24 0 0.82 0 0.12 1.06 0 0.42 0.88 0.04 0.56 0.38 0.1 0.1 0.06 0.13 0 0 0 0 0.26 0 0.36 0 0 0.51 0 0 0 0 0 0.2 0 0 1.26 0.58 0.06 0 0 0

0.06 1.34 0 0 20.64 0.23 0 0 0 11.54 0.04 0 1.31 0.04 0 0 0 1.7 0 0 0 0 0 0 0.08 0.06 0.44 0 0.04 0.36 0 0 2.08 0.2 0 0.3 0.12 0.09 1.6 0 0.44 0.7 1.5 0.4 0.04 0.6 0.4

BIODIVERSITAS

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16 (1): 10-15, April 2015

A

B

C

D

E

F

Figure 2. Average values of Simpson (A) and Shanon-Wiener (B) Margalef (C) and Menhinic (D) Camargo (E) and Smith-Wilson (F) indices for understorey.

Here we designed to investigate and compare the plant diversity in the stands of 25-year-old plantation (sequoia, maple, alder and sequoia-maple mixed). Our findings indicated that the floristic change trends were different according to the planted tree species. It is recommended to preserve biodiversity of the north forest of the country in destructed areas with planting of such species as sequoia mixed with native species. Since, this study examined a 25 year old plantation biodiversity which within this duration numerous species entered and disappeared so it is suggested such studies be conducted to document succession years and biodiversity in this area again in the following years. It is recommended that these trees planted in degraded lands and clear cut areas in small zones.

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Humphery JW, Ferris F, Quine CP (eds). 2003. Biodiversity in Britainâ&#x20AC;&#x2122;s Planted Forests. Forestry Commission, Edinburgh.

Humphrey JW, Davey S, Peace AJ, Ferris R, Harding K. 2002. Lichens and bryophyte communities of planted and semi-natural forests in Britain: the influence of site type, stand structure and deadwood. Biol Conserv 107: 165-180.

Humphrey WF, Dalke A, Schulten K. 1996. VMD-Visual Molecular Dynamics. J Mol Graph 14: 33-38.

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Kooch Y, Hosseini SM, Zaccone C, Jalilvand H, Hojjati SM. 2012. Soil organic carbon sequestration as affected by afforestation: the Darab Kola forest (North of Iran) case study. J Environ Monit 14: 24382446.

Kooch Y, Zaccone C, Lamersdorf NP, Tonon G. 2014b. Pit and mound influence on soil features in an Oriental Beech (Fagus orientalis Lipsky) forest. European J For Res 133: 347-354.

Kuksina N, Ulanova G. 2000. Plant species diversity in spruce forest after clear cutting disturbance: 16 year monitoring in Russian Tajo, proceeding of reforestation and management of biodiversity, Finland, August 21-24.

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Lambin EF, Geist HJ. 2006. Land-Use and Land-Cover Change: Local Processes and Global Impacts (Global Change-The IGBP Series).

Springer, Berlin.

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Mohammadnejad Kiasari Sh, Sagheb-Talebi Kh, Rahmani R, Akbarzadeh M. 2013. Comparison of plants diversity in natural forest and afforestation (Case Study: Darabkola, Mazandaran). J Wood For Sci Technol 19:59-76.

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Taleshi H, Akbarinia M. 2011. Biodiversity of woody and herbaceous vegetation species in relation to environmental factors in lowland forests of eastern Nowshahr. Iranian J Biol 24: 766-777.

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B I O D I V E R S IT A S Volume 16, Number 1, April 2015 Pages: 16-21

ISSN: 1412-033X E-ISSN: 2085-4722 DOI: 10.13057/biodiv/d160103

Plant species diversity among ecological species groups in the Caspian Sea coastal sand dune; Case study: Guilan Province, North of Iran MOKARRAM RAVANBAKHSH1,♥, TAYYEBEH AMINI2, SAYED MOHSEN NASSAJ HOSSEINI1 1

Environmental Research Institute, Academic Center for Education, Cultural Research (ACECR), Rasht, Iran, Tel. 0098-131-3232413, Fax. 0098-131-3242006, ♥email: ravanbakhsh@acecr.ac.ir 2 Agricultural and Natural Resources Research Center of Mazandaran (RCANRM), Noshahr, Iran Manuscript received: 3 August 2014. Revision accepted: 30 September 2014.

Abstract. Ravanbakhsh M, Amini T, Hosseini SMN. 2015. Plant species diversity among ecological species groups in the Caspian Sea coastal sand dune; Case study: Guilan Province, North of Iran. Biodiversitas 16: 16-21. Biodiversity is often discussed in terms of species diversity is concentrated. Species diversity is one of the important characteristics of biological communities and its as a function of the number and size represent populations of species in a special geographic region.The aim of this study was to identification of ecological species groups and investigates the diversity among ecological species groups. The research area comprises a coastal dune system in northern of Guilan Province, Iran. Vegetation sampling was carried out along 22 shore perpendicular transects, approximately 500-m long. A total of 62 plot of 25 square meters were taken in transects. In each sampled plot, the cover percentage value of each species was estimated using Bran-Blanquet scales. Vegetation classified using Two-Way Indicator Species Analysis (TWINSPAN). The comparison of diversity indices among groups were performed with ANOVA test. The results revealed that there were 232 plant taxa and 8 ecological species group in the region. Results of analysis of variance in species diversity indices showed significant differences among the groups in terms of biodiversity indices. The survey of variation in the groups showed that groups 5 and 6 had the highest and groups 1, 2, 3, and 7 had the lowest indices. Checking of the group's position with high diversity in comparison with other groups in this coastal area indicates that the group settled on the coastal land with stabilized soil and proper distance from the sea had higher diversity indices.

Key words: Coastal sand dune, ecological species groups, plant species diversity, Guilan, Iran.

INTRODUCTION Iran is one of the centers of plant diversity is considered old world so that nearly 22 percent of the 8000 plant species of flora are the endemic (Ghahreman 1975).

Despite to endangered state of coastal vegetation in Iran, some fragmented sandy areas are still natural. Some of these separated sandy patches often constitute parts of Caspian coastal ecosystems designed in Ramsar checklist of International Wetlands (Alagol, Ulmagol and Ajigol Lakes, Amirkelayeh Lake, Anzali Mordab (Talab) complex MR., Bujagh National Park, Gomishan Lagoon, Miankaleh Peninsula, Gorgan Bay and Lapoo-Zaghmarz Ab-bandan) and others are considered as part of protected areas, no hunting areas, wildlife refuges, biosphere reserves (Naqinezhad 2012b; Ramsar 2014).

During the last decades, a few studies were conducted on the Flora, identification of vegetation groups and ecological characters in these ecosystems. Most of these studies concentrated in the wetlands on the southern beach of the Caspian Sea (Riazi 1996; Asri and Eftekhari 2002; Ejtehadi et al. 2003; Asri and Moradi 2004, 2006; Shokri et al. 2004; Asri et al. 2007; Sharifnia et al. 2007; Khodadadi et al. 2009). Rarely floristic and vegetation grouping studies were carried out on the southern coastal area of the Caspian Sea (Frey 1974; Amini 2001; Akhani 2003; Ghahreman et al. 2004; Sobh Zahedi et al. 2005, 2007; Naqinezhad 2012b). Ecological species groups differ from

individual indicator species, in that once vegetationenvironment relationships are established the abundance of multiple species of a group may strongly indicate environmental site conditions than the abundance of individual species (Bergeron and Bouchard 1984; Spies and Barnes 1985).

This study was carried out to evaluate the significance of coastal dune habitats for biodiversity conservation. In order to achieve this gold, the vegetation of the nearly unaltered coastal sand dune between Roodsar and Astara, on the southern Caspian Sea, was described by means of ecological species groups and quantitative analysis of the vegetation by diversity indices.

MATERIALS AND METHODS Study area The research area comprises a coastal dune system in northern Guilan Province, Iran, between 48° 52´ 44´´ - 50° 35´ 59´´ E and 36° 56´ 4´´-38° 26´ 55´´ N. The study area was delimited using a Landsat 7ETM satellite image (Path 166/ Row 34) (Figure 1).The Caspian Sea constitutes the southern limit of the study area. The climate is humid and very humid with cool winter according to Eumberger climate classification (Abedi and Pourbabaei 2010). Guilan has a humid subtropical climate with by a large margin the heaviest rainfall in Iran reaching as high as 1,900 mm in

RAVANBAKHSH et al. – Plant diversity in the Caspian Sea coastal sand dune

the southwestern coast and generally around1, 400 mm.

Rainfall is heaviest between September and December because the onshore winds from the Siberian High are strongest, but it occurs throughout the year though least abundantly from April to July. Humidity is very high because of the marshy character of the coastal plains and can reach 90 percent in summer for wet bulb temperatures of over 26°C (Zarekar et al. 2012). Mean annual temperature is 15.8˚C and precipitation is 1506 mm.

Maximum and minimum temperature is 27.8°C in August and 4.1°C in February, respectively. The Alborz range provides further diversity to the land in addition to the Caspian coasts ( Zarekar et al. 2012).

Sampling methods Data collection was performed from May 2011 to May 2012. Voucher specimens were submitted in Guilan University Herbarium (GUH). Prior to the commencement of fieldwork a short reconnaissance survey was undertaken to get an overview of the area (Mashwani et al. 2011). A total of 22 site were selected and one transect was established in each site. For detailed data collection line transect survey was selected which is a very popular vegetation survey technique (Kent and Coker ,1992).

Vegetation sampling was carried out along 22 shore perpendicular transects between 100-500-m long (Figure 1). The length of transects was variable depended on the strip of the natural vegetation. Size of sampling plots was

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determined using nested plot sampling and species/area curve (Muller-Dombois and Ellenberg 1974). A total of 62 sampling areas were selected in stands of vegetation that were homogeneous to the eye in floristic composition and structure (Monserrat et al. 2012). In each sampled plot, the cover percentage value of each species was estimated using Braun-Blanquet scale (Braun-Blaunquet 1964, MullerDombois and Ellenberg 1974).

Data analysis Vegetation analysis method The floristic data matrix consists of 62 plots and 116 species. To classify vegetation types present in the study area, the vegetation data were analyzed using two-way indicator species analysis (TWINSPAN) using PC-ORD version 4.14 (Mc Cune and Mefford 1999).This analysis was used to produce a divisive classification of the stations and plant species matrix (Murphy et al., 2003). This method is a commonly employed program in ecological studies for the classification of vegetation types according to their floristic similarity (Kent and Coker, 1992).Classification was stopped at the fifth level, so that the result in groups would contain sufficient number of samples to characterize each vegetation groups (Vogiatzakis et al. 2003; Khaznadar et al. 2009). The names of identified vegetation types were derived from the dendrogram and importance of more frequent species into each group of plots (Abedi et al. 2011).

Figure 1. Location of Guilan Province in Iran and vegetation sampling in coastal sand dune.

B I O D I V E R S IT A S 16 (1): 16-21, April 2015

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Measuring plant diversity To quantify the diversity of the plant species, The Shannon-Wiener’s (H'), Simpson index (1-D), Margalef ’s richness index (DMg) and Pielou ’s evenness index (E1) were used. The formulas are as follows. s

1  D   Pi2

Pi 

i 1

D Mg 

ni N

Comparison of plant diversity Normality of the data distribution was checked by Kolmogorov -Smirnov test, and Levene’s test was used to examine the equality of the variances. One -way analyses (ANOVA) of variance were used to compare groups with normal distribution data. Duncan test was used to test for significant differences in the species richness, diversity and evenness indices among the groups. This analysis was conducted using SPSS 16.0.

S 1 LnN

RESULTS AND DISCUSSION

s

s

i 1

i 1

H   Pi ln Pi   (Pi) (log pi)

H' = Shannon-Wiener's diversity index, D = Simpson's index, DMg = Margalef 's richness, E1 = Pielou's evenness, Pi = relative frequency of ith species, S = number of species, N = Total individual of species (Ludwig and Reynolds 1988).

Vegetation groups The application of TWINSPAN analysis led to identify vegetation groups associated with the distribution of these plants in the region (Figure 2). The classification was stopped at fifth level of division, leaving only groups with a sufficient number of samples to characterize the vegetation communities. Thus, 62 sampling plots were classified into eight groups. In the first level, 62 sampling plots were divided into two groups (Eigenvalue = 0.536).

The indicator species which have been seen on the left side included: Alnus subcordata, Oxalis corniculata and Scutellaria tournefortii. The indicator species on the right side were Crypsis schoenoides and Argusia sibirica. In

Aln sub (1.9) Oxa cor (0.8) Scu tou (0.7)

Cry sch (22.5) Are sib (12.0)

Con arv (2.0)

Con per (7.0)

Are sib (11.0) Cry sch (15.1)

Oxa cor (8.0)

Phy nod (2,5)

Cen asi (5.0) Cry sch (15.1) Oxa cor (7,1)

Era bar (0.3) Sil lat (1.3)

Pun gra (1,9) Eri cau (3.4)

Jun acu (3.0) Scu tou (3.0)

Rub san (0.4) Tor lep (0.4)

Arg sib (9.2) Cry sch (3.10) Cen ibe (1,6)

Figure 2. Classification of ecological species groups by using TWINSPAN analysis.

Note: Aln sub = Alnus subcordata, Oxa cor = Oxalis corniculata, Scu tou = Scutellaria tournefortii, Cry sch = Crypsis schoenoides, Arg sib = Argusia sibirica, Con arv = Convolvulus arvensis , Phy nod = Phyla nodiflora, Con per = Convolvulus persicus, Cen asi = Centella asiatica, Pun gra = Punica granatum, Ery cau = Eryngium caucasicum, Jun acu = Juncus acutus, Era bra = Eragrostis barrelieri, Sil lat = Silene latifolia, Rub san = Rubus sanctus, Tor lep = Torilis leptophylla, Cen ibe = Centaurea iberica.

RAVANBAKHSH et al. – Plant diversity in the Caspian Sea coastal sand dune

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Table1 1. ANOVA results of diversity indices among groups and mean and standard error of diversity indices.

Diversity index Shanon diversity index Simpson diversity index Margalef richness index Pielou 's evenness index

F

P

3.756 3.074 2.769 2.679

.003* .010* .018* .021*

Mean square .606 .068 1.872 .051

the second level, 35 sampling plots were divided into two groups (Eigenvalue = 0.523) with Convolvulus arvensis (group 1) on the left side as indicator species. Also, on the right side, there was not any indicator species. In this level, 27 sampling plots were divided into two groups (Eigenvalue = 0.585) with Phyla nodiflora and Oxalis corniculata on the left and right side, respectively, as indicator species.

In the third level, 33 sampling plots were divided into two groups (Eigenvalue = 0.499); the indicator species on the left side was Convolvulus persicus (group 2) and there was not any indicator species on the right side. In this level, 18 sampling plots were divided into two groups (Eigenvalue = 0.586). The indicator species on the left side included: Centella asiatica, Crypsis schoenoides and Oxalis corniculata. The indicator species on the left side were Punica granatum and Eryngium caucasicum (group 3). Also, in this level, 9 sampling plots were divided into two groups (Eigenvalue = 0.791). The indicator species on the left side were Juncus acutus and Scutellaria tournefortii (group 4) while on the right side, there was not any indicator species.

In the fourth level, 26 sampling plots were divided into two groups (Eigenvalue = 0.527); the indicator species on the left side were Argusia sibirica and Crypsis schoenoides and those on the right side included: Eragrostis barrelieri, Silene latifolia (group 5). In this level, 12 sampling plots were divided into two groups (Eigenvalue = 0.616); the indicator species were Rubus sanctus × hyrcanus and Torilis leptophylla (group 6) on the right side while there was not any indicator species on the left side. In the fifth level, 21 sampling plots were divided into two groups (Eigenvalue = 0.540) that those indicator species on the left and right side were Argusia sibirica (group7), and Crypsis schoenoides and Centaurea iberica (group8), respectively.

Species diversity among groups First of all, based on Kolmogorov-Smirnov test it should be approved that the data are normal. .For analyzing the diversity among the groups, one-way Analysis of variance (ANOVA) was used. ANOVA results of diversity indices among groups and mean and standard error of diversity indices were listed in Table 1. ANOVA showed that there were significant differences among groups in terms of biodiversity indices (P<0.05).

Duncan's test of groups showed in figure of 3- 6. Figure 3 shows the changes of Shannon-Wiener's diversity index among ecological groups. Maximum of Shannon-Wiener's

df 7 7 7 7

Mean and standard error 1.491±0.662 0.6572±0.236 2.070±0.285 0.6572±0.236

diversity index is in group 6. While there were not significant differences among groups of 5 and 6. The lowest value of Shannon-Wiener's diversity index is in group 3. There were not significant differences among groups of 1, 2, 3, 7 also between groups of 4 and 8. Figure 4 shows the changes of Simpson's diversity index among ecological groups. Group 6 had the highest value of Simpson's diversity index. The lowest value of Simpson's diversity index was in group 3.In this index there were not significant differences among groups of 1, 2, and 7 also between groups of 4 and 8.

Figure 5 shows the changes of Margalef's richness index among groups. The highest value of Margalef's richness was in group 5.and group 1 had the lowest value of Margalef 's richness index. Figure 6 shows the changes of Pielou's evenness index among ecological groups. The highest value of Pielou's evenness index was in group 5.and group 3 had the lowest value of this richness index.

Altogether Duncan's tests showed that among the eight ecological groups, Group 5 and 6 had the highest and Group 1 and 3 had the lowest values in groups.

Discussion In the present study, TWINSPAN analysis was performed to identify plant species groups. Results showed that eight ecological species groups were found in the southwestern coastal sand dune of the Caspian Sea. The vegetation groups in the Caspian coastal regions have been analyzed by different methods such as physiognomic, Braun-Blanquet and multivariate methods led to the identification of these communities and types: Juncus, Rubus, sand dune, halophyte, hydrophyte (Shokri et al.

2004); Plantago indica-Carex nutans, pure Punica, Punica-Rubus, Punica-Juncus, Juncus-Rubus, Frankenia hirsuta-Plantago coronopus (Ejtehadi et al. 2005); Convolvulus persicus-Cakile maritima, Juncus acutus, Trifolium repens-Centaurea iberica, Tamarix ramosissima, Ruppia maritima, Typha latifolia-Phragmites australis, Schoenoplectus litoralis, Nelumbium caspicum, Ceratophyllum demersum-Myriophyllum spicatum (Naqinezhad 2012a), Potamogeton pectinatus, Ceratophyllum demersum-Azolla filiculoides, Nymphaea alba, Nelumbium nuciferum, Phragmites australis, Hydrocotyle ranunculoides, Typha latifolia, Cladium mariscus, Sparganium neglectum, Cyperus sp., Paspalum distichum, Cerastium dichotomum (Asri and Moradi 2006); Frankenia hirsuta, Juncus acutus, Juncus maritimus, Juncus littoralis, Juncus littoralis-Tamarix

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B I O D I V E R S IT A S 16 (1): 16-21, April 2015

Figure 3. Changes in Shannon-Wienerâ&#x20AC;&#x2122;s diversity index among ecological groups.

Figure 4. Changes in Simpson's diversity index among ecological groups.

Figure 5. Changes in Margalef's richness index among ecological groups.

Figure 6. Changes in Pielou's evenness index among ecological groups.

arceuthoides, Salicornia europaea, Tamarix ramosissima, Artemisia tschernieviana, Convolvulus persicus, Tournefortia sibirica, Filaginella sp., Juncus maritimusRubus sanctus, Juncus littoralis-Punica granatum, Juncus littoralis-Rubus sanctus, Mespilus germanica-Punica granatum, Punica granatum, Rhamnus pallasii-Punica granatum, Rubus sanctus-Punica granatum, Saccharum griffithii, Saccharum kajkaiense, Sambucus ebulus, Phragmites australis, Phragmites australis-Juncus acutus, Scirpus lacustris, Typha laxmannii, Typha laxmanniiPhragmites australis (Asri et al. 2007).

These groups, types and communities indicated that some of them belong to Freshwater or marginal freshwater ecosystems those were plentifully found in the wetlands and southern coastal area of the Caspian Sea. Our study showed that the vegetation types included: Convolvulus persicus, Juncus acutus, Punica granatum and Rubus sanctus have been reported in the other studies (Shokri et al. 2004; Ejtehadi et al. 2005; Asri et al. 2007; Naqinezhad 2012a). The following ecological species groups were reported firstly from Iran containing: Convolvulus arvensis, Eragrostis barrelieri-Silene latifolia, Argusia sibirica and Crypsis schoenoides-Centaurea iberica.

As was mentioned in the results, the goal of this research was to investigate diversity indices among ecological groups. The results of the ANOVA and the Duncan's tests showed that the groups differed significantly in terms of biodiversity indices. Checking of the 3-6 Figures indicated that groups 5 and 6, and 1, 2, 3 and 7 had the highest and lowest level of the indices, respectively.

The reason of high diversity in groups 5 and 6 can be interpreted that these groups belong to coastal pasture grass including helliophyta species. Also, the group 6 was a marginal sea shrub cover consisting of some sciophyta species in the under layer of canopy cover and helliophyta species in the open space between shrubs. Also, these two groups were grown on the stabilized soil with appropriate distance from the sea. In contrast, the reasons of the low species diversity in the groups 1, 2 and 7 were perhaps growing in donâ&#x20AC;&#x2122;t fixed sand dunes and being closely to the sea.

In conclusion, diversity is one of the main factors of sustainable management. Identifying plants species of a region and their biodiversity is very effective way to identify disturbance factors and develop recovery plans. It is also essential to maintain a high proportion of native species, create protection programs and preserve the area against human and livestock disturbances. Our knowledge about plant biodiversity of the southern Caspian coast is fragmentary and requires in-depth studies to reveal all of its components. The southern Caspian coast and their sand dune went through extensive man-made changes during the past decades. The growth of population followed by the land usage changing for agriculture and urban utilizing, has affected on the biodiversity. The loss of genetic and species diversity by the destruction of natural habitats would need to restore many years. It is necessary to establish certain laws and regulations in order to protect all plants species and ecosystems.

RAVANBAKHSH et al. – Plant diversity in the Caspian Sea coastal sand dune

ACKNOWLEDGEMENTS We are indebted to Dr. S.H. Saiedie, F. Vahdati and R. Shahi from Guilan University, Iran for their helps during the field studies. We would also like to thank the Environmental Research Institute, Academic Center for Education, Cultural Research (ACECR), Iran for their financial support.

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Ghahreman A. 1975. The Species Eliminated from the Native Vegetation of Iran; Proceeding of First seminar on problems of the Natural vegetation of Iran.Tehran, June 29-July 2 1975. [Iran] Ghahreman, A, Naqinezhad AR, Attar F. 2004. Habitats and Flora of the Chamkhaleh-Jirbagh Coastline and Amirkelayeh Wetland. J Environ Stud 33 (1): 46-67.

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Kent M, Cocker P. 1992. Vegetation description and analysis: A practical approach. Belhaven Press, London.

Khaznadar M, Vogiatzakis IN, Griffiths GH. 2009. Land degradation and vegetation distribution in Chott El Beida wetland. Algeria J Arid Environ 73: 369-377.

Khodadadi S, Saeidi Mehrvarz S, Naqinezhad AR. 2009. Contribution to the Flora and Habitats of the Estil Wetland (Astara) and Its Surroundings, Northwest Iran. Rostaniha 10 (1): 44-63.

Mashwani ZUR, Arshad M, Ahmad M, Khan MA. 2011. Diversity and distribution pattern of alpine vegetation along Lake Saif-ul-Mulook, Western Himalaya, Pakistan; Proceeding of International Conference on Environmental, Biomedical and Biotechnology. Shanghai 19-21 August 2011. [China] Mc Cune B, Mefford M. 1999. Multivariate Analysis of Ecological Data, version 4.17. M.J.M. Software. Glenden Beach, Oregon, USA. Monserrat AL, Cintia E. Celsi, and Sonia L. Fontana . 2012. Coastal Dune Vegetation of the Southern Pampas (Buenos Aires, Argentina) an Its Value for Conservation. J Coast Res 28 (1): 23-35 Muller-Dombois D, Ellenberg H. 1974. Aims and Methods of Vegetation Ecology. John Wiley, New York.

Murphy KJ, Dickinson G, Thomaz SM, Bini LM, Dick K, Greaves K, Kennedy MP, Livingstone S ,McFerran H, Milne JM, Oldroyd J, Wingfield RA. 2003. Aquatic plant communities and predictors of diversity in a sub-tropical river floodplain: the upper Rio Parana.

Brazil. Aquat Bot 77: 257-276.

Naqinezhad A. 2012a. A Physiognomic-Ecological Vegetation Mapping of Boujagh National Park, the First Marine-Land National Park in Iran. Advances Bio Res 3 (1): 37-42.

Naqinezhad AR. 2012b. A Preliminary Survey of Flora and Vegetation of Sand Dune Belt in the Southern Caspian Coasts, N. Iran. Res J Biol 2 (1): 23-29.

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Department of Environment, Tehran, Iran.

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B I O D I V E R S IT A S Volume 16, Number 1, April 2015 Pages: 22-26

ISSN: 1412-033X E-ISSN: 2085-4722 DOI: 10.13057/biodiv/d160104

Short Communication: Note on Excoecaria indica (Willd.) Muell.-Arg, 1863 (Euphorbiaceae), from the Andaman and Nicobar Islands, India; a data deficient species PADISAMY RAGAVAN1,â&#x2122;Ľ, K. RAVICHANDRAN2, P.M. MOHAN3, ALOK SXAENA4, R.S. PRASANTH1, R.S.C. JAYARAJ5, S. SARAVANAN1 1

Institute of Forest Genetics and Tree Breeding, R.S.Puram, P.B. No 1061, Coimbatore 641002, Tamil Nadu, India.

Tel. +91-422-2484100, Fax. +91-422-2430549, â&#x2122;Ľemail: van.ragavan@gmail.com.

2 Department of Environment and Forest, Andaman and Nicobar Administration, Port Blair, A & N Islands, India.

3 Department of Ocean studies and marine Biology, Pondicherry University, Brookshabad Campus, Port Blair, A & N Islands, India.

4 Indira Gandhi National Forest Academy, Dehradun, Uttarakhand, India.

5 Department of Environment and Forest, Arunachal Pradesh, India.

Manuscript received: 1 October 2014. Revision accepted: 27 October 2014.

Abstract. Ragavan P, Ravichandran K, Mohan PM, Sxaena A, Prasanth RS, Jayaraj RSJ, Saravanan S. 2015. Note on Excoecaria indica (Willd.) Muell.-Arg, 1863 (Euphorbiaceae), from the Andaman and Nicobar Islands, India; a data deficient species. Biodiversitas 16: 22-26. Excoecaria indica (Wild.) Muell.-Arg was recorded from Middle Andaman and Great Nicobar Island representing a new addition to the mangrove flora of the, Andaman and Nicobar islands. This species is characterized by its thorny trunk, crenulatelanceolate leaves and cherry-sized green fruits containing three seeds. Information about E. indica is inadequate, and it is recognized as data deficient species. Further studies and conservation measures are imperative for managing the mangrove diversity of the islands with regards to this species.

Key words: Andaman and Nicobar Islands, Excoecaria, India, new records.

INTRODUCTION Mangrove forests are unique plant communities of the critical interface between terrestrial, estuarine, and nearshore marine ecosystems in tropical and subtropical regions (Polidoro et al. 2010; Hanum et al. 2013). Despite its ecological and economical values, globally mangrove areas are disappearing at the rate of approximately 1% per year (FAO 2003, 2007). The mangroves of Andaman and Nicobar (A & N) Islands are probably the best in India in terms of its density and growth (Mandel and Naskar 2008, Dagar et al. 1991). According to the latest estimate by the Forest Survey of India (Anon. 2013), the total mangrove area is about 4,628 km2 in India, out of which, 604 km2 occur in A & N Islands. Out of this 601 km2 is in Andaman and only 3 km2 found in Nicobar Islands. Total 13 km2 area of mangrove stands has degraded as a consequence of massive earthquake and subsequent tsunami of 2004 in Andaman Islands in comparison with 2011 (Anon. 2013).

However, after the tsunami distribution of some mangrove species, not recorded earlier in ANI, are reported (Dam Roy et al 2009; Nehru and Balasubramanian 2012; Goutham-Bharathi et al. 2012; Ragavan et al. 2014). The present study also adds the new distributional record of Excoecaria indica (Wild.) Muell.-Arg from this island.

The tropical indo-pacific genus Excoecaria L. belongs to the family Euphorbiaceae Juss., and includes several mangrove representatives (Duke 2006). It is distinguished

from related genera by a combination of characters including dioecious condition, axillary inflorescences, male flowers with 3 stamens, and the absence of a caruncle from the seed (Tomlinson 1986). The genus includes up to 40 species in the Indo-west pacific region, from tropical Africa and Asia to the western pacific. Two species occur in mangroves, including Excoecaria indica (Wild.) Muell.Arg and Excoecaria agallocha L. In India E. indica is rare, classified as critically endangered (Kathiresan 2008) and known to occur only in Sundarban and Kerala (Kathiresan 2008). E. indica is categorized by the IUCN as data deficient (Polidoro et al. 2010), due to lack of adequate information. In contrast, E. agallocha is abundant, known to occur on both east and west coasts of India except Gujarat and it is among the mangrove species which is considered to be in the IUCN category of lesser risk (Kathiresan 2008). Though Goel and Chakrabarthy (1990) reported the occurrence of E. indica from Andaman Islands, its occurrence was not reliable as other authors like Dagar et al. (1991) and Kathiresan (2008) reported that E. indica was not found in ANI. Moreover, authentic herbariums are not available for the occurrence of E. indica in ANI. Recent floristic surveys revealed the occurrence of Excoecaria indica from Middle Andaman and Great Nicobar Island, which we report herein as a new distribution record in the ANI along with a detailed taxonomical description as follows.

RAGAVAN et al. – Excoecaria indica of the Andaman and Nicobar Islands

TAXONOMIC TREATMENT Key to Excoecaria species in ANI 1. Leaf margin serrated, bark smooth, trunk with thrones, monoecious, fruits rounded with three seeds ......…. E. Indica 2. Leaf margin entire to wavy, bark fine fissured with lenticels, dioecious, fruits three lobed .........................….. E. agallocha

Description Excoecaria indica (Willd.) Muell.-Arg. Excoecaria indica (Willd.) Muell.-Arg, Airy Shaw, H.K., The Euphorbiaceae of Borneo. Kew Bulletin Additional Series IV, Her Majesty’s Stationaery Office, London, 245 pp. (1975); Aksornkoae, S. Ecology and Management of Mangroves. IUCN Wetlands Programme.

IUCN, Bangkok, Thailand, 176 pp. (1993); Heyne, K. De Nuttige Planten van Indonesië. Publishers W. vanHoeve, TheHague/Bandung, 2 volumes, 1660 pp. & CCXLI. (1950); Ng, P.K.L and Sivasothi, N. eds. A guide to the Mangroves of Singapore. Volume 1. The Ecosystem and Plant Diversity. Singapore Science Centre, Singapore, 160 pp. (1999); Tomlinson, P.B. The Botany of Mangroves.

Cambridge University Press, Cambridge, U.K., 419 pp.

(1986). (Figure 1).

Tree 10-15m, high with upright branches and more or less drooping twigs; thorny trunk without buttress; bark fissured; greyish brown in colour; no above ground roots.

Leaves simple, alternate, elliptic or lanceolate, 7-14 x 3-4 cm, base obtuse, margins conspicuously serrate, apex subacuminate to acuminate with two small glands at the base of the blade; lateral veins 18-24; petiole 7-20 mm long and reddish. Inflorescences solitary, raceme-like, to 10 cm, axis pilose, petal absent ; flowers unisexual, male or staminate flowers are numerous with 3 prominent stamens; stamen filaments 0.5-0.6 mm at anthesis, nearly absent in bud; anthers 0.4-0.5 mm; female or pistillate flowers are 1or 2; solitary with 3 long styles; styles ca. 1.5 mm; stigmas 4-6 mm. Fruit is a round, woody capsule, 2.5-3 cm in diameter, green ; black when dries, 3-seeded. Fruit stalk 8-22mm.

Seeds 11-13 by 7-8.5 mm, keeled on the back, medium to pale brown, not spotted, without caruncle.

Distribution. From south and east India throughout Southeast Asia to the Solomon Islands. In Southeast Asia so far not (yet) recorded in the Philippines. In India it was known from west Bengal and Kerala In this study it was recorded first from ANI at Panchwati and Campbell bay.

Habitat and ecology. The distribution and habitat ecology of E. indica is poorly known. Generally it is known to occur in primary Nypa forest in sea water, tidal river banks and sea shores (Giesen et al. 2006) and in primary and advanced secondary forests of swampy and seasonally inundated places (Esser 1999). Also occurs in freshwater swamp forests, along rivers and in evergreen lowland forest up to an altitude of 250 m.

Phenology. Flowering: March to June; Fruiting: July to October.

Specimen examined. India, Middle Andaman, Rangat, Betapur (92o57’39.7” E, 12o34’31.3”N) 6/9/2013 P.Ragavan (s n 30758PBL). 16 trees were observed in the

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muddy soil, along the tidal creek on the landward margin of mangroves in association with Heritiera littoralis, Sonneratia griffithii and Dolichandrone spathacea.

Excoecaria agallocha L. Excoecaria agallocha L.; Linnaeus, C. von, Systema Naturae ed. 10: 1288 (1759). Type: Tropical Asia, Malaya and Pacific Islands. Ng, P.K.L and Sivasothi, N. eds. A Guide to the Mangroves of Singapore. Volume 1 The Ecosystem and Plant Diversity. Singapore Science Centre, Singapore, 160 pp (1999). (Figure 2) Tree up to 10-15m high, columnar or spreading, mutlistemmed, dioecious, deciduous; bark grey, rough, fissured on maturity, pustular with lenticels. Stem simple, occasionally slight buttresses. Root above ground, serpentine like. Leaves simple, alternate, elliptic, green, apex acute to acuminate, base cuneate, margin variably serrate to entire, 5-15cm L, 2-6cm W, L/W ratio is >2; petiole green, terete 1-3.5cm L, 0.2cm W. Inflorescences axillary; male inflorescence 5-10cm L, flowers arranged spirally with bract, calyx lobes 3; stamens 3, yellow, 0.30.5cm L, pistillode absent ; Female inflorescence 2-4cm L, peduncle 0.5cm L, bract glandular, basal bracteoles 2, calyx lobes 3, staminodes absent, ovary tri-locular; styles 3, 0.3cm L. Fruit 3-lobed capsule, 1-1.5cm W, green becoming brown on maturity, three seeded; seeds are rounded, black or dark brown, streaked, up to 0.5cm W. Distribution. Occurs in the Asian tropics, from India and Sri Lanka throughout Southeast Asia, to southern China, Taiwan, Southern Japan, Australia and the west Pacific. Except Gujarat it was known to occur in other places. In ANI it was recorded from both Andaman Islands and Nicobar islands.

Habitat and Ecology. It is commonly found on the landward margin of mangroves but in Andaman Islands this species was observed from downstream to upstream estuarine position and mid and high intertidal regions. Like other common mangrove species this is also flooded by tidal fluctuations.

Phenology. Flowering: June to August; Fruiting: September to November.

Specimen examined. India, Middle Andaman, Rangat, Betapur (92o57’39.7” E, 12o34’31.3”N) 6/9/2013 P.Ragavan (s n 30935 & 30936PBL).

Discussion Both E. indica and E. agallocha are characterized with simple leaves, absences of petals, trilocular ovary with 1 ovule per loculus, inarticulate laticifers with white latex, and biglandular bracts on the inflorescences (Tomlinson 1986). However, E. indica is distinguished from E. agallocha by its thorny trunk, crenulate-lanceolate leaves and green fruits the size of cherry with three seed (Duke 2006) and most importantly E. indica is monoecious whereas E. agallocha is dioecious (Table 1). Though it is native to India, Biswas et al. (2007) categorized it as a weed with potential environmental impacts to the Sundarbans because it accumulates allelopathic toxins in the soil, affecting biota, including poisoning of animals.

The present record of E. indica from Andaman and Nicobar

B I O D I V E R S IT A S 16 (1): 22-26, April 2015

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A \

C \

B

D \

E \

G \

H \

F \ F \

I

Figure 1. Excoecaria indica. A. Habitat, B. Lanceolate leaves with serrated margin, C. Characteristic thorny trunk, D. Cat-kin like inflorescences, E. Male flowers, F. Female flower, G. Small green globose fruit, H. Mature fruit black in color, I. Cross section of fruit showing three locus arrangement.

RAGAVAN et al. â&#x20AC;&#x201C; Excoecaria indica of the Andaman and Nicobar Islands

A \

B \

C \

F \

25

D \

G \

H

E \

I

Figure 2. Excoecaria agallocha. A. Habit, B. Leafy rosette, C. Male inflorescences, D. Young male inflorescences, E. Male flowers with three stamens, F. Female inflorescences, G. Female flowers with three style, H. Three lobed fruits, I. Bark.

B I O D I V E R S IT A S 16 (1): 22-26, April 2015

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Table 1. Key diagnostic characters of Excoecaria spp in ANI Characters Leaf shape Leaf apex Leaf base Leaf margin Bark Inflorescences Male inflorescence Female inflorescence

E. agallocha Broadly elliptic to lanceolate Acute to acuminate Attenuate Variably serrate to entire Pale grey, finely fissured, trunk smooth Axillary, Male and female inflorescences on different individual Long, flowers with three long stamens Small, flowers with three style

Fruits

Three lobed, three seeded

Islands represents a new addition to the mangrove flora of the islands, and highlightens its floristic affinities with Southeast Asian countries. Further, it suggests the need for periodical floristic survey to update information on the extent and status of mangroves in the Andaman and Nicobar Islands. Since E. indica is categorized as data deficient, immediate efforts should be taken to generate information about its habitat ecology, distribution and conservation.

ACKNOWLEDGEMENTS We are highly indebted to Director General, Indian Council for Forestry Research and Education for providing necessary support under NFRP. Extremely Grateful to the Principal Chief Conservator of Forests, Andaman and Nicobar Islands for his guidance and ensuring necessary support from field. Appreciate the cooperation and support provided by the CCF (Research and Working Plan), CCF, Territorial Circle and all the Divisional Forest Officers and their staff in the Department of Environment and Forests, Andaman and Nicobar Administration. Thanks are due to Dr. N. Krishnakumar, IFS, Director, Institute of Forest Genetics and Tree Breeding, Coimbatore for his support and encouragement. Special thanks are extended to DFO Campbell Bay for constant support.

REFERENCES Anon. 2013. Status of forest report. Forest Survey of India (FSI), Dehra Dun. Biswas SR, Choudhury JK, Nishat A, Matiur Rahman Md. 2007. Do invasive plants threaten the Sundarbans mangrove forest of Bangladesh?. For Ecol Manag 245 (1-3): 1-9.

Dagar JC, Mongia AD, Bandhyopadhyay AK. 1991. Mangroves of Andaman and Nicobar Islands, Oxford and IBH, New Delhi.

E. indica Lanceolate Acuminate Cuneate Serrated Grayish brown, fissured, trunk with prominent thrones Axillary, male and female flowers on same inflorescences Numerous with three long stamens One or two at the base of inflorescences with prominent style.

Rounded, three seeded

Dam Roy S, Krishnan P, George G, Kaliyamoorthy M, Goutham Bharathi MP. 2009. Mangroves of Andaman and Nicobar Islands. CARI, Port Blair Duke NC. 2006. Australia’s Mangroves: the Authoritative Guide to Australia’s Mangrove Plants. The University of Queensland and Norman C. Duke, Brisbane.

Esser HJ. 1999. A partial revision of the Hippomaneae (Euphorbiaceae) in Malesia. Blumea 44: 149-215 FAO. 2003. Status and trends in mangrove area extent worldwide. In: Wilkie ML, Fortuna S, eds; Forest Resources Assessment Working Paper No. 63. Rome: Forest Resources Division, FAO. FAO. 2007. The World’s Mangroves 1980-2005, FAO Forestry Paper 153. Forest Resources Division, FAO, Rome.

Giesen W, Wulffraat S, Zieren M, Scholten L.2006. Mangrove Guidebook for Southeast Asia. FAO and Wetlands International. RAP Publication, Bankok, Thailand.

Goel AK, Chakrabarty T. 1990. Excoecaria indica new record Euphorbiaceae new for Andaman Islands India. J Econ Tax Bot 14 (3): 738.

Goutham-Bharathi MP, Kaliyamoorthy M, Dam Roy S, Krishnan P, Grinson George, Murugan C. 2012. Sonneratia ovata (Sonneratiaceae)－A new distributional record for India from Andaman and Nicobar Islands. Taiwania 57(4): 406–409 Hanum F, Latiff A, Hakeem KL, Ozturk. M (eds.). 2013. Mangrove Ecosystems of Asia. Springer, Berlin.

Kathiresan K. 2008. Biodiversity of Mangrove Ecosystems. Proceedings of Mangrove Workshop. GEER Foundation, Gujarat, India.

Mandal RN, Naskar KR. 2008. Diversity and classification of Indian mangroves: a review. Trop Ecol 49: 131-146 Nehru P, Balasubramanian P. 2012. Sonneratia ovata Backer (Lythraceae): status and distribution of a Near Threatened mangrove species in tsunami impacted mangrove habitats of Nicobar Islands, India. Journal of Threatened Taxa 4(15): 3395–3400.

Polidoro BA, Carpenter KE, Collins L, Duke NC, Ellison AM, Ellision JC, Farnsworth EJ, Fernando ES, Kathiresan K,. Koedam NE, Livingstone SR, Miyagi T, Moore GE, Vien Ngoc Nam, Ong JE,.

Primavera JH, Salmo SG, Sanciangco JC, Sukardjo S, Wang Y, Yong JWH. 2010. The loss of species: Mangrove extinction risk and geographic areas of global concern. PLoS ONE 5 (4): 1-10.

Ragavan P, Ravichandran K, Mohan PM, Sxaena A, Prasanth R S, Jayarai RSC, Saravanan S. 2014. New distributional records of Sonneratia spp. from Andaman and Nicobar Islands, India. Biodiversitas 15: 250259 Tomlinson BP. 1986. The Botany of Mangroves. Cambridge Univ. Press, Cambridge.

B I O D I V E R S IT A S Volume 16, Number 1, April 2015 Pages: 27-43

ISSN: 1412-033X E-ISSN: 2085-4722 DOI: 10.13057/biodiv/d160105

Ferns and fern allies of District Shopian, Kashmir Valley, India SHAKOOR A. MIR1,♥, ANAND K. MISHRA1, SHAUKET AHMAD PALA2, ZAFAR A. RESHI2, M.P. SHARMA1 1

Department of Botany, Jamia Hamdard, Hamdard Nagar, New Delhi-110062, India. Tel: +91-9968172445; ♥email: shakoorsam@gmail.com.

2 Department of Botany, University of Kashmir, Hazratbal, Srinagar-190006, Jammu and Kashmir, India Manuscript received: 11 November 2014. Revision accepted: 9 December 2014.

Abstract. Mir SA, Mishra AK, Pala SA, Reshi ZA, Sharma MP. Ferns and fern allies of District Shopian, Kashmir Valley, India Biodiversitas 16: 27-43. Shopian, recently created hilly district of Kashmir valley, Jammu and Kashmir is surrounded by the lofty mountains of Pir-Panjal range. More than half area of district is occupied by different forests, subalpine, alpine and mountainous zones.

Great altitudinal variation, adequate rainfall, high forest cover, large number of streams, springs and topographic variations render the district worthy for supporting rich fern flora. Therefore, the current study was aimed to undertake in-depth systematic survey of different habitats of Shopian for the collection of diversity of pteridophytes. Specimens were collected during 2010, 2011 and 2012 growing seasons from June to November. A total 81 species of ferns and fern allies belonging 27 genera and 11 families were reported. The dominant families of the region are Dryopteridaceae (25 species) followed by Woodsiaceae (16 species), Aspleniaceae (13 species) and Pteridaceae (12 species). Similarly, the dominant genera collected from here are Dryopteris (14 species), Asplenium (13), Polystichum (11 species) and Athyrium (6 species). A list of the fern and fern allies, along with update nomenclature, their selected Synonym, diagnostic features, distributional and ecological notes have been provided here.

Key words: distribution, diversity, ferns, Kashmir valley, lycophytes, Shopian, survey.

INTRODUCTION Pteridophytes comprise a group of seedless but spore producing plants, formed by two lineages, lycophytesfronds with no leaf gap in the stem stele (Lycophylls) and Monilophytes or ferns- fronds with leaf gap in the stem stele (Euphylls) (Pryer et al. 2001, 2004; Smith et al. 2006).

They constitute an important component of earth’s flora for millions of years (Pryer et al. 2001). Presently there are about 300 genera of pteridophytes containing approximately 9600 ferns and about 1400 lycophytes worldwide (Smith et al. 2006, 2008), with highest diversity in the tropics (Jacobsen and Jacobsen 1989; Kornas 1993; Linder 2001). The current revised treatment of fern and fern allies from India revealed 1150 species (Fraser-Jenkins 2008; Fraser-Jenkins and Benniamin 2010). The west Himalayas is the fourth richest area for pteridophytes in India after the east Indo-Himalayas, the Manipur-Khasi range and south India, and harbours about 402 species, which constitute 40%, over one-third of pteridoflora of whole country (Kumari et al. 2010). Kashmir Himalaya, a picturesque south Asian region, is unique biospheric unit located in the northwestern extreme of the Himalayan biodiversity hotspot (Rodgers and Panwar 1988).

One of the main features contributing to the worldwide fame of Kashmir is the rich biodiversity that decorates its captivating landscape (Lawrence 1895). Owing to the vast variety of edapho-climatic and physiographic heterogeneity and diverse habitats including lakes, springs, swamps, marshes, rivers, cultivated fields, orchards, subalpine and alpine meadows, mountain slopes and terraces, permanent glaciers, etc., the valley harbors almost all groups of land

plants; many species are distinct from those in the rest of the country and are endemic to this region. However, the published literature on the flora of Kashmir reveals that only the Phanerogams have been well documented. The cryptogams, particularly pteridophytes have met little attention in the past with regards to their survey and inventorization (Dar et al. 2002).

The valley has been occasionally explored in the past for pteridophyte diversity by Clarke (1880), Beddome (1883, 1992) and Hope (1899-1904). Some isolated fern collections have also been made in this region by the botanists of botanical survey of India, like R.B. Keshavanand, T.A. Rao, P.K. Hajra, U.C. Bhattacharayya, B.M. Wadhwa, M.V. Vishwanathan, etc. (Wani et al. 2012) The other studies particularly relevant to the pteridophytic flora of Kashmir are of Stewart (1945, 1951, 1957 1972, 1984); Javeid (1965), Kaul and Zutshi (1966, 1967), Kapur and Sarin (1977), Dhir (1980), Kapur (1985), Khullar (1984, 1994, 2000), Razdan et al. (1986); Khullar and Sharma (1987), Fraser-Jenkins (1989, 1991, 1992, 1993, 1997, 2008) and Singh and Pande (2002). Recently, an upto-date account of fern and fern allies has been published collectively for Kashmir valley, Gurez (Kishanganag valley) and Ladakh by Wani et al. (2012) yet representing only 106 taxa of fern and fern allies. The present authors have, therefore, undertaken to explore in-depth pteridophytic wealth and possibility of new records in the Kashmir valley, and have restricted their study to district Shopian, which possesses the majority of topographical features of the large expanse of valley.

B I O D I V E R S IT A S 16 (1): 27-43, April 2015

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MATERIAL AND METHODS Shopian, one of the ten districts of Kashmir division of Jammu and Kashmir State (Figure 1) is located in the south and south-west extremity of Kashmir valley within the coordinates of 330 20′ and 340 54′ North latitude and 730 35′ and 750 35′ East longitude. The district has been created in 2006. Total area of the region is 812.70 km2, of which more than half about 442.98 km2 is occupied by alpine zone with a considerable portion under meadowlands and glaciers. The forest cover of different forests viz, deodar, kail, fir and broad leaved forests is approximately 316.60 km2. The temperature ranges from an average daily maximum of 31ºC and minimum of 15ºC during summer to an average daily maximum of 4ºC and minimum of – 4ºC during winter (Bhat et al. 2012). The long and cold winter season is dominated by the western atmospheric depressions called Western Disturbances (Jeelani et al.

2010), which cause snowfall and rainfall. The annual precipitation of is about 1,050 mm; by and large in the form of snow during the winter months and rain during the late winter and early spring (Raza et al. 1978). According to Raza et al. (1978) the district possesses rich soil diversity, having three major categories of soils namely hill soils, alluvial soils and Karewa soils. With the gradual ascending slope from its north and north-eastern sides and loft Pir Panjal mountain range falling on its south and south-west periphery, the district has most of its areas of hilly character with the altitudinal range varying from 1600 to 4562 meters. Most of the vegetation met in this

region is deciduous type. The low land zone is mostly under cultivation and habitation, supporting crop fieldskharief crop fields, orchards, kitchen gardens and natural fields- roadsides, pastures, grasslands, graveyards, rocky meadows, streams, waste-lands, ponds, small ditches and spring sites. Mountain zone which is the maximum portion of the district includes broad-leaved deciduous forests, mixed conifer forest, kail forests, scrub forests, meadows and bare snow laden mountains. Each of these habitats has a peculiar type of vegetation, thus making the whole district biologically diverse. All these remarked features support a rich flora of cryptogams, particularly pteridophytes. However, during the last decades the floras published from this region including Flora of Pulwama (Kashmir) (Navchoo and Kachroo 1995) reported only flowering plants and therefore, no keen interest has been laid on Pteridophytic flora and as such no published pterido-flora is available from this district.

Diverse habitats such as plains, streams, rivulets, rivers, small valleys, dense forests, meadows, sub-alpine, alpine, mountains etc. of study area were surveyed for the collection of fern diversity during the years 2010 – 2012.

The major research sites are shown in Figure 1. Each specimen was photographed, numbered as it was collected and the detailed notes were entered in the notebook. The collected specimens were pressed and dried in the standard way and described and identified with the relevant literature (Beddome 1976, 1983; Clarke 1880; FraserJenkins 1989; Khulllar 1994, 2000; Ghosh et al. 2004). The identity of specimens was authenticated from Botanical

1 9 8 10 11

INDIA

13 14

7 16 15

12 15

6

4 3

2

KASHMIR Figure 1. Research sites in district Shopian of Jammu and Kashmir State, India. 1. Zainapora; 2. Kaunsernag; 3. Huran; 4. Secjan; 5.

Reshi Nagar; 6. D. K. Pora; 7. Narwani; 8. Imamsahib; 9. Rambi Ara; 10. Rambi Ara; 11. Heerpur; 12. Sedow; 13. Dubjan; 14. Peergali; 15. Aharbal; 16. Lahanthoor.

MIR et al. â&#x20AC;&#x201C; Pterydophytes of Shopian, Kashmir Valley

Survey of India, Northern Circle, Dehradun. One set of voucher specimens are deposited in Department of Botany, Jamia Hamdard and another in the Herbarium, Centre for Biodiversity and Plant Taxonomy, University of Kashmir (KASH). The identified plants are listed according to the classification system of Smith et al. (2008), followed by selected Synonym, habitat and taxonomic notes, range of distribution and Specimen examined

RESULTS AND DISCUSSION An interesting feature of the Shopian is occurrence of varying topography and great altitudinal range that develops immense habitat variation, that facilitated luxurious growth of pteridophyte flora and therefore, as many as 81 pteridophyte species belonging to 27 genera, 11 families, 4 orders and 2 classes were collected from here.

The richest fern families of this area are Dryopteridaceae sharing 25 species followed by Woodsiaceae (15 species), Aspleniaceae (13 species) and Pteridaceae (12 species).

Besides these, the families Thelypteridaceae and Equisetaceae have 5 and 3 species, respectively; Marsileaceae and Osmundaceae are represented by 1 species each and Dennstaedtiaceae, Polypodiaceae and Salviniaceae with 2 species each. Similarly, the dominant genera reported from here are Dryopteris (14 species), Asplenium (13 species) and Polystichum (11 species).

The collected species also showed a range of ecological distribution varying from aquatic (Marsilea, Azolla) to terrestrial (Athyrium, Dryopteris) to lithophytic (Lepisorus, some species of Asplenium); and from low-land to forests to sub-alpine to alpine zones. Except for some lowland ferns, namely Dryopteris filix-mas and D. pulvinulifera that mature fairly earlier, the appropriate time of maturation is from mid-June to ending October. Within these 3-4 months, the ferns produce sori and complete their life cycle. However, Equisetum arvense produce cone i.e. reproductive stage earlier than the vegetative stage from earlier April to mid-May. Based on personal observation and data available from literature (Stewart 1972; Khullar 1994, 2000; Wani et al.

2012), a rough estimate of status of collected taxa has also been figured. The most common species of the area are Adiantum venustum, Asplenium trichomanes, A. pseudofontanum, Athyrium attenuatum, A. mackinnoni, Deparia allantodioides, Equisetum arvense, Polystichum prescottianum, Pseudophegopteris levingei. On the contrary, Adiantum pedatum, Asplenium viride, Athyrium strigillosum, Cyclosorus erubescens, Cryptogramma stelleri, Lepisorus clathratus, L. stewartii, Onychium plumosum, Pellaea nitidula, Polystichum lonchitis and Woodsia alpina have been reported only with 1-3 specimens, whereas other species range in status from occasional to frequent. Out of 81 taxa, 29 taxa were recorded to be rare and just only 3 taxa, namely Adiantum venustum, Asplenium trichomanes, and Equisetum arvense were abundant in this region.

The pteridophyte species were reported within the altitudinal range of 1600 to 4100 m. 51 species were found

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growing luxuriously between 1600-2500; 18 were reported within the range of 2500-3200 m; and 12 were found within the altitudinal range of 3300-4100 m. However, Adiantum venustum, Asplenium adiantum-nigrum, A. trichomanes, Athyrium mackinnoni, Cystopteris fragilis and Equisetum arvense were collected throughout the ranges of altitudes and were numerically the most abundant and wide-spread species among all pteridophytes recorded from the region. The species Asplenium viride, Cryptogramma brunoniana, C. stelleri, Lepisorus clathratus, Polystichum lachenense, P. shensiense and Woodsia alpina were rarest and restricted to higher altitudes in alpine meadows and rocky mountains.

As per the distribution of pteridophyte species recorded

by Dixit (1984), Chandra (2000) and Khullar (1994, 2000), out of 81 species, 61 (75%) are common with the list of fern and fern allies of Jammu and 67 species (82%) are common with fern flora of Himachal Pradesh. 59 species (72%) are common with the Garhwal region, 41 species (50%) with Dehradun and 42 species (52%) with Sikkim.

Summing up, 71 species (87%) and 52 species (64%) resemble with pteridoflora of Western Himalaya (Almora, Chamoli, Dehradun, Garhwal, Himachal Pradesh, Jammu, Kinnaur, Kumaun, Nanital, Pithogarh, Shimla, Uttar Pradsh) and Eastern Himalaya (Arunachal Pradesh, Assam, Darjeeling hills, Meghalaya, Mizoram, Nagaland, Sikkim, West Bengal), respectively. 16 species (19%) ferns of this region resemble those met within the South India.

The fern species collected in this region are ethnically insignificant. The crosiers of two ferns, Diplazium allantodioides and Pteridium aquilinum are used as vegetable by the tribal people. Few others such as Adiantum capillus-veneris, Adiantum venustum, Asplenium adiantum-nigrum, Asplenium pseudofontanum, Asplenium trichomanes, Athyrium wallichianum, Deparia allantodioides, Equisetum arvense, Pteridium aquilinum have various medicinal uses.

Out of 81 species, eight have been recently added by authors to the pteridoflora of Kashmir Valley as new records. These are Dryopteris caroli-hopei Fraser-Jenk., Dryopteris blanfordii subsp. nigrosquamosa (Ching) Fraser-Jenk., Dryopteris pulvinulifera (Bedd.) Kuntze and Polystichum Nepalense (Spreng) C. Ch (Mir et al. 2014a); Hypolepis polypodioides (Blume) Hook., Pteris stenophylla Wall. ex Hook. & Grev., Dryopteris subimpressa Loyal and Dryopteris wallichiana (Spreng.)

Hylander (Mir et al. 2014b).

Two species Asplenium punjabense Bir & Fraser-Jenk.

(Figure 2) and Pseudophegopteris pyrrhorachis (Kunze) Ching, subsp. distans (Mett.) Fraser-Jenk. (Figure 3) reported here has been recorded first time from the Kashmir valley. There are no earlier reports of their collection from the valley, but have been collected from Jammu division of J & K State. Asplenium punjabense differs from its closely related species Asplenium ceterach in having long stipes (more than 3 cm) with sparse scaly lamina on abaxial surface which do not fringe the edges of lamina; whereas the later has short stipes (less than 3 cm) with dense scaly lamina on abaxial surface and fringing the edges of lamina. The P. pyrrhorachis subsp. distans

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B I O D I V E R S IT A S 16 (1): 27-43, April 2015

contrasts from its close relatives, P. pyrrhorachis subsp. laterepens in having long rhizome, castaneous stipe and pinnules with acute apex, whereas the later has rhizome short, stipe stramineous with very base brownish and pinnules with rounded apex.

Enumeration of species Equisetaceae Michx. Ex De Candolle 1. Equisetum arvense L., Sp. Pl. 2: 1061 (1753).

Synonym: Equisetum calderi B. Boivin; E. saxicola Suksd.

Terrestrial, medium sized fern ally, grows along the moist stream banks, shady marshes and fields, stem erect, dimorphic, fertile stem unbranched, sterile stem branched, abundant with altitudinal range of 1650-3500 m.

Distribution: America, Bhutan, Canada, Iran, Turkey, Japan, Korea, Mongolia, Nepal, Russia, Europe, North America, India (Northern India).

Specimen examined: D. K. Pora (SAM 801), 20/07/2011, 1750 m; Aharbal (SAM 817), 02/08/2011, 2350 m; Heerpur (SAM 929), 15/04/2012, 2650 m.

2. Equisetum diffusum D. Don, Prodr. Fl. Nepal. 19 (1825).

Synonym: Equisetum diffusum var. paucidentatum C.N. Page; E. mekongense C.N. Page Medium size fern ally occurring along road in thickets or in semi-shaded places usually on wet ground in lowland, monomorphic, stem much branched bearing 6-12 ridges, occasional from 1700-2500 m altitude.

Distribution: Bhutan, Japan, Myanmar, Nepal, Pakistan, Vietnam, India (Jammu and Kashmir, Assam, Sikkim, Himalayan Mountains from Shimla to Tibet, Meghalaya, South India).

Specimen examined: Narwani (SAM 821), 20/08/2011, 1740 m.

3. Equisetum palustre L, Sp. Pl. 2: 1061 (1753).

Synonym: Equisetum palustre var. polystachion Weigel; E. palustre var. szechuanense C. N. Page.

Medium size fern-ally growing in marshes, amongst sand and boulders of river valleys, under bushes, aerial stems monomorphic, green, branched, hollow center, sheath tubes long, teeth dark, 5-9, teeth margins white, scarious, rare from 2000-3600 m altitude.

Distribution: Bhutan, China, Japan, Korea, Nepal, North America, Russia, Pakistan, Tibet, India (Kashmir).

Specimen examined: Rambi Ara (SAM 942), 17/09/2012, 1950 m.

Osmundaceae Martinov 4. Osmunda claytoniana L, Sp. Pl. 2: 1066 (1753).

Synonym: Osmundastrum claytonianum (L.) Tagawa Terrestrial, large, thick textured, hemi-dimorphic fern, naturally growing in open sunny and rocky slopes in populations, occasional from 2600-3800 m altitude.

Distribution: Bhutan, China, Japan, Korea, Nepal, North America, Russia, Pakistan, Siberia, Tibet, India (Assam, Himachal Pradesh, Kashmir, Sikkim, Meghalaya, Dehradun, Garhwal, West Bengal).

Specimen examined: Huran (SAM 840), 3350, 15/072011; Dubjan (SAM 876), 23/07/2011, 3150 m.

Salviniaceae Martynov 5. Salvinia natans (L) All., Fl. Pedem 2: 289 (1785).

Synonym: Marsilea natans L. Aquatic, free floating small fern, stem dichotomously branched, leaves in whorls of three: 2 floating green entire and one submerged finely dissected into linear root-like structure, common in water bodies with altitudinal range from 1700-2000 m.

Distribution: Africa, Asia, China, Europe, Thailand, Vietnam, India (Kashmir.

Specimen examined: Zainapora (SAM 902), 10/09/2012, 1630 m.

6. Azolla cristata Kaulf., Enum. Fil.: 274 (1824).

(Previously mis-identified as Azolla pinnata R. Br., Prodr. Fl. Nov. Holland. 167 (1810).

Very small aquatic fern, polygonal in outline, stem pseudodichotomously branched bearing long feathery roots, leaves minute, overlapping, bilobed; dorsal lobe brown-green or reddish, ventral lobe translucent submerged, fairly common in rice fields and ponds from 1700-2000 m altitude.

Distribution: India (Kashmir) Specimen examined: 10/09/2012, 1630 m.

Marsileaceae Mirb.

7. Marsilea quadrifolia L. Sp. pl. 2: 1099 (1753) Medium size aquatic/terrestrial fern bearing long, slender, creeping and branched rhizome, leaves with 4 triangular leaflets in cross-shape; common in rice fields and ponds from 1600-2200 m altitude.

Distribution: China, Europe, Japan, Korea, North America, India (Kashmir, Tamil Nadu).

Specimen examined: Herman (SAM 918), 20/06/2011, 1800 m; Narwani (SAM 824), 01/07/2011, 1750 m.

Polypodiaceae J. Presl 8. Lepisorus clathratus (C. B. Clarke) Ching, Bull. Fan Mem. Inst. Biol. Bot. 4: 71 (1933).

Synonym: Lepisorus nepalensis K. Iwats.; Pleopeltis clathrata (C.B. Clarke) Bedd.; Polypodium clathratum C.B. Clarke Lithophytic small fern having long creeping rhizome, stipe straw colored, lamina lanceolate, widest at or below middle, costa raised on both sides, veins visible, anastomosing to form areolae; rare growing from 20004300 m altitude.

Distribution: Bhutan, China, Japan, Nepal India (Kashmir).

Specimen examined: Huran (SAM 864), 15/07/2011, 3455 m; Kongwatan (SAM 940), 20/09/2012, 2650 m.

9. Lepisorus stewartii Ching, in Acta Bot. Austro Sinica, 1: 23 (1983).

Lithophytic small fern appearing in colonies on large rocks along streams, stipe very short, green, lamina narrowly linear, pale green, margin revolute, veins obscure, midrib raised abaxially, rare with altitudinal range 22004000 m.

MIR et al. â&#x20AC;&#x201C; Pterydophytes of Shopian, Kashmir Valley

Distribution: India (West Himalaya).

Specimen examined: Aharbal (SAM 895), 06/09/2011, 2500 m.

Pteridaceae E. D. M. Kirchn.

10. Adiantum capillus-veneris L., Sp. Pl. 2: 1096 (1753).

Synonym: Adiantum formosum R. Br.; A. michelii H. Christ; A. modestum Underw.; A. remyanum Esp. Bustos; A. schaffneri E. Fourn.; A. wattii Baker Lithophytic medium size fern occurring on moist walls and rocks, rhizome long-creeping, stipe castaneous black, polished, pinnules or ultimate lobes fan shaped or rhombic, sori covered with false, yellow or yellowish brown, narrowly reniform or orbicular- reniform indusia, occasional with altitudinal range of 1700-2800 m.

Distribution: China, Japan, Vietnam, widely distributed in temperate and tropical regions in Africa, America, Asia, Europe, Oceania, India (throughout India).

Specimen examined: Munad SAM (841), 14/07/2011, 1760 m; Zainapora SAM (943), 20/08/2012, 1630 m.

11. Adiantum pedatum L. Sp. Pl. 2: 1095 (1753).

Synonym: Adiantum boreale C. Presl; A. pedatum var. aleuticum Rupr.; A. pedatum subsp. aleuticum (Rupr.)

Calder & Roy L. Taylor; A. pedatum f. billingsae Kittr.; A. pedatum var. glaucinum C. Chr.; A. pedatum var. kamtschaticum Rupr.; A. pedatum f. laciniatum Weath.

Lithophytic medium sized fern growing on moist, cool, shaded areas, fronds clustered, herb green, fan shaped, dichotomously branching from end of the stipe, with 4-6 lateral pinnae, veins multi-dichotomously forked, rare from 1750-3000 m altitude among rocky seeps.

Distribution: Bhutan, Canada, China, Japan, Korea, Nepal, North America, India (Jammu and Kashmir, Himachal Pradesh, Garhwal, Kumaun, Sikkim).

Specimen examined: Secjan (SAM 898), 09/09/2011, 2520 m; Kund (SAM 946), 14/08/2012, 2260 m.

12. Adiantum venustum D. Don, Prod. Fil. Nepal 17 (1825).

Synonym: Adiantum fimbriatum, A. venustum var. wuliangense Ching & Y. X. Lin. Terrestrial or lithophytic growing on moist shady humus rich but well drained soils, rock crevices, stipes dark-purple to nearly black, glossy, lamina triangular, lightgreen, pinnules wedge shaped, margin regularly toothed, fertile lobes with 2-3 notches, each notch bearing a sorus, abundant having wide altitudinal range from 2000-3300 m.

Distribution: Afghanistan, Bhutan, Myanmar, Nepal, Pakistan, Tibet, China, India (Jammu and Kashmir, Himachal Pradesh, Garhwal, Arunachal Pradesh, West Bengal, Assam, Meghalaya, Uttar Pradesh).

Specimen examined: Aharbal (SAM 805), 14/07/2011, 1900 m; Dubjan (SAM 838), 28/08/20111, 3410 m; Heerpur (SAM 892), 02/09/2011, 2420 m.

13. Cheilanthes persica (Bory) Mett. ex Kuhn, Fil. Afr. 73 (1868).

Synonym: Notholaena persica Bory; Cheilanthes szovitsii Fish. & Meyer Lithophytic small size fern occurring in rock crevices, fronds clustered, stipe erect, ebenous, shiny, hairy, lamina with abaxial dense white hairs, pinnae triangular to deltate,

31

sessile, pinnules linear-oblong, incised into small round bead-like segments, occasional with altitudinal range of 1700-2500 m.

Distribution: Afghanistan, Asia, Europe, Iran, Italia, Pakistan, Persia, Turkey, Tibet, India (Kashmir Himachal Pradesh).

Specimen examined: Lahanthoor (SAM 887) 15/08/2011, 2000 m.

14. Coniogramme affinis Wall ex Hieron. Hedwigia, 57: 279 (1916).

Synonym: C. affinis var. pilosa H. S. Kung; C. argutiserrata Ching & K. H. Shing. Grammitis affinis Wall.; Gymnogramma affinis C. Presl; G. javanica sensu Hook.; Syngramme fraxinea (D. Don) Bedd.

Terrestrial, medium size fern naturally growing in rocky meadows, rhizome long-creeping, scaly, lamina thinly herbaceous, pinnae linear to lanceolate, base subcunate to rotundate, margin with sharp teeth, Sori exindusiate extending from costa up to near margin, frequently distributed from 2000- 3500 m altitude.

Distribution: China, Myanmar, Nepal, Tibet, India (Jammu and Kashmir, Himachal Pradesh, Garhwal, Kumaun, Meghalaya).

Specimen examined: Secjan (SAM 891), 16/09/2011, 2500 m; Kund (SAM 924), 02/09/202, 2050 m; Kongwatan (SAM 941), 20/10/2012, 2360 m.

14. Cryptogramma brunoniana Wall. ex Hook. & Grev. Icon. Fil: t. 158 (1829) Synonym: Cryptogramma crispa Bedd.; C. crispa var. brunoniana (Wall. ex Hook. & Grev.) Hook. & Baker; C. crispa f. indica Hook.; Phorolobus brunonianus (Wall. ex Hook. & Grev.) Fee; C. emeiensis Ching & K. H. Shing; C. shensiensis Ching.

Lithophytic small size plants occurring at higher altitudes in rock crevices or under rocks in areas with latelying snow, fronds dimorphic; sterile tufted, many and short, fertile fronds one or two with longer stipes, occasional with altitudinal range of 3000-4000 m.

Distribution: Afghanistan, Bhutan, China, Japan, Nepal, Pakistan, Siberia, Taiwan, India (Kashmir, Himachal Pradesh, Garhwal, Kumaun, Sikkim, Andaman & Nicobar).

Specimen examined: Dubjan (SAM 863), 10/07/2011, 3560 m, Huran (SAM 915), 12/08/2012, 3220 m.

16. Cryptogramma stelleri (S. G. Gmel.) Prantl in Bot. Jahrb. Syst. 3: 413 (1882).

Synonym: Allosorus gracilis (Michx.) C. Presl; A. minutus Turcz. ex Trautv.; A. stelleri (S.G. Gmel.) Rupr.; Cheilanthes gracilis (Michx.) Kaulf.; Pellaea gracilis (Michx.) Hook.; P. stelleri (S.G. Gmel.) Baker; Pteris gracilis Michx.; P. stelleri S.G. Gmel.

Lithophytic high altitudinal small size fern grows on cool moist and shady calcareous rocks, rock lodges and cliffs, fronds remote, dimorphic, sterile shorter than fertile ones, sori elongate, submarginal indusiate, indusium false, pale-green, occasional from 2700-4000 m altitude.

Distribution: Afghanistan, China, Japan, Nepal, Russia, North America, India (Kashmir, Kumaun).

Specimen examined: Dubjan (SAM 867); 10/07/2011; 3550 m. Huran (914), 12/08/2012, 3120 m.

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B I O D I V E R S IT A S 16 (1): 27-43, April 2015

17. Onychium contiguum Wall. ex Hope, Journ.

Bombay Nat. Hist. Soc., 13: 444 (1901).

Synonym: Cheilanthes contiguum Wall.; Onychium cryptogrammoides H. Christ; O. japonicum var. intermedia C. B. Clarke; O. japonicum var. multisecta C.B. Clarke Terrestrial medium size fern growing in open rocky meadows along stream banks, stipe long, base black, straw colored distally, lamina finely dissected 4 pinnatepinnatifid or more; sterile ultimate lobes linear with acute apex, entire margin, fertile ultimate lobes broader than sterile ones, frequent with altitudinal range of 1800-2700 m altitude.

Distribution: Bhutan, China, Cambodia, Laos, Myanmar, Nepal, Thailand, Vietnam, Pakistan, Taiwan, Tibet, India (Jammu and Kashmir, Himachal Pradesh, Garhwal, Sikkim, West Bengal).

Specimen examined: Secjan (SAM 822), 15/07/2011, 2430 m; Aharbal (SAM 831), 19/07/2011, 2340 m; Kongwatan (SAM 871), 13/09/2011, 2360 m.

18. Onychium japonicum (Thunb.) Kunze Bot. Zeitung (Berlin) 6: 507 (1848).

Synonym: Caenopteris japonica (Thunb.) Thunb.; Cryptogramma japonica (Thunb.) Prantl; Trichomanes japonicum Thunb.

Medium size fern growing on open rocky meadows near streams, rhizome long-creeping, branched, fronds slightly dimorphic (fertile fronds more coarsely divided than sterile), stipes similar in length or shorter than the lamina, lamina 3-4 pinnate, deltate-oblong, olive-green, papery when dry, rarely occurring from 1700-2500 m altitude.

Distribution: Bhutan, China, Indonesia, Japan, Korea, Myanmar, Nepal, Pakistan, Philippines, Thailand, Vietnam; Pacific islands, India (Jammu and Kashmir, Himachal Pradesh, Uttarakhand, West Bengal, Meghalaya, Assam, Nagaland).

Specimen examined: Kund (SAM 909), 27/09/2012, 2300 m.

19. Pellaea nitidula Wall. ex Hope, Journ. Bombay Nat. Hist. Soc., 13: 444 (1901).

Synonym: Allosorus nitidulus C. Presl; Cheilanthes nitidula Hook.; Mildella henryi (H. Christ) Hall & Lellinger; Pellaea henryi H. Christ; P. nitidula Wall. ex Hope; Pteris nitidula Wall.

Small terrestrial fern growing amongst large boulders, stipes dark-brown, shinny, covered with reddish-brown hairs, lamina brownish-green, ultimate segments linear to lanceolate, simple, indusiate false, continuous, brown, membranous with irregularly dentate margin, rare with altitudinal range from 1700-2500 m.

Distribution: China, Nepal, Pakistan, Taiwan, Tibet, India (Kashmir, Himachal Pradesh, Garhwal, Kumaun).

Specimen examined: Kund (SAM 905), 20/17/2011, 2017 m.

20. Pteris cretica L., Mant. Pl. 1: 130 (1767).

Synonym: Pteris serraria Sw.; P. treacheriana Baker; P. trifoliata Fee.; P. triphylla M. Martens & Galeotti; P. nervosa thumb.; Pycnodoria cretica (L.) Small.

Commonly occur in open rocky meadows having dimorphic fronds, sterile fronds usually small and bending

backwards, fertile fronds with longer and stronger stipes, lamina imparipinnate, herbaceous-sub-coriaceous, pinnae lanceolate, margin spinulose-serrate, sori continuous, submarginal covered by false indusium of reflexed pinnae margin, common from 1800-3000 m altitude.

Distribution: Afghanistan, Australia, Africa, Europe, Iran, Bhutan, Burma, China, Nepal, Sri Lanka, Pakistan, Philippines, Korea, Taiwan, Thailand, Japan, India (Kashmir, Himachal Pradesh, Garhwal, Kumaun, West Bengal, Assam, Nagaland, Madhya Pradesh, Tamil Nadu, Kerala).

Specimen examined: Secjan (SAM 830), 15/07/2011, 2400 m; Aharbal (SAM 913), 17/08/2012, 2270 m; Heerpur (SAM 942), 29/09/2012, 2520 m.

21. Pteris stenophylla Wall. ex Hook. et Grev., Icon.

Filic., t. 130 (1829).

Synonym: Pteris cretica L. var. stenophylla (Wall. ex Hook. & Grev.) Baker; P. digitata Wall.; P. pellucid C. Presl. var. stenophylla (Wall. ex Hook. & Grev.) C. B. Clarke.

Terrestrial fern growing amongst dry rocks in open forests, pinnae 3-5 clustered at stipe apex, fronds dimorphic, apex of sterile pinnae small and coarsely dentate-serrate, fertile pinnae longer and narrower than sterile, rare with altitudinal range of 2500-3000 m.

Distribution: Bhutan, Nepal, Philippines, India (Jammu and Kashmir, Himachal Pradesh, Garhwal, Sikkim, West Bengal, Dehra Dun).

Specimen examined: Kellar (SAM 820); 28/08/2011; 2230 m.

Dennstaedtiaceae Lotsy 22. Pteridium revolutum (Blume) Nakai, Bot. Mag. (Tokyo) 39: 109 (1925).

(Previously mis-identified as Pteridium aquilinum (L.) Kuhn., Bot. Ost-Afrika 3: 11 (1879).

Synonym: Pteridium aquilinum subsp. wightianum (J. Agardh) W.C. Shieh; Pteridium aquilinum var. wightianum (J. Agardh) R.M. Tryon; Pteridium capense var. densa Nakai; Pteris recurvata var. wightiana J. Agardh; Pteris revoluta Blume.

Terrestrial large fern grows in open, sunny slopes, forest floor and edges, rhizome subterranean, lamina hairy, nectaries on the underside at the base of pinnae and costae, sori continuous along margins, covered with double indusium, outer made of reflexed margin, inner obsolete, membranous, common with altitudinal range of 1800-2700 m.

Distribution: North-Australia, tropical and subtropical regions of Asia including Bangladesh, Bhutan, China, Taiwan, Indonesia, Malaysia, Philippines, Thailand, Viet Nam, Nepal, Pakistan, Sri Lanka, India (Himachal Pradesh, Jammu and Kashmir, Kumaun, Assam, Meghalaya, Tamil Nadu, Kerala, Sikkim, Arunachal Pradesh, Manipur).

Specimen examined: Aharbal (SAM 814), 05/06/2011, 2225 m; Secjan (SAM 832), 14/07/2011, 2400 m; Heerpur (SAM 930), 05/09/2012, 2540 m.

23. Hypolepis polypodioides (Blume) Hook., Sp. Fil. 2: 63 (1852).

Synonym: Cheilanthes polypodioides Blume

MIR et al. â&#x20AC;&#x201C; Pterydophytes of Shopian, Kashmir Valley

Large fern occurring on wet sandy slopes and open rocky areas near streams, rhizome long-creeping, hairy, stipes very long 50 cm or more, eglandular hairy, lamina hairy on both surfaces, sori exindusiate, round, intramarginal, partially protected by marginal reflexed teeth, rare growing from 1650-2200 m altitude.

Distribution: Bangladesh, Burma, China, Indonesia, Laos, Malaysia, Myanmar, Nepal, Philippines, Taiwan, Thailand, Vietnam, India (Arunachal Pradesh, Himachal Pradesh, Manipur, Sikkim, South India, Uttar Pradesh, Uttarakhand).

Specimen examined: Kund (SAM 907), 27/09/2011; 2025 m.

Thelypteridaceae Pichi Sermolli 24. Phegopteris connectilis (Michx.) Watt., Canad.

Nat. 29 (1866).

Synonym: Aspidium Phegopteris (L.) Baumg.; Dryopteris phegopteris (L.) C. Chr.; Gymnocarpium phegopteris (L.) Newman; Lastrea phegopteris (L.) Bory; Nephrodium phegopteris (L.) Prantl; N. phegopteris (L.) Baumg. ex Diels; Phegopteris phegopteris (L.) Keyserl.; P. vulgaris Mett.; P. polypodioides Fee; Polypodium connectile Michx.; P. phegopteris L.; Polystichum phegopteris (L.) Roth; Thelypteris phegopteris (L.) Sloss. ex Rydb.

Medium size fern growing on cool moist cliffs and woods along stream banks, rhizome black, stipe clothed with unicellular white hairs, rachis winged, lamina triangular, hairy, hairs transparent, needle-like, pinnae adnate to the rachis, lowest pair drooping down and out, frequent with distributional range from 2700-3100 m altitude.

Distribution: China, Caucasus, Europe, Japan, Siberia, Pakistan, India (Kashmir, Himachal Pradesh, Garhwal, Uttar Pradesh, Kumaun, Sikkim).

Specimen examined: Dubjan (SAM 834), 27/07/2011, 3000 m; Kund (SAM 877), 07/09/2011, 2450 m.

25. Pseudophegopteris levingei (C.B. Clarke) Ching, Acta Phytotax. Sin. 8: 314 (1963).

Synonym: D. levingei (C. B. Clarke) C. Chr.; D. purdomii C. Chr.; Gymnogramma aurita var. levingei C. B. Clarke; G. levingei (C. B. Clarke) Baker; Leptogramma aurita var. levingei (C. B. Clarke) Bedd.; L. levingei (C. B. Clarke) Bedd.; Phegopteris levingei (C. B. Clarke) Tagawa; Thelypteris levingei (C. B. Clarke) Ching; Lastrea levingei (C.B. Clarke) Copel.

Medium size fern growing along streams in thickets, forests amongst boulders, rhizome thin, fronds remote, green, delicate, lamina lanceolate or oblong-lanceolate, herbaceous, base gradually tapering, apex acuminate, pinnae sessile, alternate, common from 2100-3000 m altitude.

Distribution: Afghanistan, Bhutan, China, Pakistan, Tibet, India (Jammu and Kashmir, Himachal Pradesh, Uttarakhand, Sikkim, Darjeeling hills).

Specimen examined: Dubjan (SAM 807), 16/06/2011, 1950 m; Aharbal (SAM 828), 01/07/2011, 2180 m; Heerpur (SAM 853), 11/07/2011, 2550 m.

33

26. Pseudophegopteris pyrrhorachis (Kunze) Ching, subsp. distans (Mett.) Fraser-Jenk., New Sp. Synder.

Indian Pterid.: 214 (1997) (Figure 3).

Synonym: Phegopteris distans (D. Don) Mett.

Large fern growing in rocky meadows alongside streams, rhizome thin, long-creeping, stipe base red castaneous, hairy, rachis blackish-brown, densely hairy, lamina abaxially hairy, pinnules narrow, lanceolate, rare from 2000-2800 m altitude.

Distribution: China, Malaysia, Philippines, Taiwan, Polynesia, Sri Lanka, Vietnam, India (Jammu and Kashmir, Himachal Pradesh, Uttarakhand, Sikkim, Darjeeling hills, south India).

Specimen examined: Secjan (SAM 897), 15/09/2012, 2450.

27. Pseudophegopteris pyrrhorachis (Kunze) Ching, subsp. laterepens (Trotter in Hope) Fraser-Jenk.; New Sp. Synder. Indian Pterid. 215 (1997).

Synonym: Polypodium Laterepens Trotter in Hope; Dryopteris Laterepens (Trotter) C. Chr.; Thelypteris laterepens (Trotter) R. Stewart.

Very large fern occurring in forest under-stories near streams, rhizome shot-creeping, thick, stipe long with sparse scales, rachis stramineous, hairy, lower pinnae distant, pinnules wide, deeply lobed, occasional from 20002700 m altitude.

Distribution: Pakistan, India (Kashmir).

Specimen examined: Kund (SAM 925), 10/09/2012, 2400 m.

28. Cyclosorus erubescens (Wall. ex Hook.) C. M. Kuo, Taiwania 47: 171 (2002).

Synonym: Asplenium glanduliferum Wall., nom. nud.; Christella erubescens (Wall. ex Hook.) H. Leveille; Dryopteris braineoides (Baker) C. Chr.; D. erubescens (Wall. ex Hook.) C. Chr.; Glaphyropteridopsis erubescens (Wall. ex Hook.) Ching; Glaphyropteris erubescens (Wall. ex Hook.) Fee; Lastrea erubescens (Wall. ex Hook.)

Copeland; Nephrodium braineoides Diels; N. erubescens Diels; Phegopteris erubescens (Wall. ex Hook.) J. Sm.; Polypodium braineoides Baker; P. erubescens Wall. ex Hook.; Thelypteris erubescens (Wall. ex Hook.) Ching.

Terrestrial large fern growing amongst boulders along stream banks, rhizomes erect, woody, stipe ribbed, rachis rectangular abaxially hairy, basal pinnae deflexed and forward, margin lobed, basal pair of lobes touching the corresponding lobes above and below; rare from 20003600 m altitude.

Distribution: Bhutan, China, Indonesia, Malaysia, Taiwan, Japan, Myanmar, Nepal, Pakistan, Philippines, Vietnam, India (Jammu and Kashmir, Himachal Pradesh, Uttarakhand, Kumaun, Sikkim, West Bengal, Meghalaya, Tamil Nadu).

Specimen examined: Kund (SAM 906), 27/09/2011, 2050 m.

Aspleniaceae Newman 29. Asplenium adiantum-nigrum L. Sp. Pl. 2: 1081 (1753).

Synonym: Asplenium andrewsii A. Nelson; A. chihuahuense J. G. Baker; A. dubiosum Davenport

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B I O D I V E R S IT A S 16 (1): 27-43, April 2015

Lithophytic medium size fern growing on rocky woods, hedge banks, shady walls and rocks bearing evergreen fronds, stipes clustered, lustrous, lamina 2-3 pinnate, darkgreen, glossy, thick, coriaceous, margins acutely dentate to serrate, frequent with distributional range of 1700-3000 m altitude.

Distribution: Africa, Afghanistan, Europe, Japan, Iran, Java, Nepal, Taiwan, Turkey, Pakistan, North America, India (Kashmir, Himachal Pradesh, Garhwal, Kumaun).

Specimen examined: Secjan (SAM 815), 25/06/2011, 2600 m; Kellar (SAM 854), 16/08/2011, 2150 m. Narwani (SAM 889), 19/09/2011, 1720 m.

30. Asplenium x alternifolium Wulfen, Jacq. Misc.

Austr. Bot. 2: 51 (1781).

Synonym: Asplenium x breynii Retz; Asplenium x germanicum Hope Small lithophytic fern growing among rock crevices, a cross product of Asplenium septentrionale and A. trichomanes, lamina pinnate, up to 2x1 cm, lanceolate, pinnae alternate, petiolate, oblanceolate-cuneiform, base cunate, apex toothed; sori indusiate, linear, rare from 22003300 m altitude.

Distribution: Europe, India (Kashmir, Himachal Pradesh).

Specimen examined: Huran (SAM 869), 14/08/2011, 3100 m.

31. Asplenium ceterach L., Sp. Pl. 2: 1080 (1753) Synonym: Asplenium ceterach L; Ceterach officinarum Willd.; Scolopendrium ceterach Symons; Vittaria ceterach Bernh.; Grammitis ceterach Sw.; Gymnogramme ceterach Spring; Hemidictyum ceterach (L.) Bedd.

Small lithophytic fern growing in relatively dry conditions, stipes short, brown, densely covered with brown, concolorous scales, lamina leathery, abaxially deep green, glabrous and adaxially dense scaly, margin slightly bending upwards revealing the scales, alternately lobed, frequent from 1700-2600 m altitude.

Distribution: Afghanistan, Australia, Brazil, Scotland, Siberia, Tibet, Ireland, Pakistan, Africa, Europe, India (Kashmir, Himachal Pradesh, Uttarakhand).

Specimen examined: Kund (SAM 896), 27/09/2011, 1930 m.

32. Asplenium dalhousiae Hook. Icon. Pl. 2: pl. 105 (1837).

Synonym: Asplenium alternans Wall.; A. rupium Goodd.; Ceterach alternans Kuhn; Ceterach dalhousiae (Hook.) C. Chr. Terrestrial small fern occurring in rocky meadows, stipe brown, indumenta of scales throughout, lamina deeply pinnatifid, subcoriaceous, gradually narrowed towards base, lobes alternate, spreading, lower distant, rare from 1700-2700 m.

Distribution: Afghanistan, Africa, Bhutan, Ethiopia, Nepal, Pakistan, Africa, North America, India (Jammu and Kashmir, Himachal Pradesh, Garhwal, Arunachal Pradesh, Kumaun, Sikkim, West Bengal, Meghalaya, Manipur, Rajasthan).

Specimen examined: Kund (SAM 904), 27/09/2012, 1900 m. Zainapora (SAM 931), 15/05/2012, 1630 m.

33. Asplenium kukkonenii Viane & Reichstein, Pteridol. New Mellenium, 18 (2003).

Very small fern grows on moist shady rocks on forest floor, lamina broadest below the middle, apex acute, bipinnate at base, pinnae triangular-ovate, pinnules rounded with acute teeth, lowest acroscopic pinnule the largest, rare with 2100-3300 m.

Distribution: Nepal, India (Jammu and Kashmir, Himachal Pradesh, Uttar Pradesh).

Specimen examined: Aharbal (SAM 886), 37/08/2011, 2600 m.

34. Asplenium pseudofontanum Kossinsky, Notulae Syst. Hort. Bot. Petrop. 3: 122 (1922).

Synonym: Athyrium fontanum (L.) Roth; A. halleri Roth.; Aspidium fontanum (L.) Sw.; A. halleri (Roth) Willd.; Asplenium fontanum (L.) Bernh. subsp. pseudofontanum; A. lanceolatum subsp. fontanum (L.) P. Fourn.; A. halleri (Roth) DC.; A. leptophyllum Lag. Lithophytic small ferns commonly occur in rock crevices in forests, fronds clustered, evergreen, lamina finely dissected, widest above the middle, narrowed towards the both ends, rare with distributional range of 1800-3000 m altitude.

Distribution: Afghanistan, Pakistan, India (Jammu and Kashmir, Himachal Pradesh, Kumaun, Garhwal, West Bengal).

Specimen examined: Aharbal (SAM 810), 11/07/2011, 2350 m, Secjan (SAM 829), 21/07/2011, 2400 m; Kongwatan (SAM 899), 14/09/2011, 2650 m.

35. Asplenium punjabense Bir, Fraser-Jenk. & Lovis, Fern Gaz. 13: 53-63 (1985) (Figure 2) Lithophytic fern growing in open rocky meadows, lamina brownish-green, thick, nearly pinnate with deep rounded grooves, adaxially glabrous, abaxially scaly, scales not dense, sori with rudimentary indusia, rare from 16502100 m altitude.

Distribution: Afghanistan, Pakistan, India (Himachal Pradesh, Jammu and Kashmir).

Specimen examined: Kund (SAM 903), 24/09/2012; 2020 m.

36. Asplenium ruta-muraria L. Sp. Pl. 2: 1081 (1753) Synonym: Acrostichum ruta-muraria Lam.; Phyllitis ruta-muraria Moench; Amesium ruta-muraria Newmann; Tarachia ruta-muraria C. Presl; Asplenium murale Bernh.; A. cryptolepis Fernald; A. cryptolepis Fernald var. ohionis Fernald; A. ruta-muraria var. cryptolepis (Fernald) Wherry.

Small lithophytic fern growing in open sites in rick crevices, pinnae imparipinnate triangular, pinnules lateral, obovate or rhomboid, apex sub-rounded, margins irregularly sharp dentate, base cuneate, sori born along veins, rare from 2000-4000 m altitude.

Distribution: Afghanistan, China, Iran, Japan, Kazakhstan, Korea, Nepal, Pakistan, Russia, Tajikistan; NW Africa, SW Asia, Europe, North America, India (Kashmir, Kumaun, Arunachal Pradesh).

Specimen examined: Secjan (SAM 855), 20/08/2011, 2400 m; Dubjan (SAM 888), 06/09/2011.

37. Asplenium septentrionale (L.) Hoffman, Deutschl.

Fl. 2: 12 (1795).

MIR et al. â&#x20AC;&#x201C; Pterydophytes of Shopian, Kashmir Valley

Synonym: Acrostichum septentrionale L.; Amesium sasaki Hayata; Scolopendrium septentroinal Reth; Belvisia septentrionalis Mirb.; Acropteris septentrioonalis Link; Blahnum septentrionale Wall.; Amesium septentrionale Newm.; Asplenium septentrional var. sasakii (Hayata) C. Chr. Lithophytic fern grows higher altitudes in open sites in rocks crevices and cliffs in light forest; rhizome much branched to produce dense many stemmed, tufts or mats bearing numerous crowded leaves, lamina dichotomously 2 or 3 partite, sori confluent at maturity covering the entire surface, frequent from 2200-3500 m altitude.

Distribution: Afghanistan, China, Kazakhstan, Kyrgyzstan, W Mongolia, Nepal, Pakistan, Russia, Tajikistan; Africa, Europe, North America, India (Kashmir, Himachal Pradesh, Garhwal, Kumaun, Shimla).

Specimen examined: Aharbal (SAM 811), 10/07/2011, 2400 m; Secjan (SAM 823), 28/07/2011, 2350 m; Dubjan (SAM 843), 11/08/2011, 2800 m.

38. Asplenium tenuicaule Hayata, Icon. Pl. Formosan.

4: 228 (1914).

Synonym: Gymnogramma fauriei Christ, Asplenium subvarians Ching, A. shiobarensse Koidz Small fern growing on moist shady rocks on forest floor, lamina bipinnate, 1 cm broad, oblong-lanceolate, pinnules alternate, born on thin delicate stalks, fan shaped to rounded-ovate, rare with altitudinal range from 20003600 m.

Distribution: Bhutan, China, Japan, Korea, Nepal, Pakistan, Philippines, Russia, Thailand; Africa, Pacific islands (Hawaii) India (Kashmir, Himachal Pradesh, Garhwal, Kumaun).

Specimen examined: Aharbal (SAM 922), 05/09/2012, 2600 m.

39. Asplenium trichomanes L. Sp. Pl. 2: 1080 (1753).

Synonym: Asplenium melanocaulon Will.; A. trichomanoides Houtt.; A. minus Bl.; A. pusillum Bl.; A. densum Brack.; A. melaolepis Col. Chamaefilix trichomanes (L.) Farw. Trichomanes crenatm Gilib.; Pylilitis rotundifolia Moench.

Lithophytic fern occurring in open moist areas, fronds clustered, evergreen, stipes brown- black to coppery, lustrous, brittle, lamina deep green, linear, abundant from 1650-3200 m altitude.

Distribution: worldwide in all temperate zones, in tropics on high mountains, India (Kashmir, Himachal Pradesh, Uttarakhand, Uttar Pradesh, Sikkim, Assam, Meghalaya, Arunachal Pradesh, Manipur, Rajasthan, Tamil Nadu).

Specimen examined: Aharbal (SAM 808); 25/06/2011; 2250 m; Secjan (SAM 839), 22/08/2011, 2400 m; Narwani (SAM 872), 28/09/2011, 1780 m.

40. Asplenium varians Wall. ex Hook. & Grev. con.

Filic. 2: 172 (1830).

Synonym: Asplenium lankongense Ching Small fern grows on moss covered shady rocks on forest floor, fronds erect or arching, lamina bipinnate, lanceolate, pinnae extremely short stalked, triangular-ovate, pinnules obovate, sharply dentate on the outer margin, rare with altitudinal range of 1700-2900 m.

35

Distribution: Afghanistan, Bhutan, Burma, China, Hawaii, Japan, Korea, Sri Lanka, Philippines, Nepal, Vietnam, Africa, Taiwan, Thailand, India (Kashmir, Himachal Pradesh, Uttarakhand, Sikkim, West Bengal, Assam, Meghalaya, Tamil Nadu).

Specimen examined: Aharbal (SAM 923), 05/09/2012, 2190 m.

41. Asplenium viride Hudson, Fl. Angl. 385 (1762).

Synonym: Asplenium ramosum L.; A. trichomanesramosum L. High altitudinal fern that grows on limestone rock crevices, fronds evergreen, stipe reddish-brown at base, green distally, lustrous, shallowly grooved on abaxial surface, rare from 2800-4200 m altitude.

Distribution: Afghanistan, Japan, Nepal, Pakistan, Russia, Europe, North America, India (Jammu and Kashmir, Himachal Pradesh, Uttar Pradesh).

Specimen examined: Kaunsernag (SAM 921), 28/08/2012, 3500 m.

Woodsiaceae Herter 42. Athyrium atkinsonii Bedd. Ferns Brit. Ind., Suppl.

11. t. 359 (1876).

Synonym: Aspidium senanense Franch. & Sav.; Asplenium atkinsonii (Bedd.) C.B. Clarke; A. lastreoides Baker; Athyrium lastreoides (Baker) Diels; A. microsorum Makino; A. monticola Rosenst.; A. senanense (Franch. & Sav.) Koidz. & Tagawa; Cystopteris grandis C. Chr. in H. Lev.; Davallia athyriifolia Baker; Dryopteris senanensis (Franch. & Sav.) C. Chr.; Pseudocystopteris atkinsonii (Bedd.) Ching Middle size fern commonly grows on moist areas above tree line, Fronds single, lamina 3-pinnate-pinnatifid, broadly ovate or triangular, shortly acuminate or almost acute; pinnae distant, petiolate; frequent from 2500-3500 m altitude.

Distribution: Bhutan, Burma China, Taiwan, Nepal, Japan, Pakistan, Tibet, Taiwan, Korea India (Kashmir, Himachal Pradesh, Uttarakhand, Sikkim, Darjeeling hills, Arunachal Pradesh).

Specimen examined: Dubjan (SAM 875), 23/07/2011, 3200 m.

43. Athyrium attenuatum (Wall. ex C. B. Clarke) Tagawa, Acta. Phytotax. Geobot. 16: 177 (1956).

Synonym: Asplenium filix-femina (L.) Bernh. var. attenuatum Wallich ex C. B. Clarke, Athyrium contigens Ching & Wu. Terrestrial large fern growing on meadows and gentle mountain slopes, rhizome short-erect, covered with persistent leaf bases, stipe upward, stramineous, rachis succulent, sparsely scaly, lamina tapering at both ends, pinnae higher up congested, pinnule margin serrate, common from 2200-3500 m altitude.

Distribution: Afghanistan, Bhutan, China, Nepal, Pakistan, India (Jammu and Kashmir, Himachal Pradesh, Uttarakhand).

Specimen examined: Aharbal (SAM 826), 15/06/2011, 2400 m; Secjan (SAM 835), 27/06/2011, 2300 m; Dubjan (SAM 900), 27/08/2012, 3460 m; Huran (SAM 932), 19/09/2012, 3340 m.

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B I O D I V E R S IT A S 16 (1): 27-43, April 2015

44. Athyrium distans (D. Don.) T. Moore, Index Filic.: 152 (1859).

Synonym: Asplenium distans D. Don., Athyrium imbricatum Christ, A. fangii Ching, A. sikkimense Ching; A. decorum Ching; A. nanyueense Ching in Ching & Hsieh Medium size fern grows on forest floor near water, rhizomes apex densely scaly, stipes long, purplish red to greenish stramineous, lamina 2-pinnate-pinnatisect, grassgreen, widest just above the base, pinnules distant, base asymmetrical, occasional from 1700-2500 m altitude.

Distribution: China, Nepal, Japan, India (Kashmir, Himachal Pradesh, Uttarakhand, Sikkim, Darjeeling hills, Meghalaya).

Specimen examined: Aharbal (SAM 874), 18/07/2011, 2200 m.

45. Athyrium mackinnonii (Hope) C. Chr., Index Filic.

Fasc. 3: 143 (1905).

Synonym: Asplenium mackinnonii Hope, Athyrium nigrips sensu Bedd., A. caudipinnum Ching Medium size fern grows on forest floor, rhizomes covered with persistent leaf bases, densely scaly; scales bicolorous, blackish-brown in central part, brown at margin, stipes pinkish, lamina pale green, pinnae lowest pair deflexed and opposite, rest alternate, ascending, veins visible on both surfaces, common from 1700-2700 m altitude.

Distribution: Afghanistan, Myanmar, Nepal, Pakistan, Thailand, Vietnam, India (Jammu and Kashmir, Himachal Pradesh, Garhwal, Dehra Dun, Uttar Pradesh Specimen examined: D. K. Pora (SAM 802), 10/06/2011, 1800 m; Aharbal (SAM 845), 10/07/2011, 2300 m; Heerpur (SAM 850), 15/07/2011, 2400 m.

46. Athyrium strigillosum (T. Moore ex E. J. Lowe) Salomon, Nom. Gefasskrypt, 112 (1883).

Synonym: Allantodia denticulate Wall.; Asplenium denticulatum Wall. ex. J. Sm.; A. setudosum Wall. ex. J. Sm.; A. strigillosum T. Moore ex E.J. Lowe; A. tenuifrons Wall. ex Hope; Athyrium proliferum T. Moore; A. viviparum Christ Medium size fern growing on stream sides in valleys, stipe stramineous, thin, lamina 2-pinnate, triangularlanceolate, base hardly narrowed, apex acuminate, pinnules ascending, approximate, apex toothed, margin lobed, lobes serrate-dentate, rare from 1700-2500 m.

Distribution: Bhutan, Japan, Myanmar, Nepal, India (Kashmir).

Specimen examined: Saangran (SAM 927), 20/08/2012, 2150 m.

47. Athyrium wallichianum Ching. Bull. Fan Mem. Inst. Biol., Bot. 8: 497 (1938).

Synonym: Aspidium brunonianum Wall.; A. brunonianum Wall. ex Mett.; Lastrea brunoniana Wall. ex C. Presl.; L. brunoniana (Wall. ex Mett.) Bedd. L. brunoniana C. Presl; Dryopteris brunoniana (Wall. ex Mett.) Kuntz; Nephrodium brunonianum (Wall. ex Mett.)

Hook.

Medium size growing in alpine shrub meadows, rhizome thick, black, covered with brown scales, fronds green, clustered, rachis scaly and fibrillose, lamina thick, abruptly narrowed at the apex, pinnae crowded, sessile,

pinnules round, margin serrate, duplicato-dentate, teeth triangular, common from 3300-4600 m altitude.

Distribution: Bhutan, Myanmar, Nepal, Pakistan, India (Kashmir, Himachal Pradesh, Dehra Dun, Garhwal, Kumaun, Sikkim, Arunachal Pradesh).

Specimen examined: Dubjan (SAM 836), 16/08/2011, 3500 m; Huran (SAM 890), 10/08/2012, 3360 m.

48. Cystopteris dickieana R. Sim, Gard. Farmers J. 2: 308 (1848).

Synonym: Cystopteris fragilis (L.) Bernh. f. granulos Bir & Trikha; C. fragilis (L.) Bernh. subsp. dickieana (Sim) Hook.; C. fragilis (L.) Bernh. var. dickieana (Sim) T. Moore; C. sikkimensis Ching ex Bir Distribution: Afghanistan, China, Nepal, Pakistan, Japan, Iran, Africa, Europe, North America, Pakistan, Taiwan, India (Jammu and Kashmir, Himachal Pradesh, Uttarakhand).

Small fern growing in open slopes, forest floors and edges of forests, rhizome short- creeping, internodes beset with old petiole bases, stipes fragile, sori indusiate, round, indusium forming a hood over the sorus, spore exine rugose or verrucose, occasional with altitudinal range of 1800-2800 m.

Specimen examined: Aharbal (SAM 806), 15/06/2011, 2250 m; Secjan (SAM 852), 17/08/2011, 2350 m.

49. Cystopteris fragilis (L.) Bernh. Schrad. Neues J. Bot. 1: 26 (1805).

Synonym: Aspidium dentatum Sw.; A. fragile (L.) Sw.; A. viridulum Desv.; A. dentatum (Sw.) Gray; A. fragile (L.) Spreng.; A. fumarioides C. Presl; Cyathea anthriscifolia (Hoffm.) Roth; C. cynapifolia (Hoffm.) Roth; C. fragilis (L.) J. Sm.; Cystea angustata (Hoffm.) Sm.; C. dentata (Dicks.) Sm.; C. fragilis (L.) Sm.; Cystopteris acuta Fee.; C. baenitzii Dorfl.; C. canariensis C. Presl.; C. dentata (Sw.) Desv.

Morphologically similar to Cystopteris dickieana except in spore ornamentation, spore exine is echinate not rugose or verrucose, common from 1800-2800 m altitude.

Distribution: Afghanistan, China, Nepal, Russia, Pakistan, Japan, Iran, Africa, Europe, North America, Pakistan, Taiwan, Korea India (Jammu and Kashmir, Himachal Pradesh, Uttarakhand, Nagaland Specimen examined: Sedow (SAM 837), 05/08/2011, 2350, m; Aharbal (SAM 851), 25/08/2011, 2250 m; Heerpur (SAM 865), 15/09/2011, 2400 m.

50. Deparia acuta (Ching) Fraser-Jenk., New Sp. Syndrome Indian Pterid.: 104 (1997) Synonym: Lunathyrium acutum Ching Medium size fern growing in open rocky meadow and mountain slopes, rhizome short-erect, scaly at apex, scales red-brown, stipe and rachis densely hairy, lamina much reduced towards base, apex acuminate, pinnae sessile, slightly ascending, frequent from 2300-3800 m altitude.

Distribution: China, Pakistan, India (Kashmir, Himachal Pradesh, Uttarakhand).

Specimen examined: Secjan (SAM 842), 03/07/2011, 2400 m; Huran (SAM 849), 10/08/2011, 2900 m.

51. Deparia allantodioides (Bedd.) M. Kato, J. Fac. Sci. Univ. Tokyo, Sect. 3, Bot. 13: 393 (1984).

MIR et al. â&#x20AC;&#x201C; Pterydophytes of Shopian, Kashmir Valley

Synonym: Asplenium thelypteroide sensu Clark, Athyrium allantodioides Bedd, A. thelypteroides sunsu Bedd., Deparia sikkimensis (Ching) Nakaike & Malk, Lunathyrium allantodioides (Bedd.) Ching, L. mackinnonii Ching, L. sikkimensis Ching Large fern growing on forest floor with higher levels of moisture, stipe brown, hairy, rachis fibrillose, hairy, pinnae alternate, sessile, lower pinnae reduced, sometimes up to auricles, deflexed downwards, deeply lobed, lobes connected by a narrow wing, basal basiscopic pinnule outwards; common from 1900-3700 m altitude.

Distribution: Bhutan, China, Nepal, Tibet, Pakistan, India (Jammu and Kashmir, Himachal Pradesh, Uttarakhand, Sikkim, Arunachal Pradesh, West Bengal).

Specimen examined: Aharbal (SAM 812), 25/06/2011, 2450 m; Dubjan (SAM 819); 12/08/2011, 2500 m; Secjan (SAM 934), 25/09/2012, 2400 m.

52. Deparia macdonellii (Bedd.) M. Kato, J. Fac. Sci. Univ. Tokyo, Sect. 3, Bot. 13: 391 (1984).

Synonym: Asplenium macdonellii Bedd.; Athyrium macdonellii Bedd.; Cornopteris macdonellii (Bedd.)

Tardieu; Deparia pterorachis (Christ) kato; Dryoathyrium macdonellii (Bedd.) Morton; Lunathyrium macdonellii (Bedd.) Khullar; L. macdonellii (Bedd.) Ching Very large fern growing on forest slopes along streamlets, rhizome long-creeping, lamina basal pinnae triangular and opposite, deeply lobed near to the costa; lobes connected by a very narrow wing, sinus wide and cunate, apex rounded with sharp teeth, margin crenateserrate, occasional having altitudinal range of 1800-2700 m altitude.

Distribution: Japan, Pakistan, Japan, China, India (Kashmir, Himachal Pradesh).

Specimen examined: Dubjan (SAM 911), 02/08/2012, 2500 m.

53. Diplazium maximum (D. Don) C. Chr. Index Filic. fasc. 1: 235 (1905).

Synonym: Allantodia maxima (D. Don) Ching; Asplenium maximum D. Don; A. frondosum Wall.; Diplazium flaccidum Christ; Diplazium giganteum (Bak.) Ching in C. Chr.; Gymnogramma gigantae Baker; Diplazium frondosum (Clark) Christ.; Diplazium polymorphum Wall. ex Mett.

Terrestrial fern occurring amongst large boulders along streamlets and ravines, rhizome ascending with dense loose scales at apex, scales brown or chestnut, frond large, up to 180x90 cm, pinnules sessile, rounded apex, margin shallowly crenate, sori linear, diplazoid, common from 1800-2500 m altitude.

Distribution: Bhutan, China, Myanmar, Nepal, Pakistan, Polynesia, Vietnam, India (Jammu and Kashmir, Himachal Pradesh, Uttarakhand, Sikkim, West Bengal, Assam, Meghalaya).

Specimen examined: Secjan (SAM 848), 05/08/2011, 2400 m; Kund (SAM 908), 20/08/2012, 2100 m; Kongwatan (SAM 947), 28/09/2012, 2450 m.

54. Diplazium sibiricum (Turcz. ex Kunze) Kurata in Namegata and Kurata Enum. Jap. Pterid. 292: 340 (1961).

Synonym: Asplenium sibiricum Turcz. ex Kunze; A. crenatum Sommerf; Athyrium crenatum (Sommerf) Ruper

37

ex Nyland; Allantodia crenata (Sommerf) Ching; Cystopteris crenata Fries; Dipazium sommerfeldtii Love & Love Medium size fern growing on steep forest ravines, rhizome slender, long-creeping, black, stipe longer than lamina, lamina triangular, costae and veins below hairy; basal sori paired back to back on the same vein, others singular; frequent with 2300-2800 m.

Distribution: China, Japan, Korea, Russia, Europe, Nepal, Siberia, India (Kashmir, Himachal Pradesh, Uttarakhand).

Specimen examined: Dubjan (SAM 885), 28/07/2011, 2600 m.

55. Gymnocarpium dryopteris (L.) Newman, Phytologist 4: 371 (1851).

Synonym: Aspidium dryopteris (L.) Baumgarten; Carpogymnia dryopteris (L.) A. Love & D. Love; Currania dryopteris (L.) Wherry; Dryopteris dryopteris Britton; D. pulchella (Salisb.) Hayek; D. linnaeana (L.) C. Chr.; D. pumila V.I. Krecz.; Filix pumila Gilib.; Lastrea dryopteris (L.) Bory.; Nephrodium dryopteris (L.) Michx.; Phegopteris dryopteris Fee; Polypodium dryopteris L.; P. pulchellum Salisb.; Polystichum dryopteris (L.) Roth; Thelypteris dryopteris (L.) Sloss Medium size fern growing on damp areas and among rock crevices near moisture in forests, rhizomes longcreeping, black-brown, apex densely scaly, stipe base purplish, above stramineous, lamina pentagonal-ovate, veins visible abaxially, sori exindusiate, frequent from 2000-2900 m altitude.

Distribution: Japan, China, Tibet, Nepal, Korea, Europe, North America, Pakistan, India (Jammu and Kashmir, Himachal Pradesh, Uttarakhand, Sikkim).

Specimen examined: Aharbal (SAM 804), 23/06/2011, 2400 m; Kongwatan (SAM 816), 20/07/2011, 2450 m; Heerpur (SAM 827); 12/08/2011, 2300 m 56. Woodsia alpine (Bolton) Gray, Nat. Arr. Brit. Pl. 2: 17 (1821).

Synonym: Acrostichum alpinum Bolton; Woodsia alpina var. bellii G. Lawson; W. bellii (Lawson) A.E. Porsild; W. hyperboreum R. Br.; W. himalaica Ching & S. K.Wu.; W. ilvensis var. alpina Watt; W. ilvensis subsp. alpina Asch.

Small fern occurring among rock crevices and screes at high altitudes, rhizome compact with cluster of persistent stipe bases, stipe lustrous, fibrillose and hairy, lamina pinnate to pinnatifid, hairy along the margin, indusia hairy, rare from 2500-3800 m altitude.

Distribution: Afghanistan, Iran, Tibet, Europe, N Asia, Pakistan, India (Kashmir, Himachal Pradesh, Uttarakhand).

Specimen examined: Huran (SAM 868), 20/07/2011, 3200 m.

Dryopteridaceae Herter 57. Dryopteris barbigera (T. Moore ex Hook.) Kuntze, Revis. Gen. Pl. 2: 812 (1891).

Synonym: Aspidium barbigerum (T. Moore ex Hook.)

H. Christ; Dryopteris falconeri (Hook.) Kuntze; Lastrea barbigera (Hook.) T. Moore ex Bedd.; L. falconeri (Hook.)

Bedd.; Nephrodium barbigerum Hook.; N. falconeri Hook.

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Terrestrial fern growing in birch forests, stipe, rachis and lamina densely scaly and fibrillose; scales reddishbrown, lamina thickly herbaceous, pinnule lobes rounded with serrate and prominent acute teeth, occasional from 2800-4700 m altitude.

Distribution: Bhutan, Pakistan, China, Tibet, Taiwan, Nepal, India (Jammu and Kashmir, Himachal Pradesh, Uttarakhand, Assam, Sikkim, West Bengal).

Specimen examined: Peergali (SAM 918), 18/08/2012, 3100 m; Dubjan (SAM 934), 16/09/2012.

58. Dryopteris blanfordii subsp. blanfordii (Hope) C. Chr.; Ind. Fil. 254 (1905).

Synonym: Dryopteris gongboensis Ching; Nephrodium blanfordii Hope Terrestrial fern growing on forest floor, stipes with dark-brown base, fibrillose and scaly, scales ovate to lanceolate, brown, concolorous, glossy, margin fimbriate, lamina ca. 50x17 cm, pinnules slightly lobed or sometimes without lobes, segment apex with 2-3 sharp serratures, frequent from 1800-3500 m altitude.

Distribution: Afghanistan, China, Tibet, Pakistan, Nepal, India (Kashmir, Himachal Pradesh, Uttarakhand).

Specimen examined: Aharbal (SAM 893), 08/09/2011, 2300 m 59. Dryopteris blanfordii subsp. nigrosquamosa (Ching) Fraser-Jenk., Bull. Brit. Mus. (Nat. Hist.), Bot. 18: 388 (1989).

Synonym: Dryopteris gushaingensis Ching; Dryopteris nigrosquamosa Ching Terrestrial fern growing in steep abies forests, stipes densely fibrillose and scaly, scales broadly ovate, fuscousbrown, concolorous, crinkled, margins with filamentous projections, lamina ca. 40x12 cm, pinnules lobed, segment apex rarely sharply serrate, rare with 2600-3400 m.

Distribution: SE Tibet, W. China, Nepal, India (Kashmir).

Specimen examined: Dubjan (SAM 847), 05/07/2011, 2750 m.

60. Dryopteris caroli-hopei Fraser-Jenk., Bull. Brit.

Mus. Nat. Hist. Bot. 18: 422 (1989).

Synonym: Aspidium dilatatum var. patuloides H. Christ, Dryopteris pseudomarginata Ching; D. pseudomarginata Ching; D. marginata sensu auct. West Himalaya, non (Wall. ex C. B. Clarke) Christ; Nephrodium marginarum sensu Hope Large fern growing near stream banks under shade, rhizome thick, stipe base densely scaly, scales pale-brown, broad ovate, lamina pale-green, 2-3-pinnate, elongated ovate-lanceolate, pinnules deeply lobed to the costa (sometimes becoming pinnate), apex bluntly acuminate, occasional from 1700-2300 m altitude.

Distribution: Bhutan, China, Nepal, Tibet, Yunnan, India (Jammu and Kashmir, Arunachal Pradesh, Manipur, Meghalaya, Nagaland, Himachal Pradesh, Uttarakhand).

Specimen examined: Imamsahib (SAM 844), 15/07/2011, 1866 m; D. K. Pora (SAM 860), 29/07/2011, 1800 m.

61. Dryopteris filix-mas (L.) Schott, Gen. Fil. , sub pl. 9 1834.

Synonym: Aspidium depastum Schkuhr; A. erosum Schkuhr; A. expansum D. Dietr.; A. filix-mas (L.) Sw.; A. mildeanum Gopp.; A. nemorale (Salisb.) Gray; A. opizii Wierzb.; A. umbilicatum (Poir.) Desv.; A. veselskii Hazsl. ex Domin; Dryopteris patagonica Diem; Filix-mas filixmas Farwell; Lastrea filix-mas (L.) C. Presl; Nephrodium crenatum Stokes; N. filix-mas (L.) Rich.; Polypodium filixmas L.; P. heleopteris Borkh.; P. nemorale Salisb.; P. umbilicatum Poir.; Polystichum filix-mas (L.) Roth; P. polysorum Tod.; Tectaria filix-mas Cav.; Thelypteris filixmas Nieuwl.

Terrestrial fern growing under shade near moisture, rhizome long-erect, stipe grooved, scaly and fibrillose, lamina pale to mid-green above, pinnae shortly petiolate, pinnules lobed, lobe ending in an acute tooth, sori crowded, upper part of the lamina fertile, rare from 1700-3200 m altitude.

Distribution: Africa, China, Argentina, Afghanistan, Russia, Greenland, Malaya, Peru, Mexico, Kazakhstan, Pakistan, Russia, Iran, Jamaica, Europe, North America, India (Kashmir, Uttarakhand).

Specimen examined: Narwani (SAM 879), 25/07/2011, 1775 m.

62. Dryopteris juxtaposita H. Christ, Bull. Acad. Int. Geogr. Bot. 17: 138 (1907) Synonym: Aspidium filix-mas var. normale (C. B. Clarke) H. Christ; Dryopteris odontoloma (Bedd.) C. Chr.; Lastrea odontoloma Bedd.; L. odontoloma T. Moore; Nephrodium filix-mas var. normale C. B. Clarke.

Medium size fern growing on forest floor near water, rhizome scaly, scales brown, fronds caespitose, lamina subcoriaceous, upper surface blue-green, lower surface whitish-green, sori round, mostly upper part of lamina fertile, indusia brown, rounded-reniform, occasional from 2200-3700 m altitude.

Distribution: Afghanistan, Burma, Bhutan, Japan, Nepal, Tibet, Vietnam, China, Nepal, Thailand, India (Kashmir, Himachal Pradesh, Uttarakhand, Sikkim, West Bengal, Assam, Nagaland, Manipur, Meghalaya, Tamil Nadu).

Specimen examined: Heerpur (SAM 883), 28/07/2011, 2715 m; Secjan (SAM 910), 05/09/2012.

63. Dryopteris nigropaleacea Fraser-Jenk., Bol. Soc. Brot., ser. 2, 55: 238 (1982).

Synonym: Dryopteris pallida Subsp. nigropaleacea Fraser-Jenk.; Nephrodium filix-mas Rich. var. normalis C. B. Clarke Medium size fern growing near small streams banks, stipe very base scaly and fibrillose, lamina elongate, triangular-lanceolate, crispaceous, abaxially glaucous, adaxially blue-green, matt, glabrous; pinnae lobes rectangular with rounded-truncate and toothed apices, frequent from 1700-2800 m altitude.

Distribution: Afghanistan, Bhutan, Burma, Nepal, Pakistan, India (Jammu and Kashmir, Himachal Pradesh, Uttarakhand, Sikkim, West Bengal, Nagaland).

Specimen examined: Dubjan (SAM 857), 27/07/2011, 2700 m; Heerpur (SAM 884), 18/08/2011, 2600 m.

64. Dryopteris pulvinulifera (Bedd.) Kuntz, Revis.

Gen. Pl. 2: 813 (1891).

MIR et al. â&#x20AC;&#x201C; Pterydophytes of Shopian, Kashmir Valley

Synonym: Dryopteris harae H. Ito; D. reholttumii M. Price; Lastrea pulvinulifera Bedd.; L. sparsa var. zeylanica Bedd.; L. pulvinulifera var. zeylanica Bedd.; Nephrodium pulvinuliferum (Bedd.) Baker; N. sparsum var. squamulosum C. B. Clarke Plants growing on steep forest floor under shade, rhizome thin, stipe base with densely scaly, scales bright golden, narrow, linear, lamina dark-green, deltoidlanceolate, lower part 4-pinnate, upper part 3-pinnate, pinnules pinnate, apices acute ending in few small acute teeth, pinnulets overlapping, occasional from 1700-2800 m altitude.

Distribution: Bhutan, China, Nepal, Sri Lanka, Philippines, India (Kashmir, Sikkim, West Bengal, Assam, Meghalaya, Nagaland).

Specimen examined: Heerpur (SAM 813), 08/07/2011, 2600 m; Dubjan (SAM 858), 28/08/2011, 2800 m.

65. Dryopteris ramosa (Hope) C. Chr., Index Filic. fasc. 5: 287 (1905).

Synonym: Nephrodium ramosum Hope Terrestrial large fern growing on forest floor, rhizome densely scaly and surrounded by stipe bases, lamina 3pinnate, deltate, apex acuminate, pale-green, pinnae distant, frequent with the altitudinal range of 2000-4000 m altitude.

Distribution: Afghanistan, Bhutan, California, Caucasus, Tibet, Europe, Nepal, Pakistan, India (Kashmir, Himachal Pradesh, Uttarakhand).

Specimen examined: Dubjan (SAM 846), 18/07/2011, 2700 m.

66. Dryopteris redactopinnata S.K. Basu & Panigrahi, Indian J. Forest. 3: 270 (1980).

Synonym: Dryopteris pseudofibrillosa Ching; D. tsangpoensis Ching Medium size fern growing on forest floor, stipe densely clothed with scales, scales light to mid-brown, concolorous, not glossy, lamina ca. 30x13 cm, glossy on upper surface, upper part of lamina fertile, indusia with brown center and pale brown margin, frequent from 2400-3800 m altitude.

Distribution: Bhutan, China, Tibet, Taiwan, Nepal, Pakistan, India (Jammu and Kashmir, Himachal Pradesh, Uttarakhand, Sikkim, West Bengal).

Specimen examined: Dubjan (SAM 878); 05/08/2011; 3320 m.

67. Dryopteris stewartii Fraser-Jenk., Kalikasan Philip.

J. Biol. 7: 272 (1979).

Synonym: Dryopteris odontoloma (Bedd) C. Chr. f. brevifolia Mehra & Khullar Medium size fern growing on small stream banks, stipe base densely scaly, scales blackish-brown to brown, glossy, sometimes bicolorous, margin serrate, apex acuminate, lamina widest above the base, mid-green adaxially, green abaxially, frequent with altitudinal range of 1800-3100 m.

Distribution: Afghanistan, Pakistan, Nepal, India (Kashmir, Himachal Pradesh, Uttarakhand).

Specimen examined: D. K. Pora (SAM 856); 23/07/2011, 1850 m; Heerpur (SAM 870), 08/08/2011, 2550 m; Aharbal (SAM 895), 05/07/2012, 2400 m.

68. Dryopteris subimpressa Loyal, Nova Hedwigia 16: 467 (1968).

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Synonym: Dryopteris lancipinnula Ching; D. submarginata Loyal; D. subodontoloma Loyal Medium size fern growing on small streams banks, rhizome long-ascending, stipe long, base scaly, scales pale brown, lamina deltoid-lanceolate, pale green, thinly leathery, basal pinnae pair largest, basal basiscopic pinnule of lowest pinnae largest, rare from 1800-2700 m altitude.

Distribution: Bhutan, Nepal, China, India (Kashmir, Himachal Pradesh, Kumaun, Uttarakhand, Sikkim, West Bengal).

Specimen examined: D.K. Pora (SAM 856), 06/07/2011, 1830 m 69. Dryopteris wallichiana (Spreng.) Hylander, Bot. Notis: 352 (1953).

Synonym: Aspidium donianum Spreng.; A. paleaceum Lag. ex Sw.; A. paleaceum (T. Moore) Dalla Torre & Sarnth.; A. parallelogrammum Kunze; A. patentissimum Wall. ex Kunze; A. Wallichianum Spreng.; Dichasium parallelogrammum (Kunze) Fee; D. patentissimum (Wall. ex Kunze) Fee; Dryopteris cyrtolepis Hayata; D. doiana Tagawa; D. doniana (Spreng.) Ching; D. himalaica (Ching & S.K. Wu) S.G. Lu; D. pachyphylla Hayata; D. paleacea (T. Moore) Hand. Mazz.; D. parallelogramma (Kunze) Alston; D. patentissima (Wall. ex Kunze) N.C. Nair; D. quatanensis Ching; D. ursipes Hayata; D. Walliachiana var. himalaica Ching & S.K. Wu; Lastrea paleacea (Lag. ex Sw.) T. Moore; L. parallelogramma (Kunze) Liebm.; L. patentissima C. Presl; L. patentissima (Wall. ex Kunze) J. Sm.; Nephrodium parallelogrammum (Kunze) Hope; N. patentissimum (Wall. ex Kunze) C.B. Clarke Very large fern growing on forest floor, rhizome massive, stipe very densely scaly, scales at stipe base blackish mixed with pale ones, scales upwards along with rachis light-brown to paler, mixed with few blackish ones, scale base usually dark, rachis densely scaly and fibrillose; lamina green to deep-green, large, ca. 70x22 cm, sori indusiate, round, 2/3rd of frond is fertile, indusia darkbrown, reniform falling off at maturity, frequent from 2300-3500 m altitude.

Distribution: Argentina, Bhutan, Borneo, Burma, China, Malaysia, Myanmar, Nepal, Jamaica, Cuba, Brazil, Tibet, Nepal, Taiwan, Japan, Mexico, Vietnam, Philippines, Java, India (Kashmir, Himachal Pradesh, Uttarakhand, Sikkim, Meghalaya, Arunachal Pradesh, Nilgiri hills).

Specimen examined: Dubjan (SAM 929); 27/07/2011; 2725 m.

70. Dryopteris xanthomelas (H. Christ) C. Chr., Index Filic., Suppl. 1: 41 (1913).

Synonym: Aspidium xanthomelas Christ; Dryopteris discrete Ching & S. K. Wu; D. omeicola Ching; D. canaliculata Ching in C. Y. Wu; D. centrochinensis R. C. Ching; D. chingii Nair; D. fibrillosa (C. B. Clarke) Hand.

Mazz; D. fibrillosissima Ching in C. Y. Wu; D. hupehensis Ching; D. pulcherrima Ching; D. qandoensis Ching in C. Y. Wu; D. rosthornii (Diels) C. Chr.; D. rosthornii sensu Stewart; D. sinofibrillosa Ching; Nephrodium rosthornii Diels Large fern growing on forest floor and scrub zone, stipe black, base densely scaly and fibrillose, scales black,

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broadly lanceolate, glossy, apex acuminate, upper part together with rachis clothed with linear-lanceolate and linear, dentate scales, lamina ca. 30x10 cm, sori indusiate, upper part of lamina fertile, indusia brown, roundedreniform, frequent from 2400-3800 m altitude.

Distribution: Bhutan, Nepal, Pakistan, Tibet, Taiwan, India (Kashmir, Himachal Pradesh, Arunachal Pradesh, Uttarakhand, Sikkim, West Bengal).

Specimen examined: Dubjan (SAM 880), 27/07/2011, 3320 m; Huran (SAM 945), 25/09/2012, 3450.

71. Polystichum bakerianum (Atk. ex C.B. Clarke) Diels, Nat. Pflanzen fam. 1: 191 (1899).

Synonym: Aspidium bakerianum (Atk. ex C. B. Clarke) Atk. ex Baker; A. prescottianum var. bakerianum Atk. ex C. B. Clarke; Polystichum prescottianum var. bakerianum (Atk. ex C. B. Clarke) Bedd.

Large fern growing on steep and open bushy areas at higher altitudes, stipe and rachis densely scaly and fibrillose, lamina abaxially scaly and fibrillose, pinnule margin lobed, lobes ending in a sharp long and curved tooth, frequent from 3000-3900 m altitude.

Distribution: Afghanistan, Bhutan, China, Nepal, Pakistan, India (Kashmir, Himachal Pradesh, Uttarakhand, Sikkim, Arunachal Pradesh).

Specimen examined: Peergali (SAM 917), 18/08/2012; 3250 m.

72. Polystichum castaneum (C. B. Clarke) B. K. Nayar & S. Kaur, Comp. Bedd. Handb. Ferns Brit. India, 50 (1974).

Synonym: Aspidium prescottianum Wallich ex Mettenius var. castaneum C. B. Clarke; Polystichum prescottianum (Wallich ex Mettenius) T. Moore var. castaneum (C. B. Clarke) Beddome Medium size fern growing in alpine meadows, stipes and rachis very densely scaly and fibrillose, scales usually light-brown with few dark streaks mixed with blackishbrown, pinnae basal pairs slightly distant and reduced, margin serrate and spinulose, occasional from 3200-4600 m altitude.

Distribution: China, Myanmar, India (Jammu and Kashmir, Himachal Pradesh, Garhwal, Sikkim, West Bengal).

Specimen examined: Dubjan (SAM 919), 10/08/2012, 3300 m 73. Polystichum discretum (D. Don) J. Smith, J. Bot. 3: 413 (1841).

Synonym: Aspidium discretum D. Don; Polystichum discretum (D. Don) Diels; P. fuscopaleaceum Alston; P. indicum Khullar & S. C. Gupta; P. kathmanduense Nakaike; P. nigropaleaceum (H. Christ) Diels Large fern growing at shaded and humus rich areas, stipe densely fibrillose, scaly, scales dark-brown to almost black, lamina abaxially fibrillose, adaxially shiny, lowest pinnae pair deflexed forward and downwards, pinnule apex acute with prominent teeth curved towards pinna apex, occasional from 1700â&#x20AC;&#x201C;2700 m altitude.

Distribution: Africa, Burma, China, Japan, Malaya, Sri Lanka, Bhutan, Myanmar, Nepal, Pakistan, Thailand, India, Kashmir, Himachal Pradesh, Uttarakhand, West

Bengal, Meghalaya, Bihar, Tamil Nadu, Sikkim, Manipur, Arunachal Pradesh).

Specimen examined: Narwani (SAM 881), 25/07/2011, 1775 m; Kund (SAM 945), 24/09/2012, 2350 m.

74. Polystichum lachenense (Hook.) Bedd, Ferns Brit.

India, pl. 32 (1865).

Synonym: Aspidium lachenense Hook.; Polystichum aleuticum C. Chr. ex Holten; P. tsuchuense Ching in C. Y. Wu; P. sinkiangense Ching ex Chang Y. Yang; P. xinjiangense Ching ex C.Y. Yang.

Small fern growing under rocks, stipe dark brown to blackish-purple, glossy, rachis scaly and fibrillose, lamina fragile, abaxially fibrillose, pinnae deltoid-ovate, distant, lobed, lobes ending in acute tooth, upper half of frond fertile; rare from 3000-4200 m altitude.

Distribution: Burma, China, Japan, Bhutan, Myanmar, Nepal, Taiwan, Pakistan, Tibet, India, Kashmir, Himachal Pradesh, Uttarakhand, Sikkim).

Specimen examined: Huran (SAM 866), 10/07/2011, 3550 m; Kaunsernag (SAM 920), 28/08/2012, 3450 m; Dubjan (SAM 937), 20/09/2012, 3260 m.

75. Polystichum lonchitis (L.) Roth. Tent. Fl. Germ. 3: 71 (1799).

Synonym: Aetopteron lonchitis House; Aspidium lonchitis (L.) Sw.; Dryopteris lonchitis (L.) O. Kuntze; Hypopeltis lonchitis Tod.; Polypodium lonchitis L.; Polystichum asperum Bubani Lithophytic fern growing at higher altitudes in alpine and subalpine regions, lamina linear to linear-lanceolate, base narrowed, drooping at tip, glossy, pinnae edges with spiny teeth, auricled at anterior and cunate at posterior bases, rare from 2700-4000 m altitude.

Distribution: Afghanistan, N. America, Iran, Ireland, China, Japan, Europe, Pakistan, India (Kashmir).

Specimen examined: Huran (SAM 862), 10/07/2011, 3550 m; Dubjan (SAM 938), 20/09/2012, 3330 m.

76. Polystichum luctuosum (Kunze) T. Moore Index Fil. 95 (1858).

Synonym: Aspidium tsus-simense Hook.; A. luctuosum Kunze; A. aculeatum var. pallescens Franch.; Polystichum monotis Christ; P. falcilobum Ching; P. tsus-simense var. pallescens Franch. P. mayebarae Tagawa Terrestrial fern growing in shady areas along streams with partial to no sun exposure, rhizome scaly, scales brown with blackish apex, lower pinnae bending forward and downward, pinnules auriculate, ovate-trapezoid, rare from 1700-2500 m altitude.

Distribution: Japan, China, Korea, Madagascar, Nepal, Pakistan, Africa, Tibet, Taiwan, India (Jammu and Kashmir, Uttarakhand, Arunachal Pradesh).

Specimen examined: Kund (SAM 926), 16/09/2012, 1900 m.

77. Polystichum nepalense (Spreng) C. Chr., Index Filic. 1: 84 (1905).

Synonym: Aspidium auriculatum var. marginatum C. B. Clarke; A. marginatum Wall.; A. nepalense Spreng; Polystichum atroviridissimum Hayata; P. auriculatum var. marginatum Bedd.

Medium size fern growing in rocky meadows near stream, Stipes scaly and fibrillose, lamina pinnate,

MIR et al. â&#x20AC;&#x201C; Pterydophytes of Shopian, Kashmir Valley

narrowly lanceolate, slightly narrowed below, margin irregularly more or less entire or slightly serrate with pale colored teeth, rare from 2000-3200 m altitude.

Distribution: Afghanistan, Bhutan, Burma, china, Japan, Myanmar, Nepal, Philippines, Sri Lanka, Tibet, Vietnam, Yunnan, India (Arunachal Pradesh, Darjeeling hill, Himachal Pradesh, Manipur, Meghalaya, Mizoram, Nagaland, Sikkim, Uttarakhand).

Specimen examined: Secjan (SAM 928), 08/09/2012, 2400 m.

78. Polystichum piceopaleaceum Tagawa in Acta Phytotax. Geobot. 5: 255 (1936).

Synonym: Aspidium angular sensu Hope; Polystichum aculeatum var. fargesii H. Christ; P. bicolor Ching & S.K. Wu; P. doianum Tagawa; P. setiferum var. fargesii (H. Christ) C. Chr.; P. yunnanense var. fargesii (H. Christ) C. Chr. Terrestrial fern growing on forest floor, rocky meadows, lamina bipinnate, abaxially scaly and fibrillose, apex acuminate, lower pinnae pairs deflexed downwards; pinnules rhomboidal-ovate, frequent from 1800-3200 m altitude.

Distribution: Afghanistan, Bhutan, Burma, China, Japan, Myanmar, Nepal, Pakistan, Sri Lanka, Taiwan, India (Jammu and Kashmir, Himachal Pradesh, Arunachal Pradesh, Uttarakhand, Assam, Manipur, Meghalaya, Nilgiri hills, Nagaland).

Specimen examined: Aharbal (SAM 825) 05/07/2011, 2650 m; Heerpur (SAM 933), 24/07/2011, 2500 m; Secjan (SAM 894), 06/08/2012, 2450 m.

79. Polystichum prescottianum (Wall. ex Mett.) T. Moore. Ind. Fill.: 101 (1858).

Synonym: Aspidium mouoinense Franch.; Aspidium prescottianum Wall. ex Mett.; Polystichum erinaceum Ching & S.K. Wu.; P. mouoinense (Franchet) Bedd.

Large fern forming colonies in open meadows above the tree line, fronds clustered, stipe base dark-blue, densely scaly and fibrillose, lamina densely fibrillose, pinnae margin deeply lobed, lobes/pinnules serrate with each lobe ending in an awn or spine, upper part of the frond fertile, common from 2800-3900 m altitude.

Distribution: Afghanistan, Bhutan, China, Pakistan, Taiwan, Nepal; India (Kashmir, Himachal Pradesh, Uttarakhand, Sikkim, West Bengal).

Specimen examined: Huran (SAM 861), 08/07/2011, 3200 m; Dubjan (SAM 939), 10/08/2012, 3300 m.

80. Polystichum shensiense Christ, Bull. Acad. Geogr.

Bot. Mans. 16: 113 (1906).

Synonym: Dryopteris lichiangensis (Wright) C. Chr.; Nephrodium lichiangense C.H. Wright; Polystichum lichiangense (Wright) Ching ex H.S. Kung; P. obtusipinnum Ching & H.S. Kung; P. prescottianum var. shensiense (H. Christ) C. Chr. Terrestrial fern growing above tree line, stipe and rachis scaly and fibrillose, scales light-brown, broad-ovate intermixed with lanceolate ones, pinnae sessile, alternate, triangular-lanceolate, margin deeply segmented or becoming pinnate, segments small with few marginal teeth, rare with the range of 3200-4500 m altitude.

41

Distribution: Bhutan, China, Taiwan, Tibet, Nepal, Pakistan, India (Kashmir, Uttarakhand, Himachal Pradesh).

Specimen examined: Kaunsernag (SAM 816), 15/07/2012, 3450 m; Dubjan (SAM 936), 26/09/2012, 3350 m.

81. Polystichum yunnanense H. Christ, Notul. Syst.

(Paris) 1: 34 (1909).

Synonym: Polystichum gyirongense Ching; P. jizhushanense Ching; P. yunnanense var. submuticum C. Chr.; P. makino sensu Frase-Jenkins & Khullar Large fern growing on forest floor, stipes long brown, stipe base scaly, rachis densely fibrillose, scaly, lamina broadly lanceolate, dark bluish-green upper surface with little depressions opposite the sorus, basal acroscopic pinnule largest, margin deeply lobed, lowest lobe largest and parallel to the costa facing towards apex, margin with sharp teeth, frequent from 2000-3200 m altitude.

Distribution: Afghanistan, China, Tibet, Burma, Bhutan, Myanmar, Nepal, Pakistan, India (Jammu and Kashmir, Himachal Pradesh, Uttarakhand, Tamil Nadu, Sikkim, Arunachal Pradesh, Meghalaya, Manipur, Darjeeling hills).

Specimen examined: Aharbal (SAM 873), 23/07/2011, 2250 m; Kund (SAM 912), 16/09/2012, 2120 m.

Botanical study of flora of particular area is an important aspect as it forms baseline information for the distribution of plant species or communities and their relation with physical environment. This paper gives a broad outlook about the pteridophytes of Shopian. About 81 species of ferns and fern allies have been collected in the course of the study for three consecutive years, 20102012; indicating that agro-climatic conditions of the study area are favorable for the growth and development of pteridophytes. The pteridophytes form a vital element of the ecosystem and most of them being forest dwellers that can be taken as good indicators of the extent of problems like deforestation and habitat destruction. Botanical explorations should increase in the under-explored botanically rich areas for documenting the diversity and ecological characteristics of pteridophytes. Taxonomic reinvestigations in such areas should take place to avoid the confusions with new species and existing species.

ACKNOWLEDGEMENTS The first author is thankful to University Grants Commission (UGC), New Delhi, India for providing financial assistance to carry out the research work. Authors are also thankful to Late Dr. H. C. Pande (Sci.-'D') and Brijesh Kumar, Botanical Survey of India, Dehradun; Dr. Shweta Singh, Guru Gobind Singh Indraprastha University, New Delhi; and Prof. S. P. Khullar, Chandigarh for their help in the identification of plants and rendering me necessary literature facilities.

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Figure 2. Asplenium punjabense Bir, Fraser-Jenk. & Lovis.

Figure 3. Pseudophegopteris pyrrhorachis (Kunze) Ching, subsp. distans (Mett.) Fraser-Jenk.

MIR et al. – Pterydophytes of Shopian, Kashmir Valley

REFERENCES Beddome RH. 1883. Handbook to the ferns of British India Ceylon & Malay Peninsula. Thacker Spink & Co., Calcutta Beddome RH. 1892. Supplement to the ferns of British India Ceylon & Malay Peninsula. Thacker Spink & Co., Calcutta.

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B I O D I V E R S IT A S Volume 16, Number 1, April 2015 Pages: 44-54

ISSN: 1412-033X E-ISSN: 2085-4722 DOI: 10.13057/biodiv/d160106

Local knowledge of medicinal plants in sub-ethnic Batak Simalungun of North Sumatra, Indonesia MARINA SILALAHI1,3,♥, JATNA SUPRIATNA1, EKO BAROTO WALUJO2, NISYAWATI1 1

Departement of Biology, Faculty of Mathematics and Natural Science, Universitas Indonesia, Depok, 16424, Indonesia 2 Division of Botany, The Indonesian Institute of Sciences, Cibinong, Bogor, 16911, Indonesia 3 Departement of Biology Education, Faculty of Education and Teacher Training, Universitas Kristen Indonesia, Jakarta. Jl. Mayjen Sutoyo, No. 2 Cawang, Jakarta 13630. Tel. +62-21-8009190, 8092425. Fax. +62-21-80886882. ♥email: marina_biouki@yahoo.com Manuscript received: 29 October 2014. Revision accepted: 12 Desember 2014.

Abstract. Silalahi M, Supriatna J, Walujo EB, Nisyawati. 2015. Local knowledge of medicinal plants in sub-ethnic Batak Simalungun of North Sumatra, Indonesia. Biodiversitas 16: 44-54. Research on the local knowledge of medicinal plants by sub-ethnic Batak Simalungun of North Sumatra was conducted, using an ethnobotanical approach. The sample consisted of 8 key informants and 32 general respondents, who were grouped into two, namely those who were 30-50 years old and >50 years old. Data were analyzed both qualitatively by descriptive statistics and quantitatively by calculating the index of cultural significance (ICS) and the use value (UVs).

It was found that 239 species (170 genera, 70 families) of medicinal plants were used to cure 18 kinds of natural diseases and 2 kinds of supra natural diseases. Almost half of those plants (119 species) had leaves used as medicines. Among the diseases, gastrointestinal disorders had the highest number of medicinal plants used (72 species), followed by fever (64 species), and fractures (41 species). It seemed that younger generation had lost their knowledge in the medicinal plants because their knowledge of medicinal plants (48.19 ± 8.35 species) was lower than the that of older generation (170.19 ± 18.38 species), while our key informants had the highest knowledge of medicinal plants among respondents (202.00 ± 12.32 species).

Keywords: local knowledge, medicinal plants, North Sumatra, sub-ethnic Batak Simalungun.

INTRODUCTION Man is known to have utilized plants as a source of drugs for thousand years. The World Health Organization (WHO) estimated that about 60-80% of the world population still relies on the traditional medicine derived from plants (Joy et al. 1998), and Indonesian population (60%) relies on traditional medicinal plants for their health care. Medicinal plants are species known to have efficacy to help maintain the health or to cure a disease. Medicinal plants are grouped into three: traditional, modern, and potential medicinal plants.

The local knowledge of medicinal plants is inherited orally or in written in ancient manuscripts. The main obstacle encountered in recovering local knowledge through ancient manuscripts is the difficulty in reading, because some parts of many manuscripts have been lost or damaged (Nawangningrum et al. 2004). To overcome this, it is necessary to look for more efficient alternative methods to recover the local knowledge. Ethnomedicine is an alternative that has been used because it may use both ethnology and medicinal science (Martin 1995).

Ethnomedicine is the study of perception and conception of local communities in understanding health or research that studies the traditional ethnic medical system.

The high effectiveness of ethnomedicine research is due to reduction of time and costs in finding new chemical compounds used as drugs (Fabricant and Farnsworth 2001).

Ethnomedicinal study is done through a community perspective approach (emic approach), which is then

proved through scientific approach (ethic approach) (Walujo 2009).

The traditional medicine is related to cultural diversity, ethnic diversity, and biodiversity of plants. Indonesia has more than 300 ethnics, one of which is Batak, which consists of five sub-ethnics or tribes often referred to as sub-ethnics, namely Karo, Phakpak, Simalungun, Toba, and Angkola-Mandailing (Bangun 2010). Researches on the usage of plants by local communities or ethnic groups in Sumatra have been carried out, among others: Batak Toba (Simbolon 1994), Rejang (Darnaedi 1999), Malay (Setyowati and Siagian 2004; Setyowati and Wardah 2007; Sunesi and Wiryono 2007; Rahayu et al. 2007; Hariyadi and Ticktin 2011. Those studies show that the diversity of the medicinal plants used by local communities depends on the ethnicity, locality, age of respondent, and number of respondents.

It is really unfortunate that high deforestation in Indonesia that causes the loss of many plant species and understanding of local knowledge will hamper our efforts to find new drugs. The high rate of erosion of the local knowledge has been found everywhere in the world including Indonesia (Hoang et al. 2008). At the same time, our local knowledge of medicinal plants are kept only by the older people (>50 years old) and shamans (Darnaedi 1999). While the rate of species loss is also similar to the rate loss of local knowledge (Hoang et al. 2008).

Meanwhile, researches on the local knowledge of subethnic in Sumatra have not been intensively carried out. For that purpose, we have done our research on the

SILALAHI et al. – Medicinal plants of Batak Simalungun, Indonesia

ethnomedicine of sub-ethnic Batak Simalungun. This research had two objectives: (i) to understand the local knowledge of medicinal plants in sub-ethnic Batak Simalungun; (ii) to understand and preserve the value and cultural heritage of the medicinal plants.

MATERIALS AND METHODS Study area Our study site is located in Nagori Simbou Baru, Raya Sub-district, Simalungun District, North Sumatra, Indonesia. The total area of those villages is 2002.96 hectares, within the altitude of 650-700 m above sea level.

Simbou Baru village is geographically located at N 2o57’05’’ and E 98o57’84’’ (Figure 1).

Data Collection To obtain the local knowledge on medicinal plants in sub-ethnic Batak Simalungun, interviews were conducted with ethnobotanical approach (Martin 1995; Alexiades 1996). It was conducted through semi-structured and indepth interviews. The interviews were conducted with 8 key informants (healers, ethnic chiefs), 32 general respondents with two age groups: the first group was 30-50 years old and the second group above 50 years old with a ratio of 1:1.

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Data analysis Data were analyzed using qualitative and quantitative methods. Qualitative analysis was done by grouping plants based upon usage category. Quantitative analysis was done by determining UVs, ICS, and calculating the differences of those parameters in statistical analysis, using Anova.

The results consisted of: (i) Index of cultural significance (ICS), calculated using the formula of Turner (1988), (ii) use value (UVs) of each species was based on Prance et al.

1987, and Anova was calculated using software SPSS version 17.

RESULTS AND DISCUSSION The concept of “disease” in Sub-ethnic Batak Simalungun The local knowledge of sub-ethnic Batak Simalungun in making use of plants as medicine is related to the concept of diseases. The diseases are grouped into natural and supra natural diseases. Natural diseases are those caused by the malfunctioning of the body such as, fever, toothache, ulcers, and diarrhea. Sub-ethnic Batak Simalungun has known as many as 18 kinds of natural diseases (Table 1). The medicinal plants which have been used to cure diseases vary in number and species. The highest number of medicinal plants used were those to cure gastrointestinal disorders (72 species), followed by fever (64 species), and fractures (41 species).

Figure 1. Study site of Nagori Simbou Baru, Raya Sub-district, Simalungun District, North Sumatra, Indonesia.

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Table 1. Number of medicinal plants species used to cure the “diseases” in sub-ethnic Batak Simalungun of North Sumatra.

Characteristics Name of diseases Natural disease Hypertension Cough Asthma Diarrhea Gastrointestinal disorders Stomach ache Fractures Rheumatism Itch Ulcer Kidney disease Diabetes mellitus Aphrodisiac Injury Fever Eye infection Thrush Toothache Supranatural Busung (liver disease) disease Alogo-alogo (“malnutrition”) Traditional Tinuktuk tawar (“mashed concoction concoction” to maintain stamina) Tinuktuk paranggetek (“mashed concotion” to maternity)

Number of species 15 10 24 22 72 12 41 6 8 12 25 21 13 39 64 6 4 5 23 18 117 11

The high frequency of gastrointestinal disorder is caused by poor sanitation in their houses and in the surrounding villages, which may force local communities to explore any medicinal plants to cure the diseases.

Medicinal plants which have bitter taste such as: jambu batu (Psidium guajava), horis kotala (Eurycoma longifolia), ratusan (Ageratum conyzoides), and andor golat (Mikania cordata) have been used to cure gastrointestinal disorders. The bitter taste in those plants is due to its chemical contents, namely tannin and flavonoid.

The tannin has been proven to cure the diarrhea (de Padua et al. 1999). Tannin is able to form a thin layer on the lumen, therefore reducing irritation (Munim and Hanani 2011). Those plants can also prevent water secretion and also kill microbes (Achmad et al. 2008). Furthermore, Munim and Hanani (2011) say that the tannin causes the denaturation of protein whereas flavonoid causes the destruction of cell wall of bacteria. The extract of Psidium guajava leaves prevents the growth of Escherichia coli (Voravuthikunchai et al. 2004), Staphylococcus aureus, Bacillus subtilis, and Pseudomonas aeruginosa (Abdelrahim 2002).

A large number of plants species have been used to cure fever, a symptom caused by some deseases such as chicken pox, cough, and wounds. Those plants were identified as follows: bunga raya (Hibiscus rosa-sinensis), habu-habu (Ceiba pentandra), silundad (Impatiens platypetala), rampas binei (Drymaria cordata), jarango (Acorus calamus), and horis kotala (Eurycoma longifolia). The ability of plants to reduce fever is related to the content of bioactive compounds. Buenz et al. (2005) state that Ceiba pentandra has a chemical compound that serves as

16 (1): 44-54, April 2015

catechins, ᵝ-citosterol which serves as antipyretic and analgesic.

The local knowledge of curing fractures has presumably been adopted from the behavior of a bird called siburuk.

They have learned from siburuk bird that when their chicks are fractured and hurt by communities, the mother will bring the herbs. There were 41 species of plants used to cure fractures. To cure fractures, those plants have to be cooked using coconot oil. The examples of plants used for curing fractures were jengkol (Pithecolobium lobatum), sibaguri (Sida ungulata), ompu-ompu (Crinum asiaticum), kelapa (Cocos nucifera), and baru (Hibiscus similis). The ompu-ompu leaves contain alkaloids, glycosides, triterpenes, coumarins which can serve as an analgesic (Asmawi et al. 2011) and anti-inflamantory (Rahman et al.

2013).

Supra natural diseases are those caused by supra natural spirits, bad person, and curse (karma) such as busung (liver disease) and alogo-alogo (“malnutrition”). Local communities believed that busung disease only occurs to people who commit a theft. Busung disease will be treated by shaman, through a series of rituals. Those plants used to treat busung have been identified as: kelapa (Cocos nucifera), jarango (Acorus calamus), utte mungkur (Citrus hystrix), silinjuang (Cordyline fruticosa), bagot (Arenga pinnata), and demban (Piper betle).

Alogo-alogo (alogo means wind) disease affects the children. Local communities believe that the alogo-alogo disease is caused by sin of their ancestors. The patients are characterized by thin body, pale face, bloated abdomen, and fever at night. Plants used to cure alogo-alogo disease were, among others, demban (Piper betle), jarango (Acorus calamus), and lada (Piper nigrum). Those plants are able to warm the body, so they can expedite the blood circulation, because Acorus calamus contains high potassium, which can be used to cure fever (Motley 1994).

Sub-ethnic Batak Simalungun has traditional concoction called tinuktuk (tuntuk = mashed). It is called tinuktuk because of the producing process, namely by mashing a variety of medicinal plants. The local communities can distinguish two different kinds of tinuktuk: tinuktuk tawar (to maintain stamina) and tinuktuk paranggetek (concoction for maternity). The plants that have been used to cure tinuktuk tawar were 117 species while for tinuktuk paranggetek were 11 species.

To cure Tinuktuk concoction, the roots, leaves, and tuber of 117 species of plants have been used. Roots of 7 species of bamboo (Poaceae), 7 species of palms (Arecaceae), 7 species of rattan (Arecaceae), and 7 species of Citrus (Rutaceae) were among the listed plants. While tubers that have been used were from ground orchid (Orchidaceae). The roots to be used were firstly cut into small pieces, dried, and crushed. The materials of leaves were mashed until fine and then squeezed to get the water out. The water resulted from squeezed materials was used to boil the roots until all the water evaporated and the material became dry and salt was used as preservative.

Tuber of Orchidaceae such as: salembar satahun (Nervillia plicata), salembar sabulan (Nervilia aragoana), and gadong harangan (Goodyera rubicunda) have been used.

SILALAHI et al. â&#x20AC;&#x201C; Medicinal plants of Batak Simalungun, Indonesia

Whereas rattan from both genus Daemonorops and Calamus, have been utilized more often. Other tribes such as Anak Dalam tribe in Jambi have used Daemonorops to cure wounds and headache (Sulasmi et al. 2012).

In the process of producing tinuktuk, some local communities (7-10 people) cooperate because they have to find plants from the forests, farms, and plantations. Due to difficulties in finding the plants, the young generation (<50 years old) tend to ignore this kind of treatment and consider those practices are out of date.

Diversity of medicinal plants The sub-ethnic Batak Simalungun has used as many as 239 species (170 genera, 70 families) of medicinal plants.

Those plant species which have been used by local communities were spermatophyta (230), pterydophyta (8) and lichens (1). Figure 2 shows that the main families of medicinal plants that have been used consisted of Arecaceae (20 species), followed by Poaceae (16 species), Rutaceae (13 species) and Zingiberaceae (12 species). The highest number of genera was found in Arecaceae (13 genera), followed by Euphorbiaceae (12 genera), Poaceae (11 genera), Fabaceae and Asteraceae (10 genera).

At least 20 species of Arecaceae have been used as medicinal plants by sub-etnis Simalungun, , most of which were rattans and palms. Some of the palms mainly used for medicines were kelapa (Cocos nucifera), bagot (Arenga pinnata), and pining (Areca catechu). Cocos nucifera was used to cure fever, fractures and as a component of tinuktuk concoction because it has anti bacterial, anti fungal, antiviral, immunostimulant, antioxidant, and hypoglycemia (Debmandal and Mandal 2011).

It was found that 18 spesies of medicinal plants belong to Poaceae, especially bamboo. Most parts of bamboo both leaves and roots have been used as medicinal plants. The leaves of bamboo have been used to cure injury and fever.

The same usage of bamboo leaves is found in Chinese medicines (Wang et al. 2012).

Utte bunga (Citrus aurantium), utte mungkur (Citrus hystrix), and tuba (Zanthoxylum acanthopodium) belong to Rutaceae and they were commomly used. Out of 15 spesies of Rutaceae, 13 spesies are from genus Citrus and 2 more spesies are from other genera. The number of species of Rutaceae was relatively small in comparation to the other families, however, the frequency of species used was relatively high. Simalungun and Karo Districts are the centers of Citrus cultivation in North Sumatra.

Sub-ethnic Batak Simalungun used eight species of Lamiaceae as medicinal plants, some of those were sibabi dalu (Paraphlomis javanica), silanglang kabungan (Coleus scutellarioides), simarihur-ihur niasu (Pogostemon auricularius), and terbangun (Coleus amboinicus) which have been known to be rich in essential oils, so they can be used to cure gastrointestinal disorders and fever.

Solanaceae has been used to cure fractures latting (Solanum verbascifolium), and injury timbaho (Solanum nicotiana). In Brazil, Solanum nigrum have been used to cure anti depression (Giorgetti and Negri 2011).

Our research also found that orchids (Orchidaceae) have been used for medicinal plants such as, salembar

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sabulan (Nervilia aragoana), salembar satahun (Nervilia plicata), and gadong harangan (Goodyera rubicunda).

Tuber of orchids can be used to make traditional concoction (tinuktuk), which is to enhance stamina.

Figure 2. Composition of species and family used for medicinal plants by sub-ethnic Batak Simalungun.

Figure 3. Parts of medicinal plants used as medicines in subethnic Batak Simalungun, North Sumatra.

Figure 4. The corelation between age of respondents and means of medicinal plants known.

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Table 2. Local and scientific names of medicinal plants in sub-ethnic Batak Simalungun of North Sumatra, Indonesia.

Family Acanthaceae

Actinidiaceae Amaranthaceae Amaryllidaceae Annonaceae Apiaceae Apocynaceae Araceae Arecaceae

Scientific name

Leaves Leaves Leaves Leaves

Gas, Fev Ulc, Kid Fev, Bus Gas, Fev

Topu arang

Leaves

Ash, Aph, Fev, Tt 36.0

-

2.2

2.8

Kecibeling Pijor holing Topu ringring Sopsopan Rudang Ompu-ompu Paet tandang Papaga Rahu Jarango Suhat sabah Hau sangggir Pining Bagot Malno Hotang pulogos Hotang aek Hotang kiskisan Hotang rusrus Riman Andudur Kelapa

Gas, Kid, Tt Ash, Kid Kid Dia, Gas, Inj, Tt Fev, Bus, Alg Fra, Fev Gas, Inj Gas, Kid, Inj Dia, DM Fev, Bus, Alg Fev Fev Fra, Bus, Tt, Fra, DM, Tt Tt Fra, Tt Tt Fra, Tt Tt Tt Tt Fra, DM, Bus, Tt

30.0 42.0 33.0 12.0 13.5 45.0 9.0 45.0 24.0 30.0 6.0 12.0 60.0 30.0 9.0 6.0 6.0 6.0 6.0 24.0 24.0 60.0

0.8 0.6 1.2 1.2 0.8 1.0 0.8 1.0 0.4 0.6 0.6 0.2 1.8

1.0 1.4 0.6 1.8 1.4 1,6 0.4 2.0 1.2 1.8 0.4 0.6 1.6 0.8 0.8 0.8 0.6 0.8 0.8 0.6 1.0 1.6

0.8 1.5 0.8 2.5 2.0 1.8 0.5 2.5 1.3 2.0 0.8 0.8 1.6 2.0 0.8 1.0 1.0 1.0 0.8 1.0 1.0 2.0

Cyrtostachys lakka

Simarpiningpining Hotang rutti Boar-boar Hotang dadahanan Baluhur Biruh Nipah Hotang Boar-boar Libung Salak Simanisia Tukkok matua sabungan Tukkot matua boru-boru Ratusan Galunggung Longa begu somarittop Sihampir safari

Leaves Leaves Leaves Leaves Leaves Tuber Leaves Leaves Bark Stem Stem Tuber Fruit Root Root Root Root Root Root Root Root Root, fruit Root

Ash, Kid, Fev

4.5

-

1.6

1.3

Root Root Root

Tt Tt Fra, Tt

6.0 24.0 6.0

-

0.8 1.0 1.6

0.8 0.8 0.8

Root Root Root Root Root Fruit Leaves Leaves

Tt Tt DM, Tt Tt Fra, Tt Gas Fra, Kid, Aph, Tt Ash, Fra, Aph, Tt

24.0 12.0 24.0 6.0 15.0 18.0 12.0 109.0

1.0 0.8 1.6

0.8 0.8 0.6 0.8 1.4 1.0 0.4 2.0

1.0 1.0 1.0 1.0 1.5 0.8 2.0 3.3

Leaves

Ash, Aph, Tt

93.0

-

2.4

2.8

Leaves Leaves Leaves

24.0 24.0 45.0

2.2 1.2 1.6

1.8 1.6 2.4

2.3 2.0 2.5

Leaves, fruit Leaves Leaves Leaves

Ulc, DM, Inj Gas, Inj, Bus Dia, Gas, DM, Fev Hyp, Dia, Gas, Ulc, Fev, Inj, Tt Gas, Fra, Rhe Inj Dia, Gas, DM

106.0 -

4.8

6.0

45.0 3.0 45.0

3.0 1.8

1.8 0.8 1.8

2.5 1.0 2.5

Leaves Leaves Leaves Leaves Leaves Leaves Leaves

Gas, Kid, Inj, Fev Gas, Fev Dia, Gas Too Fev Fra, Fev Gas, Kid, Fev

45.0 42.0 55.0 9.0 9.0 9.0 6.0

1.6 2.0 2.4 0.6 0.8 -

1.8 2.0 2.2 0.8 0.8 1.0 1.2

2.5 2.0 2.5 0.8 1.0 1.5 1.8

Livistona sp.1 Livistona sp.2 Nypa fruticans Plectocomia cf. elongata Oncosperma filamentosum Salacca zalacca Hoya sp.1 Hoya sp.2

Ageratum conyzoides Blumea balsamifera Clibadium surinamense Chromolaena odorata Chromolaena sp.1 Elephantopus scaber Eupatorium inulifolium

Balsaminaseae Blechnaceae

1.0 0.8 0.6

Key informants 1.5 1.5 1.0 2.0

ICS

Siborutiktik Silastom Sangke sempilit Andalotung

Hoya sp.3 Asteraceae

Use*

UVs > 50 years old 1.2 1.2 1.0 1.6

30-50 years old

Clinacanthus nutans Graptophyllum pictum Justicia gendarussa Parastrobilanthes parabolica Psedeanthemum acumiinatisimun Strobilanthes crispus Strobilanthes sp.1 Strobilanthes sp.2 Saurauia vulcani Celosia cristata Crinum asiaticum Cyathocalyx virgatus Centella asiatica Alstonia pneumatophora Acorus calamus Colocasia esculenta Colocasia sp.1 Areca catechu Arenga pinnata Calamus caecius Calamus cf. javensis Calamus sp.1 Calamus sp.2 Calamus sp.3 Caryota cf. maxima Caryota cf. mistis Cocos nucifera

Daemonorops sp.1 Daemanorops sp.2 Korthalsia junghuhnii

Asclepiadaceae

Local name

Part used

Gynura crepidioides Gynura sp.1 Mikania cordata Spilanthes iabadicensis Impatiens platypetala Blechnum orientale Blechnum sp.1

Suwawa Malehan Longa bengu marittop Payon baru Sihorhor Andor golat Sihampir Silundad Padung-padung Pahu lipan

6.0 36.0 30.0 6.0

SILALAHI et al. â&#x20AC;&#x201C; Medicinal plants of Batak Simalungun, Indonesia Bombacaceae Caricaceae Caryophyllaceae Convolvulaceae Costaceae Crassulaceae Cucurbitaceae Cyatheaceae Cyperaceae Dilleniaceae Euphorbiaceae

Fabaceae

Gesneriaceae

Guttiferae Lamiaceae

Ceiba pentandra Ceiba sp.1 Durio zibethinus Carica papaya Drymaria cordata Ipomoea batatas Ipomoea sp.1 Costus speciosus Costus sp.1 Kalanchoe pinnata Benincasa hispida Cucumis sativus Lagenaria siceraria Cyathea sp.1 Cyperus rotundus Tetracera scandens Aleurites moluccana

Habu-habu Kabu-kabu Durian Botik Rampas binei Gadong suwawa Hanawi Sibalik humosing Tabar-tabar Hatengget Gundur Ancimun Tatabu Tanggiang Sitomu dalan Pastulan Gambiri

Leaves Leaves Bark Leaves Whole Leaves Leaves Rhizome Rhizome Leaves Fruit Fruit Fruit Leaves Root Stem Seed

Bischofia javanica

Sinkam

Claoxylon indicum Euphorbia heterophylla Homalanthus populneus Jatropha curcas Mallotus philippinensis Manihot utilissima Phyllanthus urinaria Pimeleodendron griffithianum Ricinus communis Sauropus androgynus Cassia alata Cassia juncea Leucaena leucocephala

Topu hayu Katemas Andulpak Dulang jawa Sira lada Gadong hau Tanduk erbuah Sibunbun

Root, bark Leaves Sap Leaves Leaves Leaves Leaves Whole Leaves

Mimosa pudica Mimosa sp.1 Parkia speciosa

Podom-podom Sihirput Palia

Pithecolobium lobatum

Jengkol

Pterocarpus indicus Psophocarpus tetragonolobus Uraria lagopodioides Vigna unguiculata Aeschynanthus cf. horsfieldii Cyrtandra sp.1 Aeschynanthus sumatranus Garcinia atroviridis Coleus amboinicus Coleus scutellarioides

Fev Fev Gas Dia, Gas, Fev Fev Gas, Ulc, Tt Gas, Tt Aph Fev Ulc, Fev Gas, Sto Hyp Ash, Dia, Fra, Inj Fev, Inj Hyp, Ash, Kid Eye Dia, Gas, Sto, Kid, Fev, Tt Dia, Gas, Sto, DM, Inj Ash, Alg, Tt Gas Fev, Bus Gas, Fev, Too Tt Inj Hyp, Kid Fra, Fev

24.0 24.0 6.0 54.0 18.0 99.0 3.0 30.0 9.0 18.0 24.0 24.0 9.0 24.0 9.0 9.0 129.0

0.6 0.6 1.2 0.4 1.0 0.4 1.2 1.0 0.8 0.8 0.6 2.2

1.0 0.6 0.8 1.8 0.6 3.0 0.6 0.6 0.8 1.8 1.6 1.0 1.4 1.0 1.4 0.8 3.2

1.0 1.0 0.8 2.0 1.0 3.0 1.0 1.0 1.0 1.5 2.0 1.0 1.8 1.5 1.8 1.0 4.5

102.0 2.0

2.2

3.8

36.0 6.0 4.5 18.0 4.5 6.0 24.0 3.0

1.2 0.6 -

1.4 0.8 0.8 2.4 0.6 0.8 1.6 0.4

2.3 0.8 1.0 2.5 0.5 0.8 1.5 0.8

Sona Borong

Leaves Leaves Leaves Leaves Leaves, seed Root Root Bark, leaves Root, leaves Sap Leaves

Gas, Fev Gas, Fev Itc Fra, Inj, Fev, Tt Itc

18.0 36.0 15.0 90.0 4.5

1.4 1.0 0.6 -

2.0 1.8 0.6 1.8 0,6

2.0 1.8 1.0 2.3 0.8

Fra, Kid Cou, DM Gas, Itc

15.0 1.5 13.5

0.8 0.6

1.4 1.2 1.2

1.4 1.8 1.5

Gas, Fra

15.0

-

1.6

1.5

Fev, Too Inj, Tt, Tp

4.5 9.0

-

0.8 2.2

1.0 2.5

Sibalince Kacang safari Hariari sendok

Leaves Leaves Leaves

Fev Tt Fev

4.5 30.0 9.0

0.8 -

0.4 1.0 0.4

0.8 1.0 0.8

Dilah swara Simarhappilis Galugur Terbangun

Leaves Leaves Fruit Leaves

18.0 24.0 24.0 45.0

2.4

0.8 1.4 0.6 2.6

0.8 2.0 1.0 2.8

Leaves

Fra, Tt Bus, Tt Gas Ash, Gas, Inj, Tt, Tp Gas, Inj, Fev, Tt

66.0

-

3.2

3.3

Root Root Leaves Leaves

Cou, Rhe Aph, Tt Inj Fra, Fev, Alg, Tt

24.0 30.0 12.0 24.0

0.8 1.2

1.2 1.0 1.0 1.4

1.8 2.0 1.0 2.3

Kid Alg

30.0 24.0

1.0 0.4

0.6 0.8

1.0 1.0

Kid Kid Cou, Dia, Gas, Rhe, Ulc, Inj, Fev, Bus, Alg, Tt Hyp, Dia, Gas, Itc, Ulc, DM, Fev, Tt Hyp, Dia, Bus Tt

24.0 6.0 96.0

2.2

1.0 1.0 3.6

1.0 1.0 8.0

90.0

3.8

4.8

6.0

90.0

2.6

2.4

3.0

Dulang bajora Nasi-nasi Galinggang Simarabal-abal Palia moka

Lauraceae

Orthosiphon stamineus Cinnamomum burmanii

Silanlang kabungan Ruhu-ruhu Sibabi dalu Nilam Simarihur-ihur niasu Kumis kucing Kulit manis

Liliaceae

Cinnamomun cassia Persea gratissima Allium cepa

Sabal Pokat Bawang merah

Leaves Leaves, Bark Bark Leaves Tuber

Allium chinense

Hosaya

Tuber

Allium sativum

Bawang putih

Tuber

Ocimum americanum Paraphlomis javanica Pogostemon cablin Pogostemon auricularius

49

B I O D I V E R S IT A S

50

16 (1): 44-54, April 2015

Cordyline fruticosa

Silinjuang

Leaves

Lomariopsidaceae Loranthaceae Lycopodiaceae

Bolbitis heteroclite Loranthus sp.1 Lycopodium proliferum

Leaves Leaves Whole

Lythraceae Malvaceae

Lawsonia inermis Abelmoschus moschatus

Pahu sayu Sarindan kopi Limut-limut mangolu Hatirongga hau Purba jolma

Hibiscus rosa-sinensis Hibiscus similis Sida rhombifolia Sida ungulata Urena lobata

Bunga-bunga Baru Sibaguri safari Sibaguri Sampelulut

Donax cannaeformis Angiopteris evecta Clidemia hirta Melastoma malabathricum

Banban Ingol Sanduduk Sanduduk

Melastoma sylvaticum

Maranthaceae Marattiaceae Melastomataceae

Hyp, Fev, Bus Tt Kid, DM App, Tt

Alg, 45.0

1.2

2.0

2.5

18.0 36.0 9.0

0.8 -

0.6 0.8 1.8

0.8 1.3 1.3

Leaves Root, bark Leaves Root Root Root Root, leaves Leaves Leaves Leaves Root, leaves Leaves

Gas Fra, Tt

3.0 18.0

0.6 -

0.8 0.8

1.0 1.0

Fev, Tt Fra, Tt Fra, Inj Fra, Inj Fra

57.0 18.0 30.0 39.0 9.0

1.0 0,8 1.6 0.8

1.6 1.6 1.6 1.8 1.0

2.0 2.0 2.0 2.0 1.0

Ulc, Fev Ulc Gas, Inj Gas, Inj

12.0 9.0 36.0 36.0

0.4 1.0 -

1.4 0.6 1.6 1.6

1.5 1.0 2.0 1.8

Gas, Inj

24.0

-

1.4

1.8

Gas

9.0

0.6

0.8

1.0

Gas, Kid, Inj, Tt Fra, Tt Ash, Gas, Fra, DM, Tt Gas Hyp, Ash, Gas Dia, Gas, DM, Inj Gas Ash, Tt Fev, Gas Cou, Tp Gas, Sto, Fev, DM Gas, Tt Tt, Tp Gas, Sto, DM Cou, Ash, Alg, Tt Eye Too Hyp, Fev, Tt

15.0 24.0 54.0

1.2 0.4 1.0

1.4 0.8 1.8

1.8 1.0 2.3

30.0 72.0 36.0 6.0 30.0 38.0 39.0 30.0 45.0 36.0 42.0 56.0 6.0 9.0 30.0

1.0 1.4 0.6 1.2 1.6 1.4 1.4 1.4 3.2 0.6 -

0.8 1.4 2.4 1.0 2.0 1.4 1.6 1.2 0.8 1.2 1.4 4.0 0.6 1.0 1.2

1.0 2.5 2.5 1.0 2.0 1.4 2.0 2.0 1.5 1.8 2.0 4.0 1.0 1.0 1.8

Hyp, Ash, Aph, Bus, Tt Tt Dia, Tt Tt Ash, Tt Hyp, Ash, Tt Cou, Fev, Thr, Tt

96.0

1.8

3.6

3.3

96.0 54.0 35.0 30.0 18.0 12.0

1.8 -

3.6 1.4 0.6 1.4 1.6 1.8

3.3 2.0 1.0 2.0 2.8 2.8

30.0 3.0 120.0 -

1.8 0.6 2.6

2.5 0.6 4.8

24.0 120.0 1.0

1.4 2.4

2.0 3.5

24.0 24.0 24.0 9.0 24.0

0.6 0.4 -

0.8 0.8 0.8 1.0 0.8

0.8 0.8 1.0 0.8 0.8

18.0 18.0 45.0 9.0 30.0

0.2 0.4 1.8 -

1.8 1.6 1.6 0.8 0.4

1.8 2.0 2.5 0.8 0.8

Meliaceae

Lansium domesticum

Sanduduk harangan Langsat

Menispermaceae

Cyclea barbata Cycles sp.1 Tinospora crispa

Lakkup-lakkup Andor hondali Raja panawar

Root, bark Leaves Leaves Stem

Siraja enus Sukun Torop Pinasa Siraja landong Pisang sitabar Bunga lawang Salam Murak Pala Jambu batu Cengkeh Gompang batu Takkul-takkul Sonduk-sonduk

Leaves Bark Bark Fruit Leaves Stem Flower Leaves Leaves Seed Leaves Flower Leaves Leaves Whole

Orchidaceae

Tinospora sp.1 Artocarpus communis Artocarpus elastica Artocarpus heterophyllus Ficus cf. deltoidea Musa paradisiaca Eugenia aromatica Eugenia polyantha Eugenia sp.1 Myristica fragrans Psidium guajava Syzygium aromaticum Ardisia sp.1 Nepenthes garcilis Ophioglossum pedunculosum Anoectochilus reinwardtii

Suratan ilik

Whole

Oxalidaceae

Macodes sp.1 Goodyera rubicunda Nervilia aragoana Nervilia plicata Phaius callosus Oxalis corniculata

Suratan ilik Gadong harangan Salembar sabulan Salembar satahun Sukkit katari Saripitpit gawang

Whole Tuber Tuber Tuber Tuber Whole

Phyllataceae Piperaceae

Oxalis sp.1 Breynia cerma Piper betle

Saripitpit Podom-podom Demban

Whole Whole Leaves

Piper crocatum Piper nigrum

Demban siangir Lada

Leaves Seed

Piper umbellatum Piper sp.1 Piper sp.2 Adenia cordifolia Andropogon nardus

Gombalayo Dilah horbo Bursik horbo Ancimen riris Sangge-sangge dipar Bulu bolon Bulu duri Sangge-sangge Bulu sonduk Sidayok jagur

Leaves Leaves Leaves Whole Stem

Kid, Fev, Alg, Tt Fev Itc, Inj, Fev, Eye, Thr, Bus Itc, Inj, Eye, Bus Rhe, Fev, Bus, Tt, Tp Bus, Tt Fra, Tt Fra, Tt Hyp Fev

Root Root Stem Root Root

DM, Tt Fra, Bus, Tt Alg, Tt, Tp Tt Tt

Moraceae

Musaseae Myrtaceae

Myrsinaceae Nepentheceae Ophioglossaseae

Plassifloraceae Poaceae

Bambusa horsfieldii Bambusa spinosa Cymbopogon citratus Dendrocalamus asper Lophatherum gracile

SILALAHI et al. â&#x20AC;&#x201C; Medicinal plants of Batak Simalungun, Indonesia Sarang buaya Bonang sawi Bulu hayan Bulu suling

Leaves Leaves Root Root

Inj Tt DM, Tt Tt

12.0 9.0 18.0 9.0

0.8 0.6

0.8 0.8 1.6 1.0

1.0 0.8 2.0 1.0

Root Root Root Leaves Leaves Root Leaves

DM, Tt Tt Fra, Tt Fra, Kid, Tt Inj, Tt Fra, Tt Tt

18.0 18.0 9.0 1.5 6.0 18.0 30.0

0.4 0.6 -

1.6 1.0 1.0 0.8 1.2 1.8 0.6

2.0 1.0 1.0 1.8 1.3 2.0 1.0

Platycerium coronarium Ardisia japonica Robus moluccanus Morinda citrifolia Neonauclea calycina Paederia verticillata Uncaria gambir Citrus aurantium

Bulu laga Bulu lomang Bulu balakki Ria-ria Oma-oma Bulu moria Pandukkap naburuk Raja pinayungan Sibukkar Hupi-hupi Mengkudu Algit Salaun bulung Gambir Utte bunga

Bus, Tt Tt Dia, Gas, Sto, Tt DM Fra, Kid Fev Dia, Gas, Sto, Bus Fra, Fev, Tt

3.0 3.0 54.0 9.0 33.0 9.0 96.0 36.0

0.6 0.2 0.6 1.6 1.6

0.6 0.6 2.0 0.6 1.4 0.6 1.2 2.4

1.0 0.8 2.5 0.8 1.3 0.5 3.0 1.8

Citrus hystrix

Utte mungkur

Fra, Bus, Alg, Tt

90.0

2.0

2.8

2.8

Citrus maxima

Utte bolon

Fra, Tt

18.0

-

1.4

1.5

Citrus medica

Utte gawang

Fra, Tt

12.0

-

1.0

1.0

Citrus mitis

Utte kasturi

Fra, Fev, Bus, Tt

9.0

-

1.6

1.5

Citrus nobilis

Utte puraga

Tt

9.0

-

0.8

1.0

Citrus sp.1

Utte begu

Fra, Bus, Tt

18.0

-

1.2

2.0

Citrus sp.2

Utte hajor

Tt

12.0

-

0.8

1.5

Citrus sp.3

Utte hayu

Fra, Tt

9.0

-

1.0

1.5

Citrus sp.4

Utte kejaren

Fra, Tt

9.0

-

0.8

1.0

Citrus sp.5

Utte rihit

Tt

9.0

-

0.8

0.8

Ruta angustifolia Zanthoxylum acanthopodium Nephelium lappaceum

Soriangin Tuba

Leaves Leaves Leaves Fruit Leaves Leaves Leaves Root, fruit Root, fruit Root, fruit Root, fruit Root, fruit Root, fruit Root, fruit Root, fruit Root, fruit Root, fruit Root, fruit Leves Fruit

Gas, Fev Cou, Tt

9.0 60.0

0.8 1.4

1.2 2.0

1.0 2.0

Gas, Fev

9.0

-

1.2

1.8

Scrolphulariaceae

Achas zapota Kadsura scandens Kadsura sp.1 Lindernia crustacea

Gas Gas, Fra, Inj Fev Inj, Fev, Thr, Alg

6.0 4.5 12.0 24.0

0.6 -

0.6 0.6 0.6 2.2

1.0 1.0 1.0 1.3

Simaroubaceae

Lndernia liman Lindernia viscosa Eurycoma longifolia

Sawo Sibau sira Lendir sidarih Simaragongangong Siang-siang Pogu ni tano Horis kotala

Leaves, bark Fruit Leaves Leaves Leaves Whole Leaves Whole

9.0 24.0 72.0

-

1.2 2.2 2.6

1.0 2.2 3.3

Smilax sp.1 Capsicum frutescens Solanum lycopersicum

Udut tulan Lasina Tomat

Fev Gas, Kid, Alg Ash, Fra, Aph, Fev, Tt Ash, Aph Ulc Hyp, Inj, Fev

72.0 24.0 18.0

0.8 1.4

1.0 1.0 1.2

1.0 1.0 2.3

Physalis angulata Solanum nicotiana Solanum schiffnerianum Solanum torvum

Pultak-pultak Timbaho Saur paet Rimbang

Fev Inj, Too, Bus Gas, Inj, Fev Ulc, Eye

30.0 60.0 24.0 36.0

1.4 0.8 1.2 -

1.6 1.6 1.0 1.8

1.8 2.0 2.0 2.0

Solanum verbascifolium

Latting

Gas, Fra, Tt

72.0

2.0

2.4

2.8

Eurya japonica Eurya sp.1 Elatostema strigosum

Samoja Raru Sisik naga

Tt 12.0 Gas, DM 45.0 Hyp, Ash, Gas Inj, 30.0 Fev

0.4

0.6 1.4 2.8

1.0 1.8 3.3

Polypodiaceae Primulaceae Rosaceae Rubiaceae

Rutaceae

Sapindaceae Sapotaceae Schisandraceae

Smilaceae Solanaceae

Theaceae Urticaceae

Paspalum conjugatum Scleria laevis Schizostachyum blumei Schizosstachyum brachycladum Schizostachyum sp.1 Schizostachyum sp.2 Schizostachyum sp.3 Scleria purpurascens Scleria sp.1 Thysanolaena sp.

Pyrrosia sphaerotrichia

51

Rambutan

Whole Leaves Leaves, fruit Whole Leaves Fruit Leaves, fruit Leaves, fruit Leaves Bark Leaves, fruit

B I O D I V E R S IT A S

52 Elatostema sp.1 Leucosyke capitellata Usneaceae Verbenaceae

Sihip Simarhambinghambing Usnea barbata Tois alogo Clerodendrum calamitosum Simarbakkudu

Vitaceae

Clerodendrum fragrans Stachytarpheta indica Vitex trifolia Ampelocissus thyrsiflora

Burta-burta Odor-odor Sialagundi Sibalik kortas

Zingiberaceae

Pterisanthes polita Alpinia galanga

Siporgis laga Halaos

Alpinia sp.1 Boesenbergia pandurata

Laja Sitomu hursi

Curcuma domestica

Hunik

Curcuma xanthorrhiza

Tomulawak

Etlingera eliator

Rias

Etlingera sp.1

Sihala

Etlingera sp.2 Kaempferia galanga

Kambing bajar Hasohor

Zingiber americanus Zingiber officinale

Lampuyang Pege

Zingiber purpureum

Bungle

16 (1): 44-54, April 2015 Root Leaves Whole Leaves

Kid, Alg, Tt Kid, Alg, Tt

Alg, Tt Ash, Gas, Fev, Alg, Thr Leaves Cou, Gas, Itc, Inj Leaves Fev Leaves Hyp, Gas, Kid Leaves Ash, Gas, Sto, Aph, Tt Leaves Aph, Fev, Tt Rhizome Fev, Itc, Dia, Gas, Rhe, Tt, Tp Rhizome Dia, Gas, Tt Rhizom, Fev, Gas, Tt, Tp leaves Rhizome Dia, Gas, Sto, Inj, Fev, Eye, Alg, Tt, Tp Rhizome Ash, Sto, DM, Fev, Inj, Tt Leaves, Cou, Ulc, Tt, Tp stem Rhizom, Ash, Tt stem Rhizome Tt Rhizome Cou, Ash, Gas, Sto, Rhe, Aph, Fev, Alg, Tt Rhizome Dia, Gas, Tt Rhizome Gas, Sto, Inj, Fev, Aph, Tt, Tp Rhizome Dia, Gas, Tt

18.0 30.0

1.0

1.8 1.2

1.8 1.5

12.0 9.0

-

0.8 1.6

1.5 2.3

30.0 4.5 33.0 45.0

-

3.0 0.8 2.2 2.0

3.3 0.8 2.0 4.0

30.0 56.0

2.2

1.8 2.8

2.5 3.8

112.0 30.0 1.2

2.6 1.4

2.8 2.3

142.0 3.2

2.6

4.0

108.0 3.6

3.6

4.8

54.0

1.2

1.4

2.0

30.0

2.6

4.0

3.8

30.0 90.0

1.6

0.6 3.2

1.0 5.8

18.0 1.6 112.0 2.2

2.2 2.6

2.8 4.6

114.0 2.2

1.8

2.5

Note: Alg (Alogo-alogo), Aph(Aphrodisiac), Ash (Ashma), Bus (Busung), Cou (Cough), Dia (Diarrhea), DM (Diabetes mellitus), Eye (Eye infection), Fev (Fever), Fra (Bone fractures), Gas (Gastrointestinal disorders), Hyp (Hypertension), Inj (Injury), Itc (Itchy), Kid (Kidney disease), Rhe (Rheumatism), Sto (Stomach ache), Thr (Thrush), Too (Toothache), (Tp) Tinuktuk paranggetek, Tt (Tinuktuk tawar), Ulc (Ulcer).

Plant parts used as medicinal Parts of the plants used as medicinal plants were the leaves, stems, roots, bark, sap, flowers, fruits, seeds, and whole sections. The species composition consisted of leaves (119) and roots (54), flowers and the sap (2), as it is shown in Figure 3. Bioactive compounds used as medicinal plants are produced and stored in leaves, stems, roots, flowers, and seeds. For example, asiaticoside of Centella asiatica utilized as anti-inflammantory is stored in the leaves, whereas ajmalicine of Catharanthus roseus used as antihypertensive medicine is stored in the roots (Joy et al.

1998).

The usage of medicinal plant parts depends on the purpose of curing. It seemed that the local communities knew exactly the efficacy of every part of plants. For example: flowers and leaves of sampelulut (Urena lobata) were used to cure fever, while the root was used as a medicine for fractures. One of the bioactive compound of Urena lobata leaves is acetic acid which serves as analgesic (Islam et al. 2012), so that it can be used as a fever medicine.

There were 119 species or about 50% of the medicinal plants whoseleaves were used, such as: galunggung (Blumea balsamifera), ratusan (Ageratum conyzoides), papaga (Centella asiatica), gombalayo (Piper

umbellatum), and salam (Eugenia polyantha). Leaves have been used as medicines for injuries (Ageratum conyzoides, Centella asiatica), gastrointestinal disorders (Blumea balsamifera, Coleus ambonicus, Eugenia polyantha), and kidney diseases (Strobilanthes crispus, Orthosiphon stamineus). The medicinal plants used by local communities to cure kidney diseases and injuries were those whose leaves have rough-surface. Those leaves have been identified to be able to destroy kidney stones and stop bleeding in injuries. Flanol of leaves of Ageratum conyzoides has been known to cure injuries (de Padua 1999).

The number of medicinal plants whose whole parts were used, were relatively fewer than those whose only some parts (leaves, roots, or fruits) are utilized. Factors that encourage the use of whole plants parts were the relatively small size of the plants (Anoectochilus reinwardtii, Ophioglossum pedunculosum) and the difficulty in separating the parts of plant organs (Phyllanthus urinaria).

The utilization of whole plant has resulted in the death of the plants, so that some of these medicinal plants, for example suratan ilik (Anoectochilus reinwardtii) and sonduk-sonduk (Ophioglossum pedunculosum) have been hard to find in the wild.

SILALAHI et al. – Medicinal plants of Batak Simalungun, Indonesia

There were 11 species of medicinal plants whose rhizomes were used as medicines, and 9 species whose tubers were used as medicines. Medicinal rhizomes were derived mainly from Zingiberaceae (Boesenbergia pandurata, Curcuma xanthorrhiza, Curcuma domestica, Zingiber officinale), while the tubers from Orchidaceae (Nervilia aragoana, Nervilia plicata) and Liliaceae (Allium cepa, Allium sativum). The rhizomes of Zingiber officinale contain gingerol, shagaol, and gingerdion that has strong effect to cure gastrointestinal disorder (Achmad et al. 2008).

The local communities know the growth of medicinal plants in nature. For example, Nervilia aragoana is plant in the local languange called salembar sabulan (has only one piece of leaf in a month), while Nervilia plicata is called salembar satahun (has only one piece of leaf in a year).

Naturaly both of the orchids grow very slowly; therefore utilization of these plants may lead to their extinction.

IUCN (2010) noted that Nervillia plicata and Nervillia aragona have been categorized as protected plant.

The roots of horis kotala (Eurycoma longifolia), sinkam (Bischofia javanica), andudur (Caryota cf. mitis), and pining (Areca catechu) grow above ground. Those species have only one main root; therefore taking their roots kills them. This kind of harvesting accelerates the extinction of Eurycoma longifolia. In sub-ethnic Batak Simalungun roots of Eurycoma longifolia were used as medicines for fever and aphrodisiac. Bioassay of root extract of Eurycoma longifolia improves sexual activity in mice and make their coitus longer. That makes this plant appropriate to be used as aphrodisiac (Ang and Ngai 2001).

There were 14 species of plants whose bark was used as medicines, such as raru (Eurya sp.), kulit manis (Cinnamomum burmanii), and sinkam (Bischofia javanica).

Raru and Sinkam have been used primary as medicines for diabetes mellitus. The usage of raru as medicine for diabetes mellitus has been derived from the custom of local communities habit of drinking tuak (traditional beverage of sub-ethnic Batak). After having dinner they will drink tuak with seasoning made from the bark of Sinkam. Tuak is sap of Arenga pinnata, which is mixed with the bark of raru. It is believed to decrease blood sugar level. To prove the medicinal effect of Bischofia javanica and Eurya sp., phytochemistry and bioassay analysis need to be conducted. Over exploitation of the plants, especially their bark harvested directly from the forest may cause extinctions.

Index of Cultural Significance (ICS) and Use Value (UVs) of medicinal plants The ICS of medicinal plants is a quantitative method used by ethnobotanists to determine the cultural value of plants. Based on their uses, the plants were grouped into 5 categories, namely: >200 (very high), 100-199 (high), 2099 (medium), 5-19 (low), and <5 (very low) (modified from Pieroni 2011). The medicinal plants with medium values of ICS had the highest number of species (113), and followed by low categories (98), very low categories (16), and high categories (11) (Tabel 2).

The value of medicinal plants in the sub ethnic Batak Simalungun varied between 1.5 to 142.0 (Table 2). The

53

medicinal plants with the highest value of ICS (142.0) was hunik (Curcuma domestica) and the lowest value (1.5) were ria-ria (Scleria purpurascens) and sihirput (Mimosa sp.). Medicinal plants that show high value on ICS are those which have many usages and are utilized frequently by local communities, while those having low value on ICS have fewer usage and are rarely used.

The plants with medium ICS value were, among others, garlic (Allium sativum), ompu-ompu (Crinum asiaticum), and poyon baru (Gynura crepidioides). The value of ICS on sub-ethnic Batak Simalungun was higher than that of ethnic Malays (Susiarti et al. 2005), but lower than that of sub-ethnic Batak Karo (Silalahi et al. 2013). The usage of medicinal plants is strongly influenced by culture, spiritual beliefs of local communities (Cocks and Dold 2006), and geography (Pieroni 2001).

Correlation between age and utilization One of the approaches used to determine usage value of medicinal plants is carried out by calculating the use value (UVs). The UVs depends on the number of medicinal plants used and known by respondents or the local communities. Age structure seemed to influence on the sub-ethnic Batak Simalungun UVs of the medicinal plants, the younger (30-50 years old) having lower value than the older age group (>50 years old). For example, the UVs of Allium cepa was 2.2 in the younger group, 3.6 in the older group, and 8.0 for key informants (Table 2). The differences of UVs (Table 2) shows the degradation of the local knowledge in terms of traditional medicines.

Beside having lower Uvs, the younger group knew significantly (P=0.05 on Anova) fewer species of medicinal plants (48.19 ± 8.35 species) than the older (170.19 ± 18.38 species), and key informants (202. 00 ± 12.32 species) as shown in Figure 4.

The number of medicinal plants species known by the younger group was only 28.31% of those by the older group and only 23.85% of those by the key informants.

Therefore, it is clear that local knowledge of the usage of medicinal plants has declined and has not been passed into the younger generation. This degradation is due to: (1) difficulty in passing this information through oral ways to the young generation (2) Changes in cultural value, (3) the availability of modern medical system. That the younger age had less interest in local knowledge on medicinal plants was also found by Caniago and Siebert (1998), Voeks (2007), Guimbo et al. (2011).. The documentation of local knowledge in a written form is considered to be the best alternatives to avoid the degradation of the local knowledge and as a first step for the conservation of medicinal plants (Suryadharma 2010).

A total of 239 species of medicinal plants (170 genera, 70 families) were used by sub-ethnic Batak Simalungun of North Sumatra, to cure 20 diseases (18 natural disease, 2 supra natural disease) and to make 2 kinds of traditional concoction. The local knowledge of medicinal plants was lower in the younger generation (between 30-50 years old) than that of the older group (>50 years old) and key informants (mostly shamans) both in number of known species and use

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ACKNOWLEDGEMENTS We would like to express our gratitude to the local communities Simbou Baru, Simalungun District, North Sumatra, Indonesia for permission to this research and their help in the field.

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B I O D I V E R S IT A S Volume 16, Number 1, April 2015 Pages: 55-61

ISSN: 1412-033X E-ISSN: 2085-4722 DOI: 10.13057/biodiv/d160107

DNA barcoding and phylogenetic reconstruction of shark species landed in Muncar fisheries landing site in comparison with Southern Java fishing port PREHADI1, ANDRIANUS SEMBIRING2, EKA MAYA KURNIASIH1,2, RAHMAD1, DONDY ARAFAT1, BEGINER SUBHAN1, HAWIS H. MADDUPPA1,♥ 1

Department of Marine Science and Technology, Faculty of Fisheries and Marine Science, Bogor Agricultural University (IPB), Jl. Darmaga Raya, Bogor 16680, West Java, Indonesia. Telp/Fax +62 85693558653, ♥email: hawis@ipb.ac.id 2 Indonesian Biodiversity Research Center, Jl. Raya Sesetan Gg. Markisa No. 6, Denpasar 80223, Bali, Indonesia.

Manuscript received: 31 October 2014. Revision accepted: 4 Desember 2014.

Abstract. Prehadi, Sembiring A, Kurniasih EM, Arafat D, Subhan B, Madduppa HH. 2015. DNA barcoding and phylogenetic reconstruction of shark species landed in Muncar fisheries landing site in comparison with Southern Java fishing port. Biodiversitas 16: 55-61. Sharks are one of main fisheries commodity that are currently exploited on a large scale because of their high economic value.

The identification of sharks has been a difficult one due to the specimen’s similarity in morphology and mostly have had key diagnostic features removed. This study aimed to identify and to review the status of sharks, and also to reconstruct the shark species that were landed at South Java fishing port using molecular approaches. The DNA amplification was using cytochrome oxidase I mitochondrial of locus and 600-700 basepairs. A total of seven species from 59 individuals was identified including Alopias pelagicus, Carcharhinus falciformis, C. sorrah, C. amblyrhynchos, Galeocerdo cuvier, Atelomycterus marmoratus, and Spyrna lewini. The diversity of shark species landed in Muncar during the last 2 years has been decreased. The identified sharks species in this study sites were about 18% of all Indonesian sharks. The result of this study is expected help the Government to manage shark fisheries in Indonesia.

Keywords: DNA barcoding, Muncar, phylogenetic, sharks, southern Java.

INTRODUCTION Sharks has been greatly exploited in various countries across the globe, including Indonesia, which ranked first in the list of the 20 biggest shark fishing countries above India, Spain, Taiwan and Argentina between 2002-2011 (Mundy et al. 2013). During that period, Indonesia has been exporting 10,762 tons of shark fins or 13% of the total world exports (Mundy et al. 2013).

The biological characters of shark are making them vulnerable to exploitation. In their natural habitat, sharks are known to be slow in reaching maturity stage (8-13 years) and also low reproduction level (Camhi et al. 1998).

The effect from shark fisheries is damaging and hence the fast decreasing population of shark in the wild as proved by a number of studies in Northwest Atlantic, Southeast Australia, Gulf of Mexico, South Africa and Australian Great Barrier Reef (Holden 1973; Casey and Myers 1998; Graham et al. 2001; Baum and Myers 2004; Baum et al.

2005; Dudley and Simpfendorfer 2006; Robbins et al.

2006; Burgess et al. 2005). Population decline occurred as an effect from intensive and continuous fishing due to unmanaged and unregulated fisheries (Dharmadi and Fahmi 2005). The main problem is that the data given from the port is only the volume of total production without describing the shark species and the total individuals caught, for example in Muncar fish landing site (UPPPP Muncar 2013). Muncar is one of the ports that plays an important role in shark fishing in Southern Java, aside from

Cilacap of Central Java and Palabuhanratu in West Java (White et al. 2006).

The identification of sharks has been a difficult one due to the specimen’s similarity in morphology and mostly have had key diagnostic features removed. In recent years, molecular approach has been able to give solutions in identifying shark species, especially when the taxonomy method is impossible to conduct due to insufficient morphological information such as ones that has been implemented on shark fin and fillet (Smith et al. 2001).

Hebert et al (2003) introduced DNA barcode method with mitochondrial marker for all animal species, for one chain of gene is claimed to be enough for distinguishing one species to another. DNA barcoding method uses primer in PCR (Polymerase Chain Reaction) process to amplify the DNA until around 600-700 bp on cytochrome oxidase I (COI) locus of mitochondrial. DNA barcoding method has been used to identify over 207 species of fish in Australia including 143 species of teleostean, 61 species of shark and stingrays, and 3 species of chimaerid (Ward et al. 2008).

There are around 35 species of sharks that has been fished in Indonesia based on DNA barcoding analysis (Sembiring et al. 2014). As many as 10 species of shark has been recorded in Cilacap and 6 others in Palabuhanratu through DNA barcoding method (Sembiring et al. 2014).

Phylogenetic is a description of relationship based on DNA sequence composition or protein which resembles to that of a tree to estimate the past evolution process (Baldauf 2003). Phylogenetic reconstruction is able to analyze the

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gene distance through DNA variation with Neighbor Joining Tree method (Saitou and Nei 1987) that is capable on calculating the distance which is shown with bootstrap value.

The current study was conducted to identify shark species landed in Muncar, Banyuwangi, East Java in 2013, and to reconstruct phylogenetic tree between species found in Muncar compared with Cilacap and Palabuhanratu which previously has been analyzed through DNA barcoding (Kurniasih 2013; Rahmad 2013).

MATERIALS AND METHODS Sample collection The tissue sample of shark were collected from three fisheries landing site in Java Island (Indonesia), i.e.: Muncar, Palabuhanratu and Cilacap (Figure 1). A total of 59 samples were collected in Muncar in July 2013. The tissue sample was stored in 95% ethanol. In 2012, samplings were done in 2 days covering all warehouses in Muncar, while the recent research was done within 30 days when all the catch is landed from ships.

PCR extraction, amplification, and sequencing DNA was extracted using 10% chelex (Walsh 1991).

The DNA extracts were stored at -30°C until required for laboratory analyses. The mitochondrial cytochrome oxidase

I (COI) gene was amplified by polymerase chain reaction (PCR) using the forward primer fish-BCL (5' TCA ACY AAT CAY AAA GAT ATY GGC AC '3) and reverse primer fish-BCH (5'ACT TCY GGG TGR CCR AAR AAT CA '3) (Baldwin et al. 2009). PCR amplifications were performed in 26 µL reaction mixture containing 2 µL 25 mM MgCl2, 2.5 µL 8μM dNTPs ; 1.25 µL each primer pair 10 mM; 0,125 µL Taq DNA polymerase, 2.5 µL 10xPCR Buffer, 2 µL DNA template, 14.5 µL deionize water (ddH2O). Cycling parameters were an initial denaturation at 95°C for 2 min followed by 35 cycles of denaturation (94°C for 30 s), annealing (50 °C for 30 s), and extension (72°C for 45 s) with the final extension step at 72°C for 2 min. PCR-amplified DNAs were visualized on 1% agarose gels. Prior to sequencing, excess dNTPs and oligonucleotides were eliminated from the PCR product using shrimp alkaline phosphatase and exonuclease I (ExoSAP-IT kit; Affymetrix, Santa Clara CA, U.S.A.) following the manufacturer’s protocol. Sequence reactions were performed in both directions using the BigDye terminator v3.1 cycle sequencing kit (Applied Biosystems), 8-10 μL purified PCR product, and 4-5 μL of either primer (3 μM) per reaction. Sequence-reaction products were loaded into an ABI 3130xl automated sequencer (Applied Biosystems) at the Berkeley Sequencing Facility located in the United States (Sanger et al. 1997).

Figure 1. Sampling location in three main fisheries landing site in Indonesia (Muncar, Palabuhanratu, and Cilacap), located in Jawa Island (blue circle).

PREHADI et al. â&#x20AC;&#x201C; DNA barcoding of sharks

Data analysis Forward and reverse sequences were proofread, aligned and edited using MEGA 5.2 (Tamura et al. 2011). Edited sequences were deposited in GENBANK (http://www.ncbi.nlm.nih.gov/). The nucleotide sequence data will be matched with the data contained in GenBank at the NCBI (National Center for Biotechnology Information) for species identification by using BLAST (Basic Local Alignment Search Tool) in website address http://blast.ncbi.nlm.nih.gov/Blast.cgi. A neighbor joining phylogenetic trees was reconstructed in MEGA 5.2 (Tamura et al. 2011) using Kimura-2-parameter models (Kimura 1980) with 1000 of bootstrap value. Samples that have been analyzed is then compared with samples from Palabuhanratu and Cilacap (39 and 35 samples, respectively). Furthermore, the data from July 2013 from Muncar will be compared to catch from August 2012. A review of the conservation status was determine with IUCN (2014), and the trade status was determine in CITES (2014). The near threatened species are the population that is in danger of declining in the near future if nothing can be done to prevent it. Vulnerable species are those who have a high risk of extinction in the wild (IUCN-SSC 2001).

RESULTS AND DICUSSION DNA barcoding, conservation status, and trading status A total of seven species from 59 tissue samples belongs to four different families (Alopiidae, Carcharhinidae, Scyliorhinidae, and Sphyrnidae) has been successfully identified (Table 1). Family of Carcharhinidae is the most common family found in Muncar with four species with a total of 46 individuals. 41 of those belong to species of Carcharhinus falciformis (Silky shark). Carcharhinidae Family is the most commonly caught might be due to their diversified habitat or fishing gear used by fishermen. The number of Sphyrnidae Family found in Muncar is 11 individuals which consist of a single species, Sphyrna lewini or scalloped hammerhead.

All sharks identified in Muncar have been assessed by IUCN: one species is categorized as Threatened (Sphyrna lewini), five species categorized as near threatened (Carcharhinus, C. amblyrhynchos, C. sorrah, Galeocerdo cuvier, and Atelomycterus marmoratus) and 1 other species is categorized as vulnerable (Alopias pelagicus). Most

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species are not evaluated by CITES, but Sphyrna lewini is included Appendix II. Shark species landed in Muncar in 2012 and 2013 Table 2 describes sharks species landed in Muncar for the last 2 years and were identified using DNA barcoding.

C. falciformis was the most collected species within two sampling period. There was a slight different in species identified between two period of sampling (2012 and 2013). Low number of species was recorded in 2013 (7 species) compared with in 2012 (11 species), even though sampling duration was longer in 2013 (30 days) than in 2012 (2 days).

The huge differences of duration of the sampling and the number of species observed might be indication in declining shark population in the wild. However, many factors could influence on status of exploited species, such as type of gear, location fished, biological characters of shark as migratory species (Lynch et al. 2011).

The number of shark species being exploited in Muncar reached 14 species or around 18% of the entire shark species found in Indonesia (White et al. 2006). That number can be considered as huge in shark fishing, as sharks greatly varied in terms of maturing and young being produced (Castro and Mejuto 1995). Based on these facts, it is inevitable that proper shark fishing management is urgent to be implemented in Muncar, Banyuwangi, East Java.

Table 2. The number of individual of identified shark species landed in Muncar between two period sampling (2012 and 2013) through DNA barcoding Species Carcharhinus falciformis Carcharhinus brevipinna Carcharhinus limbatus Carcharhinus sorrah Galeocerdo cuvier Hemitriakis indroyonoi Mustelus lenticulatus Alopias pelagicus Alopias superciliosus Isurus oxyrinchus Prionace glauca Carcharhinus amblyrhynchos Sphyrna lewini Atelomycterus marmoratus

2012

2013

6 5 1 2 1 4 6 3 1 1 1 -

41 2 2 1 1 11 1

Table 1. Identified shark species landed in Muncar using BLAST program, along with IUCN and CITES status

Family Alopiidae Carcharhinidae

Scyliorhinidae Sphyrnidae

BLAST analysis Alopias pelagicus Carcharhinus falciformis Carcharhinus amblyrhynchos Carcharhinus sorrah Galeocerdo cuvier Atelomycterus marmoratus Sphyrna lewini

Common name Pelagic Thresher Silky Shark Grey Reef Shark Spot-tail Shark Tiger shark Coral catshark Scalloped Hammerhead

Max. Similarity (%) 100 100 99 100 100 99 100

No. of individual 1 41 1 2 2 1 11

Red List Status (2014) Vulnerable Near threatened Near threatened Near threatened Near threatened Near threatened Endangered

CITES status (2014) Not evaluated Not evaluated Not evaluated Not evaluated Not evaluated Not evaluated Appendix II

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Phylogenetic reconstruction A total of seven clades with a bootstrap value of 100 which means they possess close kinship between individuals (Figure 2). The large group consists of species such as Carcharhinus falciformis, C. sorrah, C. amblyrhynchos, Galeocerdo cuvier, Alopias pelagicus, Sphyrna lewini, and Atelomycterus marmoratus.

Cladogram could be used to show that in every family there are species which is located very close. This indicates that the study on a species of one particular family could be described through phylogenetic reconstruction (Zuazo and Agnarson 2010).

Figure 3 describes phylogenetic reconstruction of C. falciformis from 3 locations and formed 2 different clades with both bootstrap value of 65 therefore not really strong to hold the position if added by another individuals, but these two clades will still be different nevertheless. There is one clade which the individual is a C. falciformis from Palabuhanratu, but on the other side there is one other clade. It indicates that there are two different genetic groups of C. falciformis in Palabuhanratu, which means that they come from two different populations (Zhao et al.

2013) in Southern Java and proved how wide the distribution of this species, from Southern Java to Bali Sea (White et al. 2006).

Figure 2. The Neighbour-joining tree based on COI sequence data using Kimura-2-parameter substitution model with 1000 bootstrap, from shark species landed in Muncar in 2013.

PREHADI et al. â&#x20AC;&#x201C; DNA barcoding of sharks

Phylogenetic reconstruction of Carcharhinus brevipinna landed in Muncar was divided into two clades with bootstrap value of 88-89 (Figure 4.A). Differences between these two branches are pretty strong (100% bootstrap value) and indicate these individuals might come from different population. The distribution of this species covers the central sea areas of Indonesia (White et al.

2006). The species of Carcharhinus limbatus consists of two different clades between from data pooled of Palabuhanratu and Muncar (Figure 4.B). It could be seen from the cladogarm that each of species grouped according to the locations respectively. Based on White experiment

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on 2006, C. limbatus can be found throughout Southern Java Sea until South China Sea. Sphyrna lewini was divided into two different clades between Muncar and Cilacap (Figure 4.C). This indicates that the aggregate location of this species is separated between Southern Java and Northern Java, because according to their behavior, this species is likely to seek the same shelter with their own groups, usually in underwater valley basin of sea mountains (Klimley et al. 1983). S. lewini is a species that could be found widely distributed throughout Indonesian Seas. However, it was unclear where and how fishermen catch this species.

Figure 3. Phylogenetic reconstruction of Carcharhinus falciformis landed in landed in Ports in Southern Java for location, using the Neighbour-joining tree based on COI sequence data using Kimura-2-parameter substitution model with 1000 bootstrap.

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A

B

C

Figure 4. Phylogenetic reconstruction of pooled data of Carcharhinus brevipinna (A), Carcharhinus limbatus (B), and Carcharhinus limbatus (C) landed in three ports in Southern Java.

PREHADI et al. â&#x20AC;&#x201C; DNA barcoding of sharks

The diversity of shark species landed in Muncar during the last two years has been decreased, even though sampling efforts was prolonged in 2013. The identified sharks species in this study sites were about 18% of all Indonesian sharks. The phylogenetic reconstruction of shark catchment in Southern Java Fishing Port indicated that sharks might be coming from different population. The result of this study is expected help the Government of Indonesia to manage shark fisheries in Indonesia.

ACKNOWLEDGEMENTS The study was supported by the Marine Biodiversity and Biosystematics Laboratory, Department of Marine Science and Technology, Bogor Agricultural University, Bogor and Indonesian Biodiversity Research Center, Bali, Indonesia.

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B I O D I V E R S IT A S Volume 16, Number 1, April 2015 Pages: 62-68

ISSN: 1412-033X E-ISSN: 2085-4722 DOI: 10.13057/biodiv/d160108

The diversity of non-methanogenic bacteria involved in biogas production from tofu-processing wastewater SUNARTO, HANNI TSAAQIFAH, CAHYADI PURWOKO, ARTINI PANGASTUTI Department of Biology, Faculty of Mathematics and Natural Sciences, Sebelas Maret University. Jl. Ir. Sutami 36A Surakarta 57126, Central Java, Indonesia. Tel./Fax. +62-271-663375, email: rm.sunarto@yahoo.com Manuscript received: 29 November 2014. Revision accepted: 22 December 2014.

Abstract. Sunarto, Tsaaqifah H, Purwoko C, Pangastuti A. 2014. The diversity of non-methanogenic bacteria involved in biogas production from tofu-processing wastewater. Biodiversitas 16: 62-68. The energy crisis is an important current issue, so diversification of energy is needed to solve the problem. Biogas from tofu-processing wastewater is one potential alternative energy to be developed because it has high organic matter content. The amount of methane formed by methanogens is also influenced by the presence of bacteria, so it is necessary to do research on the diversity of non-methanogenic bacterial communities. This study aimed to determine the diversity of non-methanogenic bacteria in anaerobic fermentation using ARDRA (Amplified Ribosomal DNA Restriction Analysis) method. Tofu-processing wastewater was anaerobically fermented, followed by DNA extraction, amplification of 16S rRNA marker gene, and cloning. Then, restriction was done using MspI and AluI restriction enzymes, followed by sequencing of different patterns.

The data were analyzed to know the diversity of non-methanogenic bacteria and the relative importance of each phylotype in the community using Shannon-Wiener index of diversity (H’) and evenness (E).The results showed that there were 38 positive clones of 16S rRNA genes clones. The sequencing using BLASTn analysis showed the presence of 10 species of non-methanogenic bacteria.

There were four sequences with 97% similarity, indicating that the bacteria belong to species from GeneBank data. There were 6 sequences that had 96% similarity with the closest relative in the database, indicating that these bacteria are new species. The ShanonWiener index was 1.291, categorized as low and the evenness was 0.561, categorized as low because there was dominance of a species.

Keywords: ARDRA, biogas, non-methanogenic bacteria, wastewater, 16S rRNA.

INTRODUCTION Enery is essential for every living creature. Global energy demand increases along with the increasing human population and activities. The Ministry of Energy and Mineral Resources of Indonesia reported that energy consumption per capita in Indonesia increases with the growing rate of more than 5% (MEMR 2009). Ironically, the increasing demand for energy is not balanced with its supply, so the energy stocks, especially the fossil fuels are depleted. According to Sun et al. (2013), 88% of global enery consumption comes from fossil fuels, which lead to energy crisis, because fossil fuels are non renewable, which may be gone within 50-100 years.

Many governments in the world, including Indonesian government, have realized the threat of energy crisis. The utilization of alternative energy sources is the appropriate measure to overcome the energy problem. Biogas is one the most considered alternative sources of energy. Biogass refers to the mixture of methane, carbondioxide and other gasses produced by decomposition of organic waste, such as domestic waste and manure by methanogenic bacteria (Ntengwe et al. 2010).

In Indonesia one potential source of organic waste for biogas is tofu-processing wastewater. There are 84.000 tofu production units in Indonesia with a total production of 2.56 million tons per day (Sadzali 2010). Tofu-processing wastewater can be processed to make biogas through

anaerobic fermentation, consisting of four stages of decomposition, namely hydrolysis, acidogenesis, acetogenesis and methanogenesis (Briones et al. 2007).

Each stage involves a group of different microbes which work synergically with each other, so they form microbial consortia (Griffin et al. 1998). The consortia are classified as non-methanogenic and archaic bacteria. The nonmethanogenic bacteria play important roles in organic waste degradation, namely in liquefaction or hydrolysis stage and the production of acid which provides substrate for the archaic bacteria (Madigan et al. 2009).

Because the information of the diversity of nonmethanogenic bacteria involved in the processing of biogas from tofu-processing wastewater is limited, studies are needed. In general, studies on morphology, cell structure and biochemistry of bacteria are conducted through isolation and characterization using culture (Reeve 1994).

However, there is a problem because not all microbes, including the major bacteria group, can be cultured (Suwanto 1994). Therefore, it is necessary to choose a method which can be used to study the structure of all bacteria, namely ARDRA (Amplified Ribosomal DNA Restriction Analysis). This method can be used to analyze population of bacteria and the predicted genetic changes within a period of time. The objective of this study was to know the diversity of non-methanogenic bacteria involved in the processing of biogas from tofu-processing wastewater.

SUNARTO et al. – The diversity of non-methanogenic bacteria

MATERIALS AND METHODS The materials for biogas production were substrate namely tofu-processing wastewater and inoculum in the form of active sludge of tofu. The substrate and inoculum were then fermented in anaerobic condition for 20 days in a digester. Then, the molecular research was started with the extraction of digester samples using UltraClean Soil DNA Kit (MoBio, Carlsbad, CA, USA). The result of extraction was genomic DNA of non-methanogenic bacteria. Then the 16S rRNA gene was amplified using PCR cycle, using primers 63F and 1387R. The result of amplification was subsequently purified with GeneJetTM Gel Extraction Kit (Fermentas c.q. Thermo Fisher Scientific Inc., Waltham, MA, USA). The product of purification was litigated with vector pTZ57R/T (Fermentas, MA, USA) which was then cloned using InsTAcloneTM PCR Cloning Kit (Fermentas, Waltham, MA, USA). The transformant cell culture was grown in Luria Bertani (LB) agar medium with the addition of ampicillin, X-gal and IPTG. Then, Blue- white selection was conducted. The positive clones were digested using restriction enzymes, AluI and MspI. The making of biogas First, inoculum was put into digester with concentration of 20% of 264 mL of the digester working volume or equivalent to 52.8 mL. Then, 211.2 mL (or 80% of 264 mL) of the substrate was poured into the digester. Then the digester was closed tight. The fermentation took place for 20 days until biogas was produced, which was subsequently channeled through a small tube into a waterfilled gas collecting tank (a drinking water bottle, 330 mL).

The gas pushed the water out of the bottle. The volume of water spilled out the bottle was equal to the volume of gas filling the bottle.

The samples for molecular study were taken on the 20th day. According to Sunarto et al. (2013), usually the production of biogas has taken place optimally on the 20th day, so all stages of anaerobic fermentation have occurred.

Extraction of DNA samples After 20 days of incubation, the sludge from the digester was sampled for DNA extraction using UltraClean Soil DNA kit (MoBio, CA, USA), following the procedure recommended by the manufacturer. The result of extraction was genomic DNA of non methanogenic bacteria as the DNA templates in PCR process.

Amplification of 16S rRNA Gene for ARDRA The genomic DNA of non-methanogenic bacteria was then amplified. PCR reaction was made by mixing 0.5 μL of DNA samples, 17.5 μL of ddH2O, 2.5 μL of dNTP, 2.5 mM 2.5 μL of Buffer Taq, 0.2 μL of DNA Taq Polimerase enzyme, and 1 μL of primer. The DNA amplification used primer 63F (5’ CAG GCC TAA CAC ATG CAA GTC 3’) 5 pmol pairing with 1387R (5’ GGG CGG WGT GTA CAA GGC 3’) 5 pmol (Marchesi et al. 1998). The PCR program consisted of 1 cycle of pre denaturation at a temperature of 95oC for 5 minutes, followed by

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denaturation at 94oC for 30 seconds, annealing at 55oC for 30 seconds, extension at 72oC for 30 seconds (35 cycles each), post extension 1 cycle at 72oC for 7 minutes, and it was ended with storing at 4oC. Then, the PCR product was processed in electrophoresis at 0.8% agarose gel; then it was purified with GeneJETTM Gel Extraction Kit (Fermentas, MA, USA).

Cloning of 16S rRNA of PCR Product The PCR products were cloned in accordance with procedures recommended in InsTAcloneTM PCR Cloning Kit (Fermentas, Waltham, MA, USA). Clone selection was done in LB agar medium added with 50 mg/mL Ampicillin and X-Gal substrate. Then, the white clone was picked and grown in LB agar added with 50 mg/mL Ampisilin at 37 °C, shaken for 3-5 hours. This cell-containing medium was used as a template for insert amplification using primer M13 with mixed reactant composed of 10% culture, 0.8 mM dNTP, 1μM of each primer, 0.1 U/μL Taq DNA Polymerase, bufferred 1x. The PCR condition consisted of 3 cycles at 94 °C for 70 seconds, at 56 °C for 45 seconds, at 72 °C for 90 seconds; 23 cycles at 90 °C for 15 seconds, 56 °C for 30 seconds, 72°C for 1 second; 1 cycle at 72 °C for 10 minutes, and it was ended at 4 °C. Fifty clones which positively contained insert were randomly selected to be used for the subsequent analyses.

ARDRA (Amplified Ribosomal DNA Restriction Analysis) Inserts were digested using restriction enzymes, MspI and AluI, in a mixture reaction with the following composition: 0.2 µL of enzymes, 2 µL of tango buffer, 10 µL of DNA, and 7.8 µL of ddH2O. The mixture was then incubated at 37 °C for one night. Then, the DNA resulted from the digestion was processed in electrophoresis at 2% agarose gel, at a voltage of 70 V, for 2 hours. The number of inserts having different restriction patterns was counted in percentage of the total analyzed clones. Then, the 16S rRNA marker genes of the clones were sequenced for identification.

Sequencing and identification of the16S rRNA marker genes The resulted clones were run in PCR again for sequencing by 1st Base Sequencing Services (Axil Scientific Pte. Ltd., Singapore through PT. Genetika Science Indonesia, Jakarta). Identification was done by comparing the sequences of the16S rRNA marker gene, about 1300 bp in size, with the database using the Program BLAST (Basic Local Alignment Search Tool) in the web site of National Center for Biotechnology Information (http://www.ncbi.nlm.gov/BLAST).

Data analyses The diversity index of Shannon-Wiener (H’) and the evenness index (E) were determined to show the diversity of bacteria community and the relative importance value of each phylotype in the community.

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64 The diversity index:

H’ = Diversity index S = number of species Pi = Ni/N = proportion of Species i Ni = number individuals of species i N = number of individuals of all species The criteria used to interpret the diversity index: H’ < 1: low diversity 1-3: medium diversity >3: high diversity Relative importance value:

E = Evenness; H’ = Diversity index; Hmax = ln S

RESULTS AND DISCUSSION The production of biogas from tofu-processing wastewater The sludge from tofu-processing wastewater digestion, after 20 days of anaerobic fermentation, was sampled for molecular analyses. The measurements of pH and temperature were done in day 0 and day 20. The pH in day 0 was 7, and in day 20 was 7.87. The temperature in day 0 and day 20 was the same, which was 30 oC. The increase of pH in day 20 was influenced by the archaic methanogenic bacteria activities. According to Fulford (1988), at the early phase of biogas processing, the acid-producing non-methanogenic bacteria are active, so the pH of digester becomes low.

Then, the archaic methanogenic bacteria use the acid as substrate, so the pH increases. The pH in the 20th day in this study was 7,87 which is tolerable by archaic methanogenic and non-methanogenic bacteria (NAS 1981).

Concentration of methane measured using chromatography was 655.82 ppm (R. Setioningsih, pers. comm.). The flame test showed that the production of gas was not sufficient to be lit. The biogas production refers to the reaction and interaction between the methanogenic microbes and the non-methanogenic ones (Gunnerson and Stuckey 1986). The non-methanogenic bacteria are categorized into three groups, namely hydrolitic, acidogenic, and acetogenic. These non-methanogenic bacteria groups play roles in fermentation of acid, in which they convert complex compounds into simple ones. The compounds resulted from fermentation are then used by archaic methanogenic bacteria in methanogenic fermentation and converted into methane (Sunarto et al.

2013). Generally, biogas is composed of methane 55-65%, carbon dioxide 30-40%, H2O 2-7%, H2S 2-3%, NH3 00.05%, Nitrogen 0-2%, oxygen 0-2%, and 0-1% hydrogen

16 (1): 62-68, April 2015

(Yimer and Sahu 2014).

Extraction of DNA and amplification of 16S rRNA genes Extraction of non-methanogenic bacteria DNA was done to get genomic DNA as a template for PCR process.

UltraClean Soil DNA Kit (MoBio, CA, USA) was used for the extraction of genomic DNA of non-methanogenic bacteria from the tofu sludge, following the procedures in the manual kit. The genomic DNA of non-methanogenic bacteria resulted from the extraction was then amplified.

The amplification of 16S rRNA genes used the PCR cycle which was the result of optimizing in the research of Sunarto et al. (2013). The primers used were 63F and 1387R, the universal primers for amplification of 16S rRNA gene of bacteria with an amplicon target of 1.300 bp (Marchesi et al. 1998). Primer is an important component in PCR reaction because it will determine the region of targeted genome which will be amplified (Rafsanjani 2011). The amplified DNA was separated with 0.8% agarose gel electrophoresis and then visualized using coloring agent of ethidium bromide and detected with UV light. The result of electrophoresis of amplification of PCR product of non-methanogenic bacteria 16S rRNA is shown in Figure 1.

The electropherogram shows that the size of region of 16S rRNA of non-methanogenic bacteria was 1300 bp, and it also shows single band indicating that the primer pair used was specific and attached to targeted area. The single band contained collection of 16S rRNA genes from many bacteria present in tofu waste sludge from the digester. The thickness of the band indicates the quantity of the amplified DNA templates. The electropherogram in this study was clearly visible and thick, indicating that the concentration of molecules separated in this region was high. The amplicon was then amplified using GeneJetTM Gel Extraction Kit (Fermentas, MA, USA). Purification is needed because the cloning stage requires clean gene inserts, free from contaminant such as oligonucleotide which is not used in PCR process, the residues of buffers, and dNTP. The contaminant must be removed, because if the residue of dNTP combines with residue of DNA polymerase, it will disturb the methods designed for amplified DNA during cloning stage (Sanger et al. 1977).

Cloning of 16S rRNA genes The product of purification was litigated with plasmid vector pTZ57R/T (Fermentas, MA, USA), catalyzed with T4 ligase enzyme to get plasmid recombinant. The litigation product was then transformed into E. coli JM107, using InsTAcloneTM PCR Cloning Kit (Fermentas, Waltham, MA, USA) following the procedures recommended by the manufacturer. Transformant cell culture was grown in LB agar medium with the addition of X-gal by spreading it using L rod and then incubated for 16 hours at 37°C. The result of E. coli JM 107 transformation showed the formation of blue-white colonies. The white colonies were estimated to contain fragments of insert genes, whereas blue colonies were estimated not to have fragments of insert genes.

SUNARTO et al. – The diversity of non-methanogenic bacteria

The formation of blue white colonies was caused by the presence of selection marker at vector pTZ57R. The selection marker in vector pTZ57R was gene resistant to ampicillin and additional selection marker was LacZ gene.

This ampicillin-resistant gene is the marker of -lactamase enzyme, which will degrade the ampicillin, so when the cells carrying recombinant plasmid are grown in ampicillin-containing media, the cells will grow while the cells which do not carry recombinant plasmid will die. Lac Z gene is the marker for -galactosidase enzyme which will hydrolyze X-Gal into galactoside and its derivative compounds, namely the blue 5-bromo-4-chloro-indoxyl (Susanti and Ariani 2003). If there is a fragment of inserted gene in the plasmid, the synthesis of -peptide which will serve as an activator for -galactosidase enzyme will be impeded, so the blue color will not appear (Brown and Brown 1991). The white colonies formed in selection media were then amplified with PCR and confirmed with electrophoresis. The PCR of colonies was started with the selection of separated colonies in selection media.

Previously, a number was assigned to each colony. Then, with sterile toothpicks the colonies were duplicated in selection media which had been divided into several regions. The positive white clones which were estimated to contain fragments of inserted genes were subsequently grown in LB media added with 50 mg/mL of ampicillin and X-Gal substrate. The clones were used as templates for amplification of inserts using primer M13. The electropherogram of insert amplicon from cloning is presented in Figure 2.

The insert amplicon was digested with MspI and AluI restriction enzymes which cut with high frequency, which resulted in 10 different restriction patterns. The restriction patterns of DNA of non-methanogenic bacteria 16S rRNA marker genes are presented in Figure 3.

Sequencing and identification of 16S rRNA marker genes The results of PCR amplification were sent to 1st Base Sequencing Services Singapore (via PT. Genetika Science Indonesia, Jakarta) to be sequenced using ABI Big Dye Terminator Kit (Applied Biosystems® c.q. Thermo Fisher Scientific Inc., Waltham, MA, USA). The results of sequencing were then compared with the sequences in the GeneBank database, using Basic Local Alignment Search Tool (BLAST) on-line in website: www.ncbi.nlm.nih.gov.

All ten phylotypes were identified based on the 16S rRNA marker gene and compared with the database and using BLASTn analyses.

Based on the results of BLASTn analyses, there were 4 phylotype sequences of non-methanogenic bacteria from the fermentation of tofu-processing wastewater which had partial sequences similar to those in the database of the GeneBank with percent similarity 97%. This indicated that the bacteria were of the same species with the isolates which had been identified (Larsen et al. 1993).

There were 6 phylotype sequences with percent similarity <97%, meaning that those were new species whose data were not available in the GeneBank. The high percentage of <97% similarity between 16S rRNA gene

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fragments from the 6 phylotype sequences and those in the GeneBank indicated that the identified bacteria had close kinship, but not from the same species, with those in the GeneBank. The absence of 97% similarity between 16S rRNA fragments and those registered in the GeneBank indicated that the identified bacteria were partial sequences of the new strains of those in the GeneBank.

The compared sequences were partial sequences resulted from the sequencing and each restriction pattern produced different sequences. The results of phylotype identification indicated that there were at least 10 species of non-methanogenic bacteria, the composition of which is presented in Table 2.

1 kb DNA ladder

Figure 1. Electropherogram of amplicon of genomic DNA of non-methanogenic bacteria 16S rRNA marker gene.

1 kb DNA ladder

Figure 2. The electropherogram of insert amplicon resulted from cloning of 16S rRNA gene from tofu-processing wastewater samples.

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100 bp DNA ladder

16 (1): 62-68, April 2015

1 kb DNA ladder

1 kb DNA ladder

A 1 kb DNA ladder

1 kb DNA ladder

1 kb DNA ladder

B Figure 3. Restriction patterns of DNA of non-methanogenic bacteria 16S rRNA marker gene with MspI phylotype 1-10 restriction enzymes. A. 100 bp DNA ladder, B. 1 kb DNA ladder.

Table 1. The persentage of similarity between non-methanogenic bacteria DNA in the biogas production process from tofu-processing wastewater and the sequences in the GeneBank.

Restriction pattern codes Type 1 Type 2 Type 3 Type 4 Type 5 Type 6 Type 7 Type 8 Type 9 Type 10

Closest kin Chelatococcus daeguensis strain TAD1 Uncultured bacterium clone NBBP10309_58 Brevundimonas diminuta strain R057 Ochrobactrum anthropi strain YZ-1 Devosia albogilva strain IPL15 Acinetobacter junii strain J14N Ochrobactrum oryzae strain CK1711 Olsenella umbonata Defluvibacter lusatiensis strain S1 Rhizobium borbori strain DN365

No Access HM000004.1 JQ072591.1 KC252887.1 JN248785.1 NR_044212.1 JX490072.1 KC955129.1 FN178463.2 NR_025312.1 GU356639.1

% Similarity 94 98 99 94 96 95 97 94 97 94

SUNARTO et al. – The diversity of non-methanogenic bacteria

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Table 2. The composition persentage of non-methanogenic bacteria of biogas production from tofu-processing wastewater.

Restriction pattern codes Type 1 Type 2 Type 3 Type 4 Type 5 Type 6 Type 7 Type 8 Type 9 Type 10

Closest kin Chelatococcus daeguensis strain TAD1 Uncultured bacterium clone NBBP10309_58 Brevundimonas diminuta strain R057 Ochrobactrum anthropi strain YZ-1 Devosia albogilva strain IPL15 Acinetobacter junii strain J14N Ochrobactrum oryzae strain CK1711 Olsenella umbonata Defluvibacter lusatiensis strain S1 Rhizobium borbori strain DN365

The non-methanogenic bacterium with the highest percentage in this study was Brevundimonas diminuta, which was 68.42% with 26 clones out of the total of 38 clones. Chelatococcus daeguensis, Devosia albogilva, and Ochrobactrum oryzae had the same percentage, namely 5.26%, each having two clones. Several bacteria which had the smallest percentage, only 2.63%, were uncultured bacterium, Ochrobactrum anthropi, Acinetobacter junii, Olsenella umbonata, Defluvibacter lusatiensis, and Rhizobium borbori, each having only 1 clone.

Uncultured bacterium clone NBBP10309_58 referred to bacterium that had not been cultured and identified because it had such low metabolism activities that its similarity to or difference from other bacteria was not known. Based on the results of BLASTn, this phylotype was considered to have close kinship with phylum Firmicutes they had 92% similarity. According to Sun et al. (2013) the important role of phylum Firmicutes in biogas production is its ability to hydrolyze organic substrate.

So far there has been no sufficient information about non-methanogenic bacteria which play role in the hydrolysis and fermentation of substrate from tofuprocessing wastewater. The only available information is limited to general species of non-methanogenic bacteria responsible for hydrolysis and fermentation processes, including facultative and obligate anaerobic bacteria such as Actinomyces, Bacteroides, Bifidobacterium spp., Clostridium spp, Corynebacterium spp., Desulfovibrio spp., Escherichia coli, Lactobacillus, Peptococcus anaerobius, Staphylococcus and Streptococcus (Tchobanoglous et al.

2002; Madigan et al. 2009).

Based on the available information, there are 6 phylotypes of non-methanogenic bacteria considered to play role in biogas production. Uncultured bacterium clone considered to have close kinship with Firmicutes plays role in hydrolysis stage because it is able to hydrolyze organic substrate (Sun et al. 2013). Brevundimonas diminuta plays role in acidogenesis stage because it is able to produce acetic acid (Kohyama et al. 2006). Olsenella umbonata also plays role in acidogenesis stage because it can convert glucose into acetic acid and formic acid (Isolauri et al.

2004; Wilson 2005). Acinetobacter junii, Ochrobactrum anthropi and Defluvibacter lusatiensis are reported to be able to reduce nitrate into nitrite which in turn is used by other bacteria as an acceptor of electron until it becomes

% Composition of non-methanogenic bacteria 5.26% 2.63% 68.42% 2.63% 5.26% 2.63% 5.26% 2.63% 2.63% 2.63%

nitrogen. According to Sterling et al. (2001) nitrogen sources are important as nutrient for microbes to produce biogas.

In this study, seven out of ten phylotypes were aerobic which had close kinship with Ochrobactrum oryzae, Defluvibacter lusatiensis, Devosia albogilva, Rhizobium borbori, Chelatococcus daeguensis, Brevundimonas diminuta and Ochrobactrum anthropi. The presence of aerobic bacteria in the fermented sludge indicated the presence of oxygen in the digester, so the biogas production was not maximal. The high concentration of oxygen in the digester impedes archaic methanogenic bacteria in producing methane gases.

The presence of oxygen in the digester in this study might be caused by the use of simple, laboratory-scale digester. The initial condition of digester was not anaerobic, so the aerobic bacteria still survived. This condition was correlated with the low biogas production, especially the CH4.

Analyses of non-methanogenic bacteria The analyses of non-methanogenic bacteria were based on the calculation of the number of clones. Species richness (S) was the number of clones recorded, which was 10. The Shannon-Wiener index was 1.291, indicating the low diversity in the tofu-processing wastewater processed into biogas. According to Krebs (1989), the classification of H’ are as follows: low diversity (H’ = 0-2.302), medium diversity (H’ = 2.302-6.907), and high diversity (H’ > 6.09).

The Evenness index (E) indicates evenness of a community. The evenness index in the tofu-processing wastewater processed into biogas was 0.561, categorized as low. According to Krebs (1985), the value of E ranges from 0 to 1. If E approaches 0, the evenness of community is low and the abundance of species is not uniform, and the community is dominated by certain species. If E approaches 1, the evenness is high, meaning that there is no dominant species, or the abundance of species is uniform.

The low diversity in this study was caused by the dominance of Brevundimonas diminuta. The plausible reason for the dominance of this species was that the high protein content in the tofu-processing wastewater provided nutrient for the growth of this bacterium, as Chaia et al.

(2000) states that Brevundimonas diminuta uses protein as

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source of carbon in the form of amino acid.

In this study ten bacteria were found, namely Brevundimonas diminuta, uncultured bacterium clone, Defluvibacter lusatiensis, Ochrobactrum oryzae, Devosia albogilva, Acinetobacter junii, Chelatococcus daeguensis, Ochrobactrum anthropi, Olsenella umbonata, and Rhizobium borbori. These bacteria are able to survive in mesophylic condition, namely at temperature of 30-40°C and pH of 5-8.5.

It can be concluded that 10 phylotypes were found after anaerobic fermentation of tofu-processing wastewater which had been incubated for 20 days. There were 6 phylotypes which were estimated to play role in biogas production, namely Brevundimonas diminuta, Olsenella umbonata, uncultured bacterium (considered to have close kinship with phylum Firmicutes), Acinetobacter junii, Ochrobactrum anthropi and Defluvibacter lusatiensis. The Shannon-Wiener Index was 1.291, categorized as low. The evenness index was 0.561, categorized as low due to the dominance of Brevundimonas diminuta. Further study is needed to optimize the anaerobic fermentation of tofuprocessing wastewater to get higher methane gas concentration and sampling should be done in various periods of fermentation in order to find more nonmethanogenic bacteria.

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B I O D I V E R S IT A S Volume 16, Number 1, April 2015 Pages: 69-78

ISSN: 1412-033X E-ISSN: 2085-4722 DOI: 10.13057/biodiv/d160109

Infraspecific morphological and genome size variations in Linum glaucum in Iran SEYED MEHDI TALEBI1,♥, MASOUD SHEIDAI2, MORTEZA ATRI3, FARIBA SHARIFNIA4, ZAHRA NOORMOHAMMADI5 1

Department of Biology, Faculty of Sciences, Arak University, Arak, 38156-8-8349 Iran. Tel. +98-863-4173317. ♥ email:_seyedmehdi_talebi@yahoo.com.

2 Shahid Beheshti University, GC, Faculty of Biological Sciences, Tehran, Iran.

3 Bu-Ali Sina Hamedan University, Hamedan, Iran.

4 Department of Biology, North Tehran Branch, Islamic Azad University, Tehran Iran.

5 Department of Biology, School of Sciences, Tehran Science and Research Branch, Islamic Azad University, Tehran, Iran Manuscript received: 27 December 2014. Revision accepted: 20 January 2015.

Abstract. Talebi SM, Sheidai M, Atri M, Sharifnia F, Noormohammadi Z. 2015. Infraspecific morphological and genome size variations in Linum glaucum in Iran. Biodiversitas 16: 69-78. There are many discussions about taxonomic position of Linum glaucum in different Flora. In this study, the morphological traits associated with the nuclear genome size were used for identification of infraspecific variations in the nine populations of L. glaucum. Twenty three qualitative and quantitative morphological characteristics were investigated. The Analysis of variance tests showed significant difference for some morphological features. The Canonical Correspondence Analysis of habitat ecological factors showed that each of the habitats had prominent characteristics, and also Pearson’s coefficient of correlation confirmed the significant correlations between the morphological features in relation to ecological factors. In the morphological Unweighted Paired Group using Average method tree, populations were separated from each other, so that population No. 4 was separated from others. In the flow cytometery investigations, variation of about 1.19 times was presented between the maximum and minimum averages of the genome sizes in the populations. Minimum amount of genome size was found in populations No. 4. Significant correlations occurred between the nuclear genome size with habitat elevation as well as some of the quantitative morphological features. The mentioned variations caused to difference between the populations and lead to creation of ecotype and ecophene in the studied populations.

Key words: Ecology, genome size, morphology, population.

INTRODUCTION Biodiversity is mostly investigated at the species level as well as higher taxonomic ranks. However, the basis for biodiversity and organic evolution is the genetic difference within species (Ramel 1998). Linhart and Grant (1996) suggested that Comparable developmental variation presents between different populations of a species (intraspecific), which likely resounds verifications to different natural environments and it is the source of plant species differentiation. Therefore, humans have used these infraspecefic variations for the domestication and genetic improvement of many of plant species (Diamond 2002).

Linum glaucum Boiss. & Nöe belongs to section Linum of the genus Linum L. This species is an Irano-Turanian element which naturally spread in the slops of the Zagrous Mountains in the different provinces of Iran such as Kurdistan, Kermanshah, Zanjan, Hamadan, Lurestan as well as Markazi. In addition this species are found in the neighboring countries as Iraq and Turkey (Rechinger 1974; Mobayen 1996; Sharifnia and Assadi 2001).

There are many discussions about taxonomic position of L. glaucum. This taxon was described for the first time as L. alpinum Jacq, var. glaucescens Boiss. in Flora Orientalis (Boissier 1867), then Stapf recognized it as L.

strile (Rechinger 1974). In 1967, Davis named it as L. austriacum L. subsp. glaucescens, following Rechinger (1974) recognized this taxon as a synonym of L. glaucum.

Therefore there are three different synonyms for L. glaucum in different Flora such as Flora Iranica (Rechinger 1974) and Flora of Iran (Sharifnia and Assadi 2001).

Although L. glaucum is morphologically very similar to L. austriacum, the other member of this section, but in different taxonomical treatments such as palynology (Talebi et al. 2012a), seed micromorphology (Talebi et al.

2012b), anatomy (Rashnou-Taei 2014) and also molecular investigation (ISSR) (Talebi 2013) these species placed separately. The obtained results of these studies showed that L. glaucum and L. austriacum are separate species.

In addition to morphological characteristics, variation between populations can be investigated with using 2Cvalue or nuclear genome size. One of the most important features of all living organisms is nuclear DNA amount.

Swift (1950) definite C-value as DNA content in the unreplicated haploid nucleus. Nuclear DNA amount is positively related to the number of cellular traits such as: time of mitotic cell cycle, nuclear DNA synthesis rate, the size of cell and its nuclear, through to the whole plant traits such as minimum generation time and geographic distribution (Bennett 1972, 1987; Kidd et al. 1987). Studies

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confirmed that infraspecific variation in nuclear genome size amount is most significant for plant taxonomy as an indicator of taxonomic heterogeneity (Murray 2005).

Because, the lacks of previously infraspecific study for L. glaucum in Iran as well as the world, we have used for the first time in the present work morphological characteristics as well as nuclear genome size for identification and determining kind and level of infraspecific variations between the nine populations of this species in Iran.

MATERIAL AND METHODS Plant material Nine populations of L. glaucum were randomly collected from western regions of Iran during spring 2011 (Table 1). Plant samples were identified based on the descriptions provided in accessible references such as Flora Iranica (Rechinger 1974) and Flora of Iran (Sharifnia and Assadi 2001). The voucher specimens were deposited in the herbarium of Shahid Beheshti University (HSBU).

From each population four samples were selected randomly and then examined for their nuclear DNA content and morphological features.

Morphometry Twenty three quantitative and qualitative morphological characteristics from the both of vegetative and reproductive organs of this species were examined. The studied morphological characteristics include: stem lengths and its branch number, stem diameter, shape, length and width of basal and floral leaves, the shape of apex, margin and base of basal and floral leaf blade, basal leaf diameter, calyx dimensions as well as corolla color and dimensions. Four replications were used for quantitative characters measurements and the used terminologies for qualitative morphological traits were on the basis of descriptive terminology provided by Stearn (1983).

Flow cytometry In order to preparation of nuclei suspensions, small amounts of mature fresh leaf tissue together with an equal weight of mature leaf tissue of the standard reference were used. In this study Allium cepa L. was used as standard references, which has a 2C DNA value of 33.5 pg (Greilhuber and Ebert 1994).

For preparation of the nuclear suspension two-stepped method was employed. Plant materials were chopped with a sharp scalpel with adding 400 μl nuclei isolation buffer in the plastic Petri dish at room temperature. For staining the extracted nuclei, 1600 μl DNA fluorochrome or 4´, 6Diamidino-2-phenylindole (DAPI) added and the suspensions were filtered through a 50 μm nylon mesh into a labeled sample tube. Then the suspensions of stained nuclei were analyzed with a Partec PI Flow Cytometer (Partec Germany). In the obtained histograms, some flow cytometric statistics such as: coefficient of variation (CV), mode, mean and the number of counted cells were investigated. The obtained nuclear DNA amounts were

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measured in picograms (pg) and the nuclei status described in terms of ‘C’ value (Doležel et al. 2007). Both of the Cvalue as well as the genome size can be expressed either in DNA picograms (=10-9 g) or mega base pairs (1 pg = 978 Mbp). The nuclear DNA amount of the studied samples calculated on the basis of the values of the G1/G2 peak means (Doležel and Bartoš 2005).

Ecological factors In order to compare the effect of different environmental factors on the nuclear genome size and morphological characteristics of L. glaucum populations, five factors were examined for each population’s habitat, such as: longitude (E˚), latitude (N˚), altitude (in meters), average of annual minimum and maximum temperature (in C˚). Longitude, latitude and altitude were calculated with Garmin GPS map76CSx, and the averages annual minimum and maximum temperature for each population were taken from the web site of the meteorological organization of Iran.

Statistical analysis The mean and standard deviation of the studied morphological characteristics were determined. In order to group the studied populations on the basis of morphological features, data were standardized (mean = 0, variance = 1) and used for multivariate analyses including Unweighted Paired Group using Average method (UPGMA) as well as Principal Coordinate Analysis (PCoA) (Podani 2000). Analysis of variance (ANOVA) test was employed to assess the significant quantitative morphological characteristics difference among the studied populations, and Pearson’s coefficient of correlation was used to determine significant correlations of nuclear genome size and quantitative morphological traits in relation to ecological factors, as well as, longitude, latitude, altitude, average of annual minimum and maximum temperature, so as to show possible relationship between populations. The multivariate method, Canonical Correspondence Analysis (CCA), was used to elucidate the relationship between the studied populations and their environmental factors. NTSYS ver. 2 (1998) and SPSS ver.

9 (1998) softwares were used for statistical analyses.

RESULTS AND DISCUSSION Morphometry In present study, in order to compare the effect of different environmental factors on the phenotype of L. glaucum populations, twenty three traits of the both vegetative and reproductive organs were selected and examined between nine natural populations. The mean and standard deviations of the studied characteristics are presented in Table 2. Qualitative characteristics such as: the shape of blade and also its margin, apex and basis of the

TALEBI et al. – Intraspecific variations in Linum glaucum

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Table 1 .Locality and herbarium voucher number of the studied populations.

No. Population

Habitat address

Collector

Herbarium nos.

1 2 3 4 5 6 7 8 9

Kurdistan, road of Sanandaj to Hasan Abad, 1684 m.

Kurdistan, Sanandaj, Darbandeh village, 1559 m.

Kurdistan, Sanandaj, Kani Moshkan village, 1678 m.

Kurdistan, Sanandaj, Kilaneh village, 1471 m.

Kurdistan, Sanandaj, Abidar Mountain, 1585 m.

Kurdistan, road of Saqqez to Baneh, 1546 m.

Kurdistan, 25 km Baneh to Saqqez, 1623 m.

Kurdistan, Saqqez, 1570 m.

Kurdistan, road of Saqqez to Divandareh, 1617 m.

Talebi Talebi Talebi Talebi Talebi Talebi Talebi Talebi Talebi

HSBU2011353 HSBU2011354 HSBU2011355 HSBU2011356 HSBU2011358 HSBU2011360 HSBU2011361 HSBU2011362 HSBU2011363

Table 2. Selected morphological traits of the studied population of L. glaucum (all values are in cm, details of each population are given in Table 1).

Populations 1

2

3

4

5

6

7

8

9

Mean N SD Mean N SD Mean N SD Mean N SD Mean N SD Mean N SD Mean N SD Mean N SD Mean N SD

Stem length 49.12 4 9.54 60.37 4 6.75 55.37 4 6.79 40.25 4 5.17 49.75 4 6.65 47.75 4 8.46 53.87 4 7.37 55.25 4 4.85 59.00 4 8.36

Basal Stem leaf ramification length 10.50 1.77 4 4 2.64 0.10 8.75 1.96 4 4 1.25 0.49 10.25 1.67 4 4 4.78 0.38 8.00 1.27 4 4 3.65 0.17 6.50 1.50 4 4 1.73 0.11 16.75 1.57 4 4 3.09 0.26 15.00 1.87 4 4 6.78 0.26 8.75 1.47 4 4 1.70 .37 4.50 1.60 4 4 0.57 0.14

Basal leaf width 0.19 4 0.10 0.25 4 0.04 0.21 4 0.06 0.15 4 0.00 0.22 4 0.05 0.35 4 0.05 0.18 4 0.04 0.15 4 0.03 0.23 4 0.02

Basal leaf shape LinearLanceolate LinearLanceolate Linear

Linear

Lanceolate

LinearLanceolate LinearLanceolate Linear

Lanceolate

Floral leaf length 0.87 4 0.15 0.87 4 0.09 1.07 4 0.12 1.04 4 0.13 0.80 4 0.18 1.05 4 0.23 1.37 4 0.12 1.12 4 0.25 1.02 4 0.12

both of basal and floral leaf as well as petal color were examined between the populations. Basal and floral leaf shape varied between populations and presented in the shape of linear, lanceolate or linear-lanceolate. In spite of variations in blades shape in the basal and floral leaf, blade apex, margin and base shapes were stable between populations and were in the shape of acute, entire and cuneate respectively, furthermore the petal colors were stable inter-populations and presented as blue.

The performed ANOVA test on the quantitative morphological traits between the studied populations showed significant difference (p<0.05) in characteristics

Floral leaf width 0.09 4 0.02 0.11 4 0.02 0.12 4 0.05 0.31 4 0.45 0.09 4 0.00 0.16 4 0.04 .17 4 0.05 .10 4 0.00 0.24 4 0.24

Floral leaf shape

Calyx Calyx Pedicle Sepal Sepal Petal Petal width length length length width length width

LinearLanceolate

0.37 4 0.05 0.35 4 0.05 0.38 4 0.02 0.37 4 0.05 0.33 4 0.04 0.37 4 0.05 0.45 4 0.05 .38 4 0.06 0.36 4 0.04

LinearLanceolate LinearLanceolate Lanceolate

LinearLanceolate Lanceolate

Lanceolate

Linear

Lanceolate

0.45 4 0.06 0.45 4 0.05 0.40 4 0.00 0.47 4 0.05 0.42 4 0.05 0.42 4 0.05 0.47 4 0.05 .45 4 0.05 0.52 4 0.05

0.10 4 0.00 0.09 4 0.00 0.10 4 0.00 0.10 4 0.00 0.07 4 0.02 0.10 4 0.00 0.10 4 0.00 .10 4 0.00 0.09 4 0.02

0.20 4 0.00 0.18 4 0.02 0.23 4 .04 0.27 4 0.05 0.22 4 0.02 0.22 4 0.05 0.22 4 0.02 .25 4 0.05 0.23 4 0.04

0.10 4 0.00 0.10 4 0.00 0.10 4 0.00 0.10 4 0.01 0.12 4 0.05 0.10 4 0.00 0.12 4 0.05 .13 4 0.04 0.10 4 0.00

1.55 4 0.33 1.72 4 0.22 1.55 4 0.38 2.32 4 0.37 1.65 4 0.51 1.62 4 0.15 1.97 4 0.17 1.82 4 0.22 2.00 4 0.08

1.20 4 0.14 1.25 4 0.05 1.22 4 0.17 1.60 4 0.31 1.27 4 0.15 1.35 4 0.17 1.82 4 0.17 1.35 4 0.23 1.65 4 0.12

such as: stem height and diameter, branch number, basal leaf width and thickness, floral leaf length, petal length and width (Table 3).

Highest and smallest stems lengths being recorded in populations No. 2 (62.16 cm) and No. 4 (40.25 cm), respectively. Stem branch number was between 5 (population No. 9) to 17 (population No. 6). Biggest basal leaf (1.57×0.35 cm) occurred in population No. 6, while population No. 4 had smallest basal leaf (1.27×0.15 cm).

Widest floral leaf (0.31 cm) registered in population No. 4 and narrowest (0.1 cm) belonged to population No. 1.

Biggest calyx (0.45×0.45 cm) recorded in population No. 7,

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Table 3. Results on the ANOVA analysis to assess for differences in the quantitative morphological traits in L. glaucum populations.

Characters Stem height

Stem ramification

Basal leaf length

Basal leaf width

Floral leaf length

Floral leaf width

Calyx width

Calyx length

Pedicle

Sepal length

Sepal width

Petal length

Petal width

Leaf thickness

Stem diameter

Between Groups Within Groups Total Between Groups Within Groups Total Between Groups Within Groups Total Between Groups Within Groups Total Between Groups Within Groups Total Between Groups Within Groups Total Between Groups Within Groups Total Between Groups Within Groups Total Between Groups Within Groups Total Between Groups Within Groups Total Between Groups Within Groups Total Between Groups Within Groups Total Between Groups Within Groups Total Between Groups Within Groups Total Between Groups Within Groups Total

Sum of Squares

df

Mean Square

F

Significant

1252.889 1420.250 2673.139 481.556 320.000 801.556 1.457 2.362 3.819 .118 .045 .163 .926 .747 1.674 .184 .832 1.016 .032 .070 .102 .042 .070 .112 .002 .005 .007 .021 .046 .066 .005 .022 .028 2.122 2.488 4.610 1.581 .922 2.503 .062 .067 .130 .137 .093 .230

8 27 35 8 27 35 8 27 35 8 27 35 8 27 35 8 27 35 8 27 35 8 27 35 8 27 35 8 27 35 8 27 35 8 27 35 8 27 35 8 27 35 8 27 35

156.611 52.602

2.977

.016

60.194 11.852

5.079

.001

.182 .087

2.082

.074

.015 .002

8.910

.000

.116 .028

4.184

.002

.023 .031

.748

.649

.004 .003

1.554

.186

.005 .003

2.040

.079

.000 .000

1.294

.288

.003 .002

1.552

.186

.001 .001

.838

.578

.265 .092

2.879

.019

.198 .034

5.783

.000

.008 .002

3.111

.013

.017 .003

4.974

.001

while smallest calyx (0.35×0.4 cm) was seen in population No. 2. Largest (0.27 cm) and smallest sepal (0.1 cm) were found in populations No. 4 and 6, respectively. Biggest and smallest corolla (2.32×1.6 cm) were registered in populations No. 4 and 1, respectively. Thickest basal leaf (0.2 mm) and stem (0.33 cm) were found in populations No. 2 and 3, respectively. Slimmest basal leaf (0.1 mm) and stem (0.13 cm) occurred in populations No. 7 and 4, respectively.

Five geographic and climatic factors of population’s habitat were examined. CCA analysis showed that these factors varied between habitat, and each of them had a prominent environmental feature. For example, populations No. 6 and 7 placed at coldest environments or population

No. 5 rested in eastern regions (Figure 1). CCA biplot of morphological traits and environmental factors showed that the mentioned parameters had strong effect on the plant phenotype. For example, branch number and also basal leaf shape were varied along annual minimum temperature and also variations in floral leaf length, petal width and floral leaf width were parallel with northern distribution of populations; furthermore, annual maximum temperature had strong effect on floral leaf shape (Figure 2).

In addition, Pearson’s coefficient of correlations were determined between the quantitative morphological features in relation to environmental factors of habitat, including longitude, latitude, altitude and average of annual minimum

TALEBI et al. â&#x20AC;&#x201C; Intraspecific variations in Linum glaucum

and maximum temperature, showed significant negative/positive correlations. For example, floral leaf length had a significant negative correlation (p<0.05, r=0.65) with maximum temperature of year, but this feature had significant positive correlations (p<0.05) with minimum temperature of year (r=-0.65) and northern distribution (r= 0.69) of populations. A significant negative correlation (p<0.001, r=-0.94) occurred between calyx width with annual maximum temperature, while this trait had a significant positive correlation (p<0.05, r= 0.72) with northern distribution of populations.

The studied populations were separated in the UPGMA tree of morphological features (Figure 3). In the mentioned diagram two main branches were seen, one of these had one population, namely population No. 4, which arranged separately far from others. The rest of populations placed in another branch. This branch had two sub branches; populations No. 7 and 6 became detached from others and placed in one sub branch. As seen in the box and whisker plots (Figure 4), each of the studied populations had distinct morphological trait (s).

Genome size Nuclear DNA amounts of the studied populations were calculated with flow cytometer apparatus (Table 4). In the analyzed samples, most of the leaf nuclei of the both L. glaucum and standard references were at the G1 phase of the cell division and thus represented the 2C DNA amount. The average of nuclear genome size differed between populations. Maximum average of genome size was recorded in population No. 5 with about 2.06 pg, while the

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minimum average value (1.73 pg) being registered in population No. 4, showing variation of about 1.19 times.

A significant negative correlation (p<0.05, r=-0.73) occurred between genome size with habitat altitude. This means that, L. glaucum populations that grow in higher altitude possess smaller nuclear DNA amounts, while the populations found in lower had larger nuclear DNA amounts. Not only significant correlation occurred between nuclear genome sizes in relation to environmental factors of habitat, but also morphological traits of the studied populations had significant correlations with 2c-value amount of plants. For example, nuclear genome size had significant negative correlations (p<0.05) with floral leaf width (r=-0.67) and also with petal length (r=-0.72). A significant positive correlation (p<0.05, r=0.67) found between nuclear DNA amount with stem height.

Table 4. Nuclear genome size of the studied populations (details of each population are given in Table 1).

Population nos.

2C DNA amount (picograms)

1 2 3 4 5 6 7 8 9

1.99 2.05 1.99 1.73 2.06 1.96 1.94 2.02 2.01

Genome size (mega base pairs) 1946.22 2004.9 1946.22 1691.94 2014.68 1916.88 1897.32 1975.56 1965.78

Figure 1. CCA plot of the ecological factors of L. glaucum populations. Abbreviations, minte: average of annual minimum temperature, maxtem: average of annual maximum temperature, north: latitude, altit: altitude, east: Longitude (details of each population are given in Table 1).

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Figure 2. CCA biplot of morphological traits with ecological factors of the studied populations. Abbreviations, minte: average of annual minimum temperature, maxtem: average of annual maximum temperature, north: latitude, altit: altitude, east: Longitude, balesh: basal leaf shape, flesh: floral leaf shape, fllewi: floral leaf width, fllele: floral leaf length, leadi: basal leaf dimentions, petwi: petal width, balewi: basal leaf width, bra: branch number (details of each population are given in Table 1).

Figure 3. Morphological UPGMA tree of the L. glaucum populations (details of each population are given in Table 1).

Genome size

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Petal width

Petal length

Sepal width

Sepal length

Pedicle

Calyx width

Calyx length

Floral leaf width

Floral leaf length

Basal leaf width

Basal leaf length

TALEBI et al. â&#x20AC;&#x201C; Intraspecific variations in Linum glaucum

Figure 4. Box and whisker plot of some important characteristics of the studied populations (details of each population are given in Table 1).

Mixoploidy In mixoploidy condition, the plant body consisting of diploid and polyploid cells. Genome size of all cells undergoes cyclic changes. In the resting (G0 phase) and pre-replicative stages of cells (G1 phase), each cell has 2C of nuclear DNA amount, but synthesis of new DNA occurs during S phase of cell cycle, resulting in doubled (4C) nuclear DNA content. In the post-replicative period of cell growth (G2 phase), the DNA content is maintained at 4C level. In population No. 1, in addition to peak of G1 phase nuclei (2C= 1.99 pg), the peak of G2 phase nuclei were seen and represented the 4C (DNA4C = 4.11 pg). So mixoploids can be identified in flow cytometry histograms by the presence of two peaks of Linum in the histogram (Figure 5).

Figure 5. Flow cytometry histogram of mixoploidy in L. glaucum population (peak 2: L. glaucum 2c value amount, peak 3: L. glaucum 4c value amount and peak 4: standard reference).

Discussion Alonso-Blanco et al. (2005) suggested that plant diversity has fascinated man all over history, primarily due to the great difference that presents in nature for morphological and other developmental features.

According to Cronk (2001), the effects of fitness of such naturally occurring variation exist in the different populations of a species (infraspecific) have driven plant macroevolution by means of natural selection, this developmental diversity being the principle of plant classification and phylogeny.

In the present study, in addition to morphological characters, nuclear genome size was used for identification of infraspecific variations in the L. glaucum populations.

Not only quantitative morphological traits differed between populations and ANOVA test showed significant difference for some of them, but also qualitative features such as floral as well as basal leaf shape varied among the studied samples. In the UPGMA tree of morphological features, populations were detached, not only population No. 4 placed in separated branch but also populations No. 6 and 7 separated from the rest. This subject confirmed high morphological variations between the populations. The individuals of the mentioned populations had distinct morphological characteristics, for example population No.4 had smallest stem, basal leaf and sepal and also biggest corolla with widest floral leaf in comparison to other populations. Therefore, these traits differentiated these populations from others. Different morphological studies on the populations of this species confirmed the obtained results of the present study. For example morphological characters of long-styled and short-styled plant populations

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in L. glaucum were investigated by Talebi et al. (2012c). In their study, T-test analysis of morphological characters between long-styled and short-styled plant populations, showed significant difference (p<0.05) for some characters such as basal leaf length, calyx width and length.

Understanding the sources of morphological variation in organisms such as plants is central to the understanding of natural variation and the responses of organisms to their environment. Morphological traits variability expression can show differentiation of plant populations in a particular habitat (Schlichting 1986).

The observed variations between the studied populations can be caused by two main factors: it may be related to ecological factors of the population’s habitat or variations in genetic structures of populations. Most of the existing relationships between the morphological features with environmental factors were significant; this subject showed that variation in phenotypical characteristics is predictable and had significant correlations with environmental variations. Referring again to the mentioned above populations, it can see that both of the populations No. 4 and 6 were found in habitats which had salient characteristic (s), for example population No.4 found in the highest altitude (1741 m), conversely population No. 6 was seen in the lowest elevation habitat (1546 m), with variation about 200 m. These conditions were true about other ecological factors of habitats such as annual minimum and maximum temperatures and also geographical latitude and altitude. Furthermore, significant positive or negative correlations found between environmental factors with morphological characteristics of populations individuals. For example, increase in habitat temperature shortens the length of floral leaf or northern populations have more elongated floral leaf. Different studies on the other species, either of the genus Linum or other genus/ families confirmed this condition. For example, Morphological characteristics of twenty one populations of Linum album Kotschy ex Boiss. were survived (Talebi et al. 2014a). The results of the study have shown that the different populations of L. album were adapted to their habitat and the ranges of phenotype plasticity were high between populations which led to creation of ecomorphs (Talebi et al. 2014a). Furthermore, Talebi et al. (2014b) investigated morphological traits of different populations of Stachys inflata Benth. in Iran. The results of this study showed that different populations of same species that grow under different ecological conditions, altered their morphological features for adaptation with their habitat condition.

The relationships between nuclear DNA mounts and phenotypic features have been discussed in different studies at different levels, for example effect of genome size on cell size and stomatal density in angiosperms (Beaulieu et al. 2008), effect of genome size on phenotypic traits at the cellular level i.e. guard cell length and epidermal cell area (Knight and Beaulieu 2008); relationship between genome size and production of aboveground biomass, seedling establishment success as well as seed weight and seed dormancy (Munzbergova 2009), but few studies have studied how and whether nuclear genome

16 (1): 69-78, April 2015

size can alter phenotypic features at the population scale (but see Lavergne et al. 2010).

The obtained results showed that, in addition to morphological features, nuclear genome size showed variations of about 1.19 times between populations. These variations are widespread phenomenon in this genus.

ANOVA test revealed a significant variation in genome size among the Iranian populations of Linum austriacum L. (Sheidai et al. 2014a) as well as Linum album (Sheidai et al. 2014b). Some of Linum species are known as a good example of plastic genome in plant (Evans et al. 1966; Evans 1968a, b; Durrant and Jones 1971; Joarder et al.

1975), for example different cultivars of L. sativum Hasselq. had a 10 % difference in genome size. Some degree of chromosomal variation within populations such as duplications and deletions, spontaneous aneuploidy or polyploidy, heterochromatic segments, B-chromosomes and sex chromosomes will naturally cause some interindividual DNA content variation (Greilhuber 1998).

Results of this study showed that nuclear genome size had strong effect on morphological characteristics of the studied populations, and significant positive/negative correlations recorded between morphological characteristics with 2c-value amounts. Furthermore, population’s arrangements in the morphological UPGMA tree were coinciding with 2c-value amounts of the populations. Population No. 4 which separated of others had smallest amount of nuclear genome size. This condition was true about populations No. 6 and 7. These populations placed together and approximately had equal nuclear genome size. Quantitative differences in nuclear DNA contents within a species result in changes in cell cycle and cell size in many plant species such as Zea mays L. (McMurphy and Rayburn 1992), Festuca arundinacea Schreb. (Minelli et al. 1996) and Poa annua L. (Mowforth and Grime 1989). These changes at the cellular level have multitudinous effects on various phenotypic traits through allometric effects of the changes in cell dimension (Cavalier-Smith 1985). DNA is effected growth and development of each organism in two ways, directly through its informational and gene content, and indirectly by the physical-mechanical effects of nuclear DNA mass.

Although the amount of nuclear DNA does not involve expression and regulation of single genes, it has great effects on cellular metabolic processes (Cavalier-Smith 1985).

The term ‘nucleotype’ was used to describe those conditions of the nucleus, which affect the phenotype (Bennett 1972). The infraspecific changes in genome size, being therefore subject to natural selection is naturally related with those leading to the divergence and evolution of species. Therefore, a thorough appraisal of such changes is required for the application of genome size data to demarcate infrageneric taxa and in microsystematics.

In the studied populations, habitat ecological factors influenced nuclear genome size, as significant negative correlations occurred between genome sizes with habitat altitude. Different studies confirmed this condition for other Linum species. For example, various genotrophs of L. usitatissimum L. created by a process of environmentally

TALEBI et al. – Intraspecific variations in Linum glaucum

induced changes in genomes structure (Cullis et al. 1999).

The mentioned heritable alternations are due to specific rearrangements at prominent positions of the genome, furthermore Cullis et al. (1999) stated that some changes in genome structure such as highly repetitive, middlerepetitive, as well as low-copy-number sequences are involved in the polymorphisms detected, and sequence alterations of specific subsets of 5SrDNA have been distinct, in addition different varieties of L. usitatissimum which grew in different environmental conditions for example: high nitrogen concentrations or high temperatures increased 10% in nuclear DNA amounts (Evans 1966).

Price et al. (1981) studied DNA contents of 222 plant samples of Microseris douglasii (DC.) Sch. Bip. representing geographically, ecologically and morphologically diverse populations in California which showed a 14% variation between population means, with those having higher and lower DNA amounts restricted to mesic and xeric sites, respectively. Similarly, Ceccarelli et al. (1992, 1993) recorded a 32.3 % difference covering 35 natural populations of hexaploid Festuca arundinacea was positively correlated with mean temperature during the year, with the coldest month at stations and with generation time, while it was negatively correlated with latitude and germination power of seeds and growth rate. A negative significant correlation was occurred between C-value and altitude in Dactylis glomerata L. The finding results showed that plants growing at lower altitudes have larger C-values than those at higher altitudes, which suggest that there was selection for smaller C-values with increasing altitude (Reeves et al. 1998).

The results of our study demonstrated the difference in nuclear DNA content between different populations of L. glaucum, and suggested that this variation play important roles in determining phenotypic differences between the populations in this species. Based on all the examined data, it can be concluded that populations No. 4, 6 and 7 detached of others and had unique morphological characteristics as well as nuclear DNA amount, which lead to difference of the mentioned populations from the others.

Therefore high inter-population variations obviously seen between the studied populations of L. glaucum. Clausen et al. (1948) suggested that individuals of a given species typically varied in morphological features. Although some of the mentioned variations may be random, but ecological theory posits that many of these variations may represent adaptive matching of phenotypes to a variable environmental condition. As Sultan (1995) believed this matching can occur either through natural selection process that create genetically-differentiated ecotypes, or through phenotypic plasticity, in which various phonotypical characters are produced from the same genotypes in different ecological condition.

The term ecotype is definite as a preferable set of populations within a species, resulting from conformity to positional environmental situations, able of interbreeding with other ecotypes of the same species (Hufford and Mazer 2003). In ecotype, populations are adapted to local conditions, at a variety of spatial and temporal scales.

Based on above definition, at least one ecotype was

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distinguished between the studied populations. Population No. 4 had distinct morphological characteristics with least nuclear DNA amount, which with these traits adapted to its habitat, so this population could be definite as ecotype.

Creation of ecotype is very important because this type of variation had adaptive value for each species and increase biodiversity in occurring ecosystem. Matching ecotypes to local conditions increases restoration success. A practical value of recognizing ecotypic variation in grasses is in allowing recognition of the most suitable ecotypes for conservation, restoration, renovation, landscaping, and bioremediation (Gibson 2009).

Other interpopulation variation was phenotypic plasticity (accommodation ecophene) which found between the studied populations, for instance populations No. 7 and 6 that morphologically separated from others, because no appreciable difference registered in nuclear DNA amount between these in comparison with other populations. The ability of plant to alter its morphology in response to changes in environments can be regarded as phenotypic plasticity or accommodation ecophene (Bradshaw 1965; Schlichting 1986). As Thompson (1991) suggested, the resistor of population to a variable environment is usually achieved by plastic phenotypic answer because it has been agreed that plastic reactions are required for species for the possession of damaged habitats.

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B I O D I V E R S IT A S Volume 16, Number 1, April 2015 Pages: 79-83

ISSN: 1412-033X E-ISSN: 2085-4722 DOI: 10.13057/biodiv/d160110

Short Communication: Occurrence of leaf spot diseases on Aloe vera (L.) Burm.f. caused by Curvularia species from Madhya Pradesh, India SHUBHI AVASTHI1, AJAY KUMAR GAUTAM2, REKHA BHADAURIA1,♥ 1

Mycology and Plant Pathology Laboratory, School of Studies in Botany, Jiwaji University, Gwalior 474011, Madhya Pradesh, India, ♥ email:_rekhabhadauria@yahoo.com 2 Faculty of Agriculture, Abhilashi University, Mandi 175045, Himachal Pradesh, India Manuscript received: 8 January 2015 Revision accepted: 31 January 2015.

Abstract. Avasthi S, GautamAK, Bhadauria R. 2015. Occurrence of leaf spot diseases on Aloe vera (L.) Burm.f. caused by Curvularia species from Madhya Pradesh, India. Biodiversitas 16: 79-83. During 2010-2011, occurrence of leaf spot diseases was observed on Aloe vera plants grown in various nurseries of Gwalior, Madhya Pradesh, India. The typical disease symptoms were observed on the abaxial surface, tips and spiny margins of leaves. Disease spots were sunken, dry, necrotic, dark maroon to dark brown in color. On the basis of morphological and microscopic characteristics of the fungus, two species of Curvularia i.e. Curvularia lunata and Curvularia ovoidea, were found to be associated with the leaf spot diseases. Koch’s postulate was applied to confirm the causal organisms of the diseases.

According to the literature, this is the first report of leaf spot disease on A. vera caused by Curvularia lunata from Madhya Pradesh, and the first report of Curvularia ovoidea from India.

Key words: Aloe vera, Curvularia lunata, Curvularia ovoidea, leaf spot, India

INTRODUCTION Aloe vera (L.) Burm.f. belongs to family Aloeaceae, is a perennial, succulent, monocotyledonous plant with an average height of 60-100 cm. There are about 400 species of Aloe, but the most commercially cultivated species is A. vera. It is a native of warm tropical regions, especially North Africa, the Mediterranean region of southern Europe and the Canary Islands. This species was first described by Carl Linnaeus in 1753. Aloe vera belongs to a large group of plants known as Xeroids because of its ability to close its stomata completely to avoid loss of water and help in retaining a large amount of water in its tissues (Akinyele and Odiyi 2007). It is commonly referred as a “miracle plant” and has a long history of economic and medicinal uses that spans thousands of years (Daodu 2000). More than 200 chemical components have been identified from the leaf pulp and exudates of A. vera (Reynolds and Dweck 1999; Choi and Chang 2003; Ni et al. 2004). Its gel is widely used for the treatment of sores, wounds, skin cancer, cold, constipation, asthma, ulcer, diabetes and fungal infection (Joseph and Justin 2010). The leaf pulp exhibited inhibition of AIDS virus by accemannan and inhibition of the prostaglandin synthesis by anthraquinonetype compound (Hassannuzzaman et al. 2008). In the cosmetic industries A. vera is used in the production of soap, shampoo, toothpaste and body lotions (Daodu 2000).

Despite its therapeutic and antimicrobial potential, A. vera is susceptible to various fungal diseases. Leaf spot is an important disease that not only affect the leaf texture but also reduce the quality and quantity of mucilaginous gel used for medicinal and commercial purposes. Therefore, it was the objective of this study to identify the causal

agent of leaf spot symptoms on A. vera.

MATERIALS AND METHODS Sample collection and study of symptoms Various nurseries in Gwalior, India were surveyed for the presence of leaf spot diseases on A. vera during the rainy and winter seasons of 2010 and 2011. Ten infected leaves were collected randomly from each nursery, placed into labeled zip lock bags, and brought into the laboratory.

The morphology of the symptoms was studied with the help of hand lens and dissecting microscope.

Isolation and purification of pathogen from diseased leaves Diseased leaves of A. vera were taken into the laboratory and washed thoroughly with running tap water to remove the surface dirt. The leaves were cut into small pieces using sterile scalpel blades. These small pieces were then surface sterilized with 2% sodium hypochlorite solution (NaOCl) for 2 mins and then washed three-four times in sterile distilled water. These surface sterilized pieces were then placed between blotting papers and aseptically inoculated onto petridishes containing Potato Dextrose Agar media. The plates were incubated at 25±2 °C for 5 to 6 days, and the growth of fungal colonies were recorded every day.

Characterization and identification of the pathogens The isolated fungal species were identified on the basis of morphological and cultural characteristics as described

by Ellis (1971) and Gilman (2012). Identification of

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pathogens was further confirmed at the Indian Type Culture Collection (ITCC), IARI, New Delhi and the National Fungal Culture Collection of India (NFCCI), Agharkar Research Institute, Pune, Maharashtra, India.

Pathogenicity test of the pathogens Pathogenicity test was carried out in vitro on the healthy leaves of A. vera, according to the method described by Kamalakannan et al. (2008). The isolated fungal species were cultured on Potato Dextrose Agar (PDA) medium at 25±2°C for 8-10 days in an incubator.

Conidial concentration was subsequently adjusted to 1×109 per ml by using hemocytometer to make a spore suspension. Healthy leaves were surface sterilized for 1 min with 2% sodium hypochlorite solution (NaOCl).

Artificial pricks approximately 2 mm deep on the abaxial surface of leaves were made by sterilized needle. Spore suspension of the test organisms was delivered through a sprayer and lined with moist blotting paper. Leaves sprayed with sterile distilled water served as control. Leaves were incubated at 25±2 °C for 8-10 days.

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Identification of fungal pathogens The fungal colony isolated from diseased tissue was subfloccose, dark olive-gray in color and on the reverse side was greyish black on PDA. Hyphae septate, branched, 3.0-3.6 µm in diameter. Conidiophores were erect, long and unbranched. Conidia borne more or less in whorl at the tip of conidiophores, curved and brown in color. Generally conidia have four transverse septa and 18-29 × 10-8 µm in size. The central cell is typically darker and larger as compared to the end cells (Figure 1C & D). Based on morphological and cultural characteristics the fungus was identified as C. lunata (Walker) Boedijin (# ITCC 8185.11).

The growth of another fungal colony on PDA was initially light grey and circular, becoming velvety, pale brown to dark brown in color. The reverse side of the PDA turned into black color. Conidiophores were pale brown, straight, cylindrical and multiseptate. Conidia were ovoid, three septate, straight or curved, 16-29 × 10-17 µm, brown with paler end cells (Figure 2C-D). Based on morphological and cultural characteristics the fungus was identified as C. ovoidea (Hiroe & N. Watan.) Munt.-Cvetk. (# NFCCI-3053).

RESULTS AND DISCUSSION Results of the survey revealed that all the nurseries cultivated only a single species of Aloe, i.e. A. vera and none of the nursery was found free from leaf spot diseases.

Microscopic and cultural analysis of the isolated fungi indicates the association of two Curvularia species, Curvularia lunata and Curvularia ovoidea. Isolation result indicates that C. lunata and C. ovoidea were isolated from all the infected leaf samples. Minor differences were observed in disease symptoms caused by both fungi. The disease symptoms and microscopic characteristics of the pathogens are described as follows.

Symptoms of leaf spot diseases Leaf spot disease caused by C. lunata began in the form of circular, water soaked lesions on the tip and abaxial surface of the leaves. Gradually the size of the lesions expands and became light red in color bordered by water soaked tissues. As the disease progressed, the lesions were sunken, maroon color with an average size of 0.5-1.4 × 0.41.0 cm. In later stages, lesions became dry, necrotic and turned into dark maroon in color (Figure 1A & B). The disease was observed during both the rainy and winter seasons.

Symptoms of leaf spot disease caused by C. ovoidea appeared as elongated, water soaked lesions, generally occurred on the spiny margins and abaxial surface of the leaves. Progressively, these lesions became sunken and light brown in color. Later, these lesions were enlarged, centre of the lesions dark brown in color with maroon red margins, and measured about 0.7-1.7 × 0.6-1.3 cm in size.

In severe infection, the spiny margin of the leaves was twisted inside due to necrosis of the tissues (Figure 2A & B). Interestingly, the disease was observed only in the winter season.

Pathogenicity test Curvularia lunata and C. ovoidea were pathogenic under in vitro conditions. The symptoms of leaf spot diseases recorded during the pathogenicity test were almost similar to the natural symptoms. Symptoms of leaf spot infection appeared on fourth day of infestation. Initially, round, water soaked lesions were appeared on the abaxial surface of leaves. As the infection progressed, spots became sunken and reddish maroon in color. On the thirteenth day, the lesions become necrotic and turned into dark brown in color. The fungi were re-isolated from the infected leaves and were compared with the original culture of C. lunata and C. ovoidea.

Discussion Curvularia is a filamentous fungus reported to cause various diseases on different plant hosts. Leaf spot disease caused by C. lunata and C. ovoidea affects the quality and quantity of A. vera leaf gel. Jat et al. (2013) reported severe form of leaf spot disease on A. vera caused by C. lunata and its management from Jaipur. Curvularia lunata has been found to cause leaf spot disease on Amaranthus spinosus (Sharma et al. 2011), Clerodendrum indicum (Mukherjee et al. 2013), Grewia optiva (Gautam et al.

2011), Hordeum vulgare (Kumar and Singh 2002), Nelumbo nucifera (Cui and Sun 2012) and Zea mays (Akinbode 2010). Similarly, C. ovoidea has been reported as a leaf spot pathogen on Capsicum annuum (Singh and Tondon 1970) and Psophocarpus tetragonolobus (Awurum and Emechebe 2001). Other pathogenic fungi have also been reported to cause leaf spot diseases on A. vera, such as Alternaria alternata (Kamalakannan et al. 2008), Colletotrichum gloeosporioides (Avasthi et al. 2011), Nigrospora oryzae (Zhai et al. 2013) and Phoma betae (Avasthi et al. 2013). Although, C. lunata has already been reported from Jaipur, India, there are no reports available

AVASTHI et al. – Leaf spot diseases on Aloe vera caused by Curvularia species

for C. ovoidea. To the best of our knowledge, this is the first report of leaf spot disease on A. vera caused by C.

A

C

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lunata from Madhya Pradesh, while by C. ovoidea from India.

B

D

Figure 1. Leaf spot disease on A. vera caused by Curvularia lunata. A. Initiation of disease symptoms, B. Mature symptoms, C. Ten days old colony of C. lunata incubated at 25±2 °C on PDA, D. Conidia of C. lunata

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A

B

C

D

Figure 2. Leaf spot disease on A. vera caused by Curvularia ovoidea. A. Initiation of disease symptoms, B. Mature symptoms, C. Ten days old colony of C. ovoidea incubated at 25±2 °C on PDA, D. Conidia of C. ovoidea

ACKNOWLEDGMENTS

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The authors are thankful to Head, School of Studies in Botany, Jiwaji University Gwalior, Madhya Pradesh, India for providing essential laboratory facilities and support to conduct this research work successfully.

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ISSN: 1412-033X E-ISSN: 2085-4722 DOI: 10.13057/biodiv/d160111

The interaction between diversity of herbaceous species and history of planting, Masal’s plantations, Guilan Province, Iran ♥

HASSAN POURBABAEI1, , MASOUMEH ESKANDARI1, MEHRDAD GHODSKHAH DARYAEI1, MEHDI HEYDARI2 1

Department of Forestry, Faculty of Natural Resources, University of Guilan, Somehsara, Guilan, Iran. P.O. Box 1144, Tel.: +98-182-3220895, Fax.: +98-182-3223600, ♥email: H_pourbabaei@guilan.ac.ir 2 Faculty of Agriculture and Natural Resources, Ilam University, Ilam, Iran.

Manuscript received: 6 November 2014. Revision accepted: 2 February 2014.

Abstract. Pourbabaei H, Eskandari M, Daryaei MG, Heydari M. 2015. The interaction between diversity of herbaceous species and history of planting, Masal’s forests, Guilan Province, Iran. Biodiversitas 16: 84-88. The purpose of this study was to determine the relationship between the plantation age and diversity of herbaceous species. The study area is located in the Masal’s plantations of Guilan in north of Iran. For this purpose, 60 plots were sampled by a randomized-systematic method throughout the study area in order to record the herbaceous species, the Whitaker′s nested plot was applied and the minimal area was obtained 64 m2 (8 × 8 m). In order to evaluate the plant diversity, some indices including Shannon-Wiener’s diversity (H′), species richness (S) and Smith-Wilson’s evenness (Evar) were utilized. Results indicated that 31 herbaceous species were found in two plantations. There were significant differences between two sites regarding H′ and Evar. Whereas, there was no significant difference in view of species richness between two sites. In addition, results revealed that older plantation has not caused to increase richness and evenness of species.

Keywords: Age, diversity, herbaceous species, plantation, Masal’s plantations.

INTRODUCTION There is a very close relation between plants and other living organisms in an ecosystem. Observing appearance of land’s plants shows that these species choose their habitat according to their intrinsic ecological needs (Heydari and Mahdavi 2009). Plants with more complicated structure than climate and soil become stable in lands and waters and have a major role on life of living things, preserving nature and balance of ecosystem as a resources of local system.

Therefore, vegetation is always as an integral part of ecosystem. Although forests have covered only 30% of the land, but current process of destruction and subversion of forest is worried (Mosaddegh 1994). On the other hand, regarding increased population and daily increasing requirements of wood products and other services of forest, developing forests through afforestation is inevitable in present and future (Shabanian et al. 2009). In other words, destruction and declining natural forests have made it is necessary to afforest in order to reconstruct and develop forests. Plantation forests, or planted forests, are cultivated forest ecosystems established by planting or seeding in the process of afforestation and reforestation, primarily not only for wood biomass production but also for soil and water conservation or wind protection (Carnus et al. 2006).

Presently, the objectives of plantations not only for the production of lumber and wood products but also include the provision a habitat for wildlife and non-commercial plants and the conservation of biological diversity (Nagaike et al. 2003). Plant species diversity in plantations has a prominent role in order to preserve genetic resources, assessing ecological succession and identifying species

which are at risk (Brockerhoff et al. 2003). Plantations can promote regeneration of understorey by shading out grasses, increasing soil nutrients, improving micro-climate, and generally increasing the chance for seed germination and establishment, which is difficult in highly degraded sites (Senbeta and Teketay 2001).

Plantations can make an important contribution to the conservation of native biodiversity, but not if their establishment involves the replacement of native natural or semi-natural ecosystems. While a plantation stand will usually support fewer native species than a native forest at the same site, plantations are increasingly replacing other human-modified ecosystems (e.g., degraded pasture) and will almost always support a greater diversity of native species. In addition, plantations can play an important role in sustaining native biodiversity in production landscapes and indeed be an opportunity for biodiversity (Brockerhoff et al. 2008). Stand age is one of the most important factors that affects on plant species diversity (Nagaike et al. 2010, 2003; Li et al. 2014). In areas where primary or old-growth forests have already disappeared or diminished considerably, long rotation plantations are thought to mitigate the detrimental effects on biological diversity (Ratcliffe and Peterken 1995). With increasing rotation age in plantations allows to restore old-growth habitats, which can be cause structural and biological diversity (Ferris et al.

2000; Humphrey et al. 2000; Peterson and McCune 2001).

The plantations also have an important role in ecological reconstruction and create various environmental services.

Therefore, a comprehensive study about plantation effect on all part of ecosystems (living and non-living) is necessary. Today, survey in biodiversity is crucial in

POURBABAEI et al. – The interaction between species diversity and history of planting

terrestrial ecosystems due to extinction of plant species and reduction of their population (Pourbabaei et al. 2004).

The purpose of the study was to determine floristic list and to compare herbaceous species diversity in two different age plantations with the same species.

MATERIALS AND METHODS Study area The study area is located in west of Guilan Province and in Talesh mountain range, Iran (Figure 1). The mean annual precipitation and temperature are 989.7 mm and 16oC, respectively. The climate is very humid with cool winters based on Emberger method. Soil of the region is brown calcic along with brown calcimorphic soil (FRWO 2008). General altitude of the region is 150-200 m asl. and the average slop is from 20 to 25 percent.

Two plantations areas (i.e., Ahaklan and Tabarsa) with an area 100 ha and located in Masal City, 55 kilometers from the center of Guilan Province were selected. The two sites have the same conditions in terms of physiographic factors and dominated species were as follows: Alnus subcordata C.A. Mey, Acer insigne Boiss, Fraxinus excelsior L., Quercus castaneifolia C.A. Mey, Populus deltoids Marsh., Robinia pseudoacacia L. The Ahaklan and Tabarsa sites had 9 and 22 years old, respectively.

Figure 1. Study areas location in Masal City, Guilan Province, Iran.

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Data collection The study design was a random-systematic method with grid dimension of 150 m × 200 m and 60 sample plots were taken from the two sites. The size of the sample plot was obtained 68 m2 using Whitaker′s nested plots and species/area curve. The factors such as percentage of canopy cover, herbaceous species and light, litter depth were recorded in each sample plot. The herbaceous species were identified in herbarium of Faculty of Natural Resources, Guilan University, Iran.

Data analysis To measure diversity, Shannon-Wiener index (Shannon and Weaver 1949) was used, which is computed as following equation: s

H'  Pi ln pi i1

Pi is relative frequency of ith species.

To measure richness Margalef’s index (Clifford and Stephenson 1975) was used, which is computed as following equation:

R1  S  1 ln N S is total number of species and N is total population in sample plot.

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To measure evenness, Smith-Wilson’s index (Smith and Wilson 1996) was used, which is computed as following equation:

E var 

2 s   arcton    log e ( ni )   log e ( nj ) / s ) 2 j 1  i 1 s

/ S

  

Where, ni is population of species i in sample plot, nj is population of species j in sample and S is numbers of species in all samples. Mentioned indices computed by Ecological Methodology software for Windows version 6.0 (Krebs 1989).

To compare structural characteristics and averages of diversity and evenness in the two sites, student t-test was used. Before doing the test, Kolmogorov-Smirnov test was used to be certain about the normality of data. These statistical analyses were done using SPSS 16.0.

16 (1): 84-88, April 2015

RESULTS AND DISCUSSION Our findings revealed that there are significant differences in percentage of canopy cover and herbaceous species (P<0.05), so that percentage of canopy cover, and light were higher in 22 years than in 9 years old plantation.

However, percentage of herbaceous species was more in 9 years old than in 22 years old plantation.

In 9 years old plantation, species which had percentage of cover more than 10% are as follow: Rubus hyrcanus 20.38, Asplenium adiantum-nigrum (14.72), Carex sylvatica (13.83), Microstegium vimineum (11.75), Potentilla reptans (11.54), Sambucus ebulus (11 ) and Calystegia sepium (10). In addition, in 22 years old plantation species which had percentage of cover more than 10% are as follow: Oplismenus undulatifolius (16.58), Carex sylvatica (15), Viola alba (13.09), Sambucus ebulus (12.5), Microstegium vimineum (11.67). Totally, there were 31 species belonging to 26 families in the two plantations.

The 21 species were common in the both sites, 4 species were only in Ahaklan (9 years old) and 6 species were found only in Tabarsa (12 years old) as well as 4 species had 1% or less coverage the in both sites (Table 1).

Table 1. List of species [(-) herbaceous absence; (+): presence] Family name

Scientific name

Aspleniaceae Asplenium adiantum-nigrum L. Euphorbiaceae Acalypha australis L. Convolvulaceae Calystegia sepium L. Cyperaceae Carex divulsa L. Cyperaceae Carex sylvatica L. Asteraceae Conyza bonariensis L. Umbelliferae Eryngium caucasicum Trautv.

Geraniceae Geranium robertianum L. Asteraceae Helianthus tuberosus L. Hypericaceae Hypericum androsaemum L. Hypericaceae Hypericum perforatum L. Asteraceae Inula caspica Bium.

Lamiaceae Lamium album L. Lamiaceae Mentha aquatica L. Poaceae Microstegium vimineum Trin.

Poaceae Oplismenus undulatifolius (Ard.) P.Beauv.

Oxalidaceae Oxalis acetosella Boiss.

Plantaginaceae Plantago major L. Aspleniaceae Phyllitis scolopendrium (L.) Scop.

Polygonaceae Polygonum hydropiper L. Rosaceae Potentilla reptans L. Asclepiadaceae Periploca graeca L. Pteridiaceae Pteris cretica L. Rosaceae Rubus hyrcanus Juz. Caprifoliaceae Sambucus ebulus L. Lamiaceae Satureja hortensis L. Asparaginaceae Smilax excelsa L. Solanaceae Solanum nigrum L. Caryophyllaceae Stellaria media (L.) Cyr. Asteraceae Taraxacum syriacum Boiss.

Violaceae Viola alba L. Note: *F.: Frequency, **P.C.: Percent Coverage.

F.* 22 22 17 4 26 26 7 13 3 2 3 21 21 19 12 2 3 6 9 19 5 6 2 17 3 21

F. 9 18 4 1 30 30 8 10 8 2 21 1 17 20 17 8 6 13 2 18 13 10 2 24 1 2 23

P.C.** 22 9.83 9.5 2.75 4.7 15 1.86 4.31 1 0.25 3.5 11.67 11.67 16.58 3.89 0.75 5 9.25 6.11 9.76 0.5 12.5 7.62 6.94 6.67 13

P.C. 9 14.72 7.5 10 4.6 13.83 2.84 5.5 6.87 1 4.69 5 5.29 11.75 9.7 4 6.67 11.54 1 7.51 20.38 11 3 5.83 1 3 7.83

Plantation age (year) 22 9 + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +

POURBABAEI et al. – The interaction between species diversity and history of planting

Table 2. Diversity indices in herbaceous layer Plantation age 9 3.01 0.66 2.09

Indices H´ EVar R1

Sig.

22 2.63 0.51 1.88

0.03 0.04 0.20

Table 3. Relationships between the herbaceous diversity indices and environmental factors in 9 and 22 years old stands Age 9

Index R1 H´ EVar

22

R1 H´ EVar

Pearson r P r P r P

Litter 0.10 0.57 0.18 0.33 0.17 0.35

Light -0.10 0.58 0.01 0.93 0.10 0.59

Canopy 0.01 0.95 -0.12 0.52 -0.17 0.34

r P r P r P

-0.10 0.56 -0.15 0.41 -0.15 0.40

0.37 0.04 0.42 0.03 -0.17 0.36

-0.4 0.03 -0.39 0.02 -0.07 0.68

Plant diversity Regarding to diversity and evenness, there were significant differences between the two sites (P<0.05), but there was not any significant difference in view of richness (P>0.05). Average cover herbaceous was higher in species such as Plantago major and Microstegium vimineum in 9 years of plantation, but Oplismenus undulatifolius had the most average cover in 22 years of plantation (Table 2).

Relationships between the herbaceous diversity indices and canopy cover, light and litter depth The result showed that there were significant positive correlations between Shannon-Wiener and richness indices with light in 22 years stand. In other hand, ShannonWiener and richness indices had negative correlation with canopy cover. There were not any correlations between diversity indices with canopy cover, light and litter depth in 9 years stand. Also, there was not any significant correlation between the herbaceous diversity indices and litter in the both stands (Table 3).

Discussion Our findings in the studied sites showed that percentage of herbaceous species in 9 years old plantation was significantly more than 22 years old. This is probably due to opening canopy of 9 years old site. On the other hand, opening canopy cover creates situation to enter more light in the environment so that it is a suitable environment for herbaceous species. Rees and Juday (2002) stated that richness of plant species in forests is affected by open or closed canopy of overstorey. Also, there was significant difference in diversity and evenness between two sites and these values were more in 9 years old plantation. However, there is not significant difference between two plantations

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in view of richness. In spite of, richness mean was a few more in Tabarsa site. Nagaike et al. (2003) in three different ages of a plantation concluded that plantation with higher age had less diversity and richness than younger plantation and opening canopy, obtaining much light and pruning weeds are reasons for high diversity in younger plantation. The results of Nagaike’s studies indicated that the relationship between stand age and plant species diversity and richness of forest floor plants have been varied in plantations. Several studies have shown a positive correlation between plant species diversity and plantation age (Kirby 1988; Kiyono 1990), whereas Hill and Jones (1978) and Sykes et al. (1989) demonstrated that the richness of herbaceous species in Picea sitchensis, P. abies, and Tsuga heterophylla plantations decreased with increasing stand age. Peterken and Game (1984) found that the richness of herbaceous species was not correlated with plantations age that were planted between 1600 and 1947 in England (Nagaike et al. 2003).

The result showed that there was significant positive correlation between Shannon-Wiener and richness indices with light in 22 years stand. On other hand, ShannonWiener and richness indices had negative correlation with canopy cover. There were not any correlations between diversity indices with canopy, light and litter depth in 9 years old stand. Canopy cover values are closely related to understorey light levels (Machado and Reich 1999) and they are effective on net primary productivity and nitrogen cycling rates (Reich et al. 2001). Higher light availability in younger plantation is favorable for the growth of heliophytes; lower light availability in older plantation is more favorable for neutrophilous or shade-tolerant plants (Christian et al. 1994).

It seems light availability in 22-year-old stand was more favorable for plant species diversity. This result can be justified because the light in young plantation is intense and direct. Light is commonly considered to be the major limiting factor of forest vegetation cover and (or) richness (Barbier et al. 2008).

Higher opening canopy cover provides condition for a higher abundance of Rubus hyrcanus in 9 years old site where had significant difference with 22 years old site in view of percentage of canopy cover of Rubus hyrcanus. If the salvage cutting does not apply until canopy is closed, tree regeneration is disturbed and shadows destroyed it.

Also, Asplenium adiantum is found wherever light is very much so it creates a competitive condition for other species and it is more successful in this competition. In addition, Calystegia sepium and Potentilla reptans were more in 9 years old site which is due to high light requirement these species. There were six species (Eryngium caucasicum, Geranium robertianum, Helianthus tuberosus, Periploca graeca, Solanum nigrum and Taraxacum syriacum) which they only found in nine years old plantation. On the other hand, Oplismenus undulatifolius had a high percentage in 22 years old plantation and this species is indicating high compacted soil. This is due to presence of wild and domestic animals of adjacent regions come to this area of plantation and cause a compacted soil. In addition, the other reason for less percentage of vegetation in 22 years

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old plantation than 9 years old site is closed canopy cover and shortage of light and higher regeneration of Buxus hyrcana species in this site. Since Buxus hyrcana is a shade tolerant species, consequently it prevents to establish shade intolerant herbaceous species and it is decreased percentage of vegetation. Also, Pourbabaei et al. (2004), stated the same reason for low percentage cover of some species in the Tanian region in Somesara city. Veinotte et al. (1998) and Humphrey et al. (2000), found that plant diversity in plantation increases rapidly in early ages of plantation due to intensity of light and sometimes invasive species become dominant species. When the age of plantation is increased and the light intensity decreased, as a result it causes to decrease herbaceous diversity. Kuksina and Ulanova (2000) stated that when canopy cover is closed in plantations, plant species diversity declines.

Kryshen (2000) found about 90% vascular species in a Aspen plantation during 1 to 2 first years, but 3 to 4 years after planting when canopy cover was closed, presence of vascular plant declined to 58% and annual plants declined to 40%. In general, plantations in a long-term help to maintaining ecological processes in ecosystems. Also, different managements have major effects on diversity of plant species in forest ecosystems (Nagaike et al. 2003).

Regarding to the aim of plantation whether is for restoration or economic exploitation; we should achieve our purposes using different managements in different plantations. Regarding that this research has conducted in two sites with different plantation ages, it is recommended that such studies which conducted in consecutive years with pure and mixed species being compared and surveyed.

ACKNOWLEDGEMENTS We hereby offer our profound thanks and appreciation to all dears who were cooperated us in different stages of this research.

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B I O D I V E R S IT A S Volume 16, Number 1, April 2015 Pages: 89-94

ISSN: 1412-033X E-ISSN: 2085-4722 DOI: 10.13057/biodiv/d160112

Feeding habit and length weight relationship of keureling fish, Tor tambra Valenciennes, 1842 (Cyprinidae) from the western region of Aceh Province, Indonesia ZAINAL A. MUCHLISIN1,â&#x2122;Ľ, AGUNG S. BATUBARA1, MOHD N. SITI-AZIZAH2, MUHAMMAD ADLIM3, AFRIZAL HENDRI4, NUR FADLI1, ABDULLAH A. MUHAMMADAR1, SUGIANTO SUGIANTO4 1

Department of Aquaculture, Faculty of Fishery and Marine Sciences, Syiah Kuala University, Banda Aceh 23111, Tel. +62-651-7411323, Fax. +62-6517552370 Indonesia. â&#x2122;Ľemail: muchlisinza@unsyiah.ac.id 2 School of Biological Sciences, Universiti Sains Malaysia, 11800 Penang, Malaysia.

3 Department of Chemistry, Faculty of Education and Teacher Training, Syiah Kuala University, Banda Aceh 23111, Indonesia.

4 Faculty of Fisheries and Marine Sciences, Teuku Umar University, Meulaboh, Indonesia 5 Faculty of Agriculture, Syiah Kuala University, Banda Aceh 23111, Indonesia Manuscript received: 8 December 2014. Revision accepted: 5 February 2015.

Abstract. Muchlisin ZA, Batubara AS, Siti-Azizah MN, Adlim M, Hendri A, Fadli N, Muhammadar AA, Sugianto S. 20015. Feeding habit and length weight relationship of keureling fish, Tor tambra Valenciennes, 1842 (Cyprinidae) from the western region of Aceh Province, Indonesia. Biodiversitas 16: 89-94. The objective of the present study was to describe the aspects of feeding habit and lengthweight relationship of keureling fish Tor tambra, this information is crucial to plan a conservation startegy for this species. A series of samples were taken between June and September 2012 and February 2014 in the two main rivers of western Aceh i.e. the Sikundo and Nagan Rivers. A total of 48 and 38 fish were caught during the study in Nagan and Sikundo Rivers, respectively. Stomach content analysis suggested that freshwater green algae and earthworms were the main food items for T. tambra, indicating an omnivorous feeding habit. In addition, the length-weight relationship revealed that T. tambra has an allometric negative growth pattern from all populations, and the condition factors indicate the rivers are still in good condition and support fish life.

Key words: Allometric, LWS, green algae, threatened, proponderance

INTRODUCTION The Aceh Province Indonesia has many aquatic resources including coastal waters, marshes, rivers and lakes and rain forests in the Leuser and Ulu Masen ecosystems (Muchlisin et al. 2012). According to Muchlisin and Siti-Azizah (2009) there are at least 114 freshwater fishes in Aceh waters Indonesia, of these 14 species have high economical value. In addition, a total of 73 species were recorded in the Tripa peat swamp forest in the western coast of Aceh Province where 46 species are categorized as fish consumption and 17 have potential for aquaculture, for example Tor tambra, Anabas testudineus, Anguilla bicolor, Channa spp., Clarias spp., Anematichthys repasson, Cyclocheilichthys spp., Osteochilus spp., Oxyeletris sp., and Barbonymus sp. (Muchlisin et al. 2015). In particular, the Genus Tor or locally know as keureling, it is belonging to the family Cyprinidae, forms the basis of a wild fishery and has high potency for aquaculture industry (Muchlisin 2013). This genus is an important group of freshwater cyprinids in Indonesia and Malaysia which inhabits fast flowing stream throughout the transHimalayan and South-east Asian regions (Ambak et al.

2007). There are 20 species of Tor in Asia (Kiat 2004) and at least four (T. soro, T. tambra, T. douronensis dan T. tambroides) are found Indonesian (Haryono 2006), three of these were recorded in Aceh; T. soro, T. tambroides and T.

tambra (Muchlisin et al. 2009) and presumed one other species of T. douronensis was also occurred in Aceh waters (Muchlisin et al. 2014). Of these four species, T. tambra is is the most abundant and the most highly target fish due to its good taste and higher price of upto USD35-kg.

The keureling is one of the largest freshwater fishes in Aceh, reaching upto 30-45 kg. Therefore, this species has always been the predominant target of local fishermen using various fishing gears, including destructive fishing gears, such as electric fishing, poison and dynamite. This has resulted in declining wild population over the last ten years (Personal communication with local fishermen).

According to local fishermen, it is very difficult to catch large wild mahseer any more. Presently, T. tambra is currently listed as endangered in the IUCN red list (IUCN 1990). According to Kottelat et al. (1993) and Singh (2007) the Genus Tor is threatened by over-explotation, pollution and ecological perturbation. This is supported by Decamps (2011) who stated that most large freshwater fish species are threatened by overfishing and habitat degradation on a global scale.

Fishing keureling in the wild should be reduced and possibly be prohibited in the future to protect this species; hence the fishermen have to shift their business to aquaculture. For this purpose, breeding and feeding technologies need to be developed. Therefore, information on biological aspects such as feeding, reproduction and

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growth are crucially important to support these programs.

Information of feeding habit of fish in their natural habitat is crucial in order to support the domestication process and to develop feeding practices and breeding technologies to support the aquaculture industry and to plan an effective conservation strategy. Indonesia is known as a mega biodiversity country second in fish species richness only to Brazil (Muchlisin and Siti-Azizah, 2009); however, there is very limited information on the biology and ecology of fishes in particular threatened species from Aceh waters for example within genus Tor. To date only two threatened species of Rasbora tawarensis and Poropuntius tawarensis from this region have been studied comprehensively (Muchlisin et al. 2010a; 2010b; 2011b). Hence, the objective of the present study was to desribe important aspects of the biology of T. tambra in particular, diet and growth pattern from western region of Aceh Province, Indonesia.

MATERIALS AND METHODS Study site and sampling The study was conducted for four months between June and September 2012 plus February 2014 at two different locations in western region of Aceh Province, Sumatra,

Indonesia. The first location was Nagan River, Nagan Raya District at seven sampling sites: (1) 040.14’.34,9”N, 960.29’.36,7”E; (2) 040.14’.39,6” N, 960.30’.44,1” E; (3) 040.14’.40,20”N, 960.30’.24,20”E; (4) 040.14’.44,3”N, 960.31’.00,1” E; (5) 040.16’. 50,7” N, 960.26’. 0”E; (6) 040.16’.34,2” N, 960.27’.13,7”E; (7) 040.16’.19,4”N, 960.27’.42,6” E, and the second location was Sikundo River, Aceh Barat Distric at two sampling sites: (8) 040.

28’.36,5”N, 960.21’.52,1”E; (9) 040.13’.29,6”N, 0 ’ 96 .10 .24,4”E. Site selection was based on information by local residents. Gillnets (mesh size of 0.75, 1, 2 and 3 inchnormally metric), hooks and casting nets (mesh size of 1, 2 and 3 inch) were used to collect the fish samples.

The collected fishes were counted, rinsed and anesthetized in a solution of tricaine methanesulfonate (MS 222), prepared by dissolving 4 g of MS 222 in 5L tap water, then preserved in 10% formalin in a plastic bag with which was tagged with the location, date and habitat characteristics. The fish samples longer than 10 cm in total length were injected with absolute formalin in their body cavity prior to preservation in 10% formalin to unsure that internal organs (gonad and alementary organs) did not decay. Fishes were identified based on Kottelat et al.

(1993). Samples were transported to the laboratory for further evaluation.

8

5 67

1 32 2 4

9

Figure 1. Map of Aceh Barat and Nagan Raya Districts of Aceh Province showing sampling sites

MUCHLISIN et al. â&#x20AC;&#x201C; Tor tambra of the western Aceh

Diet analysis The specimens were abdominal dissected by using a surgical scissors, and then their stomachs were taken and weighed nearest to 0.01 g. The stomachs were dissected and the contents emptied into a petri-dish. The larger foods were isolated, counted and weighed. The food items were observed by the nake eye.

Food occurrence The occurrence of each food item was scored and then converted to a percentage by multiplying the ratio of the number of times an item occurred to the total number of stomachs analyzed by a hundred. The percentage abundance of each food item was also computed by multiplying the ratio of the number of a particular item in the stomach to the total number of items in the stomach by a hundred.

Index of proponderance Index of preponderance is a combination of volumetric and frequency of occurrence methods, and it is used to identify the most important food items eaten by fish. The index proponderance of fish was calculated based on Biswas (1993) as follow:

Where Ii= Index of preponderance, Vi = Percentage of volume of each food item, Oi = percentage of occurence of each food item, â&#x2C6;&#x2018;Vi is total percentage of occurence of food items, â&#x2C6;&#x2018;Oi is total percentage of volume for all food items.

Dietary shifts To evaluate the ontogenic shift in dietary preference, the sampled fishes were divided into three length classes i.e. 100-175 mm, 176-250 mm, and 251-325 mm. The food compositions of each class were evaluated and compared.

Lenght weight relationship and condition factor The Linear Allometric Model (LAM) was used to estimate the parameters a and b by log-transformed weight and length measurements. A correction for bias attributable to the back-transformation of mean weights from logarithmic units was applied to predict weight at length from parameters fitted to the allometric equation following De-robertis and William (2008): W = e0.56 (aLb) where W is the weight (g), L is the length (mm), a the intercept of the regression, b the regression coefficient and e is the variance of the residuals from the LAM regression.

0.56 is the correction factor of the data sets.

The relative weight (Wr) and Fulton condition coefficient (K) were used to evaluate the condition factor of T. tambra. Relative weight (Wr) was determined according to Rypel and Richter (2008) as follows: Wr = (W/Ws) x 100 where Wr is relative weight, W the weight of a particular fish and Ws the predicted standard weight for the

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same fish as calculated from a composite of length-weight regression throughout the range of the species: Ws = aLb. The Fulton condition coefficient (K) was determined according to Okgerman (2005) as follows: K = WL-3 x 100 where K is the condition factor, W is weight (g), L is length (mm), and -3 is coefficient of length to ensure that the K value tend to one.

RESULTS AND DISCUSSION Diets A total of 48 and 38 keureling fish were caught from Nagan and Sikundo Rivers, respectively, of these stomach content was examined for 45 and 35 fishes. The stomach contents of fish from Nagan containing 69.6% of earthworms, 27.8% of green algae, 2.1% of leafage and 0.5% of sands. The keureling from Sikundo has stomach contents of green algae (77.60%) and earthworms (22.4%) (Table 1). The analysis of food occurance showed that earthworms found in 80.8% and 81.3% of the fish samples from Nagan River and Sikundo, respectively. While, green algea was found in 73.1% and 93.8% of fish samples from Nagan and Sikundo, respectively (Table 2). However, the preponderance index analysis showed that green algae was the most important food item for keureling fish from both populations (Table 3).

Th results revealed that the younger fish prefer earthworms, but this shifted to green algae after the fish reached bigger size (Table 4). Change in feeding habit was associated with an increase in the length of the alimentary tract. For example, fish 100-175 mm in lenght (137.5 mm average) had an alimentary tract length of157 mm (1.2 times of alimentary tract). This increased to 377 mm with an average total length of 288 mm (1.3 times of alimentary tract).

Our results showed that the T. tambra eat both plants and aquatic animal, indicating an omnivorous diet. Further evidence in support of omnivory is the fact that their alimentary tract length was slightly longer than the body length (1.25 times of the total length). According to Smith (1989),omnivorous fish have a digestive tract 1.5 to 2 times their total body length. Haryono (2006) studied the feeding habit of T. tambroides from the Barito River, Central Kalimantan, Indonesia and the alimentary tract of this species was 1.5 times of the total body length. Green algae and earthworms were the foremost food items for mahseer in Sikundo and Nagan Rivers, Western Aceh, Indonesia.

According to local fishermen, in addition to green algae mahseer also eats figs, small crabs and mollusks. In addition, the local fishermen in Sikundo River frequently use chopped cassava for catching mahseer. The diet of the keureling fish also changed with ontogeny where smaller fish fed preferentially on earthworms, and this changed to a diet of mainly green algae above 175 mm body length.

According to Kolkovski (2001) and Mai (2005) that at early life of fish, the alimentary tract is rudimentary and insufficient digestive enzyme capacity so they are unable to digest algae. However, in several marine fishes the

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alimentary tract has been present at first day hatching larvae, but not well developed (Infante and Cahu 2001; Putra et al 2012).

16 (1): 89-94, April 2015 Table 1. Volume of food items consumed by keureling fish (T. tambra) in Nagan and Sikundo Rivers, Aceh Province, Indonesia.

Food items

Length weight relationship and condition factors The total lengths ranged between 117 mm to 194 mm (mean 149.89±19.33 mm) and 120 mm to 505 mm (mean 228.32±99.66 mm) for Nagan and Sikundo population, respectively. There were no fish greater than 200 mm lengthin Nagan River. The relative weight condition factors were closed to 100, while the Fulton’s condition factor ranged from 1.75 to 2.67.

The length weight relationship showed that the tambra has b value between 2.34 to 2.84 (Table 5). Growth is considered isometric when b value is equal to 3 or allometric if otherwise (positive allometric if b>3 and negative allometric if b<3). However, generally exponent b values lie between 2.5 to 3.5 (Froese 2006). The exponent b values irrespective populations were lower than 3 (b<3).

Thus, both populations could be categorized as displaying allometric negative growth. The allometric negative growth pattern has been observed in several freshwater fish species in Southeast Asia for example; Garra cambodgiensis from Ulu Dungun, Malaysia (Mazlan et al. 2007), Rasbora sumatrana in Bukit Rengit, Malaysia (Shukor et al. 2008), R. tawarensis and Poropuntius tawarensis (Muchlisin et al.

2010b). On the other hand, Mystacoleucus marginatus (Mazlan et al. 2007) displayed allometric positive growth pattern. In contrast, isometric growth pattern was observed in Leiognathus jonesi and L. decorus sampled from the coastal waters of Pulau Sibu-Tinggi, Malaysia (Mazlan and Seah 2006) and serandang (Channa pleurophthalmus) in Musi River, Indonesia (Said 2007).

The predicted growth was relatively similar to actual growth trend (Figure 2), indicate the fish is growing in optimum performance in both Sikudo and Nagan Rivers.

However, unfortunately, no large mahseer were caught, suggesting the species is overfished in these rivers. In general, population b values are dependent on physiological growth condition such as gonad development and food availability (Jenning and Kaiser 2001), biological and environmental conditions, geographical, temporal and sampling factors (Froese 2006). According to Shukor et al. recorded in this study where the Sikundo and Geumpang Rivers have higher speed of water flow compared to Nagan (2008) fast flowing streams result in lower b value as River resulted in lower in b values relatively. Besides the above stated factors, it is speculated that the b value is also

Green algae Earthworm Leafage Sand Total

Nagan River n=45 Total volume (%) (mL) 5.3 27.8 13.3 69.6 0.4 2.1 0.1 0.50 19.1 100

Sikundo River n=35 Volume (mL) 20.1 5.8 25.9

(%) 77.6 22.4 100

Table 2. Food items occurance in keureling fish (T. tambra) stomach from Nagan and Sikundo Rivers, Aceh Province, Indonesia.

Foo items Green algea Earthworm Leafage sand

Nagan River n= 45 Occurance % 19 73.1 21 80.8 3 11.5 1 3.9

Sikundo River n=35 Occurance % 15 93.8 13 81.3 -

Table 3. Index preponderance of keureling fish (T. tambra) fish from western region of Aceh Province (Nagan and Sikundo Rivers).

Vi

Food item Green algea Earthworm Total

mL 20.1 5.8 25.9

% 77.6 22.4 100

Food item occurance Occur. % 15 93.8 13 81.3 -

Vi × Oi

Ii (%)

7275.94 1819.19 9095.13

78 22 100

Table 4. Dietary shift of the keureling fish (T. tambra) according to the length classes Food occurance Fish size (mm) 100 - 175 176 - 250 251 - 325

Erathworm (%) 81.25 100 33.3

Green algae (%) 75 100 100

Others (%) 12.5 -

Length of alimentary tract Range (mm) 118-214 190-294 312-396

Average (mm) 157 273 377

Table 5. Lenght weight relationship and condition factors of two populations of keureling fish (T. tambra) from western region of Aceh Parameter Total lenght, TL (mm) Measured weight, W (g) Predicted weight, Ws (g) Relative weight, Wr (g) Fulton condition factor, K b value

Population Nagan River (n=48) 117.0 - 194.0 (149.9±19.3) 17.4 - 70.9 (33.4±13.2) 15.7 - 66.1 (33.2±12.4) 74.5 - 123.9 (100.6±10.9) 2.5 - 2.9 (2.7±0.1) 2.84

Sikundo River (n= 38) 120.0 - 505.0 (228.3±99.7) 28.0 - 1390.0 (201.7±340.4) 22.8 - 1000.1 (177.0±243.6) 55.9 - 139.0 (102.3±20.8) 2.7 - 3.1 (3.0±0.1) 2.63

MUCHLISIN et al. â&#x20AC;&#x201C; Tor tambra of the western Aceh

Figure 2. Comparison between observed and predicted growth for keureling fish (T. tambra) catches in Nagan and Sikunod Rivers (growth curve of predicted, y= 0.723x-71.50, R2= 0.983 and observed growth, y= 0.648x-63.86, R2=0.906)

affected by fish behaviour, for example active swimming fish (mostly pelagic fishes) have lower b value compared to passive swimming fishes (mostly demersal fishes). This is related to the energy allocation for movement and growth (Muchlisin et al. 2010b). Our results suggest that T. tambra is an active fish.

The relative weight condition factors were closed to 100 indicating the population is stabile with sufficient food and a lack of predation. The condition factor is regularly calculated to assess the overall health, productivity and physiology of fish population (Richter 2007; Blackwell et al. 2000). It reflects the fish physiological characteristic such as body morphology, lipid content and growth rate (Bister et al. 2000; Rypel and Richter 2008; Stevenson and Woods, 2006). The b value and condition factor of T. tambra from Sikundo River were relatively lower than those Nagan River (Table x). According to Anderson and Neumann (1996), values of Relative Weight (Wr condition factor) falling below 100 for an individual or population suggest problems such as low prey availability or high predatory density, whereas values above 100 indicate prey surplus or low predatory density. In addition, Porath et al.

(2003) stated that changes in body condition could be interpreted as a measure of predation success i.e. fish of greater weight body condition and thus greater utilization of prey. Besides the availability of feed and predator, biotic and abiotic factors and fisheries management also affect various condition factors (Murphy et al. 1991; Blackwell et al. 2000). The results showed that the condition factor values of T. tambras were moderate (tend to 100). This reflected that the availability of feed relative to the presence of predator is balanced in this community indicating that the water quality of the lake is still adequate to support fish communities specifically the species investigated. A similar finding was found in others freshwater fishes in Aceh waters such as R. tawarensis and P. tawarensis (Muchlisin et al. 2010b).

Generally the mahseer fish caught in Nagan and Sikundo Rivers were young and small, much smaller than

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average size reported elsewhere (Asih and Setijaningsih 2011; Sarkar et al. 2012; Haryono 2006). According to the local fishermen of Nagan Raya and Aceh Barat districts it is very difficult to find larger fish over the last decade, suggesting the keureling fish has been over exploited and is highly threatened in Nagan and Sikundo Rivers. This finding is in agreement with Haryono and Subagja (Haryono and Subagja 2008) who studied the T. tambroides in Muller Mountains, Central Kalimantan where the the population were dominated by juvenile fish.

Similarly, Ogale (2002) reported that the mahseer (Genus Tor) population in India has been declining in number and size and is in serious danger of extinction due to indiscriminate fishing of brood and juvenile fish and the adverse effect on the habitat of of the river valley development projects. Clearly, the mahseer has a number of life history traits that make if vulnerable to overfishing and accordingly much more information on the ecology and biology of these species are required to ensure the sustainability of the resource.

The stomach content analysis indicated that earthworms and freshwater green algae were the most important food for keureling T. tambra in western Aceh region, indicates an omnivorous feeding habit. In addition T. tambra had an allometric negative growth pattern, both relative weight index and Fultons condition factor showed that the rivers can still support the presence of T. tambra.

ACKNOWLEDGEMENTS The work reported in this paper was supported by a grant from the Directorate General of Higher Education (DGHE), Republic of Indonesia (Project No. 139/UN11/A.01/APBN-P2T/2012). We express appreciate to anonymous reviewers for their critical comments and suggestions. The technical assistance by all members of Aquaculture Research Group of Syiah Kuala University, Banda Aceh, Indonesia are also acknowledged.

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B I O D I V E R S IT A S Volume 16, Number 1, April 2015 Pages: 95-101

ISSN: 1412-033X E-ISSN: 2085-4722 DOI: 10.13057/biodiv/d160113

Comparison of woody species diversity between managed and unmanaged forests considering vertical structure in Hyrcanian forests, Iran ZAHRA NOURI♥, MAHMOUD ZOBEIRI, JAHANGIR FEGHHI, GHAVAMALDIN ZAHEDI AMIRI, MOHAMMAD REZA MARVIE MOHADJER Tehran University, Faculty of Natural Resources, department of Forestry and Forest economics, Shahid Chamran Blvd., Karaj, Elborz Province, Iran.

Tel.: +989111584369, Fax: +982632249312, ♥email: znouri9@gmail.com Manuscript received: 27 January 2015. Revision accepted: 13 March 2014.

Abstract. Nouri Z, Zobeiri M, Feghhi J, Amiri GZ, Marvie Mohadjer MR. 2015. Comparison of woody species diversity between managed and unmanaged forests considering vertical structure in Hyrcanian forests, Iran. Biodiversitas 16: 95-101. Biodiversity conservation is increasingly considered a key to sustainable forest management and understanding biodiversity in forest ecosystems is essential to reveal forest dynamics, so this study aims to compare tree and shrub species diversity and vertical species distribution between two managed and unmanaged forest stands. The study area was consisted of two adjacent districts in which two different management practices were implemented. In 1000 m2 circular plots, type of species and diameter at breast height (D.B.H) of all trees larger than 2.5 cm as well as type of species of all shrubs and vertical distribution of tree species were recorded. In addition to richness, evenness and heterogeneity indices were calculated for whole tree and shrub species and also for each tree layer. The results showed that tree species diversity indices were significantly greater in the unmanaged area, whereas richness and heterogeneity indices of shrubs were greater in the managed area. Comparing the biodiversity indices indicated that richness index had the greatest value in the understorey and heterogeneity indices were greatest in the middle storey in both regions. Greater amounts of evenness were detected in the middle- and over storey of unmanaged and managed areas, respectively. As forestry operations result in change in various forest characteristics such as species diversity, it is essential to assess and monitor the effect of logging operations on forest diversity Hyrcanian forests of Iran.

Keywords: Forest stories, heterogeneity indices, Iran, species richness, sustainable management.

INTRODUCTION Sustainable management of forest is one of the recent expressions of sustainability in forestry. It adopts an ecosystem approach to forest management, while operating in an interdisciplinary context (Hahn and Knoke 2010).

One of the comprehensive definitions of sustainable forest management is managing forest stands in such a way that their productivity, regeneration, vitality and biodiversity is preserved in accordance with ecological, economic and social functions in local, national and global levels, currently and in the future (Bernasconi 1996). Nowadays, it is increasingly acknowledged that biodiversity, as a main aspect of ecosystem structure and functioning as well as a key issue of sustainable forest management, needs to be considered in forest management plans in conjunction with other environmental and economic criteria (Bertomeu and Romero 2001). Biodiversity has a substantial role in forest ecosystems for the preservation of ecosystem functioning (Bengtsson et al. 2000). Indeed biologically diverse systems serve a variety of human needs; it provides us with a wealth of products and services. There is concern that the loss of biodiversity within forests is jeopardizing these services (Aerts and Honnay 2011).

It is also related to the delivery of many ecosystems services (Balvanera et al. 2006), and results in

geomorphological, hydrological, ecological stability in forest ecosystems (Noss 1999). Loss of habitat resulting from human land-use practices is the single largest factor causing the decline of biodiversity (Millennium Ecosystem Assessment 2005; Mönkkönen et al. 2014). Various ecosystem functions might be affected negatively by biodiversity loss, and long term reduction of species numbers may lead to a decrease of the ecosystem’s ability to confront with disturbances (Ishii et al. 2004). As a consequence, any sustainable forest management should be in accordance with environmental policies including biodiversity conservation.

Biodiversity of forest ecosystems encompasses many different aspects, such as species diversity, ecosystem diversity and genetic diversity. Species composition can alter ecosystem properties through functional traits and interactions (Hooper et al. 2005; Schmidt et al. 2015). At the stand level, species diversity is often associated with stand structural complexity (e.g. vertical and horizontal structure and other spatial components), as an appropriate indicator of habitat diversity (Ishii et al. 2004), and has been reported to be a major factor determining forest vegetation (Cardinale et al. 2012).

Forest structure is directly related to the habitat of many different animal and plant species (Kint 2000) and the alterations in forest structure through the intervention of

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man and natural disturbances can affect the conditions needed for the survival of many species (Guner 2002).

Moreover, natural and human disturbances influence different components of biodiversity in many communities by their direct impact on species and stand structural diversity (Huston 1994; Robert and Gilliam 1995; Lindgren and Sullivan 2001). Species diversity as well as its vertical differentiation within stands may thus be used as indicators of disturbances regime in forest ecosystems (Larsson and Danell 2001).

Over the last 300 years, the world population has increased considerably. This rapid development brings increased, widened and varied demands for timber and non-timber products (Baskent 2000). Timber extraction is one of the most important disturbances occurring in forests throughout the world, and there is an increasing debate over the global effect of forest management for timber production on biodiversity (Siitonen 2001). At the local scale, unmanaged forests in general are said to contain more species than managed forests (Okland et al. 2003), but the results of some studies failed to confirm this idea for particular taxa (Graae and Heskjaer 1997; Bobiec 1998). Hyrcanian (Caspian) forests located in the north of Iran comprise a narrow band of temperate deciduous forests and are contiguous to a larger forest block extending across eastern Turkey and Caucasia. These forests cover the north-facing slopes of the Elborz

16 (1): 95-101, April 2015

Mountain, and extend from the Northwest to the Northeast of Iran with a total area of about 1.9 million hectares. Most parts of the forests within this range are managed for timber production as these forests are the only source of wood production in Iran and the main revenue of forest management plans is the income of logging (Forest and Rangelands Organization of Iran 1997). We hypothesize that the intensive logging in Hyrcanian forests has considerably decreased the species diversity in managed stands compared to natural forest relicts. Therefore this study aims at analyzing tree and shrub species diversity and comparing vertical species distribution between two managed and unmanaged forests using different diversity indices.

MATERIALS AND METHODS Study area This research was conducted in the Kheyrud Forest Research Station of University of Tehran (KFR), an 8000 hectare forest which is a portion of Hyrcanian uneven-aged forest, located approximately 7 km east of Nowshahr (or Noshahr) (51°32ʹN, 36°27ʹE), Mazandaran Province, within the Elborz mountain range, northern Iran (Figure 1).

A

B

C

Figure 1. Study site in the Kheyrud Forest Research Station of University of Tehran (KFR) (C), east of Nowshahr (B), Mazandaran Province, northern Iran (A).

NOURI et al. – Woody species diversity considering structure

The elevation in the region ranges from 50 to 2200 m above sea level, composed of mostly broadleaved species.

The climate of the area, according to the Emberge climate system, is wet with cold winters. The mean annual temperature is 15.9ͦC, the highest mean monthly temperature of 29 ͦ C occurs in June and July and the lowest of 7.1 ͦC in February. The mean annual precipitation is 1354.5 mm, with maximum and minimum amounts in September and June, respectively. KFR is divided into 7 districts; however, only the first district, Patom, was surveyed. After field inspection, two adjacent areas characterized by the same soil type, plant community and site quality were selected. The southern area which is a protected forest has never been harvested and characterized as a reference forest for sustainable forest management, whereas in the northern area, logging is performed.

Procedures Using systematic random sampling method, 40 0.1 ha circular sampling plots were laid out in each study area.

Within each plot, D.B.H of all trees larger than 2.5 cm and the crown cover of all shrubs were recorded.

Considering that Hyrcanian forests consist of three stories (understorey, middle storey and overstorey), vertical layers were determined by visual estimation. Slope, altitude and aspect were measured for each plot using a clinometer, altimeter and compass, respectively.

Data analysis Many different indices have been proposed for the evaluation of species diversity. During their historical development, the indices have been separated into three categories: indices of (i) species richness: the oldest and the simplest understanding of species diversity expressed as a number of species in an ecosystem, (ii) species evenness: a measure of the equality in species composition in a community, and (iii) species heterogeneity: a characteristic encompassing both species richness and evenness (Ludwig and Reynolds 1988; Krebs 1989). In this study, species richness S is the number of species recorded at each sampled plot (Magurran 1988). For species heterogeneity Shannon (Shannon 1949) and Simpson (Simpson 1949) diversity indices were used, as these are successful tools for the evaluation and quantification of plant diversity (Dale et al. 1994).

Shannon’s index was calculated as: s

H    ( pi )(log 2 pi ) i 1

Where pi is the relative abundance of species i.

Simpson’s index (D), a diversity index heavily weighted towards the most abundant species in the sample while being less sensitive to species richness, was calculated as:

s

D  ( i 1

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ni (ni  1) ) N ( N  1)

Where ni is the number of individuals in the ith species and N the total number of individuals. When D increases, diversity decreases and therefore Simpson’s index is usually expressed as 1.D -1 this expression (i.e. 1.D -1) was also used in this study. Two indices of evenness, Smith and Wilson and Simpson’s (E), were used. The Simpson’s evenness index (E) is calculated as the ratio of the Simpson’s diversity index D to the maximum possible index of diversity, given S species and N individuals.

Smith and Wilson (1996) proposed an index of evenness based on the variance in abundance of the species. According to Smith and Wilson, this is the best index of evenness because it is independent of species richness and is sensitive to both rare and common species in the community, the index calculated as:

EVar

2 s   s       log n  log n / S    e i e j       2 j 1  1    arctan  i1   S            

Where, Evar= Smith and Wilson’s index of evenness ni= Number of individuals in species i in sample (i = 1, 2, ..., s) nj= Number of individuals in species j in sample (j = 1, 2, ..., s) S = Number of species in entire sample The indices were calculated for tree and shrub species separately, as well as for vertical tree layers within each plot. If the number of trees N is used, the size of the tree is neglected for calculating species diversity indices, while using the basal area, the tree size is taken into account through its diameter, or more precisely by the square of its diameter (Merganic and Smelko 2004). The sum of basal area of all individuals for each species in sample plots was used instead of species number in calculating evenness and heterogeneity indices of trees. Ecological Methodology software package was employed for calculating diversity indices. The normality of calculated indices for shrub and tree species was examined by using Kolmogorov-Smirnov test. Since some geomorphological features like elevation, aspect and slope were different between the two studied areas and might influence the diversity indices, these features were considered as covariates and an Analysis of Covariance (ANCOVA) was applied for removing their effects and then comparing indices between logged and unlogged areas and also among vertical layers. Significant differences among treatment averages for different parameters were tested at p ≤ 0.05.

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RESULTS AND DISCUSSION There were only a few shrub species and 15 tree species in this study area. Among the species, four tree species and one shrub species were only found in the unlogged area.

Carpinus betulus L., Diospyros lotus L., Parrotia persica (Dc.) C.A.M., Acer velutinum Boiss., Fagus orientalis Lipsky, Quercus castaneifolia C.A.M., Acer cappadocicum Gled., Alnus subcordata C.A.M., Ulmus glabra Huds., Cerasus avium (L). Moench, Rhamnus frangula and Tilia begonifolia Stev., Ulmus carpinifolia Borkh., Ficus carica L., and Sorbus torminalis (L.) Crantz were only recorded in the unlogged area. Tree species, stem density and basal area were common species in both areas, whereas of all tree species in both areas are presented in Table 1.

Crataegus microphylla C. Koch and Mespilus germanica L. were common shrub species in both forest sand Prunus divaricata Ledeb was only observed in the logged area (Table 2).

Comparison of indices between two areas and among tree layers using ANCOVA showed that diversity indices were not significantly influenced by geomorphological features. The p-values related to covariates in all tests were  0.05 . Since community, climatic and edaphic conditions were similar in two regions and geomorphological characteristics did not show significant influence on diversity indices, it seems the difference between diversity indices of two areas were the output of management practices.

Tree species diversity indices Species richness The comparison between both study areas showed that species richness of trees was significantly higher in unlogged area (Table 3). Species richness values calculated for vertical layers were also greater in unlogged area, but the difference was only significant in the middle- and understorey (Table 3). Species richness indicated significant difference among layers, and the highest species richness was found in the understorey in both areas (p < 0.05).

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Evenness indices Both evenness indices showed significant differences between the areas, with higher values for unlogged area.

Evenness indices calculated for the overstorey were higher in unlogged area (Table 4), but these differences were not significant. In the middle- and the understorey, evenness indices were significantly higher in the logged area (Table 4). Highest values in the logged region were observed in the middle storey (p < 0.05), but in unlogged area evenness values were the highest in the overstorey (p < 0.05).

Table 2. Stem density of observed shrub species in the managed and unmanaged areas.

Shrub species

Crataegus monophylla Mespilus germanica Prunus divaricata

Stem density (per ha) Managed Unmanaged area area 145 8 57 6 3 -

Table 3. Mean values ± standard error of richness index and result of statistical tests in both study areas Logged area Total tree species Overstorey Middle storey Understorey

3.923 ± 1.156 0.911 ± 2.100 0.811 ± 2.020 0.811 ± 2.600

Unlogged area 1.156 ± 0.925 1.196 ±2.575 1.500±3.575 1.500 ± 3.950

p-value 0.000 0.077 0.000 0.000

Table 6. Mean values of diversity indices of shrubs Diversity indices Richness Evenness Heterogeneity

S Simpson Smith and Wilson Simpson Shannon

Managed area 2.143 0.771 0.689 0.408 0.911

Unmanaged area 1.307 0.799 0.708 0.332 0.705

Table 1. Stem density and basal area of observed tree species in the managed and unmanaged areas.

Tree species Carpinus betulus Diospyros lotus Parrotia persica Acer velutinum Fagus orientalis Quercus castaneifolia Acer cappadocicum Alnus subcordata Ulmus glabra Rhamnus frangula Tilia begonifolia Prunus avium Ulmus carpinifolia Ficus carica Sorbus torminalis

Managed area Stem density (per ha) Basal area (m2.ha -1) 167 26.23 51 0.50 42 2.30 14 2.04 8 0.94 4 1.86 4 0.45 3 0.28 3 <0.01 1 <0.01 1 <0.01 -

Unmanaged area Stem density (per ha) Basal area (m2.ha -1) 153 11.29 51 0.72 490 11.30 3 2.50 7 0.90 57 6.62 29 1.79 1 <0.01 5 0.11 1 <0.01 17 1.26 4 0.13 1 <0.01 1 <0.01 1 <0.01

NOURI et al. – Woody species diversity considering structure

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Table 4. Mean values ± standard error of evenness indices and result of statistical tests in both study areas

Total tree species Overstorey Middle storey Understorey

Managed area Smith and Simpson Wilson 0.438 ± 0.162 0.228 ± 0.215 0.141±0.650 0.538 ± 0.230 0.721 ± 0.164 2.568 ± 0.980 0.175 ± 0.533 0.262 ± 0.350

Unmanaged area Smith and Simpson Wilson 0.501 ± 0.098 0.236 ± 0.168 0.687 ± 0.141 0.633 ± 0.202 0.167 ± 0.611 0.488± 1.919 0.179± 0.424 0.230 ± 0.264

p-value Simpson 0.020 0.147 0.006 0.004

Smith and Wilson 0.000 0.096 0.007 0.093

Table 5. Mean values ± standard error of heterogeneity indices and result of statistical tests in both study areas

Total tree species Overstorey Middle storey Understorey

Managed area Simpson Shannon 0.338 ± 0.186 0.842 ± 0.446 0.181 ± 0.331 0.422 ± 0.809 0.387 ± 0.151 0.899 ± 0.348 0.174 ± 0.284 0.705 ± 0.390

Shannon’s diversity index The Shannon diversity index for all tree species was significantly higher in unlogged area (Table 5). The same is observed for tree layers, but the difference was not significant in understorey (Table 5). Regarding to vertical structure, this index had the highest value for middle storey, but the difference was only significant difference between layers in unlogged area (p < 0.05).

Simpson’s diversity index The Simpson diversity index for tree species showed significant differences between both areas with higher diversity value for unlogged area (Table 5). The same holds for all tree layers, but only in the upper and middle storey the differences were significant (Table 5). Regarding to vertical structure, this index had the highest value in middle storey but only in the unlogged area a significant difference was found between layers (p < 0.05).

Shrub diversity indices Since shrub species were recorded in 35 sample plots in logged area and only in 3 sample plots in unlogged area, the difference of diversity indices of shrub species between two areas were obvious. As presented in Table 6, in contrary to tree species, richness and heterogeneity (Simpson and Shannon) indices of shrubs were greater in the logged area but evenness indices values were greater in unlogged area.

Discussion Considering overall tree species diversity indices, all species richness, evenness and heterogeneity indices showed significant differences between unlogged and logged areas with greater values for unlogged area. This difference is most probably related to management differences between both regions and selective logging.

These findings are in accordance with Brown and Gurevitch (2004) who reported that logging operation

Unmanaged area Simpson Shannon 0.628 ± 0.104 1.727 ± 0.406 0.196 ± 0.450 0.528 ± 1.143 0.173 ± 0.482 0.523 ±1.263 0.178 ± 0.298 0.441 ± 0.804

p-value Simpson Shannon 0.000 0.000 0.021 0.010 0.023 0.003 0.748 0.331

decreases species diversity; independent of how many years ago clear cutting or selective cutting were done.

Forest utilization by logging may result in better conditions for invasive species (Drake et al. 1989) and poses a threat to biodiversity by possibly displacing native plants or reducing their diversity. Even though logging might reduce competition, it increases light by creating gaps in canopy and increases the chance of soil contact for incoming propagates (Robert and Dong 1993), in long term managed forests do not retrieve their primary tree species diversity.

Other studies showed a positive effect of management on the total species richness of vascular plants (Schmidt 2005), which could be a specific response to the disturbance that causes an increment in species diversity. In fact, the increase in diversity in some harvested forests has been related to a short-term response of the system to disturbance (Pitkanen 2000; Peltzer et al. 2000; Roberts and Zhu 2002). Some studies showed that in the first 20 years of management practices, species richness was higher in managed than to unmanaged forests; after the 20-year cutoff, higher species richness is observed in unmanaged forests (Paillet et al. 2010).

Regarding to tree structural diversity, the results also indicated that all diversity indices in the overstorey were greater in unlogged area, whereas in the middle- and understorey only species richness and heterogeneity were higher in the unlogged area, but evenness values were greater in the logged area. In most sample plots in unlogged area the middle storey consisted of semi shade tolerant species such as Carpinus betulus and Parrotia persica, which hindered presence of other species and resulted in uneven distribution of species in these layers. In analyzing tree structural diversity it was observed that species richness decreased from understorey to overstorey in the unlogged area. It seems in parallel with tree individual’s diameter and height increment and transition to higher layers, some species were outcompeted because of competition for ecological conditions like light, moisture, nutrients, etc. In contrary, the trend of species

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richness changes was different in the managed area; it decreased from understorey to middle storey and then increased to overstorey, in other words this index had its highest value in the top layer. Heterogeneity indices had their highest values in the middle storey layer, in this study three factors played important roles in calculating heterogeneity indices: i) number of species in each layer, ii) summation of basal area of tree individuals iii) distribution of basal area among species. Greater value of heterogeneity indices in the middle storey compared to understorey was related to higher value of basal area of trees in this layer not to the more number of tree species; because species richness values were greater in the understorey. These findings may explain the essence of using the variable related to size of trees (e.g. diameter, basal area or volume) rather than number of individuals for calculating species diversity in forest stands. Diversity indices calculated for shrubs showed significant differences between two regions with higher value for the managed area. It seems that gaps created by logging allowed the understorey to outspread more because canopy closure restricts spread of most herbs and shrubs. Frequent disturbances in managed forests such as canopy openings, litter removal, and soil disturbance, all strongly favor understorey plants, especially shade intolerant and competitive species. Structural diversity which is proved to be a good indicator of forest structure has also significantly affected by disturbance in this study.

Hence, development of indicators based on only one dimension of diversity may not be sufficient to assess the effect of management on biodiversity (Roberts and Gilliam 1995).

Biodiversity conservation is increasingly considered a key to sustainable forest management and understanding of biodiversity in forest ecosystems is essential to reveal forest dynamics and providing methods and approaches to manage forest stands. Growing empirical evidence demonstrates that biodiversity loss can affect major ecosystem properties such as primary productivity and nutrient cycling (Cardinale et al. 2012; Isbell et al. 2011; Ratcliffe 2015).

Trees are the most important components in forest ecosystems and provide resources and habitats for many other forest species which depend on tree species. Tree species diversity has the potential to increase productivity in a temperate forest through facilitation and/or complementary resource use if the resource considered limits productivity (Schmidt et al. 2015).

So in forests in which a selection cutting system is applied for timber production, these species and consequently forest species diversity may be endangered in long term, especially if only some specific species and diameters are selected. In the most cases some improvement in design and management of forests can conserve biodiversity better, often with little or no reduction in timber production (Hartley 2002). Finally, as forestry operations result in changes in various forest characteristics including plant composition, forest structure, soils and species diversity, in management plans for Hyrcanian forests of Iran, assessing and monitoring the

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effect of management programs like logging operations on forest diversity is essential.

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B I O D I V E R S IT A S Volume 16, Number 1, April 2015 Pages: 102-107

ISSN: 1412-033X E-ISSN: 2085-4722 DOI: 10.13057/biodiv/d160114

Nest temperatures of the Piai and Sayang Islands green turtle (Chelonia mydas) rookeries, Raja Ampat Papua, Indonesia: Implications for hatchling sex ratios RICARDO F. TAPILATU1,2,♥, FERDIEL BALLAMU3 1

Marine Science Laboratory and Department, University of Papua (UNIPA). Marine and Fisheries Hall Room Ikn-4, Jl. Gunung Salju, Amban, Manokwari 98314, Papua Barat, Indonesia. Tel./Fax.: +62-986-212156/211455, ♥email: rf.tapilatu@unipa.ac.id 2 Pacific Marine Resources Research Center, University of Papua (UNIPA). Manokwari 98314, Papua Barat, Indonesia 3 Papua Sea Turtle Foundation, Sorong 98413, Papua Barat, Indonesia Manuscript received: 25 January 2015. Revision accepted: 31 March 2015.

Abstract. Tapilatu RF, Ballamu F. 2015. Nest temperatures of the Piai and Sayang Islands green turtle (Chelonia mydas) rookeries, Raja Ampat Papua, Indonesia: Implications for hatchling sex ratios. Biodiversitas 16: 102-107. Sex determination and hatching success in sea turtles is temperature dependent. Warmer sand temperatures may skew sea turtle population sex ratios towards predominantly females and high sand temperatures may also decrease hatching success. Therefore, understanding nest temperatures is important for conservation programs, including the evaluation of the potential impact of global climate change. Nest temperatures were monitored during the 2013 nesting season of the green sea turtle, Chelonia mydas, at Piai and Sayang Islands, Raja Ampat, West Papua, Indonesia.

Nest temperatures increased from 29oC early in the incubation to 34-36oC in the middle, before decreasing again. Monitored nest temperatures were similar across all beaches. Nest temperatures increased 2-4oC during the middle third of incubation due to metabolic heating. Hatchling sex ratio inferred from nest temperature profiles indicated a strong female bias. This finding is consistent with the relatively warm thermal profiles of the majority of the nesting beaches. This also included some extremely warm nest temperatures that were associated with lower hatching success. Information from this study provides a foundation for developing conservation strategies for enhancing hatchling production with optimal sex ratios at the most important nesting beaches for the western Pacific green sea turtle.

This study is the first comprehensive assessment of sex ratios for green sea turtles in Raja Ampat and represents the initiation of a longterm database that can be used at a local level to develop strategies that could potentially offset the impact of long-term climate change on the western Pacific green sea turtle.

Keywords: Chelonia mydas, hatching success, Piai, Sayang, sex determination.

INTRODUCTION Sea turtle populations, globally, are experiencing dramatic population decline. This is due, primarily, to nesting habitat destruction and disturbance, the incidence of commercial fishing by-catch, hunting, shell marketing and raiding of nests by villagers in subsistence economies.

Reviews of the status of sea turtle populations in Southeast Asia by Limpus (1994; 1997) determined that all marine turtle populations in the Indo Pacific region, outside Australia, are severely depleted through overharvesting and excessive incidental mortality. Limpus (1997) further estimated that the rate of turtle harvest exceeds the replacement capacity of existing populations in the entire Pacific region. Raja Ampat is a unique site, located on the northwestern side of West Papua province, Indonesia. The archipelago contains a full range of marine and coastal habitats that are important for the breeding, foraging and migration of several species of sea turtles. Similar to other areas in the Indo Pacific region, the commercial harvest is identified as one of the major threats to turtle populations in Raja Ampat. Exploitation of sea turtles for both subsistence and commercial purposes is a long-standing practice in Raja Ampat. Sea turtles have long been a source

of protein for local villagers. Further, the expansion of the Balinese turtle fishery towards eastern Indonesia in the mid 1970s caused the depletion of populations in Green turtle rookeries in Sulawesi, Maluku and Irian Jaya (Polunin and Nuitja 1982). Hunting for subsistence and poaching for commercial benefit are most likely to occur during the nesting season abundance.

Like all sea turtles, the green turtle possesses temperature-dependent sex determination (TSD).

Incubation temperature determines the sex of hatchling sea turtles during the middle third of embryonic development, with low temperatures producing males, and high temperatures producing females (Yntema and Mrosovsky 1982). Because the environmental conditions at the nest site are a major determinant of nest temperature, it may be expected that females choose their nest site carefully.

However, even within a rookery there must be some variation in nest temperature in order to maintain phenotypic diversity. As such, it is of interest to evaluate naturally occurring sex ratios in green turtle populations. In this study, nest temperature profiles were reported from green turtle rookeries on Piai and Sayang Islands of Raja Ampat. Nest temperature profiles were also used to predict the sex ratio of turtles emerging from nests. Documenting

TAPILATU & BALLAMU – Nest temperature of green turtle

variation in nest temperatures within a rookery is the first step toward a detailed understanding of how incubation temperature influences sea turtle populations. The results provide insight on spatial and temporal dynamics of nest temperatures and predicted hatchlings sex ratios produced on the primary nesting beaches for the western Pacific green turtles at Raja Ampat. These data provide an initial step in establishing a long-term database of hatchling sex ratios for assessing the ecological and conservation implications of TSD in this population, including the potential impact of global climate change. This type of information can prove valuable when attempting to understand the reproductive ecology of sea turtles and when developing conservation strategies for enhancing the recovery of threatened and endangered populations (Coyne et al. 2007; Wibbels 2007).

MATERIALS AND METHODS This study took place on Piai and Sayang Islands (Figure 1) of Raja Ampat, West Papua, Indonesia during the 2013 green turtle (Chelonia mydas) nesting season.

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Sayang island is the largest island with approximately 9km of beaches, which are fragmented by karsts. A large number of nests were found on the western beach. Piai island, which is much smaller than Pulau Sayang, is also an important rookery for Green turtles. Sandy beaches, with an approximate length of 3km, are situated on the northern and southern parts of the island. Nest temperatures below the surface were monitored every hour at two sites on Piai and Sayang islands at two different beach zones, open area and under vegetation with temperature data loggers from January to April 2013. In general, the topography of the beaches includes an intertidal zone, then a gently sloping zone, followed by a vegetative zone. Most of the nesting typically occurs well above the high tide line on the gently sloping zone of the beach. Due to logistical difficulties related to placing and retrieving data loggers, they were used primarily during the main nesting season (i.e. the austral summer, January to April). A type of temperature data loggers (HOBO Pendants, Onset Computer Corporation, Pocassete, MA) was used to record sand and nest temperatures. The datalogger accurately records temperatures to approximately ± 0.3-0.4°C.

Figure 1. Map of study area in the Piai and Sayang Islands, Raja Ampat, West Papua, Indonesia

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A

B

Figure 2. Nest temperature traces. A. Nests constructed in the open beach section. B. Nests constructed close and or under vegetation zones

Figure 3. Nests temperatures (Mean±SE) during middle third of development in nests that were monitored with dataloggers at open and vegetation zones at Piai and Sayang Islands

Nesting females were located during the night and the nests were excavated after nesting had finished. Eggs were removed and counted and a temperature data logger was positioned in the centre of the egg mass during egg replacement. The nest was reburied and the eggs incubated naturally without further interference. Temperature data loggers were used to record incubation temperatures during incubation period of in situ nests. Three nests and four nests were opportunistically selected from open beach zones and under/close to vegetation zones respectively for data logger placement. Nests were excavated after hatching when hatchlings hatched from eggs and dug upwards to retrieve the data logger. The data loggers were programmed in the laboratory to record temperature every hour. The hour measurements were averaged over 24 h to obtain a single daily temperature.

Nest temperature traces from the time of oviposition to the time of emergence were used to model the thermal reaction norm of embryonic development using the R package, embryo growth (5.2, http://cran.r-project.org) (Girondot and Kaska 2014). Hatchling sex ratios were

predicted from incubation temperature, knowing that incubation at a constant temperature of 26oC produces 100% males, incubation at a constant temperature of 29 oC produces 100% females, and assuming a linear transition from all males to all females within this temperature range (Booth and Astill 2001; Miller and Limpus 1981). Further, to evaluate the effect of nest temperature on hatchling sex ratio accurately, the mean nest temperature during the middle third of incubation was calculated for each nest.

The middle third of development for each nest was estimated from the model of embryonic growth to determine the TSP duration. Hatchling sex ratio was estimated by the mean temperature, weighted by embryonic growth, during the middle third of development for each nest. The mean nest temperature during the middle third in each nest was compared to available pivotal temperature for western Pacific green turtle population. Previous studies indicate that mean nest temperature during the middle third of incubation represents an accurate method for predicting sex ratio in nest that do not experience large daily fluctuations in temperature (Georges et al. 2004; Georges et al. 1994). No direct validation of the sex predictions at this reporting period was made because this would have involved sacrificing the hatchlings for dissection to determine their sex.

RESULTS AND DISCUSSION Results The profiles of nest temperatures could be divided into three distinct phases: early, heating, and cooling (Figure 2).

During the January-April period, nest temperatures at nests laid both open and vegetation zones are likely to track the sand temperature at 60 cm for the early portion of incubation and then increased to be 2-4oC above sand temperature at middle portion (Figure 2A-B). The climb in nest temperatures was relatively smooth with temperature fluctuations within any given week being less than 0.9oC.

TAPILATU & BALLAMU – Nest temperature of green turtle

Fluctuations in nest temperatures over a period of a few days probably due to rainfall events. Nest temperature increased slightly so that at the time of middle incubation period, nest temperatures were 2-4oC distinctively warmer than sand temperature. An increase in nest temperature above surrounding sand temperature during middle portion of incubation is commonly reported in sea turtle nests (Bustard 1972; Booth and Astill 2001; Broderick et al.

2001; Godley et al. 2002), and is caused by the metabolic heat produced by developing embryos which increases greatly during the final stages of incubation.

The patterns of nest temperature profiles early nesting season 2013 were consistent across two monitoring zones.

Nest temperatures for nests laid at open beach zones are relatively similar with zones close to vegetation with the exception of Nest with YPP-09 datalogger. During the early phase, nest temperatures of this nest tracked the beach temperatures at 27oC while majority of nests initiated at 29oC. Furthermore, mean nest temperatures recorded in the current study were typically above the pivotal temperature (PT, Figure 3) that had been reported for green turtles in Costa Rica, 28oC (Morreale et al. 1982), in Mediteranean, 29oC (Kaska et al. 1998), and tend to cluster at 29oC (Mrosovsky 1988) in all sea turtles.

An extreme female-biased sex ratio was predicted by the mean temperature during the middle third of development regardless of whether temperatures were weighted by embryonic growth or time. However, the mean temperature of nests during the TSP weighted by embryonic growth was higher ( = 32.14°C±0.69) than when weighted by time ( = 31.71°C±0.59). In addition, a female-biased sex ratio was also predicted for all but one nest when TSP length in this study was compared to TSP length where hatchling sex ratios were known (Miller and Limpus 1981). It is possible that a few intersex individuals may have been produced in these nests (Miller and Limpus 1981); however, the overall sex ratio would remain strongly female biased.

Discussion It has been suggested that numerous factors affect nest temperatures (Binckley et al. 1998). These vary from large scale annual and seasonal differences in climate (patterns of precipitation and air temperature) during nesting seasons to mesoscale factors such as nest placement on a beach (sun versus shade, distance to high tide) or sand color (black versus white) down to factors at the individual nest such as depth, egg position (bottom versus top) and metabolic heat generated by developing embryos (Godfrey et al. 1997; Binckley et al. 1998; Mickelson and Downie 2010).

Sexual differentiation in sea turtles is strongly influenced by ambient incubation temperature or TSD (Standora and Spotila 1985; Mrosovsky 1994).

Specifically, the sustained temperature to which the embryo is exposed during the middle trimester of incubation determines the eventual gonadal differentiation and sex of the hatchling (Wibbels 2003). The pivotal temperature may vary with species and locale. The pivotal

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temperatures for green turtles have been estimated to cluster at 28oC in Costa Rica (Morreale et al. 1982) and at 29oC in Mediterranean (Kaska et al. 1998). The simple method predicted that all successfully monitored nests during the January-April 2013 period should produce female hatchlings, even in a nest (YPP-20) that initiated incubation temperature at 27oC. The overall prediction from monitored nests was an extreme female bias. In addition, all nests monitored with dataloggers, mean temperatures were above the pivotal temperature (Figure 3) suggesting female biased sex ratios. Thus, collectively these results support the hypothesis that female-biased hatchling sex ratios may predominate on the green turtles nesting beaches of Piai and Sayang Islands. This female biased hatching ratio is similar with reports of green turtle nests on Heron Island (Limpus et al. 1983; Booth and Astill 2001), but the bias found in this current study is more extreme than reported in Heron Island. This difference might be attributed to a warmer than average nesting season in 2002-2003on Heron island. There is a trend for a female hatchling bias from sea turtle rookeries worldwide (Spotila et al. 1987; Mrosovsky 1994; Broderick et al.

2001; Godley et al. 2001, 2002). The evolutionary reason(s) for the apparent strong female hatchling bias in most sea turtle rookeries remain unexplained (Mrosovsky 1994).

In an evolutionary context, a female visiting a particular rookery has the potential to influence the sex ratio of her offspring by varying her nest timing, nesting beach and nest depth. Given the importance of nest temperature in determining hatchling attributes, a prediction might be that once ashore females use cues to choose nest sites of optimal thermal characteristics. Studies investigating the role of thermal cues in nest site choice have not been done for green turtles from Piai and Sayang, but green turtles nesting at Tortugero, Costa Rica do not actively select nest sites based on temperature (Bjorndal and Bolten 1992).

Global climate change (IPCC 2013) could have a significant impact on reptiles with TSD (Janzen 1994; Mitchell and Janzen 2010) including sea turtles (Hawkes et al. 2007; Chaloupka et al. 2008; Poloczanska et al. 2009; Fuentes et al. 2009, 2010; Hays et al. 2010; Witt et al.

2010; Patino-Martinez et al. 2012). It has been suggested that increases in sand temperature associated with climate change will affect both leatherback sex ratios (Binckley et al. 1998; Hulin et al. 2009; Patino-Martinez et al. 2012) and hatchling fitness (Mickelson and Downie 2010). These changes are predicted to increase the proportion of female leatherback hatchlings and reduce hatching success and fitness primarily at sites already experiencing biases toward female-producing temperatures (Binckley et al. 1998; Mickelson and Downie 2010; Patino-Martinez et al. 2012).

On Piai and Sayang islands, nest temperatures may indicate the production of a range of sex ratios but with an overall female bias. This could potentially pose a problem if future temperatures increase due to global climate change.

Considering that green sea turtles on these islands are already producing a female biased hatchling sex ratio and that some nest temperatures reach as high as 35 oC, the

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projected increases (IPCC 2013) could result in extreme female biases and potentially impact hatching success.

This initial study suggested that the potential of hatchling sex ratio produced on Piai and Sayang is femalebiased and the finding adds to the general paradigm emerging for sea turtles that female biases are prevalent (Mrosovsky 1994; Wibbels 2003). There are several implications for the conservation of the western Pacific sea turtle population at Birdâ&#x20AC;&#x2122;s Head since this population has significantly declined due to over-harvesting (Hitipeuw 2003). It is plausible that possessing TSD and producing female-biased sex ratios from Piai and Sayang islands could be advantageous to the recovery of the Raja Ampat green sea turtle population. It has been proposed that biased sex ratios could significantly impact the recovery of sea turtle populations in both positive and negative ways (Vogt 1994; Mrosovsky and Godfrey 1995; Wibbels 2003; Coyne et al. 2007; Wibbels 2007). Coyne (2007) provides a model showing that female-biased sex ratios could potentially increase recovery rates, since increasing the number of females will increase the egg (and therefore juvenile) production in future years. As an example, the recovery rate of the Kempâ&#x20AC;&#x2122;s ridley turtle may have been accelerated by the artificial skewing of sex ratios (Wibbels 2007).

However, these projections are based upon the assumption that lack of males does not become a limiting factor as the proportion of females increases.

The concept of manipulating sex ratios has drawn some controversies (Vogt 1994; Mrosovsky and Godfrey 1995).

In particular the impact of biased sex ratios on the reproductive ecology of sea turtles is not well understood and it is plausible that extreme biases could have a negative impact (Mrosovsky and Godfrey 1995). Extreme biases could potentially affect reproduction in regards to fertility, probability of nesting, and multiple paternities (Wood and Wood 1980; Harry and Briscoe 1988; Chan and Liew 1991). It is plausible that an extreme female bias could lead to the Allee effect due to reduction in number of males. For example, it has been suggested that low hatch rates of leatherback in Malaysia could have related to insufficient number of males to fertilize clutches (Chan and Liew 1991). However, it has been suggested that the Allee effect may not impede the recovery of relatively small population of sea turtles (Hays 2004).

ACKNOWLEDGEMENTS Our sincere thanks and appreciation are to sea turtle patrollers from villages of Salio and Selpele of Waigeo Sub-District who assisted in the experiment and gave access to use dataloggers in nests of green for this study at Piai and Sayang Islands. Taylor Roberge and Dr. Thane Wibbels of Biology Department University of Alabama at Birmingham (UAB)-USA assisted in data analysis and Petrus Dimara of University of Papua (UNIPA) assisted with graphic. We also thank the anonymous reviewers for their insightful comments. Laure Katz and Debbie Jacobs of Conservation International Indonesia Program who solicited the Walton Family Fund (WFF) grant application,

Dr. Mark Erdmann who provided advices in obtaining WFF grant.

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Title of the article should be written in compact, clear, and informative sentence, preferably not more than 20 words. Name of author(s) should be completely written. Name and institution address should also be completely written with street name and number (location), postal code, telephone number, facsimile number, and email address. Manuscript written by a group, author for correspondence along with address is required. First page of the manuscript is used for writing above information.

Abstract should not be more than 200 words. Keywords is about five words, covering scientific and local name (if any), research theme, and special methods which used; and sorted from A to Z. All important abbreviations must be defined at their first mention. Running title is about five words.

Introduction is about 400-600 words, covering the background and aims of the research. Materials and Methods should emphasize on the procedures and data analysis. Results and Discussion should be written as a series of connecting sentences, however, for manuscript with long discussion should be divided into subtitles. Thorough discussion represents the causal effect mainly explains for why and how the results of the research were taken place, and do not only re-express the mentioned results in the form of sentences.

Concluding sentence should be given at the end of the discussion.

Acknowledgments are expressed in a brief; all sources of institutional, private and corporate financial support for the work must be fully acknowledged, and any potential conflicts of interest are noted.

Figures and Tables of maximum of three pages should be clearly presented. Title of a picture is written down below the picture, while title of a table is written above the table. Colored figures can only be accepted if the information in the manuscript can lose without those images; chart is preferred to use black and white images. Author could consign any picture or photo for the front cover, although it does not print in the manuscript. All images property of others should be mentioned source. There is no appendix, all data or data analysis are incorporated into Results and Discussions. For broad data, it can be displayed on the website as a supplement.

References Author-year citations are required. In the text give the authors name followed by the year of publication and arrange from oldest to newest and from A to Z. In citing an article written by two authors, both of them should be mentioned, however, for three and more authors only the first author is mentioned followed by et al., for example: Saharjo and Nurhayati (2006) or (Boonkerd 2003a, b, c; Sugiyarto 2004; El-Bana and Nijs 2005; Balagadde et al. 2008; Webb et al. 2008). Extent citation as shown with word "cit" should be avoided. Reference to unpublished data and personal communication should not appear in the list but should be cited in the text only (e.g., Rifai MA 2007, pers. com. (personal communication); Setyawan AD 2007, unpublished data). In the reference list, the references should be listed in an alphabetical order (better, if only 20 for research papers). Names of journals should be abbreviated. Always use the standard abbreviation of a journal's name according to the ISSN List of Title Word Abbreviations (www.issn.org/222661-LTWA-online.php). The following examples are for guidance.

Journal: Saharjo BH, Nurhayati AD. 2006. Domination and composition structure change at hemic peat natural regeneration following burning; a case study in Pelalawan, Riau Province. Biodiversitas 7: 154-158.

Book: Rai MK, Carpinella C. 2006. Naturally Occurring Bioactive Compounds.

Elsevier, Amsterdam.

Chapter in book: Webb CO, Cannon CH, Davies SJ. 2008. Ecological organization, biogeography, and the phylogenetic structure of rainforest tree communities. In: Carson W, Schnitzer S (eds) Tropical Forest Community Ecology. Wiley-Blackwell, New York.

Abstract: Assaeed AM. 2007. Seed production and dispersal of Rhazya stricta. 50th annual symposium of the International Association for Vegetation Science, Swansea, UK, 23-27 July 2007.

Proceeding: Alikodra HS. 2000. Biodiversity for development of local autonomous government. In: Setyawan AD, Sutarno (eds) Toward Mount Lawu National Park; Proceeding of National Seminary and Workshop on Biodiversity Conservation to Protect and Save Germplasm in Java Island.

Sebelas Maret University, Surakarta, 17-20 July 2000. [Indonesian] Thesis, Dissertation: Sugiyarto. 2004. Soil Macro-invertebrates Diversity and Inter-Cropping Plants Productivity in Agroforestry System based on Sengon. [Dissertation].

Brawijaya University, Malang. [Indonesian] Information from internet: Balagadde FK, Song H, Ozaki J, Collins CH, Barnet M, Arnold FH, Quake SR, You L. 2008. A synthetic Escherichia coli predator-prey ecosystem.

Mol Syst Biol 4: 187. www.molecularsystemsbiology.com

Front cover: Fagus orientalis

(PHOTO: Farzam Tavankar)

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