Biodiversitas vol. 10, no. 4, October 2009 by Biodiversitas Unsjournals

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

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

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BIODIVERSITAS Volume 10, Number 4, October 2009 Pages: 163-167

ISSN: 1412-033X (printed edition) ISSN: 2085-4722 (electronic) DOI: 10.13057/biodiv/d100401

Comparison Analysis of Genetic Diversity of Indonesian Mangosteens (Garcinia mangostana L.) and Related Species by Means Isozymes and AFLP Markers 1,♥

2,♥♥

SOBIR , SOALOON SINAGA

, ROEDHY POERWANTO , RISMITASARI , RUDY LUKMAN

Departement of Agronomy and Horticulture, Institut Pertanian Bogor (IPB), Bogor 16680 2 Kopertis Wilayah III, Jakarta 13640 3 Centre for Plant Conservation Bogor Botanic Gardens, Indonesian Institutes of Sciences (LIPI), Bogor 16122 4 Bisi International & SEAMEO BIOTROP, Bogor 16720 Received: 2nd March 2009. Accepted: 26th June 2009.

ABSTRACT Mangosteen (Garcinia mangostana) belongs to a large genus of Garcinia that native in South East Asia, as well as Indonesia, and in order evaluate genetics diversity of mangosteen and their close relatives, we employed isoenzyme and AFLP marker on 13 accessions of mangosteen and their close relatives. Isoenzyme marker using four enzyme systems produced 25 bands and 88% out of them were polymorphic and elucidate genetic variability at similarity level ranged between 0.38-0.89. AFLP markers with three primer system produced 220 polymorphic bands and revealed genetic variability at similarity level ranged between 0.38-0.89 successfully produced high polymorphism bands and elucidates genetic variability at similarity coefficient ranged between 0.21-0.77. Both markers exhibited similar clustering pattern, and group successfully G. mangostana accessions in one clustering group. Furthermore G. malaccensis and G. porrecta consistently showed closer genetic relationship to G. mangostana clustering group in both markers, in comparison to G. hombroniana, which implies the assumption they may be the progenitor of G. mangostana, and should be reviewed with more accurate data. © 2009 Biodiversitas, Journal of Biological Diversity Key words: genetic diversity, mangosteen, isozymes, AFLP.

INTRODUCTION Mangosteen (Garcinia mangostana L.) belongs to family Guttiferae, genus Garcinia (Verheij, 1991). Garcinia is a large genus that consists of about 400 species, and originated from East India, Malay Peninsula and South East Asia, as well as Indonesia (Campbell 1966). Based on morphological and cytological studies, Yaacob and Tindall (1995) suggested that mangosteen originated from South East Asia; subsequently Almeyda and Martin (1976) proposed that mangosteen is an inhabitant Indonesian fruit. Some species of Garcinia, including G. mangostana produce fruit without pollination, the phenomenon is referred to as agamospermy, which is the production of seed without fusion of gametes (Koltunow et al., 1995; Thomas 1997). The process of embryo formation in G. mangostana was first studied Corresponding address: Kampus IPB Darmaga Bogor 16680 Tel./fax: +62-251-8326881 email: rsobir@yahoo.com  Jl. SMAN 14, Cawang (Samping BAKN), Jakarta Timur 13640 Tel +62-21-8005610, Fax +62-21-8094679. email: horas97@gmail.com 

by Treub (1911) who reported that the early development of woodiness in the endocarp soon after anthesis made the observation of embryo development difficult (Tixier, 1955). However, Lan (1989) provided a detailed account of mangosteen embryology and reported that the embryo of G. mangostana is derived from tissue of integument instead of from the egg. An understanding of genetic diversity and its phylogeny among cultivated plant accession significantly influence on the quality increase and the results, and it also improves the management of germplasm conservation (Roldan-Ruiz et al., 2001). Plant genetic improvement highly depends on the available genetic resources. Wide genetic diversity will give higher opportunity in the selection process of the best characters. Some research on the genetic diversity using some markers could explain the phylogeny within and among population (Fajardo et al., 2002; Hurtado et al., 2002; McGregor et al., 2002). Genetic variability analysis can be done by using many manner of markers, such as morphology (Talhinhas et al., 2006), isoenzymes (Ayana et al., 2001), and molecular markers (Assefa et al., 2003; Cavagnaro et al., 2006), such as AFLP marker (Vos

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et al., 1995). Recently, due to burgeoning in biotechnological technique, the molecular markers have been widely used to elucidate genetic information in the molecular level (Roy et al., 2006). Each marker system has the advantages and disadvantages, so that the assessment of the markers system is an important step to decide the most suitable marker regarding to research purpose. The comparison of several markers has been done with comparative study of some molecular markers with PCR base such as Palombi and Damiano (2002) which compared RAPD and SSR markers to detect genetic variability of kiwi plant, Ferdinandez and Coulman (2002), compared the efficiency of RAPD, SSR, and AFLP to identify plant genotypes. Saker et al. (2005), has used different markers to characterize the barley. The study is aimed to distinguish the advantages of isoenzyme and AFLP markers in elucidating genetic variability and phylogenetic relationships among the mangosteen (Garcinia mangostana L.) and the close relatives, and to study the suitable molecular to develop specific molecular markers in characterization of mangosteen and its close relatives.

MATERIALS AND METHODS Plant material This research was conducted in the laboratory of Biotechnology and Tree Breeding BIOTROP Bogor, Molecular Laboratory and Plant Biology the Research Center for Biological Resources and Biotechnology IPB Bogor, and Laboratory of Tropical Fruit Research Center IPB Bogor. Thirteen (13) leaf samples of mangosteen and its close relatives were collected from several locations in Indonesia, namely: Pandeglang (Banten), Sukabumi, Purwakarta (West Java), Ponorogo (East Java), Lampung Regency, Palangkaraya (Central Kalimantan), Kendari (South East Sulawesi), Ambon (Maluku), G. rigida, G. hombroniana, and G. celebica (Bogor Botanical Gardens), and G. malaccensis, G. porrecta, and G. benthami (Mekarsari Tourism Park Bogor). Isoenzymes analysis Thirteen fresh samples were taken for isozyme analysis following Soltis and Soltis (1989). The enzymes analyzed are peroxidase (PER), phosphatase acid (ACP), malic dehydrogenase (MDH), and esterase (EST). The separation of isoenzyme bands was done with electrophoresis by using agarose gel with concentration of 10% for 4 hours, and 100 volt. AFLP analysis Extraction and DNA purification The DNA of Leaf samples were extracted for AFLP analysis the same as for isoenzyme analysis. DNA extraction followed CTAB (Doyle and Doyle, 1987)

with some modifications. DNA concentration was tested with electrophoresis and immigrated with standard DNA (DNA lambda) 10 and 100 ng/mL on agarose gel 1.2%. Restriction-ligation Approximately 0.5 µg genomic DNA was cut 1 unit MseI and 5 unit EcoRI. At the same time it is ligated with 5 pmol EcoRI and 50 pmol MseI adaptor with 1 U T4 DNA ligase. The adaptor sequence EcoRI is 5´CTCGTAGACTGCGTACC-3´, 3´CTGACGCATGGTTAA-5´ and the adaptor sequence MseI is 5´-GACGATGAGTCCTGAG-3´, 3´TACTCAGGACTCAT-5´. Preselective amplification Primers for preselective amplification are EcoRI+A and MseI+C as homologous adaptor EcoRI and MseI, each with one additional nucleotide at 3’ end. PCR reactions were carried out in reaction mix containing of 4 µl restriction-ligation DNA, 2.5 pmol primer EcoRI +A and 2.5 pmol MseI primer +C, 0.4 U Taq polymerase DNA, 0.2 mM each dNTP and 1x buffer PCR 20 µL. The PCR amplification was programmed for 20 cycles at 94° (1 second), 56°C (30 seconds), and 72°C (2 minutes). The PCR products 10 uL was tested w on 1.5% agarose gel. The amplified fragments range from 100-1500 bp. Selective amplification The selective amplifications were conducted by using primer EcoR1+ ANN and Mse1+CNN. The PCR reaction was performed using DNA pre-amplification 3 µL, 1 pmol primer EcoRI + ANN, 5 pmol primer MseI + CNN without labeling, 0.4 U Taq polymerase DNA, 0.2 mM each dNTP and 1 x buffer PCR with a total volume of 20 µl. PCR reaction was programmed with 1 cycle for 30 seconds at 94°C, 30 seconds 65°C, 2 minutes at 72°C, followed by eight cycles of variable annealing temperature with a decrease of 1°C each cycle, and terminated with 23 cycles of 1 second at 94°C, 30 seconds at 56°C, 2 minutes at 72°C. PAGE electrophoresis The selective amplification products were displayed using PAGE electrophoresis, and presented as a diagram. Approximately 2 µL PCR product mixed with 0.15 µL 6-carboxy-Xrhodamin (ROX)-labeled internal standard length GeneScan500 ROX and dye 0.85 µL formamide, denaturized for 3 minutes at 90°C and cooled in ice. Electrophoresis using 5% gel denaturing polyacrylamide (Long RangerTM, FMC Bioproducts) in buffer electrophoresis 1x TBE by using ABI PrismTM 377 DNA sequencer (Applied Biosystems) at 2500 V for 4 hours. The raw data was obtained using ABI PRISMTM V.1.1 software. Next, the AFLP fragments were analyzed with GENESCANTM version 2.1 (Applied Biosystems).

SOBIR et al. â&#x20AC;&#x201C; Isozymes and AFLP diversity of Indonesian mangosteen

Data analysis The bands of the isozyme technique and AFLP were translated into the binary data. These data were used to arrange the genetic similarity matrix based on the formula of Nei and Li (1979) with UPGMA (Unweighted Pair-Group Method Arithmetic) method using NTSYS (Numerical Taxonomy and Multivariate System) version 2.02 (Rolf, 1998). Genetic similarity between all pairs of accessions was calculated according to Nei and Li (1979).

RESULTS AND DISCUSSION

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Variation in apomictic plants occurred faster in mutation (Hughes and Richards, 1985). This results indicated that isozyme analysis successfully grouped mangosteen out of their close relatives, and G malaccensis closer to mangosteen than other close relatives. However, further analysis showed that G. porrecta has closer genetic relationship to G. mangostana clustering group at 0.61 of similarity coefficient, compare to G. hombroniana which is assumed as another progenitor of mangosteen (Richards, 1990), indicated that isozyme assay not yet confirmed G. hombroniana as G mangostana progenitor. Lampung

Variability analysis with isozyme marker Isozymes analysis on 13 accessions of mangosteen and their close relatives showed that the four isoenzyme systems of esterase (EST), peroxidase (PER), acid phosphatase (ACP), and malic dehydrogenase (MDH) produced 25 bands and 22 bands (88%) out of them were polymorphic band (Table 1).

G.malaccensis_b Kalteng Kusu-kusu Banten Wanayasa Sukabumi Ponorogo

Table 1. The number of bands and polymorphism level of 5 isoenzyme on 13 accessions of mangosteen and their close relatives. Isoenzymes EST-1 EST-2 EST-3 PER-1 PER-2 PER-3 ACP-1 ACP-2 MDH-1 MDH-2

Band number 4 3 3 2 3 1 1 3 1 4 25

Polymorphic bands 4 (100%) 3 (100%) 3 (100%) 2 (100%) 3 (100%) 0 (0%) 1 (100%) 2 (66,7%) 0 (0%) 4 (100%) 22 (88%)

Monomorphic Band 0 0 0 0 0 1 0 1 1 0 3

Cluster analysis based on isoenzyme assay revealed, that genetics distance among 13 accessions of mangosteen and their close relatives ranged between 0.38-0.89 of similarity coefficient (Figure 1). The similarity matrix correlation value MxComp r = 0.902 indicated that the dendrogram produced with goodness of fit highly compatible which depict the cluster (Rolf, 1998). Presentation accumulation of the three main first components on the 13 accessions of mangosteen and its relatives represent 63,5% genetic diversity that explained by 25 isozyme characters, and 70% genetic diversity was obtained from accumulation of four main components. Subsequently, isozyme analysis showed that mangosteen accessions and G malaccensis are clustered at 0.68 of similarity coefficient (32%) separated to other close relatives (Figure 1). The genetic diversity resulted from similarity analysis was relatively high for the obligate apomictic compared to Taraxacum (19%) (Ford and Richards, 1985).

G.porrecta G.rigida G.hombroniana G.celebica G.benthami 0.38

0.51

0.63

0.76

0.89

Koefisien kemiripan

Figure 1. Dendogram of 13 accessions based on isozyme marker.

Variability analysis with AFLP AFLP analysis on 13 accessions of mangosteen and their close relatives using three primer combinations of ACC_CAG, ACT_CAA and ACT_CAC produced 220 polymorphic bands at band size ranged between 50-500 bp. The number of bands resulted from each primer combination varied between 19-94 bands or at average 73.3 bands for each primer combination. The primer combination of ACT_CAA produced the highest number of polymorphic (94 bands) followed by primer combination of ACT_CAA 70 bands and primer ACC_CAG 56 bands (Table 2). Cluster analysis results based on AFLP markers, showed that genetics distance among 13 accessions of mangosteen and their close relatives ranged at between 0.21-0.77 (Figure 2). Based on the AFLP dendrogram, this hypothesis can be accepted. With value r = 0.977, meaning that the dendrogram resulted with goodness of fit very suitable to depict the grouping. Principle component analysis indicated that the three main first components represented 47.2% genetic diversity, and 70% genetic diversity of 612 characters was obtained from accumulation of six main components.

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Table 2. The number of bands and polymorphism of 3 pairs of primer AFLP on 13 accessions of mangosteen and close relatives.

compare to 62% that explained by isozyme marker. Cophenetic correlation value of both markers as high as 90% showed that the dendrogram generated from both markers have equal clustering pattern Primer AFLP Band number Polymorphic bands descended from the symqual matrix. The highest ACC_CAG 94 100% cophenetic correlation resulted by AFLP marker was ACT_CAA 70 100% 0.978. This value showed correlation between ACT_CAC 56 100% grouping and similarity matrix was fit, and gave best Total 220 100% value to construct the grouping and arrangement similarity matrices (Table 3). However, grouping Lampung pattern in isozyme marker was slightly different to G.porrecta those of AFLP marker, in terms of the number of groups, since isoenzymes generated four clustering Kalteng groups compared to AFLP marker that generated six Sukabumi clustering groups (Table 3). The occurrence of genetic variability between and Ponorogo within individuals, within population and between Banten cultivars in cultivated species occurred by mutation, Wanayasa introgression, recombination, adaptation to new G.malaccensis_b environment, and selection which occurs continually (Geleta et al., 2007). Genetic diversity within Kusu-kusu cultivated and wild plants is important to prevent G.hombroniana some problems associated with cultivation failure. Cultivated plants can be improved by introduction of G.benthami wild relatives especially in the center of distribution, G.celebica such as the mangosteen which is distributed in G.rigida Indonesia and Malay Peninsula (Harlan and de Wet, 1971; Hawkes, 1977). 0.21 0.35 0.49 0.63 0.77 Koefisien kemiripan High genetic diversity as represented by polymorphic band percentage is not common for Figure 2. Dendogram of 13 accessions based on AFLP marker. mangosteen as an apomictic obligate, this might due to several factors as accumulation of natural Further analysis on dendrogram constructed from mutation, repeated hybridization among mangosteen AFLP marker indicated that mangosteen accessions progenitors Carman (2001), and ploidy developmental clustered in one group with G. porrecta, separated processes. High variation among mangosteen with other close relatives at similarity coefficient of genotype is a genetic potential to obtain high potential 0.58. Subsequently, AFLP marker results confirmed genotypes for specific purpose, which could be done that among evaluated close relatives of mangosteen through selection approach among superior trees in G. malaccensis and G. porrecta consistently closer to the field (Sobir and Poerwanto, 2007). mangosteen accessions clustering group compare to Since G. malaccensis consistently showed closer other close relatives. genetic relationship with G. mangostana clustering group in isozyme and AFLP markers, we conducted Discussions bands similarity proportion analysis that contributed Since AFLP markers produced higher polymorphic by G. malaccensis, G. porrecta and G. hombroniana characters (220 bands) compare to those of resulted which were estimated as mangosteen progenitor by isozyme marker (22 polymorphic bands), AFLP against the mangosteen based AFLP markers. G. marker revealed higher genetic diversity 79% ma l ac c ens is shared 53% similar band with G. Table 3. Similarity coefficient value, cophenetic correlation, mangosteen group and close their relatives with isoenzyme and AFLP markers in similarity 58%. Isoenzim AFLP Similarity coefficient Value Group Accession Similarity coefficient Value Group Accession Polymorphism (%) 88% I M, GM, GP Polymorphism (%) 100% I M, GP Highest value (%) 0.889 II GR Highest value (%) 0.773 II GM (Accessions) GM vs. L III GH & GC (Accessions) GP vs. L III MK Lowest value (%) 0.2 IV GB Lowest value (%) 0.169 IV GH & GB (Accessions) GB vs. W (Accessions) GR vs. S V GC Cophenetic correlation (r) 0.902 Cophenetic correlation (r) 0.978 VI GR Notes: M = mangosteen (G. mangostana), GM = G. malaccensis, L = Lampung mangosteen, GB = G. benthami, W= Wanayasa mangosteen, GP = G. porrecta, GR = G. rigida, GH = G. hombroniana, and S = Sukabumi mangosteen.

SOBIR et al. – Isozymes and AFLP diversity of Indonesian mangosteen

G. porrecta shared 61.5 % similar band with G. mangostana, while G. hombroniana shared 50% similar band with G. mangostana. Moreover, if G. malaccensis and G. hombroniana simulated as progenitor of G. mangostana, 33% of G mangostana bands could not explained by G. malaccensis and G. hombroniana, while if G. malaccensis and G. porrecta simulated as progenitor of G. mangostana, 29 % of G. mangostana bands could not explained by G. malaccensis and G. porrecta. These result of above indicated that the proposal of G. malaccensis and G. hombroniana were progenitor of G. mangostana should be reviewed carefully with more accurate evidences, since fruit morphology of G. mangostana to fruit morphology of G. porrecta, compare to those of G. hombroniana fruit characters (Sobir et al., 2009, unpublished data).

CONCLUSION Isoenzyme assay employed four enzyme systems and three primer combinations of AFLP marker on 13 accessions of mangosteen and their close relatives successfully produced high polymorphism band and elucidate genetic variability at similarity coefficient of 0.38 and 0.21 respectively. Both markers exhibited similar clustering pattern, and grouping G mangostana accessions in a clustering group. G. malaccensis and G. porrecta consistently in both markers showed closer genetic relationship to G. mangostana clustering group compare to G. hombroniana that implies the assumption of progenitor of G. mangostana, should be reviewed with more accurate data.

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Ferdinandez, Y. and B. Coulman. 2002. Evaluating genetic variation and relationship among two bromegrass species and their hybrid using RAPD and AFLP markers. Euphytica 125: 169-176. Ford, H. and A.J. Richards. 1985. Isozyme variation within and between Taraxacum agamospesies in a single locality. Heredity 55: 289-291. Geleta, M. 2007. Genetic diversity, phylogenetics and molecular systematics of Guizotia Cass. (Asteraceae). Alnarp: Swedish University of Agricultural Sciences. Harlan, J.R. and J. de Wet. 1971. Toward a rational classification of cultivated plants. Taxon 20: 509-517. Hawkes, J.G. 1977. The importance of wild germplasm in plant breeding. Euphytica 26: 615-621. Hughes, J. and A.J. Richards. 1985. Isozyme in heritance in diploid Taraxacum hybrid. Heredity 54: 245-249. Hurtado, M.A., A. Westman, E. Beck, G.A. Abbott, G. Llacer, and M.L. Badenes. 2002. Genetic diversity in apricot cultivars based on AFLP markers. Euphytica 127 (2): 297-301. Koltunow, A.M., R.A. Bicknell and A.M. Chaudhury. 1995. Apomixis: Molecular strategies for the generation of genetically identical seeds without fertilization. Plant Physiology 108: 13451352. Lan, L.A. 1989. The embryology of Garcinia mangostana L. (Clusiaceae). Garden Bulletin Singapore 37: 93-103. McGregor, C.E., R. van Treuren, R. Hoekstra, and Th. J.L. van Hintum. 2002. Analysis of the wild potato germplasm of the series Acaulia with AFLPs: Implications for ex situ conservation. Theoretical and Applied Genetics 104: 146-156. Nei, M., and W.H. Li. 1979. Mathematical model for studying genetik variation in terms of restriction endonucleases. Proceeding of the National Academic of Sciences of the USA 76 (10): 5269-5273. Palombi, M. and C. Damiano. 2002. Comparison between RAPD and SSR Molecular markers in detecting genetic variations in kiwi fruit (Actinidia deliciosa A. Chev.). Plant Cell Report 20 (11): 1061-1066. Roldan-Ruiz, I., F.A. van Eeuwijk, T.J. Gilliland, P. Dubreuil, C. Dillmann, J. Lallemand, M. de Loose, and C.P. Baril. 2001. A comparative study of molecular and morphological methods of describing relationships between perennial ryegrass (Lolium perenne L.) varieties. Theoretical and Applied Genetics 103: 1138-1150. Rolf, F.J. 1998. NTSys-pc; Numerical Taxonomy and Multivariate Analysis System. Version 2.02. New York: Exerter Software. . Roy, J.K., R. Bandopadhyay, S. Rustgi, H.S. Balyan, and P.K. Gupta. 2006. Association analysis of agronomically important traits using SSR, SAMPL, and AFLP markers in bread wheat. Current Science 90 (5): 683-689. Saker, M., M. Nagchtigall, and T. Kuehne. 2005. A comparison study of DNA fingerprinting by RAPD, SSR, and AFLP in genetic analysis of the some barley genotypes. Egypt Journal of Genetic and Cytology 34: 81-97. Sobir, dan R. Poerwanto. 2007. Mangosteen genetics and improvement. International Journal of Plant Breeding 1 (2): 105-111. Soltis, D.E. and P.S. Soltis. 1989. Isoenzymes in Plant Biology. Portland, Oregon: Discorides Press. Talhinhas, P., J. Leitao, and J. Neves-Martins. 2006. Collection of Lupinus angustifolius L. germplasm and characterization of morphological and molecular diversity. Genetic Resources and Crop Evolution 53: 563-578. Thomas, S.C. 1997. Geographic parthenogenesis in a tropical forest tree. American Journal of Botany 84, 1012-1015. Tixier, P. 1955. Contribution a’ l’ etudes des Garcinia. Fruits 10: 209-212. Vos, P., R. Hogers, M. Bleekers, M. Reijans, T. van de Lee, M. Hornes, A. Frijters, J. Pot, J. Peleman, E. Jacobsen, J. Helder, and J. Bakker. 1995. AFLP: a new technique for DNA fingerprinting. Nucleic Acids Research 23: 4407-4414. Verheij, E.W.M. 1991. Garcinia mangostana L. In: Verheij, E.W.M. and R.E. Coronel (eds.). Plant Resources of South East Asia No. 2, Edible Fruit and Nuts. Wageningen: Pudoc. Yaacob, O and H.D. Tindall. 1995. Mangosteen Cultivation. FAO Plant Protection Paper 129. Rome: Food and Agriculture Organization of the United Nations..

BIODIVERSITAS Volume 10, Number 4, October 2009 Pages: 168-174

ISSN: 1412-033X (printed edition) ISSN: 2085-4722 (electronic) DOI: 10.13057/biodiv/d100402

Genetic Relationship of Sago Palm (Metroxylon sagu Rottb.) in Indonesia Based on RAPD Markers 1,♥

BARAHIMA ABBAS , MUHAMMAD HASIM BINTORO , SUDARSONO , MEMEN SURAHMAN , HIROSHI EHARA 1

Faculty of Agriculture and Technology, State University of Papua (UNIPA), Manokwari 98314, Indonesia 2 Faculty of Agriculture, Bogor Agricultural University (IPB), Bogor 16680, Indonesia 3 Faculty of Bioresources, Mie University, 1577 Kurimamachiya,Tsu-city, Mie-Pref. 514-8507, Japan Received: 8th March 2009. Accepted: 20th July 2009.

ABSTRACT The areas of sago palm (Metroxylon sagu Rottb.) forest and cultivation in the world were estimated two million hectares and predicted 50% of that areas located in Indonesia. Distribution of sago palm areas in Indonesia is not evenly distributed as well as their diversities. Information of plant genetic diversities and genetic relationship is very important to be used for germplasm collection and conservation. The objectives of research were revealed the genetic relationships of sago palm in Indonesia based on RAPD molecular markers. Fragments amplification PCR products were separated on 1.7% agarose gel, fixation in Ethidium Bromide, and visualized by using Densitograph. Genetic relationships of sago palm in Indonesia showed that sample in individual level were inclined mixed among the other and just formed three groups. Genetic relationship of sago palm population showed that samples populations from Jayapura, Serui, Sorong, Pontianak, and Selat Panjang were closely related each others based on phylogenetic analysis and formed clustered in one group, event though inclined to be formed two subgroups. Populations from Manokwari, Bogor, Ambon and Palopo were closed related each others, they were in one group. Genetic relationships in the level of island were showed sago palm from Papua, Kalimantan, and Sumatra closely related. Sago palms from Maluku were closed related with sago palm from Sulawesi whereas sago palm from Java separated from the others. Based on this observation we proposed that Papua as centre of sago palm diversities and the origin of sago palm in Indonesia. This research informed us the best way to decide sago palm places for germplasm of sago palm conservation activity. © 2009 Biodiversitas, Journal of Biological Diversity Key words: genetic relationships, population, sago palm, RAPD, Indonesia.

INTRODUCTION Indonesia has the biggest sago palm (Metroxylon sagu Rottb.) forest and cultivation as well as its rich of genetic diversities. The areas of sago palm forest and cultivation in the world were predicted two million hectares and estimated 50% of that area located in Indonesia. Kertopermono (1996) reported that sago palm areas in Indonesia were larger than proposed by Flach (1983). According to measurement of Kertopermono (1996), sago palm areas in Indonesia were 1,528,917 ha and it was distributed into several locations in Indonesia. The locations of sago palm areas in Indonesia were observed in the previous studied, namely: Irian Jaya 1,406,469 ha, Ambon 41,949 ha, Sulawesi 45,540 ha, Kalimantan 2,795 ha, West Java 292 ha, and Sumatra 31.872 ha. The distribution of sago palm areas in Indonesia was not

 Corresponding address: Jl. Gunung Salju Amban, Manokwari 98314, Papua Barat Tel. +62-986-212095, Fax.: +62-.986-212095 e-mail: barahimabas@yahoo.com

evenly distributed as well as their diversities. Flach (1983) predicted that sago palm diversities in Indonesia were found higher in Papua islands (New Guinea) than other islands in Indonesia. Information of plant genetic diversities is very important to be used for germplasm collection and conservation. When germplasm conservation activity is done, information on genetic diversities are needed, especially from the natural habitat to carried out germplasm conservation efficiently. A popular DNA markers used for revealing genetic diversities and genetic relationships are Random Amplified Polymorphism DNA (RAPD) markers. The RAPD marker is one of many techniques used for molecular biology research. The advantages of RAPD markers are simpler in their preparation than other molecular markers. The other RAPD markers are easy applied for examining the diversities of organism (Powel et al., 1995; Colombo et al., 1998; Ferdinandez et al., 2001), because it is not using radioactive and relatively chief (Powel et al., 1995). Research which carried out for revealing genetic relationships by using RAPD markers were reported for Sorghum bicolor L. (Agrama and Tuinstra, 2003),

ABBAS et al. â&#x20AC;&#x201C; Genetic relationship of sago palm in Indonesia

Brassica oleracea L (Graci et al., 2001), and Medicago sativa L. (Mengoni et al., 2000). Whereas, study for genetic structure of population was reported for Acacia raddiana Savi (Shrestha et al., 2002), Pimelodus spp. (Almeida et al., 2004), and Primula elatior (L.) Oxlip (Jacquemyn et al., 2004).

MATERIALS AND METHODS Sago palm samples were collected from several islands in Indonesia. A total 100 samples of sago palm were collected from six islands and nine populations of sago palm centre in several islands in Indonesia. Location and geographical range of the selected sago palm stands were presented in Figure 1. The populations and the numbers of samples that were used in this experiment were presented in Table 1. Leaf samples were collected and preserved by using silica gel granules in zip lock plastic according to previous reported procedures (Chase and Hill,

169

1991). Isolation and extraction of total DNA from dried sago palm leaf samples were conducted using procedures as described in Qiagen DNA extraction kit o (Qiagen, 2003). The total DNA was stored in -20 C in freezer until ready for using. PCR Amplification RAPD primers used in this research were as follows: P01 (GCG GCT GGA G), P02 (GTG ACG CCG C), P04 (CGT CTG CCC G), P06 (TTC CGC GGG C), P17 (ATG ACG ACG G), OPG02 (GGC ATC GAG G), OPA04 (AAT CGG GCT G), OPAB04 (GGC ACG CGT T), OPAA17 (GAG CCC GAC T), and OPAB18 (CTG GCG TGT C). PCR mixtures and cycles condition were followed procedures described by Ehara et al. (2003) which has a little bit modification such as 0.12 ÎźM, 0.63 U Ampli Taq TM Gold , 10 ng DNA genome, 1.7% agarose gels for separating amplification fragments, and visualization by using Densitograph, Bioinstrument ATTA.

A G

F D

Figure 1. The map of sampling sites of sago palm used (scale 1: 39,800,000). The cycles represent the population sampling. A. Selat Panjang, B. Bogor, C. Pontianak, D. Palopo, E. Ambon, F. Sorong, G. Manokwari, H. Serui, I. Jayapura.

Table 1. The populations and the numbers of sample used Island Papua

Population Jayapura

Maluku Sulawesi Kalimantan Jawa Sumatra

Serui Manokwari Sorong Maluku Palopo Pontianak Bogor Selat Panjang

Numbers of sample 6, 7, 9, 11, 14, 24, 27, 34, 35, 49, 49, 50, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 1, 3, 5, 12, 18, 25, 26, 38, 43, 44, 47, 48, 73, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85 2, 4, 9, 20, 21, and 22 8, 13, 17, 28, 69, 70, 71, 72, 74 10, 41, 45 36, 37, 39, 40 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68 15, 16 23, 29, 30, 31, 32, 33, 42

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Data analysis Dissimilarity matrix was calculated by using distance coefficient. The dissimilarity matrix was employed to construct phylogenetic by the Unweighted PairGroup Method Arithmetic Average (UPGMA), using the Sequential Agglomerative Hierarchical Nested Cluster Analysis (SAHN-clustering, Sneath and Sokal, 1973) and TREE program from NTSYS-pc, version 2.02 packages (Rohlf, 1998). Bootstrap analysis with permutation 10,000 times were performed by using software Tools for Genetic Analysis (TFPGA 1.3). Ordinate analysis calculated by using Multidimensional Scaling (MDS) and performed by using NTSYS 2.02 Package (Rohlf, 1998). RESULTS AND DISCUSSIONS RAPD Polymorphism Polymorphisms of RAPD amplification fragments by using ten RAPD primers and performed in the PCR tools were resulted 86 numbers of polymorphic fragments and two to seven genotype numbers per population. Samples DNA Fragments resulted by PCR were shown in Figure 2. High numbers of RAPD polymorphisms and genotypes were found in this observation. These results were similarly with genetic diversity of sago palm in the previous study, by Ehara et al. (2003) by using RAPD markers utilizing small amount individual sago palm samples from Indonesia and Malaysia. Fig 2 showed that the performance samples of DNA bands were amplified by using 10 primer sets. Numbers of fragment DNA band were amplified from each primer, and it was ranging from 6 to 12 polymorphic bands per primers and no monomorphic DNA band was observed. The averages polymorphic DNA bands were calculated 9 per primer. Primer P17 was resulted the highest numbers of polymorphic DNA bands that was 12 DNA bands, whereas primers OPA04 and P06 produced the lowest numbers of polymorphic DNA bands that were produced 6 polymorphic DNA bands per primers. Base pairs sizes of DNA bands produce by 10 primer sets were ranging from 150 bp (base pairs) to 1800 bp. Overall primers used in this observation

were suitable for studying genetic of sago palm. The previous of this observation applied more than 100 RAPD primers sets. Genetic relationships in the level of individuals Genetic relationships in individual levels showed that the samples divided into three groups based on phylogenetic construction (Figure 3) and three clusters based on multidimensional scaling analysis (Figure 4). Numbers of individual samples associated in group I were the sample number 2, 10, 13, 15, 16, 17, 20, 21, 22, 23, 33, 34, 39, 40, 42, 43, 44, and 62; group II were the sample number 6, 9, 14, 24, 25, 26, 27, 41, 49, 51, 58, 75, 95, and 97; group III were the sample number 1, 3, 4, 5, 7, 8, 11, 12, 18, 19, 28, 29, 30, 31, 32, 35, 36, 37, 38, 45, 46, 47, 48, 50, 52, 53, 54, 55, 56, 57, 59, 60, 61, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91`, 92, 93, 94, 96, 98, 99, and 100. The individual samples in group I and group III were associated individual samples from overall populations. Group II individual samples associated with population from Jayapura, Serui, Manokwari, Ambon and Pontianak. These grouping were similarly with sago palm grouping by Ehara et al. (2003) which divided sago palm samples from Indonesia and Malaysia into two groups and sub group based on RAPD markers. Papua islands in Indonesia were shown that individual samples divided into three groups also based on cp-DNA markers (Barahima et al., 2005). Based on our observation, we proposed that sago palm in Indonesia classified into three groups. Individuals grouping in the phylogenetic construction were based on genetic distances, grouping methods, and coefficient used or bootstrapping levels. In our observation showed that the different genetic markers used did not change grouping pattern of sago palm. Some cases in the molecular analysis, the dissimilarities grouping pattern, by using the same markers or different markers, were found frequently in the studied of genetic relationships (Ishikawa et al., 1992; Viard et al., 2001; Panda et al., 2003).

10 12 14 17 18 21 22 25 M 24 27 28 29 30 36 37 39 49 50 51 52 53 54 55 56 M 57 58 59 60 61 62 63 64

Figure 2. Performance of RAPD fragment by using OPAA17 primers on 1.7% agorose gels. Marker (M) and the number of well (10 to 64) indicated number of sago palm samples.

ABBAS et al. â&#x20AC;&#x201C; Genetic relationship of sago palm in Indonesia

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0.74 0.80 0.73

III

0.64 0.48 0.55 0.77 0.85 0.87 0.87

0.53 1.00

Figure 3. Phylogenetic of samples in the level of individuals based on 86 loci and 10 RAPD primers of 100 individuals samples by using UPGMA clusters and bootstrap by using 10,000 permutations.

Figure 4. Ordinate analysis of individual level by using MDS based on 86 loci, 10 RAPD primers, and 100 individuals of sago palm. Two dimension scales (4A) and three dimensional scales (4B). Individual samples from Jayapura ( ), Serui ( ), Manokwari ( ), Sorong ( ), Ambon ( ), Palopo ( ), Potianak ( ), Bogor ( ), Selat Panjang ( ).

Genetic relationships in the level of populations Phylogenetic construction show that sago palm samples in the population levels was divided into two groups, those were group I and II. The group I was inclined to form two sub groups because bootstrap value was high (0.99) in one of finger phylogenetic (Figure 5) and two clusters based on MDS analysis (Figure 6). The group I included population sample from Jayapura, Serui, Sorong, Pontianak, and Selat Panjang. The group II was associated population sample from Manokwari, Ambon, Palopo, and Bogor. The group I will be divided into two sub groups. The subgroup I included population from Jayapura, Serui, and Sorong and the subgroup II included population from Pontianak and Selat Panjang. The genetic relationships in the level of population showed the same pattern with individual levels, even though samples in the level of population just inclined to form

three groups, but solid pylogenetic construction only showed two groups (Figure 4). Variation levels were detected in this observation similarly with genetic variation of Cynara scolymus L. by using RAPD markers (Lanteri et al. 2001) and Medicago sativa L. (Mengoni et al. 2000). The differences of relationships among population probably were caused by out breeding, so that populations become different. Population differences may be owing to pollen migration (Latta and Mitton 1997). Generally, pollination of sago palm occurred a cross pollination since male and female flower mature in different of time period (Jong, 1995). Cross pollination process in sago palm may cause population different. Association sample population from Jayapura, Serui, Sorong, Pontianak, and Selat Panjang to form one group in the phylogenetic construction probably owing to sago palm interchange from one population

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0.94 0.99 0.62 0.96 1.00

Jayapura

Subgroup I

Serui Group I

Sorong Pontianak

0.40 0.37

Subgroup II

Selat Panjang Manokwari Group II

Bogor 0.60

Ambon Palopo

Figure 5. Phylogenetic of samples in the level of populations based on 86 loci and 10 RAPD primers of 100 individuals samples by using UPGMA clusters and bootstrap by using 10,000 permutations.

Figure 6. Ordinate analysis of population level by using MDS based on 86 loci, 10 RAPD primers, and 100 individuals of sago palm. Two dimension scales dimension (6A) and three dimensional scales (6B). Populations from Jayapura ( ), Serui ( ), Manokwari ( ), Sorong ( ), Ambon ( ), Palopo ( ), Potianak ( ), Bogor ( ), Selat Panjang ( ).

to another population which carried by people. In this research we do not know exactly, when sago palm came of exchange and where sago palm population originated. Based on sago palm diversities and natural stand we found that the largest variation and the largest natural stand in the population from Papua. Sago palm population from Jayapura we found the largest variation and the largest vernacular name was given by local people. Matanubun et al. (2005) reported that there were 96 sago palm varieties in Papua based on morphology characteristic and Yamamoto (2005) reported that there were 15 sago palm varieties in Jayapura based on morphological characters. Population from Jayapura has the largest variation of sago palm. Based on that data, we can estimate that the population origin of population in group I came from Jayapura population. Populations formed in group II, we predicted also caused by interchange individual of sago palm in the past through people mobilization from one place to another place. Therefore, the population in one group such as group II have average genetic distance closed each others. We

have no sufficient data to estimate the population origin in group II. This research gives us information for the best way to chose sago palm places for germplasm of sago palm conservation activities. Genetic relationships in the level of islands The genetic relationships of sago palm in the level of island showed that it also formed three groups as shown on individual levels. Sago palm sample from Papua, Kalimantan and Sumatra were observed and show genetic distance closed each others, and formed Group I. Sample from Ambon and Sulawesi formed Group II, and sample from Java formed Group III in the phylogenetic construction. The genetic relationships based on phylogenetic construction (Figure 7) and MDS analysis (Figure 8) showed that samples in island levels were closely related between samples from Papua, Kalimantan and Sumatra. Samples from Sulawesi islands were closely related with samples from Ambon. Samples from Java island were separated with samples from the others island based on RAPD markers. There was very interesting phenomenon, at which we should pay attention,

ABBAS et al. â&#x20AC;&#x201C; Genetic relationship of sago palm in Indonesia

samples in the island levels formed the same group with samples from other islands, which have distances far away each other. Those shown by Papua island were in the same group with Sumatra island (Figure 7) at group I. This phenomenon may be occurred owing to samples in individual levels from Papua have genetic distances more closely than individual samples from Sumatra, which made total genetic distance between Papua and Sumatra closed each others. If we estimated through migration aspects, probably individual of sago palm from Papua mixed with sago palm individual from Sumatra in the past by people mobilization/migration. During the Dutch colonization in Indonesia, people already moved from Sumatra to Papua or the other way around, with probably people carrying sago palm plant and growing at new places for anticipating food crisis in the future. Features of sago palm in Papua have highest variation, largest sago palm forest, many wild types, and semi cultivated. Sago palm features in another island in Indonesia Such as Sumatra, Kalimatan, Java, Sulawesi, and Maluku were found sago palm cultivated, semi cultivated, low

0.54 0.99

variation, no wild types, and no sago palm forest. Therefore we estimated the origin of sago palm in Indonesia come from Papua. The genetic distances of sago palm from Papua were assayed closed with sago palm from Sumatra. Probably, sago palm from Papua moved to Sumatra which carried out by people when they moved from Papua to Sumatra in the past and formed a new population in the new places. This prediction may occur because RAPD markers which used did not show as conservative as cpDNA markers which uniparental inherited (Ishikawa et al., 1992; Savolainen et al., 1995). RAPD markers are molecular nuclear genome which related with DNA recombinant process and biparentally inherited (Viard et al., 2001). Therefore, RAPD markers are molecular markers which it have no longer conservative periods time rather than cpDNA markers. In the previous studies at different plants showed that higher variation were found by using nuclear genome markers (RAPD, AFLP, ISSR, and nuclear SSR), then using chloroplast genome markers such as cpDNA markers (Hultquist, 1996; Viard et al., 2001; Cronn et al., 2002; Panda et al., 2003).

Papua Kalimantan

0.89

173

Group I

Sumatra Ambon

1.00 0.41

Group II Sulawesi Jawa

Group III

Figure 7. Phylogenetic of samples in the level of islands based on 86 loci and 10 RAPD primers of 100 individuals samples by using UPGMA clusters and bootstrap by using 10,000 permutations.

Figure 8. Ordinate analysis of island level by using MDS based on 86 loci, 10 RAPD primers, and 100 individuals of sago palm. Two dimension scales dimension (8A) and three dimensional scales (8B). Samples from Papua ( ), Ambon ( ), Sulawesi ( ), Kalimantan ( ), Jawa ( ),Selat Panjang ( ).

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CONCLUSIONS Genetic relationships of sago palm in Indonesia showed that sago palm in individual level were inclined to mix among the others, and just formed three groups. Sago palm population from Jayapura, Serui, and Sorong were closely related; sago palm from Manokwari, Bogor, Ambon, and Palopo were closely related; and sago palm from Pontianak was closely related with sago palm from Selat Panjang. In the level of Islands which has long geographical distance showed that sago palm from Papua island closed related with sago palm from Kalimantan and Sumatra island. Sago palm from Ambon closely related with sago palm from Sulawesi, and sago palm from Jawa island not formed cluster with sago palm from the other islands. Thus, we proposed that Papua is as centre of sago palm diversity, and the origin of sago palm in Indonesia. This research informed us the best way to decide sago palm places, for germplasm and sago palm conservation activity.

REFERENCES Agrama, H.A and M.R. Tuinstra. 2003. Phylogenetic diversity and relationship among sorghum accessions using SSRs and RAPDs. African Journal Biotechnology 2 (10): 334-340. Almeida, F.S.D, L.M.K. Sodre, and E.P.B. Contel. 2004. Population structure analysis of Pimelodus maculates Pisces, Siluriformes) from the Tiete and Paranapanema Rivers (Brazil). Genetic and Molecular Biology 26 (3): 301-305 Barahima, A, M.H. Bintoro, Sudarsono, M. Surahman, and H. Ehara. 2005. Haplotype diversity of sago palm in Papua based on chloroplast DNA. In: Karafir, Y.P., F.S. Jong, and V.E. Fere (eds). Sago Palm Development and Utilization. Proceeding of the Eighth International Sago Symposium in Jayapura, Indonesia. Japan Society for the Promotion Science, Jayapura, 4-6 August 2005. Chase, M. and H. Hill. 1991. Silica gel: an ideal material for field preservation of leaf samples. Taxon 40: 215-220. Colombo, C., G. Second, T.L. Valle, and A. Charrier. 1998. Genetic diversity characterization of cassava cultivars (Manihot esculenta Cranz.) RAPD markers. Genetic and Molecular Biology 21: 69-84. Cronn, R.C., R.L. Small, T. Haselkorn, and J.F. Wendel. 2002. Rapid diversification of the cotton genus (Gossypium: Malvaceae) revealed by analysis of sixteen nuclear and chloroplast genes. American Journal of Botany 89 (4): 707-725. Ehara, H., S. Kosaka, N. Shimura, D. Matoyama, O. Morita, H. Naito, C. Mizota, S. Susanto, M.H. Bintoro, and Y. Yamamoto. 2003. Relationship between geographical distribution and genetic distance of sago palm in Malay Archipelago. Sago Palm 11: 8-13. Ferdinandez, Y.S.N., D.J. Somers, and B.E. Coulman. 2001. Estimating the genetic relationship of hybrid bromegrass to smooth bromegrass and medow bromegrass using RAPD markers. Plant Breeding 120: 149-153. Flach, M. 1983. The Sago Palm. Domestication, Exploitation, and Product. Rome: FAO Plant Production and Protection. Graci, A., I. Divaret, F.M. Raimondo, and A.M. Chevre. 2001. Genetic relationships between Sicilian wild populations of

Brassica analyses with RAPD markers. Plant Breeding 120: 193-196. Hultquist, S. J., K.P. Vogel, D.J. Lee, K. Arumuganathan, and S. Kaeppler. 1996. Chloroplast DNA and nuclear DNA content variations among cultivars of Switchgrass, Panicum virgatum L. Crop Science 36: 1049-1052. Ishikawa, S., S Kato, S. Imakawa, T. Mikami, and Y. Shimamoto. 1992. Organelle DNA polymorphism in apple cultivars and rootstocks. Theoretical and Applied Genetics 83: 963-967. Jacquemyn, H., O. Honnay, P. Galbusera, and I.R. Ruiz. 2004. Genetic structure of forest herb Primula elatior in a changing landscape. Molecular Ecology 13: 211-219. Jong, F.S. 1995. Research for the Development of Sago Palm (Metroxylon sagu Rottb.) Cultivation in Sarawak, Malaysia. Kuching, Sarawak: Department of Agriculture, Malaysia. Kertopermono, A. P. 1996. Inventory and evaluation of sago palm (Metroxylon sp.) distribution. Sixth International Sago Symposium. Pekan Baru, 9-12 December 1996. Lanteri, S., I.D. Leo, L. Ledda, M.G. Mameli, and E. Portis. 2001. RAPD variation within and among population of globe artichoke cultivar â&#x20AC;&#x2DC;Spinoso sardoâ&#x20AC;&#x2122;. Plant Breeding 120: 243-246. Latta, R.G. and J.B. Mitton. 1997. A comparison of population differentiation across four classes of gene marker in limber pine (Pinus flexilis James). Genetics 146: 1153-1163. Matanubun, H., B. Santoso, M. Nauw, A. Rochani, M.A.P. Palit, D.N. Irbayanti, and A. Kurniawan. 2005. Feasibility study of the natural sago sago forest for the establishment of the commercial sago palm plantation at Kaureh District, Jayapura, Papua, Indonesia. Sago Palm Development and Utilization. Proceeding of the Eighth International Sago Symposium in Jayapura, Indonesia. Japan Society for the Promotion Science, Jayapura, 4-6 August 2005. Mengoni, A., A. Gori, and M. Bazzcalupo. 2000. Use of RAPD and micro satellite (SSR) variation to assess genetic relationships among populations of tetraploid alfalfa, Medicago sativa. Plant Breeding 119: 311-317. Panda, S., J. P. Martin, and I. Agunagalde. 2003. Chloroplast and nuclear DNA studies in a few members of the Brassica oleracea L. group using PCR-RFLP and ISSR-PCR markers: a population genetic analysis. Theoretical and Applied Genetics 106: 1122-1128 Powel, W., C.O. Castillo, K. J. Chaluers, J. Provan, and R. Waugh. 1995. Polymerase chain reaction based-assays for the characterization of plant genetic resources. Electrophoresis 16: 1726-1730. Rohlf, F. J. 1998. NTSYS-pc. Numerical Taxonomy and Multivariate Analysis System. Version 2.02. New York: Exter Sift Ware.. Savolainen, V., R. Corbaz, C. Moncousin, R. Spchiger, and J.F. Manen. 1995. Chloroplast DNA variation and parentage analysis in 55 apples. Theoretical and Applied Genetics 90: 1138-1141. Shrestha, M. K., A.G. Goldhirsh, and D. Ward. 2002. Population genetic structure and the conservation of isolated population of Acacia raddiana in the Negev Desert. Biological Conservation 108: 119-127. Sneath, P. H. and R. R. Sokal. 1973. Numerical Taxonomy. San Francisco: Freeman. Viard, F., Y.A.E. Kassaby, and K. Ritland. 2001. Diversity and genetic structure in populations of Pseudotsuga menziesii (Pinaceae) at chloroplast micro satellite loci. Genome 44: 336344. Yamamoto Y, Yoshida T, Miyazaki A, Jong FS, Pasolon YB, and Matanubun H. 2005. Biodiversity and productivity of several sago palm varieties in Indonesia. Proceeding of Eighth International Sago Symposium in Jayapura, Indonesia. Japan Society for the Promotion Science, Jayapura, 4-6 August 2005.

BIODIVERSITAS Volume 10, Number 4, October 2009 Pages: 175-180

ISSN: 1412-033X (printed edition) ISSN: 2085-4722 (electronic) DOI: 10.13057/biodiv/d100403

Mycorrhizal Association of Terrestrial Orchids of Cycloops Nature Reserve, Jayapura ♥

VERENA AGUSTINI , SUPENI SUFAATI, SUHARNO Biologi Department, Faculty of Mathematics and Natural Science, Cenderawasih University (UNCEN), Jayapura 99358, Papua Received: 14th December 2008. Accepted: 15th June 2009.

ABSTRACT Study on exploration of mycorrhizal association of terrestrial orchid of Cycloops Nature Reserve, Jayapura was done. The aims of this study were to collect terrestrial orchid and to isolate orchid mycorrhiza associated with it. Survey method was used in this study. Isolation of orchid mycorrhiza was based on modified methods of Masuhara and Katsuya (1989). The result showed that there were 10 species of terrestrial orchid in this area. Eleven orchid mycorrhizal fungi were isolated from five terrestrial orchids. Among them, 6 isolates were associated with Geodorum sp. From the seventeen mycorrhizal fungi, 3 isolates were identified, namely Rhizoctonia sp., Tulasnella sp., and Ceratorhiza sp, while the last fourteen isolates have not been identified yet. Mostly, each isolate has a specific orchid host, except species G (sp. G) which associated with Phaius sp. and Plocoglottis sp. © 2009 Biodiversitas, Journal of Biological Diversity Key words: terrestrial orchid, orchid mycorrhiza, Mt. Cycloops Nature Reserve, Jayapura.

INTRODUCTION Papua contains very high level of plant diversity. It may have at least 20.000-25.000 species of vascular plants including orchids. 3000 species of orchids are found in Papua, mostly are epiphytic. The exploration of orchids in Papua remains uncompleted due to a complicated geographic mosaic. Some orchid species have restricted ranges as a consequence of the complex geologic history of the island and its numerous barriers to dispersals. One area which should be explored is Cycloops Nature Reserve (Cagar Alam Pegunungan Cycloops, CAPC). It is totally 22.520 ha (SK Mentan No.05/KPTS/UM/1978). All orchids, epiphytic or terrestrial and autotrophic or heterotrophic heavily dependent on fungi for their existence. It is different with the symbiosis between plant and fungi called AM (arbuscular mycorrhizal), and also ECM (ectomycorrhizal). The role of the fungi is unique, which known to serve as orchids mycorrhiza. Terrestrial orchids, in their habitats require the presence of suitable fungi in the living cells of the plant embryo and development of multicellular absorptive structures in order to develop and mature successfully (Currah et al., 1990)

 Corresponding address: Kampus UNCEN WAENA, Jl. Kamp Wolker, Jayapura 99358 Tel./Fax.: +62-967-572115 email: verena_agustini@yahoo.com.

Many researches have been done for mycorrhizal of terrestrial orchids in temperate area, but only a few in tropical area. Mycorrhizal fungi are associated with root systems of more than 90% of terrestrial plant species in a mutual symbiosis. In nature, all orchids utilize endomycorrhizal fungi to initiate seed germination and seedling development. The availability of each fungi, therefore, is an absolute requirement of orchid life cycle The orchids-fungus symbiosis is initiated when orchid seeds are infected by a suitable fungus (Arditti, 1982; Rasmussen, 1995) Orchids inoculated by fungi isolated from other plant did not show any positive effect on seedling development (Agustini and Kirenius, 2002). Dendrobium seedling inoculated with orchidmycorrhiza isolated from other orchids shown a better growth than non inoculated one (Agustini, 2003). Most of orchid-mycorrhiza is endomycorrhiza. Fungi associate with photosynthetic orchid mostly belong to subdivision Basidiomycotinae, class Hymenomycetes, genus Rhizoctonia. Otero et al. (2002) reported that among 9 species of Puerto Rican orchids there were 108 Rhizoctonia like fungi which belong to Tulasnella, Ceratobasidium, and Thanatephorus. Studies by Taylor and Bruns (1997) and Taylor et al. (2004) shown that 17 to 22 fungi species of Russulaceae are associated with Corallorhiza maculata. Limodorum abortivum, an orchid grown in Mediterranean is also associated with fungi belong to family Russulaceae (Girlanda et al., 2006). Russulaceae is basidiomycetes-ectomycorrhizal.

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Orchids of CAPC still remain as one of the least studied (Pokja Cycloops, 2003), therefore some areas such as surrounding Jayapura city, 6 species of terrestrial-orchids were found (Numberi, 2005). Study for the rest of CAPC areas is needed. Because of virtuallyl lack of knowledge in the biodiversity of the mycorrhizal fungi of tropical Orchidaceae, the distribution and identification of fungi from variety of terrestrial orchids were examined. The objectives were to make an inventory of terrestrial-orchids of CPAC area, and to isolate and identify the naturally occurring mycorrhizal fungi of terrestrial orchids from various habitats. MATERIALS AND METHODS The status of orchids-mycorrhiza Survey on status of orchids-mycorrhiza was done due to determine whether any infection of fungi in the living cells of the roots of the developing orchids or not. Healthy roots of terrestrial-orchids were collected from CPAC areas, namely: (i) UNCEN Campus at Waena (Kamp Walker and Buper), (ii) Sentani (Kemiri and Kampung Harapan), and (iii) downtown city of Jayapura. They were taken, wrapped in tissue paper, put in plastics bag and bring to the laboratory. Collection and identification of terrestrial orchids Sample of terrestrial orchids were collected from various locations of CPAC areas. They were collected ex situ on sterile media due to collect fungi from the orchids. The samples were identified at Botany Laboratory, Cenderawasih University, Jayapura. Some references like Becker and Bakhuizen van den Brink (1963, 1965, 1968); Comber (1990); Segerback (1992); Schuiteman (1995); Mahyar and Sadili (2003); Banks (2004) were used. Fungal isolation The root segment taken from root tip was washed with distilled water to remove any soil. For surface sterilization they were treated with ethanol 70% for 30 second. The roots were then cut into transverse section about 300 Âľm thick and observed for the presence of hyphal coil (polotons) on a glass slide under a stereo-microscope in sterile condition. Then they were culture onto potato dextrose agar (PDA) media in petridishes, three pieces in each petridish. (Irawati, 2006; pers. comm). Mycorrhizal fungi were isolated using a modification of Masuhara and Katsuya Methods (Manoch and Lohsomboon, 1991). After 1-2 days incubation, the hyphae will grow. If it shows any differences in shape and pattern of growth, then they will be isolated and culture on PDA in order to separate the variety of the fungi. Pure cultures were maintained on PDA slant. Fungal identification Macroscopic features examined were colony growth pattern, color, and mycelia formation. Fungal

growth rate was measured from the colony on PDA. For microscopic examination, fertile hyphae were mounted in sterile water on a microscopic slide, covered with a cover slip, and examine under light microscope. Hyphal and monilioid cells were measured. Literatures used in the fungal identification were Sharma et al. (2003) and Athipunyakom et al. (2004).

RESULTS AND DISCUSSION Terrestrial orchid species Ten terrestrial orchids were found in this study. Among the tenth species, 3 are found in campus areas, seven others are found in Sentani areas (Table 1). Table 1. The location of terrestrial orchids of CPAC No. of collection WAE-01 WAE-02 SEN-01 SEN-02 WAE-03 WAE-04 WAE-05 SEN-10 SEN-04 SEN-05 SEN-06 SEN-07 SEN-08 SEN-09 JAP-06

Name of species

Location

Spathoglottis plicata L.

Campus Campus (Buper) Sentani (Kemiri) Sentani (Harapan) Phaius tankervilleae Campus Campus Geodorum sp. Campus Sentani (Harapan) Calanthe sp.1 Sentani (Harapan) Calanthe sp.2 Sentani (Harapan) Plocoglottis sp.1 Sentani (Harapan) Plocoglottis sp.2 Sentani (Harapan) Plocoglottis sp.3 Sentani (Harapan) Paphiopedilum violascens Schltr. Sentani (Harapan) Macodes petola Jayapura

The result is slightly different with Numberiâ&#x20AC;&#x2122;s studied on 2005. She found 6 species of terrestrial orchids growth at city of Jayapura areas including campus surrounding areas. 4 species namely Spathoglottis sp., Phaius sp., Geodorum sp., and Macodes sp. are found in Jayapura surrounding areas. Two of them Spathoglottis sp. and Geodorum sp. were found in broader areas of study. Spathoglottis sp. can be found in many habitats; from open areas to shading areas in forest either secondary or tertiary. The variety of habitat of Spathoglottis sp. make the flowers have different colors which were identified by Numberi (2005) as different species. It is stated in some literatures that wide ranges of habitat might caused the variety of color of flower. Among the tenth species, some mycorrhizal fungi has been isolated just from 8 species, fungi from the 2 species remains unisolated. The results of the present study showed that one orchid was associated with a number of mycorrhizal fungi, for example Geodorum densiflorum associated with 6 mycorrhizal fungi; Plocoglottis sp.3 associated with 3 mycorrhizal

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Table 2. Mycorrhizal fungi isolated from terrestrial orchids of CPAC. No. of Mycorrhizal Species Habitat fungi collection WAE-01 Spathoglottis sp. Rhizoctonia Open areas sp.

Location

Isolate characteristics

Campus On PDA, colony growth slow, 4,5 cm in diameter after 4 days incubation, thin mycelia, hyalin, globose, hyphae septate. Tulasnella Open areas Campus On PDA colony growth slowly but faster than Rhizoctonia sp., mycelium white dense, hyphae sp. branches. Diameter 5,5 cm after 4 days incubation. Hyphae septate, rare, some of hyphae have branches, upright angles thicken. Conidia globe to ellipsoidal. WAE-05 Geodorum Sp.A *) Shading/ Campus On PDA colony growth rapidly, mycelia dense, hyphae densiflorum canopy areas branches rarely, end of hyphae bearing abundant conidia, globe. Sp.B *) Shading/ Campus On PDA colony growth slowly, thin mycelia, hyphae canopy areas branching often at nearly upright angles but more o commonly at 40-60 Ceratorhiza Shading/ Campus On PDA colony growth slowly, mycelia thin, hyphae sp. canopy areas branching, end of hyphae bearing abundant monilloid cells with typical mode of attachment in a chain 5-6 globes. Sp.D *) Shading/ Campus On PDA colony growth more slowly, dense, circle canopy areas growth, black-white. Mycelia thin, septate, abundant globulus oil. Fungi isolated from terrestrial orchid Sp.E *) Shading/ Campus On PDA colony growth slow, circle growth, black and canopy areas white. Concentric zonation, mycelia dense, septate dense, a few number of globulus oil. Sp.F *) Shading/ Campus On PDA colony growth slowly. Mycelia hyaline, canopy areas branched, single globulus. A few number of coil. WAE-03 Phaius sp. Sp.G *) Shading areas, Campus On PDA colony growth rapidly. Hyphae dense, next to creek. branches, peloton. Hyphae septate, multinucleate, a few number of globulus. SEN-04 Calanthe sp.1 Sp.H *) Secondary Harapan, On PDA colony growth rapidly, densely hyphae, forest Sentani branches. Characteristics globulus with stem on right and left of main hyphae. SEN-05 Calanthe sp.2 Sp.J *) Secondary Harapan, On PDA colony growth rapidly, white until optimal forest Sentani growth, turn on black color. Diameter 1 cm after 1 day incubation, and 5.5 cm after 5 days of incubation. Hyphae dense, globulus branches. SEN-06 Plocoglottis sp.1 Sp.I *) Secondary Harapan, On PDA colony growth rapidly, dense, parallel growth forest Sentani hyphae, on tip of hyphae abundant granule. SEN-07 Plocoglottis sp.2 unisolated Secondary Harapan, forest Sentani SEN-08 Plocoglottis sp.3 Sp.G *) Secondary Harapan, On PDA colony growth rapidly. Hyphae dense, forest Sentani branches, peloton. Hyphae septate, multinucleate, a few number of globulus. Sp.K *) Secondary Harapan, On PDA colony growth rapidly, white, dense at center, forest Sentani hyphae thin, branching upright, smaller branches loosely arrange. Bearing abundant globulus, especially at the tip of growing hyphae. Sp.L *) Secondary Harapan, On PDA colony growth slowly after 15 days of forest Sentani incubation the color turn in black, concentric zonation, dense and thin alternately. Mycelia growth fast and dense. Abundant granular. SEN-09 Phapiopedilum Sp.M *) Secondary Harapan, On PDA colony growth rapidly, white, with dense aerial violascens Schltr. forest Sentani mycelium in the centre. Hyphae septate, dense, granular rare. Host: Paphiopedilum violascens Schltr. Sp.N *) Secondary Harapan, On PDA colony growth rapidly, white, hyphae dense, forest Sentani branching densely, with abundant globular. Host: Paphiopedilum violascens Schltr. JAP-06 Macodes sp. unisolated Secondary Jayapura forest Note: *) unidentified.

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Figure 1. Six isolates taken from the root of terrestrial orchid Geodorum sp.: A. Sp.A, B. Sp.B, C. Ceratorhiza sp., D. Sp.D, E. Sp.E, and F. Sp.F (400x).

Figure 2. Reproductive portion of mycorrhiza. A. Hyphae of Rhizoctonia sp. 4 days on PDA. 400 x. B. Hyphae Sp.G taken from Phaius sp., septate, multinucleate, globulus oil, 1000x. C. Conidia of Tulasnella sp., 400 x. D. Hyphae supporting conidiospores, 100x.

Figure 3. Colony of mycorrhiza. A. Sp.I: colony on PDA showing concentric zonation, 100x; B. Sp.I: abundant granule on tip of hyphae, 100x; C. Sp.J: colony on PDA; D. Sp.J: branches hyphae with globulus, 400x; E. Sp.L: colony on PDA showing concentric zonation and black in color after 15 days incubation; F. Sp.L: hyphae with abundant granule, 100x; G. Sp.M: dense and septate hyphae with few granule, 1000x; H. Sp.N: dense and branching hyphae with abundant granule, 100x.

AGUSTINI et al. â&#x20AC;&#x201C; Mycorrhiza of Cycloops terrestrial orchids

fungi (sp.G, sp.K, and sp.L); Spathoglottis and Paphiopedilum violascens associated with 2 mycorrhizal fungi each. The study also found that a number of mycorrhizal fungi were associated with a single orchid host, for instance Rhizoctonia sp. and Tulasnella sp. are associated with Spathoglottis whereas Mycorrhizal fungi sp.M and sp.N are associated with Paphiopedilum violascens (Figure 3).The other orchids are associated with single fungus. This study indicated that mycorrhiza sp.G was isolated from Phaius sp. and also from Plocoglottis sp.3. This study showed that from the two orchids Plocoglottis sp.2 and Macodes sp., mycorrhizal fungi remained unisolated. There are 17 mycorrhizal fungi isolated from 8 orchids of CPAC areas (Table 2). Athipunyakom et al. (2004) did a similar study, and found 14 mycorrhizal fungi from eleven terrestrial orchids in Thailand, among them are Rhizoctonia globularis, Ceratorhiza sp., and Tulasnella sp. Association of mycorrhiza and terrestrial orchid The results of the study shown that a number of mycorrhizal fungi were associated with only one orchid. There were six mycorrhizal fungi associated with Geodorum sp. (Figure 1). Rhizoctonia sp. and Tulasnella sp. are symbiont of Spathoglottis.(Figure 2). According to Athipunyakom et al. (2004), Tulasnella sp. Is also associated with Cymbidium tracyanum, in root of Calanthe sp. was found fungi Epulorhiza and Ceratorhiza, while in this study Ceratorhiza fungi were isolated from Geodorum sp. Fungi sp.G are associated with both Phaius and Plocoglottis sp.3. Kristiansen et al. (2004) also found a similar situation that many fungi were associated with one specific orchid. Almost all mycorrhiza associated with terrestrial orchid is Rhizoctonia including anamorphic of Tulasnella, Ceratobasidium, and Thanatephorus (Otero et al., 2002; Bonnardeaux et al., 2007). Fungi known to be associated with Spathoglottis sp. is Rhizoctonia, similar to study done by Tan et al. (1998) using terrestrial orchid Spathoglottis plicata. He inoculated Rhizoctonia AM9 on the media to germinate seed. The study showed that there is important role of the fungi on Spathoglottis plicata seed germination. Hayakawa et al. (1999) showed different results, there was no significant effect of the isolate on in vitro seed germination. In contrast, this study indicated that one fungi merely associated with a single orchid host. Calanthe sp.1 and Sp.H and Calanthe sp.2 and Sp.J which both remained unidentified (Table 2). Study of symbiosis of fungi and orchid is extremely unique, mainly Pterostylis, Caladenia and Thelymitra. These fungi grow in very close habitat but they have different host, namely Ceratobasidium, Sebacina and Tulasnella (Andersen and Rasmussen, 1996). The Fungi Rhizoctonia is known as a symbiont of Spathoglottis sp, and is found in Pinus radiata root as

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well. Furthermore, Bidartondo et al. (2004), reported that orchids grow under canopy can be associated both with fungi mycorrhiza and trees at the surrounding habitat. Fungi which are associated with orchid are basidiomycetes. Some of them are saprophytic, ectomycorrhiza, and plant parasitic (Rasmussen, 2004), while Yamato et al. (2005) said that achroroplilous Epipogium roseum also known associated with Coprinus which is saprophytic fungi. The specific relationship between fungi and orchid also reported by Rasmussen (2004). The specificity was found from taxon species to subtribe (Warcup, 1981). Photosynthetic orchids showed a high specificity in association of mycorrhizal-orchids (Shefferson et al., 2005). Specificity possibly leads to high rates of orchid seed germination and a more efficient physiological association when the interaction is fully functional (Bonnardeaux et al., 2007). Mycoheterotrophic orchid Corallorhiza maculata was associated with twenty two fungi belong to family Russulaceae which is ectomycorrhizal in plants (Taylor et al., 2004). In this study, transversal root segments of terrestrial orchids were used as a source of mycorrhizal fungi, this method were also used by Shan et al. (2002) and Bonnardeaux et al. (2007). Other isolates source of orchids-fungi were hyphal coils (peloton) taken from longitudinal sections of roots (Athipunyakom et al., 2004; Bonnardeaux et al., 2007).

CONCLUSION Ten terrestrial orchids were found in Cagar Alam Pegunungan Cycloops, Jayapura. From eight of the ten orchids there were seventeen mycorrhizal orchids, three of them are identified as Rhizoctonia sp., Tulasnella sp., and Ceratorhiza sp. Dari Among them six fungi isolated from a single orchid Geodorum sp. In this study the specificity was found in most mycorrhizal-fungi except Sp.G which found in two different terrestrial orchid species namely Phaius sp. and Plocoglottis sp.

ACKNOWLEDGEMENTS We thank to the Directorate General of Higher Education, Ministry of National Education through Fundamental Research Grant, 2007 and Indonesia Managing Higher Education for Relevance and Efficiency Project (I-MHERE Project) Sub-Component B.1. Batch II, through Research Grant Program 2007 for financial support, and we also would like to thank to Ira Aldila Putri and Dewi Katemba for their laboratory work.

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REFERENCE Arditti, J. 1982. Orchid seed germination and seedling culture-A manual. In: Arditti, J. (ed.) Orchid Biology: Reviews and Perspectives II. London: Cornell University Press. Agustini, V. 2003. Peranan mikoriza-anggrek pada pertumbuhan anggrek Dendrobium sp. Sains 3 (2): 39-42. Agustini, V. dan M. Kirenius. 2002. Pengaruh mikoriza pada pertumbuhan dan perkembangan awal anggrek Dendrobium sp. Sains 2 (2): 55-60. Andersen, T.F. and H.N. Rasmussen. 1996. The mycorrhizas species of Rhizochtonia. In: Sneh, B., S. Jabaji-Hare, S. Neate and G. Dijst (eds.). Rhizoctonia Species: Taxonomy, Molecular Biology, Ecology, Pathology and Disease Control. London: KAP. Athipunyakom, P., L. Manoch, and C. Piluek. 2004. Isolation and identification of mycorrhizal fungi from eleven terrestrial orchids. Natural Science 38 (2): 216-228. Backer, C.A and R.C. Bakhuizen van de Brink, Jr. 1963. Flora of Java. Vol. 1. Groningen: Wolter-Noordhoff N.V. Backer, C.A and R.C. Bakhuizen van de Brink, Jr. 1965. Flora of Java. Vol. 2. Groningen: Wolter-Noordhoff N.V. Backer, C.A and R.C. Bakhuizen van de Brink, Jr. Flora of Java. Vol. 3. Groningen: Wolter-Noordhoff N.V. Banks, D.P. 2004. Orchid Grower's Companion: Cultivation,

Propagation, and Varieties. Portland, Or.: Timber Press. Bidartondo, M.I., B. Burghardt, G. Gebaner, T.D. Bruns, and D.J. Read. 2004. Changing partners in the dark: isotopic and molecular evidence of ectomycorrhizal liaisons between forest orchids and trees. Proceeding of Biological Science 271 (1550): 1799-1805. Bonnardeaux, Y., M. Brundrett, A. Batty, K. Dixon, J. Kock, and K. Sivasithamparam. 2007. Diversity of mycorrhizal fungi of terrestrial orchid: compatibility webs, brief encounters, lasting relationships and alien invasion. Mycological Research 111: 51-61. Comber, J.B. 1990. Orchid of Java. London: Royal Botanic Gardens Kew, England. Girlanda, M., M.A. Selosse, D. Cafasso, F. Brilli, S. Defline, R. Fabbian, S. Ghignone, P. Pinelli, R. Segreto, F. Loreto, S. Cozzolino, and S. Perotto. 2006. Inefficient photosynthetis in the Mediterranean orchids Limodorum abortivum is mirrored by specific association to ectomycorrhizal Russulaceae. Molecular Ecology 15 (2): 491-504. Hayakawa, S., Y. Uetake, and A. Ogoshi. 1999. Identification of symbiotic rhizoctonias from naturally occuring protocorms and roots of Dactylorhiza aristata (Orchidaceae). Journal of Faculty of Agriculture, Hokkaido University 69 (2): 129-141. Kristiansen K.A., J.V. Freudenstein, F.N. Rasmussen, H.N. Rasmussen. 2004. Molecular identification of mycorrhizal fungi

in Neuwiedia veratrifolia (Orchidaceae). Mol Phylogenet Evol. 33 (2): 251-258. Mahyar, W. dan A. Sadili. 2003. Jenis-jenis anggrek Taman Nasional Gunung Halimun. Bogor: LIPI-JICA-PHKA. Manoch, L. and P. Lohsomboon. 1991. Isolation of mycorrhizal fungi from orchid roots. ASEAN Regional Training Course on Mycorrhiza. SEAMEO BIOTROP & Chiang Mai University. Chiang Mai, 17-19 March 1991. Numberi, A. 2005. Jenis-Jenis Tanaman Anggrek Terestrial di Kotamadya Jayapura. [Skripsi]. Jayapura: FMIPA Universitas Cenderawasih. Otero, J.T., J.D. Ackerman and P. Bayman. 2002. Diversity and host specificity of endophytic Rhizoctonia-like fungi from tropical orchids. American Journal of Botany 89 (11): 18521858. Pokja Cycloops. 2003. Potret Penataan Hutan Kawasan Cagar Alam Cycloops. Jayapura: WWF. Rasmussen, H.N. 2004. Recent development in the study of orchid mycorrhiza. Plant and Soil 244 (1): 149-163. Schuiteman, A. 1995. Key to the genera of Orchidaceae of New Guinea. Flora Malesiana Buletin 11 (6): 401-424. Segerback, L.B. 1992. Orchid of Malaya. Rotterdam: A.A. Balkema Publisher. Shan, X.C., E.C.Y. Liew, M.A. Weatherhead, and I.J. Hodgkiss. 2002. Characterization and taxonomic placement of Rhizoctonia-like endophytes from orchid roots. Mycologia 94 (2): 230 - 239. Sharma, J., L.W. Zettler, and J.W. van Sambeek. 2003. A Survey of mycobionts of federally threatened Platanthera praeclara (Orchidaceae). Symbiosis 34: 145-155. Shefferson, R.P., M. Weib, T. Kull, and D.L. Taylor. 2005. High specificity generally characterizes mycorrhizal association in rare ladyâ&#x20AC;&#x2122;s slipper orchids, genus Cypripedium. Molecular Ecology 14 (2): 613-621. SK Mentan No. 05/KPTS/UM/1978 tentang Penentuan Kawasan Konservasi Cagar Alam Cycloops. Tan, T.K., W.S. Loon, E. Khor, and C.S. Loh. 1998. Infection of Spathoglottis plicata (Orchidaceae) seeds by mycorrhizal fungus. Plant Cell Reports 18: 14-19. Taylor, D.L., and T.D. Bruns. 1997. Independent, specialized invasions of ectomycorrhizal mutualism by two nonphotosynthetic orchids. Proceeding of the National Academic of Science of the USA 94 (9): 4510-4515 Taylor, D.L., T.D. Bruns, and S.A. Hodges. 2004. Evidence for mycorrhizal races in a cheating orchid. Proceeding of Biological Science 271 (1534): 35-43. Yamato, M., T. Yagame, A. Suzuki, and K. Iwase. 2005. Isolation and identification of mycorrhizal fungi associating with an achlorophyllous plant, Epipogium roseum (Orchidaceae). Mycoscience 46 (2): 73-77. Warcup, J.H. 1981. The mycorrhizal relationships of Australian orchids. New Phytologist 87: 371-381.

BIODIVERSITAS Volume 10, Number 4, October 2009 Pages: 181-186

ISSN: 1412-033X (printed edition) ISSN: 2085-4722 (electronic) DOI: 10.13057/biodiv/d100404

Growth Rate of Acropora formosa Fragments that Transplanted on Artificial Substrate Made from Coral Rubble NUR FADLI

♥

Marine Science Department, Faculty of Science, Syiah Kuala University, Banda Aceh 23111, Indonesia Received: 25th March 2009. Accepted: 16th June 2009.

ABSTRACT Coral reefs play an important physiological and ecological role in coastal ecosystems such as providing natural breakwaters that protect shorelines and human settlements from waves and storm. Corals killed by tsunami, waves and storms are often degraded into rubble. This rubble is dynamic, easily shifted by currents and storms, which effectively forms ‘‘killing fields’’ for coral juveniles, hindering coral recovery. In order to rehabilitate coral reefs, artificial substrates are used both for coral transplantation and recruitment. Unfortunately, most artificial substrates are expensive and use land-based material such as concrete/cement-bases. In order to develop a new low-cost artificial substrate that can replace concrete/cement-base as a media for coral transplantation, modified coral rubble was tested in a pilot study in Seribu Island, Jakarta. Two different nets (nylon and polyethylene) were used to form rubble into a compact shape, stable and strong substrate. The stability of the rubble and the complexity of the surface which is created by the net make this substrate suitable for coral transplantation. Additionally, from an economic perspective the nets are very cheap and locally available. In a number of experiments, modified coral rubble successfully replaced the concrete/cement-base as a media for coral transplantation. The coral transplants were growing over time. With this method, we can try to rehabilitate the degraded coral reef destroyed by tsunami or other factors with material that already is available at the site and with less money. However, this approach requires testing at additional sites and for longer periods, to determine the replicability of the results. © 2009 Biodiversitas, Journal of Biological Diversity Key words: cement base, coral, rubble, transplantation.

INTRODUCTION Coral reefs are regarded as one of the most diverse, complex (Buddemeier et al., 2004; Veron, 1995) and productive ecosystems (Burke et al., 2002; Tomascik et al., 1997) on earth. At least, 794 species of scleractinian corals are known to build coral reefs (Spalding et al., 2001). The majority of coral reefs are located in tropical and subtropical regions between 22. 5°N and 22. 5°S latitude, with the center of maximum coral diversity in the Southeast Asian region (Buddemeier et al., 2004). The Indonesian archipelago with more than 17,500 islands and a coastline in excess of 80,000 km, is one of the largest countries with coral reefs in the region (Burke et al., 2002; Nontji, 2004; Tomascik et al., 1997). Approximately 18% of the world’s coral reefs are located in Indonesia. More than 480 species of hard corals (which represents 60% of the described coral in the world) are located in Indonesia (Burke et al., 2002). Unfortunately, in 2003, it was estimated that  Alamat korespondensi: Jl. Syech Abdul Rauf, Darussalam, Banda Aceh 23111 Tel. +62-0651-7410248, Fax. +62-0651-7551381 e-mail: ivad29@yahoo.com

only 7% of Indonesia’s reefs remained in an excellent state. Over 27% were in fair condition, and more than 36% were reported to be in poor condition (Nontji, 2004). There are many factors involved in degradation of coral reef ecosystem in Indonesia. Some of them are natural, but several factors are anthropogenic. One of the common threats is blast fishing. In remote areas, were law enforcement is minimal, blast fishing is more commonly practiced (Erdmann, 1998; Kunzmann, 2002). Blast fishing has serious negative impacts on corals because the blast shatters the coral skeleton, which leads to mass fragmentation. Some of the fragments may initially survive for several months but eventually die (Fox et al., 2003). A typical 1 kg beer bottle bomb can create a rubble field of 1-2 m in diameter (Burke et al., 2002). Furthermore, blast fishing may leave fields of rubble that shift in the current, abrading or burying new coral recruits, and thereby slow down or prevent the reef from recovery even when an area is protected from further blasting (Fox et al., 2005). In the Philippines, many rubble fields virtually show no hard coral cover upon 20-30 years post-blasting (Raymundo et al., 2007). Due to this reason, artificial substrates are always used in coral transplantation in areas of unconsolidated

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sediment and water movement. Unfortunately, most rehabilitation techniques are expensive and labor intensive (Clark and Edwards, 1995; Edwards and Clark, 1998). Researchers comparing various coral restoration methods found that costs could range from US$13,000 to more than US$100 million/ha (Spurgeon and Lindahl, 2000). Unsurprisingly, the methods are not suitable for developing countries like Indonesia (Fox et al., 2005). Artificial substrates should possess the ability to avoid the abrasion, dislodgement and transport due to water movement (Lindahl, 2003), and be placed high enough above the bottom substrate to minimize burial and abrasion (Fox et al., 2005). As long as the artificial substrate can accommodate these problems, different material can be used. In order to develop a new low-cost method of artificial substrate as a media for coral transplantation and coral recruitment a net is used to modify coral rubble into suitable media for transplantation. The net will tie and make the rubble a compact shape, stable and strong substrate. The stability of the rubble and the complexity of the surface shape, which are created by the net may increase the natural coral recruitment (Edwards and Clark, 1998; Raymundo et al., 2007). Furthermore, coral rubble provides an appropriate biofilm for larvae to settle (Harrington et al., 2004; Mundy, 2000). Additionally, from an economic perspective the nets are very cheap and locally available.

MATERIALS AND METHODS The main study was carried out from the first week of September 2007 until the second week of January 2008. The experiment was located on the reef-flat on the western side of Panggang Island (located in the mid region of Seribu Island); about 300 m from the island (Figure 1). The western part of Panggang Island is a sandy plateau, which is part of the reef-flat zone. The reef-flat, which is composed of limestone, is covered with coral rubble and sand. The coral in this site is considered in poor condition (coral cover â&#x2030;¤ 2 25%) 4 x 20 m line intercept transect in each depth, (English et al., 1997), with dead coral cover reaching almost 50% in both (6 and 10 m) depths. In addition, the rubble covers 25% of the site. The immense availability of rubble in this site provides enough material for the experiment. Additionally, most of the coral farmers place their coral farming in this area, indicating that this site is suitable for coral transplantation. For the experiment, three different artificial substrates with approximately equal size (size 3 ~30x50x10 cm ) were used: the cement base (cement-base), rubble which was tied together with nylon net (nylon + rubble) and rubble which was tied together by polyethylene net (polyethylene + rubble) (Figure 2 and 3). The cement-based substrates were considered as the control since this substrate was

commonly used as a medium for coral transplantation in Indonesian waters especially in the Seribu Island. In addition, the nets that were used in this experiment were locally available with a 2 cm diameter mesh size. The rubble which was used to form the rubble substrates was taken from the water on the western part of the Panggang Island. The rubble was filled into the net on the land and left for 2-3 days. The iron sticks (diameter = 10 mm) were used as a media to attach the coral fragment onto the substrate. These different artificial substrates constituted three different treatments. Afterward, on each artificial substrate, five of coral fragments were interspersed at approximately equal intervals. The coral were tied directly to the iron sticks using plastic cable ties. The artificial substrates were placed into two different depths (6 m and 10 m), representative of shallow and deep water. An exception was the cement-base substrates, where 8 substrates in 6 m and 10 substrates in 10 m were deployed due to technical problems. The substrates were deployed in the experimental site by using a fisherman boat. The total sample size was 180 fragments in 6 m and 190 fragments in 10 m. The SCUBA gear was used to put the constructions in position. The species used during the experiment was identified as Acropora formosa. Fragments for the experiments were collected from a donor site about 100 m away from the experimental site. Donor colonies were located at about 2-3 m depth. For every surviving fragment, the change of the main branch in linear extension (the total length from the apical tip to the net) was measured with plastic Vernier calipers (0.01 mm error margin) for every sampling interval and the overall duration. For the calculation of growth rates per day, the mean increment of all surviving fragments per treatment per time was used, divided by the number of days between two samplings. Fragments showing negative growth between surveys were not considered in calculation of average growth rates to make sure that only growth was considered. In order to determine if the growth rates of the fragments were different between substrate, the mean increment per day of transplants for each type of substrate was tested by Wilcoxon Rank Sum test. Each depth was examined separately. All data used in the statistical analysis were analyzed using JMP 7.0.1 software (trial version).

RESULTS AND DISCUSSION Results In a number of experiments, the transplants were increasing their length over time. Some fragments were also increasing the number of branches indicating that they can grow on the substrates. The stability of the rubble which is created by the net is

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Figure 1. Location of the study site, (pink circle = experimental site, located in reef flat of Panggang Island; rectangular = location of rubble and sand mining; black star = location of rubble and sand reclamation).

Figure 2. Cement-base (left) and net + rubble substrate (right)

Figure 3. The rubble substrates after deployment.

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suitable for coral transplantation. The average initial length of the coral transplants on the nylon + rubble substrate in 6 m was 55.6 ± 6.0 mm (n = 70). In January 2008 or at the end of the experiment, the average length was 84.1 ± 13.3 mm (n = 18), an increase of about 1.5 fold. The average length of the coral transplants on the polyethylene + rubble substrate was 60.9 ± 9.4 mm (n = 70) at the start of the experiment (September 2007), and 77.2 ± 14.7 mm (n = 26) in January 2008, an increase of about 1.2 fold. For the cement-base, the average initial length of the coral transplants was 55.6 ± 10.2 mm (n = 40), an increase of about 1.3 fold over the observation period (72.9 ± 3.4 mm, n = 23). There was no significant difference in term of growth rate between the transplants on the cement-base with the fragments on the nylon + rubble substrate and polyethylene + rubble substrate (P >0.1978, Table 1). The average increment of transplants on different type of substrates in 6 m is depicted in Figure 4. 0.6

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difference of transplant growth between 6 and 10 m. The growth rate was better in 6 m (Table 1). Table 1. Comparison of growth rate by depths and substrates (*significant difference (p<0.05).

I. Comparing between depths 6 m vs. 10 m

II. Comparing substrates per depth Cement-base vs. nylon+rubble vs. Polyethylene+rubble (6 m) Cement-base vs. nylon+rubble vs. Polyethylene+rubble (10 m) 0.6

Non-parametric comparisons Wilcoxon/Kruskal-Wallis test P Statistic 137070 0.0002* Wilcoxon/Kruskal-Wallis test P Statistic 3.24

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Figure 4. Mean increment of transplants in the different substrates and sampling intervals in 6 m standardized to length increase in mm per day. Error bars are standard deviations.

In 10 m, the average initial length of the coral transplants on the nylon + rubble substrate was 62.8 ± 11.4 mm (n = 70) in September 2007. In January 2008 or at the end of the experiment, the average length was 87.4 ± 23.0 mm (n = 5), an increase of about 1.5 fold. At the start of the experiment (September 2007), the average length of the coral transplants on the cement-base substrate was 66.7 ± 10.5 mm (n = 40), and 79.2 ± 9.1 mm (n = 20) in January 2008, an increase of about 1.2 fold. For the polyethylene + rubble, the average initial length of the coral transplants was 61.7 ± 7.7 mm (n = 70), an increase of about 1.3 fold over the observation period (77.7 ± 4.4 mm, n = 2). There was no significant difference in growth rate between the transplants on the cement-base with the fragments on the nylon + rubble substrate and polyethylene + rubble substrate (P >0.8276, Table 1). The average increment of transplants on different type of substrates in 10 m is depicted in Figure 5. Overall, there was a significant

30/10/2007

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Figure 5. Mean increment of transplants in the different substrates and sampling intervals in 10 m standardized to length increase in mm per day. Error bars are standard deviations.

Discussion The transplants were increasing their length over time. In addition, some fragments were also increasing the number of branches indicating that they can grow on the substrates. There were no differences in terms of growth detected among the substrate in 6 m or in 10 m. Overall, the transplants grew by 0.46 cm/month in 6 m and 0.33 cm/month in 10 m. Compared to other studies which used the same species in Seribu Island, this result was relatively low. It was reported that A. formosa increased their length by 0.88 cm/month (Sadarun, 1999), 1.1 cm/month (Alhusna, 2003), 1.14 cm/month (3 m) and 0.76/month (10 m) (Yarmanti, 2002). This result only corresponds with Johan (2001). He reported that the fragments increased by 0.374 cm/month. The growth of the transplants was higher in shallow depth (6 m). This result corresponds with the result of other studies (Yap et al., 1998; Freytag, 2001; Yarmanti, 2002; Ferse, 2003). A. nobilis fragments in shallow depths had higher growth rates than in deeper areas (Freytag, 2001). The depth had a significantly negative effect on A. gomezi (Ferse,

FADLI – Artificial substrate for Acropora formosa transplantation

2003). Depth also had negative effects on Porites cylindrical and P. rus in the Philippines, the growth of the coral transplants were lower in deeper depth. The reduced light was the important factor for the difference (Yap et al., 1998). However, it is difficult to quantify the effect of the material used in this experiment on the growth of transplants. The quality of the water surrounding Seribu Island was assumed to be responsible for the differences. The water quality some years ago was better than the current water quality in this area. The transplants have higher survival and growth when the water quality is good (Clark and Edwards, 1995). Yap and Molina (2003) stated that environmental parameters like light, sedimentation, eutrophication, chemical pollution or coastal development activities may have negative impact to the growth and survival of coral especially if these parameters exceed the threshold. The transplants have higher survival when the water quality is good (Clark and Edwards, 1995). Most studies about transplants also reported that most of the reasons for transplant mortality were dominantly related to environmental impact, for example: sediment (Yap and Gomez, 1985); wave action (Clark and Edwards, 1995); algal competition and strong water movement (Yap et al., 1998); sediments, algal bloom and grazing pressure by Drupella sp. (Schuhmacher et al., 2000); water circulation, overgrowth by macroalgae due to increasing nutrient level and the absence of grazers (Yap and Molina, 2003); and combination of increasing temperature and algal overgrowth (Yap, 2004). In this study, the combination of the turf/macro algae overgrowth and the sedimentation were assumed to be the cause of the mortality of the transplants. The algae started to grow on the substrates 3 weeks after deployment and overgrew the substrates in the next weeks. The algae that invaded and overgrew the transplants differed between the depths. Dictyota sp. invaded and covered the fragments in 10 m, while Padina sp. covered the fragments in 6 m. The algae in 10 m had relatively smaller size than that covered the fragments in 6 m. These small size algae overgrew and covered all the animal tissue or polyps of the coral easily and gave no opportunity for the coral to survive, while canopies of large/leathery algae like Padina sp. shade or whiplash corals (McCook et al., 2001). This result corresponds with Yap and Molina (2003). They reported that their coral transplants (P. cylindrica and P. rus) died due to alga overgrowth. The higher number of algae in the experimental site was assumed to be a result of the combination of nutrient enhancement and removal of the herbivores. Nutrients can affect the algal growth. In addition, the abundance of the algae may or may not increase depending on herbivory rates (McCook et al., 2001). Even at the experimental site which is a part of a Marine National Park, evidence of fishing activities by the islanders is easily observed. Some of them are

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working as marine ornamental fisher. The exact numbers of the fish that are caught by the fisherman is difficult to quantify; the fishermen catch any fish that has economic value including herbivorous fish. This activity is presumed to be responsible for the decreasing the numbers of herbivores. Although sand and rubble mining are forbidden in this area, as a result of its inclusion in the Marine National Park; these activities were observed by the author in August 2007. Rubble and sand mining occurred in the eastern part of Semak Daun Island. This rubble was used for the reclamation of Gosong Pramuka, a sandy plateau between Panggang and Pramuka Island (Figure 1). It is estimated that more than 10.000 tons of rubble and sand (400 sacks x 60 boat per day x 20 kg per sack x 21 days) were mined within 3 weeks. This rubble and sand mining appeared to influence the high sedimentation levels in the water. The visibility of the water surrounding the Seribu Island, especially in the experimental site were also low (average ± 2-3 meters) indicating the sedimentation was high. Rachello-Dolmen and Cleary (2007) also indicated that the sedimentation problem is becoming one of the common problems across Seribu Island. During their study, they found that the corals and other benthic organisms such as sponges were often covered by sediment. Corals are not well adapted to pronounced sedimentation and produce mucus to remove the sediment that settles on their polyps (McCook et al., 2001). Sediments overlying coral tissues can cause tissue death from smothering or bacterial infection, reduce the amount of available light and the capacity to capture food, and increase the energy demand for active sediment rejection (Rogers, 1983; Okubo et al., 2005). Yusri and Estradivari (2007) also reported that coral diseases have already invaded corals in the Seribu Island. The sediment may also easily cause occlusion on the coral that have small corallites such as Acropora (Rachello-Dolmen and Cleary, 2007). In addition, the algae have indirect effects on coral due to sedimentation. Together with the mucus which produced by the coral for removing the sediment that settles on their polyps, the algae could become a sediment trap for sediment on the coral surface. This effect may significantly increase damage to underlying coral tissue (McCook et al., 2001).

CONCLUSION In terms of growth, there were no differences detected among the substrates, indicating that modified rubble can be used as an alternative media for coral transplantation. Furthermore, for future development, the shape of the layout still can be improved, the layout of the rubble in the water can be arranged into different shapes and creating more three dimensional substrate, for example pyramid-like “rubble piles” (Figure 6). However, this approach

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Figure 6. Example of the future layout of the rubble in the water introduced term ”rubble piles”; pyramid like (a), rectangular (b) and square (c).

requires testing at additional sites, to determine the replicability of the results.

REFERENCES Alhusna, I.S. 2003. Studi Laju Pertumbuhan Induk Koloni dan Fragmen Transplant pada Transplantasi Karang Spesies Acropora formosa dan Hydnophora rigida di Perairan Pulau Pari, Kepulauan Seribu. [Thesis]. Bogor: Bogor Agricultural University. Buddemeier, R. W., J. A. Kleypas, and R. B. Aronson. 2004. Coral Reefs and Global Climate Change. Potential Contributions of Climate Change to Stresses on Coral Reef Ecosystems. Arlington, USA: Pew Center on Global Climate Change. Burke, L., E. Selig, and M. Spalding. 2002. Reefs at Risk in Southeast Asia. Washington D.C., USA: World Resources Institute (WRI). Clark, S., and A. J. Edwards. 1995. Coral transplantation as an aid to reef rehabilitation: evaluation of a case study in the Maldives Islands. Coral Reefs 14:201-213. Edwards, A. J., and S. Clark. 1998. Coral transplantation: a useful management tool or misguided meddling? Marine Pollution Bulletin 37: 474-487. English, S., C. Wilkinson, and V. Baker 1997. Survey Manual for Tropical Marine Science. Townsville: Australian Institute of Marine Science. Erdmann, M. V. 1998. Destructive fishing practices in the Pulau Seribu archipelago. In: Soemodihardjo, S. (ed.) Proceedings of the Coral Reef Evaluation Workshop, Pulau Seribu, Jakarta, Indonesia, October 1998. Ferse, S. 2003. Growing Corals in An Ocean-based Nursery: The Use of Cages. [Thesis]. Bremen: University of Bremen. Fox, H. E., P. J. Mous, J. S. Pet, A. H. Muljadi, and R. L. Caldwell. 2005. Experimental assessment of coral reef rehabilitation following blast fishing. Conservation Biology 19: 98-107. Fox, H. E., J. S. Pet, R. Dahuri, and R. L. Caldwell. 2003. Recovery in rubble fields: long-term impacts of blast fishing. Marine Pollution Bulletin 46:1024-1031. Freytag, I. 2001. Growth and Mortality of Acropora nobilis Anthozoa: Scleractinia Fragments in Shallow Waters of the Seribu Islands, Indonesia, Using Different Attachment Methods. [Thesis]. Bremen: University of Bremen.

Harrington, L., K. Fabricius, G. De’ath, and A. P. Negri. 2004. Recognition and selection of settlement substrata determine post-settlement survival in corals. Ecology 85: 3428-3437. Johan, O. 2001. Tingkat Keberhasilan Transplantasi Karang Batu di Pulau Pari, Kepulauan Seribu Teluk Jakarta. [Thesis]. Bogor: Bogor Agricultural University. Kunzmann, A. 2002. On the way to management of West Sumatra’s coastal ecosystems. Naga, the ICLARM Quarterly (January-March) 25: 4-10. Lindahl, U. 2003. Coral reef rehabilitation through transplantation of staghorn corals: effects of artificial stabilization and mechanical damages. Coral Reefs 22: 217-223. McCook, L. J., J. Jompa, and G. Diaz-Pulido. 2001. Competition between corals and algae on coral reefs: a review of available evidence and mechanisms. Coral Reefs 19: 400-417. Mundy, C. N. 2000. An appraisal of methods used in coral recruitment studies. Coral Reefs 19:124-131. Nontji, A. 2004. COREMAP Tahap I: Upaya Anak Bangsa dalam Penyelamat dan Pemanfaatan Lestari Terumbu Karang. Jakarta: Kantor Pengelola Program COREMAP, Pusat Penelitian Oseanografi, LIPI. Okubo, N., H. Taniguchi, and T. Motokawa. 2005. Successful methods for transplanting fragments of Acropora formosa and Acropora hyacinthus. Coral Reefs 24:333-342. Rachello-Dolmen, P. G., and D. F. R. Cleary. 2007. Relating coral species traits to environmental conditions in the Jakarta Bay/Pulau Seribu reef system, Indonesia. Estuarine, Coastal and Shelf Science 73:816-826. Raymundo, L. J., A. P. Maypa, E. D. Gomez, and P. Cadiz. 2007. Can dynamite-blasted reefs recover? a novel, low-tech approach to stimulating natural recovery in fish and coral populations. Marine Pollution Bulletin 54: 1009-1019. Rogers, C. S. 1983. Sublethal and lethal effects of sediments applied to common Caribbean reef corals in the field. Marine Pollution Bulletin 14: 378-382. Sadarun. 1999. Transplantasi Karang Batu Stony Coral di Kepulauan Seribu Teluk Jakarta. [Thesis]. Bogor: Bogor Agricultural University. Schuhmacher, H., P. van Treeck, M. Eisingert, and M. Paster. 2000. Transplantation of coral fragments from ship groundings on electrochemically formed reef structure. Proceedings of the 9th International Coral Reef Symposium 2: 983-990. Spalding, M. D., C. Ravilious, and E. P. Green. 2001. World Atlas of Coral Reefs. Berkeley, USA: University of California Press. Spurgeon, J. P. G., and U. Lindahl. 2000. Economics of Coral Reef Restoration. In Cesar, H. S. J. (ed.). Collected Essays on the Economics of Coral Reefs. Sweden: CORDIO, Kalmar University. Tomascik, T., A. J. Mah, A. Nontji, and M. K. Moosa, editors. 1997. The Ecology of the Indonesian Seas, Part One and Two. Singapore: Periplus Editions HK Ltd. Veron, J. E. N. 1995. Corals of the World. Townsville, Australia: Australian Institute of Marine Science. Yap, H. T. 2004. Differential survival of coral transplants on various substrates under elevated water temperature. Marine Pollution Bulletin 49: 306-312. Yap, H. T., R. M. Alvarez, H. M. Custodio III, and R. M. Dizon. 1998. Physiological and ecological aspects of coral transplantation. Journal of Experimental Marine Biology and Ecology 229: 69-84. Yap, H. T., and E. D. Gomez. 1985. Growth of Acropora pulchra. III. Preliminary observations on the effects of transplantation and sediment on the growth and survival of transplants. Marine Biology 87: 203- 209. Yap, H. T., and R. A. Molina. 2003. Comparison of coral growth and survival under enclosed, semi-natural conditions and in the field. Marine Pollution Bulletin 46: 858-864. Yarmanti, K. D. 2002. Studi Pertumbuhan dan Tingkat Keberhasilan Hidup Karang Batu Spesies Acropora nobilis dan Acropora formosa pada Dua Kedalaman yang Berbeda di Pulau Pari, Kepulauan Seribu. [Thesis]. Bogor: Bogor Agricultural University. Yusri, S., and Estradivari. 2007. Distribution of infection by White Syndrome and coral bleaching diseases to coral communities in Petondan Timur Island, Kepulauan Seribu. Berita Biologi 8 (4): 223-229.

BIODIVERSITAS Volume 10, Number 4, October 2009 Pages: 187-194

ISSN: 1412-033X (printed edition) ISSN: 2085-4722 (electronic) DOI: 10.13057/biodiv/d100405

Forest Dynamics of Peat Swamp Forest in Sebangau, Central Kalimantan EDI MIRMANTO

♥

Botany Division, Research Centre of Biology, Indonesian Institute of Sciences (LIPI), Cibinong-Bogor 16911. Received: 18th March 2009. Accepted: 5th June 2009.

ABSTRACT Forest dynamics were studied from 1999 to 2001 for individuals > 15 cm in girth of 24 most common species in six 0.25-ha plots. The plots were set up in natural peat swamp forest in the upper catchments of Sebangau, Central Kalimantan. Aim of the study is to understand the dynamics and vegetation changes of forest studied during period of study. The peat swamp forest in the study site might be categorized as moderately forest dynamic in term of rate of growth, mortality and recruitment. Annual relative growth rate and mortality rate was comparable to previous study but recruitment rate relatively higher. There was significant effect of diameter class on annual growth rate, but not to mortality rate. Even not too strong two environment factors (peat depth and distance to river) were significant correlated with rate of mortality and recruitment. During two-year period study there was no significant changes in vegetation structure. © 2009 Biodiversitas, Journal of Biological Diversity Key words: Central Kalimantan, dynamic, growth, mortality, peat swamp forest, recruitment, Sebangau.

INTRODUCTION It was often assumed that a tropical rain forest characteristics is diversity in term of plant species and vertical and horizontal dynamics complexity. Although the coexistence among individuals of tree species whom living together have been observed (Kohyama, 1992; 1993). The dynamics of forest naturally was steady state (Kohyama et al., 2001), but impact of human activities and disturbance events might change the pattern and process of forest dynamics. Studies on forest dynamics approach have been widely used to compare the undisturbed and disturbed forests in order to evaluate the effect of disturbances. Forest dynamics in term of rate of diameter growth, mortality and recruitment have been studied in many temperate and tropical forests with various aims and aspects. Studies on forest dynamics following impact of logging, silviculture treatment and natural disturbance (Silva et al., 1995; Pelissier et al., 1998; Graff et al. 1999; Nebel et al., 2001; Vandermeer et al., 2001; Coates, 2002; Sist et al., 2002; Olano and Palmer, 2003; Wolf et al., 2004), and based on long-term observation (Takahashi et al., 2003; Laurance et al., 2004) have been studied. In addition some studies have been also conducted

on the level of population (Primack et al., 1985; Roger, 1999; Fuhr et al., 2001; Guedje et al., 2002; Sanford et al., 2003), and have been used to estimate age of forest (Martínez-Ramos and Alvarez-Buylla, 1998). Almost of those previous forest dynamics studies have been conducted in dry forest, whereas in wet forest has been reported by Visser and Sasser (1995), and also Nebel et al. (2001). Study on forest dynamics of peat swamp forest, on the other hand so far is undocumented. Aim of the present paper is to provide information on forest dynamics and vegetation changes of natural peat swamp forest in Sebangau, Central Kalimantan, Indonesia. The forest is one of remain relatively undisturbed peat swamp forest in this area and has been declare as Natural Laboratory of Peat Swamp Forest. So far there have little report on forest ecological study based on some permanent plots (Rieley et al., 1996; 1997; Shepherd et al., 1997; Page et al., 1999; and Mirmanto et al., 2003). Forest dynamics were studied for individual > 15 cm in girth breast height of 24 most common species in six 0.25ha plots. The rate of growth, mortality and recruitment were calculated and compare to others studies, and the effects of related factor were described.

MATERIALS AND METHODS  Corresponding address: Jl. Raya Bogor Km 46, Cibinong Bogor 16911 Tel.: +62-21-8765066/7, Fax.: +62-21-8765063 e-mail: emirmanto@yahoo.co.id

Study site The study has been done in the upper catchments o of Sebangau river, which is situated at 2 18’ 24” S

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and 113 55â&#x20AC;&#x2122; 4.1â&#x20AC;? E, at about 10 m above sea level. This area is a small part of peat-covered landscape between Katingan river to the west and Kahayan river to the east. The study area belongs to Kereng Bangkirai village about 20-km southwest of Palangkaraya the capital city of Central Kalimantan, Indonesia. The vegetation here is characteristic of peat-swamp forest, with condition in general was flooded, especially during rainy season and with the peat layer depth varied from 2 to 10 m up. In general the condition of vegetation is a primary forest, although in some sites have been disturbed due to the selective logging. According to Schmidt and Ferguson (1957), the climates in those three study areas are belonging to type of A, with mean annual rainfall generally exceeds 2.500 mm (Page et al., 1999) The mean daily o o temperature varied from 25 up to 33 C, with high in humidity (up to 90%). Methods Six permanent plot of 50-m x 50-m was established in a relatively undisturbed forest with distance at least 500 m up to 1 km each other and then each plot was divided into 25 sub-plots of 10m x 10m. First enumeration of all six plots was made in 1999. All trees with girth breast high (gbh) over 15 cm were tagged with aluminium tag on 1.4 m above ground. Trunk girth measurement was conducted on 10 cm below tagged or above buttress and the position of measurement was recorded. The density and dominance (expressed with basal area) and their relative value for each species were calculated follows Mueller-Dombois and Ellenberg (1972). In second enumeration on 2001, tree recruitment (new individual that grow up to 15-16 cm gbh) and tree death within each plot were recorded. The similar trunk measurement was also conducted for all survive and recruited trees. All species recorded were colected as voucher specimen for scientific identification. Data analysis The growth was analyzed based on girth increment of survive trees. Growth rate was calculated as relative growth rate (RGR) follow formula of Kohyama and Hotta (1986): RGR= (ln G00-ln G99)/t where G99 and G00 are girth breast high in first and second measurement respectively, and t is period of study. Mortality rate (M) was calculated based on individuals with dbh 6 cm up, and calculated following formula of Condit et al. (1995) and Sheil et al. (1995). M= (ln N0-ln N1)/t where N0 is number of individuals at beginning measurement and N1 is survive individual at the second measurement, and t is period of study.

Recruitment rate per unit area is defined as the number of recruitâ&#x20AC;&#x2122;s trees, which reaches up to minimum size (16 cm) in gbh during two-years (19992001) period. Recruitment rate (R) calculated as formula of Nebel et al. (2001). R= Nr/ (N0-Nm) Nr is number of recruited trees, N0 is number of trees at beginning measurement and Nm is number of mortal trees. Growth rate and mortality rate were calculated for each plot and each diameter class per plot. Relationships between the above parameter were analyzed in Spearman correlation analysis and multiple regression analysis, in which diameter class, total density and total basal area per plot, relative density and dominance per species, diversity, peatdepth and distance to river were chosen as independent variables. The values of total density, total basal area, diversity, peat depth and distance to river followed Mirmanto et al. (2003).

RESULTS AND DISCUSSION Vegetation features The total number of species among 4.656 trees (dbh > 4.8) recorded within 6 plots was 126 trees. The most abundant 24 species were analyzed (Table 1), and these accounted for 66.41% of number of tree, 74.13% of basal area but only 18.25% of number of species. Almost all of those 24 species were occurs in 6 plots, except Combretocarpus rotundatus and Calophyllum biflorum in 5 plots, and Palaquium sp.1 and Shorea cf. parvifolia in 4 plots. However the contributions in density and basal area of those 4 species were relatively high. Figure 1 shows the similar pattern in distribution of diameter of those 24 species, except for Combretocarpus rotundatus. Growth-rates Growth rate, mortality rate, recruitment rate of 24 most common species recorded within 6 plots of 0.25 ha were shown in Table 2. Growth-rate was significantly positive correlated with relative density but was negatively correlated with diameter class and relative dominance (r =-0.884, p = 0.000; r = 0.567, p = 0.000; r =-0.229, p = 0.000 (Table 3). That mean growth rate to increase with increasing in population density, but to decrease with increasing in size of trees. In other words that growth rate of the dense young population is high, which indicated by a lot of small size individuals, tended higher. In addition according to multiple regression analysis, growth rate also affected by diameter class and relative density of each species but there is no significant effect of relative dominance (Table 4). Analysis of one-way ANOVA seems to fit these results. Although there is any variation among species in growth-rate within

MIRMANTO et al. â&#x20AC;&#x201C; Peat swamp forest dynamic in Sebangau

each class diameter (Figure 2), but in general the growth rate of small trees were significantly higher than the big trees (ANOVA; Tukey test, p < 0.001).

species, and most of them may be able to be categorized as uncommon species, which only occur on 1 or 2 plots with relatively lower in number of individual. The mean annual mortality rates of 24 species -1 analyzed varied from 0.0 to 6.6% yr with mean 1.1% -1 yr (Table 2). This variation in mortality rate among 24 species was not significant (ANOVA; P = 0.207). However, even not significant Lithocarpus elegans tended the highest in mortality rate, followed by

Mortality Total number of tree death recorded over one year period was 70 individuals within 6 plots, with total 2 basal area of 0.66 m /ha. They belong for about 25% of tree species, which occur in at least 4 plots with number of individual relatively higher. On the other hand, there is no mortality for about 75% of tree

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Figure 1. Number of individual of 24 most common species according to diameter class.

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Table 1. Frequency (F= number of plot where a species occur), diameter minimum (D-min= cm), diameter maximum (D2 max= cm), total basal area (BA= m /ha), total density (trees /1.5 ha), relative dominance (RDo=%) and relative density (RD=%) of 24 most common species recorded within 6 plots of 0.25 ha. Species Acronychia porteri Blumeodendron elateriospermum Calophyllum biflorum Calophyllum teysmannii Campnosperma coriaceum Combretocarpus rotundatus Cratoxylum glaucum Diospyros dajakensis Eugenia castaneum Eugenia clavatum Eugenia densinervium Gonystylus bancanus Gymnacranthera eugeniifolia Horsfieldia crassifolia Lithocarpus elegans Neoscortechinia philippinensis Palaquium leiocarpum Palaquium sp.1 Sandoricum emarginatum Shorea guiso Shorea parvifolia Stemonurus secundiflorus Xanthophyllum palembanicum Xylopia fusca

F 6 6 5 6 6 5 6 6 6 6 6 6 6 6 6 6 6 4 6 6 4 6 6 6

D-min 4.8 4.8 4.8 4.8 4.8 4.8 4.8 4.8 4.7 4.8 4.8 4.8 4.8 4.8 4.8 4.8 4.8 4.8 4.8 4.8 4.8 4.8 4.8 5.1

D-maks 62.7 29.5 56.0 29.1 45.6 74.5 22.5 29.1 42.9 31.1 44.3 62.8 19.2 28.2 26.5 33.2 39.4 44.0 24.6 38.2 30.8 29.1 41.8 33.7

BA 0.92 0.77 1.25 1.03 0.83 2.26 0.30 0.53 1.39 0.46 2.07 1.00 0.42 0.68 0.30 1.09 4.34 0.59 0.53 0.84 0.38 0.41 0.83 0.57

D 197 155 136 144 84 27 67 97 217 79 238 80 100 107 59 196 624 45 96 124 72 86 86 62

RDo 2.90 2.43 3.96 3.27 2.63 7.15 0.96 1.69 4.39 1.47 6.54 3.18 1.34 2.14 0.94 3.46 13.76 1.87 1.69 2.66 1.22 1.30 2.64 1.81

RD 4.23 3.33 2.92 3.09 1.80 0.58 1.44 2.08 4.66 1.70 5.11 1.72 2.15 2.30 1.27 4.21 13.40 0.97 2.06 2.66 1.55 1.85 1.85 1.33

Table 2. Growth rate, mortality rate, recruitment rate of 24 most common species recorded within 6 plots of 0.25 ha. Species Acronychia porteri Blumeodendron elateriospermum Calophyllum biflorum Calophyllum teysmannii Campnosperma coriaceum Combretocarpus rotundatus Cratoxylum glaucum Diospyros dajakensis Eugenia castaneum Eugenia clavatum Eugenia densinervium Gonystylus bancanus Gymnacranthera eugeniifolia Horsfieldia crassifolia Lithocarpus elegans Neoscortechinia philippinensis Palaquium leiocarpum Palaquium sp.1 Sandoricum emarginatum Shorea cf. parvifolia Shorea guiso Stemonurus scorpioides Xanthophyllum palembanicum Xylopia fusca All species

< 10 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.02 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03

Growth rate yr-1 < 20 < 30 < 40 0.02 0.01 0.00 0.02 0.01 0.00 0.02 0.01 0.01 0.02 0.01 0.00 0.02 0.01 0.01 0.02 0.02 0.01 0.02 0.01 0.00 0.02 0.01 0.00 0.02 0.01 0.01 0.02 0.01 0.01 0.02 0.01 0.01 0.02 0.01 0.01 0.02 0.00 0.00 0.02 0.01 0.00 0.02 0.01 0.00 0.02 0.01 0.01 0.02 0.01 0.01 0.02 0.01 0.01 0.02 0.01 0.00 0.02 0.02 0.01 0.02 0.01 0.01 0.02 0.01 0.00 0.02 0.01 0.01 0.01 0.01 0.01 0.02 0.01 0.01

Combretocarpus rotundatus, Diospyros dajakensis and Xanthophyllum palembanicum. Mortality rate was significantly correlated with relative density and relative dominance of each species and distance to

> 40 0.01 0.00 0.01 0.00 0.01 0.01 0.00 0.00 0.01 0.00 0.01 0.01 0.00 0.00 0.00 0.00 0.00 0.01 0.00 0.00 0.00 0.00 0.01 0.00 0.01

< 10 0.00 0.00 0.00 0.00 0.00 0.69 0.03 0.05 0.03 0.00 0.00 0.03 0.00 0.00 0.00 0.02 0.02 0.00 0.00 0.00 0.00 0.01 0.03 0.00 0.01

Mortality rate yr-1 < 20 < 30 < 40 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.12 0.00 0.00 0.00 0.00 0.07 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.18 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.06 0.00 0.00 0.00 0.00 0.04 0.00 0.00 0.00 0.00 0.00 0.20 0.00 0.00 0.01 0.00 0.00 0.01 0.02 0.00 0.00 0.18 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.09 0.00 0.00 0.00 0.18 0.00 0.01 0.03 0.00

> 40 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

Rect-rate -1 yr 0.07 0.01 0.01 0.01 0.03 0.00 0.02 0.02 0.01 0.07 0.03 0.03 0.00 0.04 0.07 0.01 0.01 0.04 0.02 0.01 0.01 0.04 0.01 0.02 0.02

river even not strong (Table 3). However according to multiple regression analysis, the effect of both those parameters above were not significant (Table 4).

MIRMANTO et al. â&#x20AC;&#x201C; Peat swamp forest dynamic in Sebangau

Change in tree density and basal area During one-year period there are any changed both in density and basal area of 24 tree species within 6 plot (Table 5). Overall the basal area 2 -1 2 -1 increase from 20.91 m ha to 21.44 m ha and -1 density increase from 1895 to 1905 individualsâ&#x20AC;&#x2122; ha . The tree density increase because the increase of tree density from recruitment was greater than lost of tree by death. The increase of tree density also followed by increase of basal area, because the increase of basal area of survived trees and from recruited trees were greater than the lost by death. However this differences both in density and basal area were not significant (ANOVA; p > 0.9). 0 .0 4

0 .0 3

Growth rate per species

Recruitment During one-year period, there are 48 individual of trees were reach up to 15-17 cm in girth breast high, -1 -1 or about 41 trees ha yr . Out of 24 species, 22 of them were recruited and 2 others (Combretocarpus rotundatus and Gymnacranthera eugeniifolia) were not recruited. Recruitment rate among those 24 -1 species varied from 0.0 to 7.41% yr with mean value -1 was 1.65% yr . There is no significant difference in recruitment rate among 22 species (ANOVA; p = 0.132). However the Acronychia porteri and Lithocarpus elegans were tended the highest in recruitment rate. Recruitment rate was positive correlation with diversity but negative correlation with total density per plot (Table 3). Even not strong, recruitment rate was also negative correlated with peat depth and distance to river. This may be explaining that within community with condition low in density but high in diversity recruitment rate tends to be higher. According to multiple regressions analysis, recruitment even not strong was affected by total basal area, relative dominance and distance to river (Table 4). However based on Gf (recruitment estimation) as expected the recruitment rate was strong affected by relative density and relative dominance (Table 4).

191

0 .0 2

0 .0 1

0 .0 0

Table 3. Correlation coefficients between various measurements (growth rate, mortality rate,) and some parameter factors (diameter class, total density, total basal area, relative density, relative dominance, diversity, distance to river and peat depth).

0 .0 4

Dependent variable Independent variable Grw-rate Mor-rate Diameter class (cm) -0.884 *** -0.068 Total density (ind./ha) -0.045 -0.066 Total basal area (m2/ha) -0.023 -0.070 Diversity 0.045 0.066 Distance to river (km) -0.012 -0.102 * Peat depth (m) -0.030 -0.072 Relative density (%) 0.564 *** 0.152 ** Relative dominance (%) -0.229 *** 0.130 ** * p < 0.05; ** p < 0.01; *** p< 0.001

Rec-rate -0.265 -0.135 0.265 -0.182 -0.205 0.138 0.029

** ** * *

Growth rate per plot

0 .0 3

0 .0 2

0 .0 1

0 .0 0

D ia m e te r c la s s (c m )

Figure 2. The value of growth rate among 24 most common species according to diameter class Table 4. Results of multiple regression analysis between various measurements (growth rate, mortality rate, recruitment rate and Gf) and some parameter factors (diameter class, total density, total basal area, relative density, relative dominance, diversity, distance to river and peat depth). Dependent variable Independent variable Grw-rate Mor-rate Diameter class (cm) -0.799 *** 0.017 Total density (ind./ha) 0.068 0.286 2 Total basal area (m /ha) 0.180 0.832 Diversity 0.194 0.848 Distance to river (km) -0.077 -0.902 Peat depth (m) 0.007 0.413 Relative density (%) 0.111 ** -0.039 Relatif dominance (%) -0.012 -0.028 F 123.23 *** 1.65 ns * p < 0.05; ** p < 0.01; *** p< 0.001

Rec-rate -0.243 2.074 1.201 -1.786 0.722 0.165 -0.252 2.860

* *

* ***

Overall the mean diameter increment of 24 -1 species was 2.11 mm yr with range from 0.32 to -1 5.73 mm yr . This range value is comparable those reported in some tropical rain forest of southeast Asia (Nicholson, 1965; Manokaran and Kochummen, 1993; and Sist et al., 2002), but little bit lower than Amazonian rain forest (Condit et al., 1995; Clark and Clark, 1999; Nebel et al., 2001; Silva et al., 2002; and Laurance et al., 2004). Some of those previous studies (Silva et al., 2002; Clark and Clark, 1999 and Sist et al., 2002) reported that growth pattern was highly dependent on tree size. Based on absolute growth rate was found that big trees tended faster with assumption because larger trees occupy the

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canopy, and have more energy supply and higher photosynthetic rates. In this study we use relative growth rate and detect there is negative significant effect of diameter class to relative growth rate (Table 4). As have been observed in some previous studies, the relative growth rate of 24 species analyzed tended decrease with increasing in diameter class (Kohyama and Hotta, 1986; Yoneda et al., 1994; Simbolon et al., 2000). In addition Kohyama et al. (2003) stated that growth rate is exponential function of size of tree trunk. This support our finding that growth rate was significantly affected by diameter class. This study found that means annual mortality in total was 1.1% with slightly varied among 24 species 1 (0.0-6.6% yr ). This is comparable to those reported in some previous studies both tropical and temperate forest (Sist et al., 2002; Silva et al., 2002; Guedje et al., 2002; Olano and Palmer, 2003; and Takahashi et al., 2003). However, Finegan and Camacho (1999) found that the annual mortality rates was 2.0-2.3% and stated similar to those the most dynamics forest at La Selva. The differences in mortality rate might be associated with differences in environmental and climate conditions, for example higher mortality rate in La Selva was impact of unfamiliar climate and environment (Lieberman and Lieberman, 1987). Some previous studies reported that there are higher mortality rates of small trees (Guedje et al., 2002; Pelissier et al., 1998; Olano and Palmer 2003). In contrast this study detect that there is no significant effect of diameter class in mortality rate similar to those reported in mixed dipterocarp forest (Wyatt-

Smith, 1966; Primack et al., 1989; Sist et al., 2002) and others studies in tropical forest (Philips and Gentry, 1994; Lieberman et al., 1985; Swaine et al., 1987; Manokaran and Kochumen, 1987; Sheil et al., 1996). According to Pelissier et al. (1998) higher mortality of small trees was common in temperate forest but rarely documented for tropical forests. Competition, for light, water or other nutrients, among trees of the smaller size classes is the major factor determining tree death or survival (Wolf et al., 2004). Martinez-Ramos and Alvarez-Buylla (1998) on the other hand, assumed that mortality rate might be higher in older individuals because they lose vigor and become more liable to predation by natural enemies and physical damage. Unfortunately trees with same size may have different in age. In addition, the fact that most tropical rain forest species do not lose vigor with age, except in short-lived species (Sarukhรกn, 1980). In this study there is no significant different in mortality rate among size class, even each species was calculated separately. This study found that means annual recruitment -1 rate of 24 species was 1.94% yr with slightly varied -1 among species (0-7.41% yr ). The value is relatively higher compared to those reported in some previous studies (Takahashi et al., 2003; Pelissier et al., 1998). The differential in recruitment rate might be associated with seed availability and suitable habitat condition. Coates (2002) observed that difference in abundance of parent tree species might influence difference in its recruitment density. In this study however, there is no significant correlation between recruitment and relative density of each species.

Table 5. Density and basal area change of 24 most common species during period of study. Species Acronychia porteri Blumeodendron elateriospermum Calophyllum biflorum Calophyllum teysmannii Campnosperma coriaceum Combretocarpus rotundatus Cratoxylum glaucum Diospyros dajakensis Eugenia castaneum Eugenia clavatum Eugenia densinervium Gonystylus bancanus Gymnacranthera eugeniifolia Horsfieldia crassifolia Lithocarpus elegans Neoscortechinia philippinensis Palaquium leiocarpum Palaquium sp.1 Sandoricum emarginatum Shorea cf. parvifolia Shorea guiso Stemonurus scorpioides Xanthophyllum palembanicum Xylopia fusca

1999 131 145 117 132 69 23 58 80 195 43 201 72 94 87 62 155 622 58 89 91 117 73 74 55

Density (trees /1.5 ha) % M % R % 2000 % 100 -1 -0.8 7 5.3 137 104.6 100 0 0.0 1 0.7 146 100.7 100 -1 -0.9 0 0.0 116 99.1 100 -2 -1.5 3 2.3 133 100.8 100 -4 -5.8 0 0.0 65 94.2 100 -1 -4.3 0 0.0 22 95.7 100 -1 -1.7 1 1.7 58 100.0 100 -3 -3.8 2 2.5 79 98.8 100 0 0.0 3 1.5 198 101.5 100 0 0.0 3 7.0 46 107.0 100 -1 -0.5 6 3.0 206 102.5 100 -3 -4.2 2 2.8 71 98.6 100 0 0.0 1 1.1 95 101.1 100 -1 -1.1 3 3.4 89 102.3 100 -3 -4.8 0 0.0 59 95.2 100 -1 -0.6 2 1.3 156 100.6 100 -5 -0.8 5 0.8 622 100.0 100 -1 -1.7 3 5.2 60 103.4 100 -2 -2.2 1 1.1 88 98.9 100 -1 -1.1 1 1.1 91 100.0 100 0 0.0 1 0.9 118 100.9 100 0 0.0 1 1.4 74 101.4 100 -2 -2.7 2 2.7 74 100.0 100 -1 -1.8 0 0.0 54 98.2

1999 0.70 0.64 1.10 0.93 0.75 1.62 0.31 0.40 1.19 0.35 1.79 0.67 0.40 0.50 0.39 0.88 4.42 0.53 0.46 0.45 0.84 0.34 0.69 0.55

% 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100

-1

Basal area ( m ha ) GR % M % R % 0.02 3.3 -0.02 -2.6 0.009 1.3 0.03 3.9 0.00 0.0 0.001 0.2 0.04 3.3 -0.04 -3.4 0.000 0.0 0.03 3.4 0.00 -0.3 0.004 0.5 0.02 3.0 -0.03 -3.6 0.000 0.0 0.03 1.9 0.00 -0.1 0.000 0.0 0.01 4.2 0.00 -0.7 0.001 0.4 0.02 3.8 -0.01 -1.8 0.003 0.6 0.04 3.8 0.00 0.0 0.004 0.4 0.01 3.2 0.00 0.0 0.004 1.1 0.06 3.4 0.00 -0.2 0.008 0.5 0.02 2.8 -0.01 -0.9 0.003 0.4 0.02 4.4 0.00 0.0 0.002 0.4 0.02 3.9 0.00 -0.4 0.004 0.7 0.01 3.5 -0.02 -5.1 0.000 0.0 0.03 3.9 -0.02 -1.8 0.003 0.3 0.16 3.7 -0.05 -1.1 0.012 0.3 0.02 3.5 -0.02 -4.7 0.004 0.7 0.02 4.0 0.00 -0.9 0.001 0.3 0.02 3.9 0.00 -0.6 0.001 0.3 0.03 3.5 0.00 0.0 0.001 0.1 0.01 3.9 0.00 0.0 0.001 0.4 0.02 3.1 -0.01 -1.6 0.009 1.3 0.02 2.9 -0.02 -4.3 0.000 0.0

2000 0.72 0.67 1.10 0.97 0.74 1.65 0.32 0.41 1.24 0.36 1.86 0.69 0.42 0.52 0.38 0.90 4.55 0.52 0.48 0.47 0.87 0.35 0.71 0.55

% 102.0 104.1 99.9 103.6 99.3 101.7 104.0 102.6 104.1 104.3 103.6 102.3 104.8 104.3 98.4 102.4 102.9 99.5 103.3 103.6 103.6 104.2 102.8 98.6

MIRMANTO et al. â&#x20AC;&#x201C; Peat swamp forest dynamic in Sebangau

In addition there is negative correlated with total density. This may suggests that there is presence competition among individuals of seedling during early growth rather than among species. In all the forest studied in term of growth, mortality and recruitment may be able to categorize as moderately dynamics forest. Relatively higher in growth rate may suggest that poor nutrient habitat condition was not as major limited factor. This -1 indicated by increasing in basal area about 4% yr -1 -1 (0.71-m2 ha yr ) of 24 most abundance species. Annual recruitment rate was relatively higher, with contribution in basal area about 0.4%. Lower mortality rate compare to some Amazonian forest may suggest that there is no any significant disturbance during study period. The higher mortality rate was only found for uncommon species (not include in this analysis) such as Macaranga sp., which its mortality rate was 26.2%. There is indication that mortality rate of uncommon species was relatively higher but lower in recruitment rate, which may influence diversity of tree species.

CONCLUSION There is significant effect of diameter class on annual growth rate, but not to mortality rate. Even not too strong two environment factors (peat depth and distance to river) were significant correlated with rate of mortality and recruitment. In addition, annual relative growth rate and mortality rate was comparable to previous study but recruitment rate relatively higher. During two-year period study there is no significant changing in vegetation structure

AKNOWLEDGEMENT This work was financed by counterpart budget of Research Center for Biology under LIPI-JSPS Core University Program on Environmental Management of Wetland Ecosystems in Southeast Asia. We wish to thank Messer Suwido H. Limin, Patih, Agung and Adi of Palangkaraya University, Dirman, Wardi Yosman and Aden Muhidin of Herbarium Bogoriense and anonymous reviewer for much help.

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BIODIVERSITAS Volume 10, Number 4, October 2009 Pages: 195-200

ISSN: 1412-033X (printed edition) ISSN: 2085-4722 (electronic) DOI: 10.13057/biodiv/d100406

Diversity and Abundance of Beetle (Coleoptera) Functional Groups in a Range of Land Use System in Jambi, Sumatra 1,♥

FRANCISCUS XAVERIUS SUSILO , INDRIYATI , SURYO HARDIWINOTO

2, ♥♥

Faculty of Agriculture, Lampung University (UNILA), Bandar Lampung 35145, Indonesia 2 Faculty of Forestry, Gadjah Mada University (UGM), Yogyakarta 55281, Indonesia Received: 11th April 2009. Accepted: 10th June 2009.

ABSTRACT Degradation of tropical rain forest might exert impacts on biodiversity loss and affect the function and stability of the related ecosystems. The objective of this study was to study the impact of land use systems (LUS) on the diversity and abundance of beetle functional groups in Jambi area, Sumatra. This research was carried out during the rainy season (May-June) of 2004. Inventory and collection of beetles have been conducted using winkler method across six land use systems, i.e. primary forest, secondary forest, Imperata grassland, rubber plantation, oilpalm plantation, and cassava garden. The result showed that a total of 47 families and subfamilies of beetles was found in the study area, and they were classified into four major functional groups, i.e. herbivore, predator, scavenger, and fungivore. There were apparent changes in proportion, diversity, and abundance of beetle functional groups from forests to other land use systems. The bulk of beetle diversity and abundance appeared to converge in primary forest and secondary forest and predatory beetles were the most diverse and the most abundant of the four major functional groups. © 2010 Biodiversitas, Journal of Biological Diversity Key words: beetle, land use system, diversity, abundance, Sumatra.

INTRODUCTION Land uses are changing (Vitousek, 1994) as linked with deforestation and agricultural intensification (GCTE, 1997). Tropical forests in Indonesia which are known as high biodiversity areas have been declining steadily, with estimated deforestation rate of about 2 million hectares per year (Suparna, 2005). Parts of the forested areas, for instance those in Sumatra, are cleared and changed into various land use systems, including oilpalm plantations, rubber plantations, cassava gardens, and Imperata grassland (van Noordwijk et al., 1995). Changes tropical rain forest to other land-uses might exert impacts on forest fragmentation and degradation, cause loss of diversity and affect the function and stability of the ecosystems. There has been a perception that land use change affects soil biological diversity but more investigations are needed to collect the evidence (Giller et al.,



Corresponding address: Jl. Sumantri Brojonegoro No. 1, Bandar Lampung 35145 Tel.: +62-721-785665, Fax:+62-721-702767, email: fxsusilo2000@yahoo.com

 Jl. Agro-Bulaksumur, Yogyakarta 55281 Tel.: +62-274-512102, 901420, Fax: +62-274-550541 email: suryohardiwinoto@yahoo.com

1997). Jones et al. (2003) reported a reduction in diversity of termites as related to deforestation and agricultural intensification in Jambi, Sumatra. Does land use change affect diversity of soil-related beetles? Beetles are taxonomically diverse and common components of soil community which dwell mainly in litter (Lavelle and Spain, 2001). Soil beetles may play important roles in the ecosystem through their activities as predators, herbivores, and scavengers (Brussaard et al., 1997). Herbivorous beetles may cause crop injury and yield loss while, in contrast, predatory beetles can perform as biological control agents against the crop pests (Kalshoven, 1981). Scavenger beetles comminute and decompose soil organic matters. In agroecosystems beetles are often exposed to soil tillage, chemical pesticide, inorganic fertilizer application, and monoculture planting system. Tillage could damage beetle microniches and foraging sites while insecticide could toxify them. Meanwhile, monoculture system could in one hand limit food access for a number of species but in the other hand allow excessive exploitation for only few other species of herbivorous beetles. This study was aimed at inventorying the diversity and abundance of beetle functional groups in a range of land use systems of different intensity gradient in Jambi, Sumatra.

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MATERIALS AND METHODS Beetle field sampling was done during the end of rainy season (May-June) of 2004 across six land use 2 systems (LUS) distributed over ca. 6 km area in Jambi, Sumatra where a stratified-grid procedure (GW2, 2003) was used to select the sample points. The observed LUS were primary forest (Forest Less Intensive/FLI), secondary forest (Forest Intensive/FI), rubber plantation (Tree-based Intensive/TBI-1), oilpalm plantation (Tree-based Intensive/TBI-2), cassava garden (Crop-based Less Intensive/CBLI), and Imperata grassland (Shrubs/Shrb). Descriptions for each LUS can be found in Prastyaningsih (2005). The sampling area was first delimited into three 2 windows of 2-3 km size each, i.e. Muara Kuamang o o (South 01 34’12.1’’-01 34’52.8’’ and East o o 102 15’05.4’’-102 15’59.7’’) consisted of FLI and FI; o o Kuamang Kuning (South 01 36’32.2’’-01 37’06.1’’ and o o East 102 17’01.7’’-102 17’42.3’’) consisted of TBI-1, TBI-2, Shrb, and CBLI; and Rantau Pandan (South o o o 01 39’03.2’’-01 40’07.6’’ and East 101 56’05.4’’o 101 56’52.0’’) dominated by FLI, FI, and TBI-1. Next, undivided grid of 200 m x 200 m points was set over each window resulting in 64-72 prospective points per window. The grid points were then each groundchecked for feasibility. Feasible criteria included the ease of access and the minimum patch size. A minimum of 20 m x 20 m sampling area of the same patch of land use type should be fit somewhere in a feasible point. Finally, five out of existing feasible points were selected randomly per LUS in a window and taken to be the sample points. That way, the observed LUS and windows where the sample points were selected were as follow: FLI (Muara Kuamang), FI (Muara Kuamang), TBI-1 (Kuamang Kuning), TBI-2 (Kuamang Kuning), Shrb (Kuamang Kuning), and CBLI (Kuamang Kuning). Winkler method (Chung and Jones, 2003) was used to collect beetles from litter in the sample points. In each sample point gross litter was taken from three Winkler quadrates of 1m x 1m along transect laid out 12 m from the center point (GPS grid). The distance between quadrates in transect was 6 m. The litter was sieved, weighed, and incubated (Susilo and Karyanto, 2005). The sieving was done by two persons for five minutes per quadrate using a Winkler sieve (Jones, 2003). The litter materials passing the sieve, i.e. fine 2 litter (sized < 1 cm ) was collected in situ into the Winkler collecting bag for further handling in the incubation room. The fine litter was weighed and placed in the Winkler sieves which were then suspended inside the Winkler bag for incubation for 72 hours under room temperature. During the incubation period the litter dried out, causing beetles to leave it and drop off into the collecting bottle containing 70% alcohol at the base of the Winkler bag. Beetle specimens were then transferred into vials containing 75% alcohol for labeling, storage, and identification. Identification up to family (and some to

subfamily) level was done under a dissecting microscope using Chung (2003) and Borror et al. (1981). The documented data included the beetle diversity and abundance. The diversity was the number of families and subfamilies found in three 1-m quadrates of litter (a sample point). The abundance was taken to be the number of individuals of each family per three 1-m quadrates (i.e. per sample point). Having five records of diversity as well as abundance data, a land use type had its mean diversity and mean abundance data and their corresponding standard errors. Mean diversity was the number of family averaged from five replications (five sampling points) of the same land use. Mean abundance was defined as the number of individuals of beetles (all species combined by sample point) averaged from five replications. The similarity in beetle communities between land use system was determined using Bray-Curtis measure of dissimilarity (B) as follow (Krebs, 1989)

B

X  X

ij ij

 X ik

 X ik  th

X ij , X ik  number of individuals in i

beetle family

in each sample (i.e. land use system), j = one land use system (1, 2, 3, 4, 5, 6), and k = other land use system (1, 2, 3, 4, 5, 6) being compared with j. The resulting B values were used to compose a dendrogram and grouping of land use systems. The collected beetles were then grouped by their functional groups. Six feeding groups identified in Chung et al. (2000) which followed Hammond (1990) were simplified into four, i.e. herbivores, predators, scavengers, and fungivores. The mean diversity and abundance data were calculated by land use systems, by beetle functional groups, and by combination of land use system-functional group. The mean values were compared between land use systems, between beetle functional groups, and between combination of land use system-functional group using ANOVA and LSD at 5% level of significance (Snedecor and Cochran, 1980). In addition, the diversity data, i.e. the number of beetle families of each functional group, was also pooled within each land use system and their proportion of each functional group was plotted by land use systems.

RESULTS AND DISCUSSION The beetles recovered from litter of six land use systems in Jambi consisted of 47 families and subfamilies with four major feeding groups (Table 1). Figure 1 shows three groups of land use systems in Jambi based on dissimilarity (similarity) in their beetle community composition at (sub) family level, namely

0.60 0.54

0.49

0.40 0.37 0.20 FLI

TBI-1

Shrb

TBI-2

CBLI

Land use grouping

Figure 1. Dendrogram of dissimilarity in beetle communities in a range of land use systems in Jambi (FLI = primary forest, FI = secondary forest, TBI-1 = rubber plantation, TBI2 = oilpalm plantation, Shrb = Imperata grassland, CBLI = cassava garden).

Based on the total diversity and total abundance, the functional groups can be arranged in a descending order, as follows: predator, scavenger, herbivore, and fungivore (Figure 2). One family, Byrrhidae, is the moss feeders. The pooled data showed apparent changes in proportion of some functional groups from forests to other land use systems (Figure 3 and Figure 4). Herbivore diversity and abundance were of higher proportion in agroecosystem or Imperata grassland as compared with those in the forest. Similar pattern held for scavengers. Predator’s proportion, however, showed different pattern; while no apparent change occurred in its diversity proportion (Figure 3) the abundance proportion of predator pool decreased in non-forest land use systems (Figure 4). As for fungivores, there was no clear pattern of increase or decrease in their proportion relative to the other functional groups. 70

Diversity (% families) Abundance (% indiv.)

60 50 40 30 20 10 0 Predator

Scavenger

Fungivore

Herbivore

Other

Beetle functional group

Figure 2. Total diversity and abundance of four major functional groups of beetles in Jambi 100% 80%

Herbivore

60%

Fungivore

40%

Scavenger

20%

Predator CBLI

Shrb

TBI-2

TBI-1

FLI

No. Family and sub-family Functional group* 1 Anthicidae SCAVENGER 2 Brentidae HERBIVORE 3 Byrrhidae MOSS FEEDER 4 Carabidae PREDATOR 5 Cerambycidae HERBIVORE 6 Alticinae HERBIVORE 7 Eumolpinae HERBIVORE 8 Galerucinae HERBIVORE 9 Coccinellidae PREDATOR 10 Colydiidae PREDATOR 11 Corylophidae FUNGIVORE 12 Cryptophagidae FUNGIVORE 13 Cucujidae SCAVENGER 14 Chryptorhynchinae HERBIVORE 15 Otiorhynchinae HERBIVORE 16 Rhynchoporinae HERBIVORE 17 Elateridae HERBIVORE 18 Histeridae PREDATOR 19 Hydrophilidae PREDATOR 20 Languriidae FUNGIVORE 21 Leiodidae SCAVENGER 22 Lymnichidae HERBIVORE 23 Mycetophagidae FUNGIVORE 24 Mycteridae SCAVENGER 25 Nitidulidae FUNGIVORE 26 Pselaphidae PREDATOR 27 Ptiliidae SCAVENGER 28 Scaphididae FUNGIVORE 29 Aphodiinae SCAVENGER 30 Melolonthinae SCAVENGER 31 Valginae SCAVENGER 32 Scirtidae SCAVENGER 33 Scolytidae FUNGIVORE 34 Scydmaenidae PREDATOR 35 Silvanidae SCAVENGER 36 Aleocharinae PREDATOR 37 Euaesthetinae PREDATOR 38 Osoriinae PREDATOR 39 Oxytelinae PREDATOR 40 Paederinae PREDATOR 41 Protopselaphinae PREDATOR 42 Tachyporinae PREDATOR 43 Staphylininae PREDATOR 44 Asidinae SCAVENGER 45 Tentyriinae SCAVENGER 46 Tenebrioninae SCAVENGER 47 Throscidae FUNGIVORE Note: *) based on Hammond (1990), Chung et al. (2000), and other sources (including Borror et al., 1981 and Kalshoven, 1981)

0.80

0.00

Percentage

Table 1. Taxonomic and functional group diversity of beetles collected using Winklers in Jambi, Sumatra, MayJune 2004

197

1.00

Diversity of beetles (nos families)

(i) the primary forest (FLI)-secondary forest (FI), (ii) the rubber plantation (TBI-1)-oilpalm plantation (TBI2), and (iii) Imperata grassland (Shrb)-cassava garden (CBLI). The beetle community assemblages in the second group are more similar to the third than to the first group. In other words, the beetle assemblages in the non-forested land use systems are less similar to those in the forested systems. However, the grouping might be clearer if the beetle community composition was delimited down to the lower taxonomic level, i.e. genus or species.

Bray-Curtis Dissimilarity

SUSILO et al. – Coleoptera in Jambi, Sumatra

Land use system

Figure 3. Total diversity proportions of four major functional groups of beetles in a range of land use systems in Jambi (FLI = primary forest, FI = secondary forest, TBI-1 = rubber plantation, TBI-2 = oilpalm plantation, Shrb = Imperata grassland, CBLI = cassava garden)

B I O D I V E R S I T A S Vol. 10, No. 4, October 2009, pp. 195-200

Total abundance of beetle functional groups (indiv.)

198

100% 80%

Herbivore

60%

Fungivore Scavenger

40%

Predator

20% 0% FLI

TBI-1

TBI-2

Shrb

CBLI

Land use system

Figure 4. Total abundance proportions of four major functional groups of beetles in a range of land use systems in Jambi (FLI = primary forest, FI = secondary forest, TBI-1 = rubber plantation, TBI-2 = oilpalm plantation, Shrb = Imperata grassland, CBLI = cassava garden)

The overall bulk of beetle diversity appeared to converge in primary forest (FLI) and secondary forest (FI) (Table 2). Table 2 shows significant decrease in the beetle overall mean diversity (number of family) as the primary forest (FLI) were changed into nonforest systems (Shrb, TBI-1, TBI-2, CBLI). The predatory beetles were the most diverse of the four major functional groups (Table 3). Figure 5 separates the beetle mean diversity by land use systems and the beetle functional groups. It depicts the following information. The highest diversity of predatory beetles was found in primary forest. The diversities within other functional groups (scavengers, fungivores, or herbivores) did not significantly fluctuate across land use systems but the diversity between the functional groups varied within a common land use system. In primary forest, the diversities of scavenger, fungivore, and herbivore beetles were comparatively of the same level but less than that of predatory beetles. In secondary forest, the diversity of beetle functional groups could be clustered in three classes, i.e. relatively high diversity (predators), relatively low diversity (fungivores and herbivores), and in between the two (scavengers). Three classes of beetle diversity could also be seen in rubber plantation (TBI1) and oilpalm plantation (TBI-2), i.e. relatively high diversity (predators), relatively low diversity (herbivores), and in between the two (scavengers and fungivores). No variation in diversities was shown between beetle functional groups either in Imperata grassland (Shrb) or cassava garden (CBLI). The diversities of each beetle functional group in the later two land use systems were unfluctuated and relatively of low level. The highest beetle abundance was found in primary forest (FLI) and secondary forest (FI) (Table 2). Table 2 shows significant decrease in the beetle overall mean abundance as the primary forest (FLI) were changed into non-forest systems (Shrb, TBI-1, TBI-2, CBLI). Predatory beetles were the most abundant functional group (Table 3). Figure 6 shows differences in abundance between beetle functional

groups within land use systems but no differences in abundance within beetle functional groups across land use systems (except those of predators). The highest abundance of predatory beetles was found in primary forest; while the second highest was in secondary forest. No abundance differences across land use systems were detected for scavenger, fungivore, and herbivore beetles. In primary forest and secondary forest, predatory beetles were more abundant than scavenger, fungivore, or herbivore beetles. However, no differences in abundance of all four functional groups were detected in rubber plantation (TBI-1), oilpalm plantation (TBI-2), Imperata grassland (Shrb), and cassava garden (CBLI). The collapse of beetle assemblage along a land-use intensification gradient from less disturbed (forested) to more disturbed (non-forested) land use systems as shown in this study is in accordance with the results of other soil insect studies done previously in Sumatra, including Susilo et al. (2006) on beetles in Lampung, Susilo and Hazairin (2006) on ants in Lampung, Susilo and Aini (2005) on termites in Lampung, and Jones et al. (2003) on termites in Jambi. Table 2. Mean diversity and mean abundance of beetles in a range of land use systems in Jambi Mean diversity Mean (number of abundance Land use system family/sample (indiv./sample point) point) FLI 2.2 a 8.6 a FI 1.7 ab 5.8 ab Shrb 1.0 b 2.0 c TBI-1 1.2 b 2.3 c TBI-2 1.3 b 2.9 bc CBLI 1.3 b 4.2 bc LSD0.05 0.8 3.1 Note: mean values followed by different letters are significantly different using LSD test at 0.05 level (FLI = primary forest, FI = secondary forest, TBI-1 = rubber plantation, TBI-2 = oilpalm plantation, Shrb = Imperata grassland, CBLI = cassava garden)

Table 3. Mean diversity of four major functional groups of beetles in Jambi Mean diversity Mean (number of abundance Functional groups family/sample (indiv./sample point) point) Predator 2.7 a 10.9 a Scavenger 1.4 b 3.0 b Fungivore 0.8 b 1.8 b Herbivore 0.8 b 1.4 b LSD0.05 0.6 2.5 Note: mean values followed by different letters are significantly different using LSD test at 0.05 levels

Beetle mean diversity (nos. family per sample point)

SUSILO et al. – Coleoptera in Jambi, Sumatra

CONCLUSIONS

6 5

Predator

Scavenger

Fungivore

Herbivore

4 3 2 1 0 FLI

TBI-1

TBI-2

Shrb

CBLI

Land use system

Figure 5. Mean diversity of four major functional groups of beetles in a range of land use systems in Jambi (FLI = primary forest, FI = secondary forest, TBI-1 = rubber plantation, TBI-2 = oilpalm plantation, Shrb = Imperata grassland, CBLI = cassava garden)

The inventory resulted in 47 families and subfamilies of beetles with four major functional groups, i.e. herbivores, predators, scavengers, and fungivores. There was an apparent difference of the beetle familial assemblages between land uses or groups of land uses based on bray-curtis indices. Changes of beetle assemblages were also detected in proportion and abundance of beetle functional groups from forests to other land use systems. There were differences in diversity and abundance between beetle functional groups within land use systems but no differences in diversity and abundance within beetle functional groups across land use systems (except those of predators). The bulk of beetle diversity and abundance appeared to converge in primary forest and secondary forest while predatory beetles were the most diverse and the most abundant of the four major functional groups.

ACKNOWLEDGMENTS

35 Beetle mean abundance (indiv. per sample point)

199

Predator Fungivore

Scavenger Herbivore

25 20 15 10 5 0 FLI

TBI-1 TBI-2 Shrb Land use system

CBLI

Figure 6. Mean abundance of four major functional groups of beetles in a range of land use systems in Jambi (FLI = primary forest, FI = secondary forest, TBI-1 = rubber plantation, TBI-2 = oilpalm plantation, Shrb = Imperata grassland, CBLI = cassava garden)

This publication presents part of the findings of the international project “Conservation and Sustainable Management of Below-Ground Biodiversity (CSMBGBD)” implemented in seven tropical countries, namely Brazil, Mexico, Cote d’Ivoire, Kenya, Uganda, India, and Indonesia. The project is coordinated by Tropical Soil Biology and Fertility Institute of CIAT (TSBF-CIAT) with co-financing from the Global Environment Facility (GEF) and implementation support from the United Nations Environment Programme (UNEP). A.M. Hariri and L. Wibowo deserve our special appreciation for their assistance in the identification and tallying the beetles in the laboratory. Our sincere gratitude also goes to Dr. Shahabuddin Saleh for critically reviewing this manuscript.

REFERENCES Environmental disturbance may lead to reorganizations of local resources available to consumers which in turn affects the food web structure (Chung et al., 2000). This process explains changes in tropic group proportion, especially in the most disturbed habitats. The disturbance is usually suitable for herbivores (Lawrence, 1996; Chung et al., 2000) but unfavorable for predators (Brown and Southwood, 1983; Pimm et al., 1991; Chung et al., 2000). Figure 4 seems to conform to the theory (decrease the proportion predator abundance and increase the proportion of herbivore abundance), as are Figure 5 and Figure 6 (decrease the mean diversity and mean abundance of predatory beetles). It is interesting to note, however, that the disturbance also seems to favor scavengers (Figure 4, increase the proportion of scavenger abundance).

Borror, D.J., D.M. De Long, and C.A. Triplehorn. 1981. An Introduction to the Study of Insects. 5th ed. Philadelphia: Saunders College Publ. Brown, V.K. and T.R.E. Southwood. 1983. Tropic diversity, niche breadth and generation times of exopterygote insects in a secondary succession. Oecologia 56: 220-225. Brussaard, L., V.M. Behan-Pelletier, D.E. Bignell, V.K. Brown, W. Didden, P. Folgarait, C. Fragoso, D.W. Freckman, V.V.S.R. Gupta, T. Hattori, D.L. Hawksworth, C. Klopatek, P. Lavelle, D.W. Malloch, J. Rusek, B. Soderstrom, J.M. Tiedje, and R.A. Virginia. 1997. Biodiversity and ecosystem functioning in soil. Ambio 26, 563-570. Chung, A.Y.C. and D. T. Jones. 2003. Extraction rates of soil arthropods using the winkler bag sampling technique. In: Jones, D.T. (ed.). Tools for the Rapid Assessment of Soil Invertebrate Biodiversity in the ASEAN Region; Handbook to Accompany the Training Course, 13-25 October 2003. Kota Kinabalu: Universiti Malaysia Sabah. Chung, A.Y.C. 2003. Manual for bornean beetle (Coleoptera) family identification. In: Jones, D.T. (ed.). Tools for the Rapid

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Assessment of Soil Invertebrate Biodiversity in the ASEAN Region; Handbook to Accompany the Training Course, 13-25 October 2003. Kota Kinabalu: Universiti Malaysia Sabah. Chung, A.Y.C., P. Eggleton, M.R. Speight, P.M. Hammond, and V.K. Chey. 2000. The diversity of beetle assemblages in different habitat types in Sabah, Malaysia. Bulletin of Entomological Research 90: 475-496. GCTE (Global Change Terrestrial Ecosystem). 1997. Living with Global Change: Linking Science and Policy in South East Asia. Workshop Summary and Synthesis. GCTE Report No. 14/BIOTROP-GCTE Southeast Asian Impacts Centre (IC-SEA) Report No. 2. Canberra. Giller, K.E., M.H. Beare, P. Lavelle, A.M.N. Izac, and , M.J. Swift. 1997. Agricultural intensification, soil biodiversity and agroecosystem function. Applied Soil Ecology 6: 3-16. GW2 (Global Workshop II Report). 2003. Conservation and Sustainable Management of Below-ground Biodiversity. UNEPGEF TSBF-CIAT Project GF/2715-02-4517 GF/1030-02-05. February 24-28, 2003. Sumberjaya-Lampung, Indonesia. Hammond, P.M. 1990. Insect abundance and diversity in the Dumago-Bone National Park, North Sulawesi, with special reference to the beetle fauna of lowland rainforest in the Toraut region. In: Knight, W.J. and J.D. Holloway (eds.). Insects and the Rainforests of South East Asia (Wallacea). London: The Royal Entomological Society of London. Jones, D.T. 2003. In: Jones, D.T. (ed.). Tools for the Rapid Assessment of Soil Invertebrate Biodiversity in the ASEAN Region; Handbook to Accompany the Training Course, 13-25 October 2003. Kota Kinabalu: Universiti Malaysia Sabah. Jones, D.T., F.X. Susilo, D.E. Bignell, S. Hardiwinoto, A.N. Gillison, and P. Eggleton. 2003. Termite assemblage collapse along a land-use intensification gradient in lowland central Sumatra, Indonesia. Journal of Applied Ecolology 40: 380-391. Kalshoven, L.G.E. 1981. Pests of Crops in Indonesia. Rev. and Transl. by P.A. van der Laan. Jakarta: P.T. Ichtiar Baru-van Hoeve. Krebs, C.J. 1989. Ecological Methodology. New York: Harper Collins Publ.

Lavelle, P. and A.V. Spain. 2001. Soil Ecology. Amsterdam: Kluwer Academic Publisher. Lawrence, W.T., Jr. 1996. Plants: the Food Base. In: Reagan, D.P. and R.B. Waide (eds.). The Food Web of a Tropical Rainforest Chicago: The University of Chicago Press. Pimm, S.L., J.H. Lawton, and J.E. Cohen. 1991. Food web patterns and their consequences. Nature 350: 669-674. Prastyaningsih, S.R. 2005. Diversitas Makrofauna Tanah pada Berbagai Tipe Penggunaan Lahan di Jambi. [Tesis]. Yogyakarta: Program Studi Kehutanan Jurusan Ilmu-ilmu Pertanian, Sekolah Pascasarjana Universitas Gadjah Mada. Snedecor, G.W. and W.G. Cochran. 1980. Statistical Methods. 7th ed. Ames: The Iowa State Univ. Press. Suparna, N. 2005. Meningkatkan produktivitas kayu dari hutan alam dengan penerapan silvikultur intensif di PT Sari Bumi Kusuma Unit Seruyuan-Kalteng. Dalam: Hardiyanto, E.B. (ed.) Prosiding Seminar Nasional: Peningkatan Produktivitas Hutan. Proyek ITTO PD 106/01 Rev. 1 (F). Yogyakarta: Fakultas Kehutanan UGM & International Tropical Timber Organization. Susilo, F.X. and F.K. Aini. 2005. Diversity and density of termites in a range of land use types in the Rigis Hill Area, SumberjayaLampung. Jurnal Sains dan Teknologi 11(3): 129-136. Susilo, F.X. and M. Hazairin. 2006. Forest conversion into coffeebased agroforestry systems in Sumberjaya reduces abundance of predatory Myrmicine ants. Agrivita 28 (3): 238-251. Susilo, F.X. and A. Karyanto. 2005. Methods for Assessment of Below-ground Biodiversity in Indonesia. Bandar Lampung: Universitas Lampung. Susilo, F.X., A.M. Hariri, Indriyati, dan L. Wibowo. 2006. Keanekaragaman dan populasi kumbang pada berbagai sistem penggunaan lahan di Bukit Rigis Sumberjaya, Lampung Barat. Jurnal Sains dan Teknologi 12 (3): 143-148. van Noordwijk, M., T.P. Tomich, R. Winahyu, D. Murdiyarso, Suyanto, S. Partoharjono, and M. Fagi (eds.) 1995. Alternative to Slash-And-Burn in Indonesia. Summary Report Phase 1. Bogor: ASB-Indonesia Report No. 4 Vitousek, P.M. 1994. Beyond global warning ecology and global change. Ecology 75: 1861-1876.

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ISSN: 1412-033X (printed edition) ISSN: 2085-4722 (electronic) DOI: 10.13057/biodiv/d100407

Population and Microhabitat Characteristic of Homalomena bellula Schott in Mount Slamet, Central Java, Indonesia ♥

YAYAN WAHYU CANDRA KUSUMA , INGGIT PUJI ASTUTI Center for Plant Conservation-Bogor Botanic Garden, Indonesian Institute of Sciences (LIPI), Bogor 16003. . Received: 3rd March 2009. Accepted: 16th June 2009.

ABSTRACT The population and habitat characteristics of Homalomena bellula Schott in Java have received little attention. In this present study, the authors estimated the population density and habitat characteristic of H. bellula in Mount Slamet, Central 2 Java, by means of random samplings of 2 x 2 m plots. Two distinct populations were studied at two different altitude levels, i.e. below and above 1000 m asl. We used to suspect that there was a difference in both populations. But our result revealed that these two populations were actually similar in abundance, population density, and dispersion pattern and even in microhabitat characteristics. © 2009 Biodiversitas, Journal of Biological Diversity Key words: Homalomena bellula, Araceae, population density, population structure, microhabitat. Introduction

Homalomena has a wide range of distribution throughout the world. About 150 species occur in Indo-Malesia to South China and Solomon Islands. Another eight species are found in the neotropics (Hay, 1999), although recent molecular evidence strongly questioning the generic status of the neotropical species (Gauthier et al., 2008). In Indonesia, Homalomena comprises of 42 species, distributed from Sumatra to Papua Barat and only eight of which are recorded in Java (Govaerts and Frodin, 2000). Another report stated that about 18 species of Homalomena exist in Java (Hay et al., 1995). However, all of these numbers must be regarded as provisional since there has been no critical revision of Homalomena for over a century. Recently initiated work in Sarawak suggests that for North Borneo alone the number of Homalomena species may exceed 200, with virtually all endemic. At the present time no Homalomena species are a major priority for conservation and as none have been included in The Red List IUCN Data Book (IUCN, 2007). This may be loosely interpreted as that no Homalomena are threatened. However, considering the rate of forest degradation in Indonesia, some exception might be applied for species that have

 Correcponding address: Jl. Ir. H. Juanda No. 13, Bogor 16122. Tel. +62-251-8322187; Fax. +62-251 8322187 e-mail: yayanwahyu@gmail.com

limited distribution such as Homalomena bellula Schott. Homalomena bellula is distinct from other Javanese Homalomena in having a shoot organization that resembles a ginger in that flowering is both functionally and anatomically terminal, with the axis renewal taking place by the release of a dormant lateral bud. Thus the architecture of the rhizome resembles that of many gingers and most Schismatoglottis species allied to S. calyptrata (Roxb.) Zoll. & Moritzi. This shoot organization is in marked contrast to that of other Homalomena species where the shoot consists of a physiognomically unbranched sympodium. In common with most other Homalomena, the rhizome and aerial shoot tissue smells strongly of ginger when cut. Homalomena bellula is ca. 40 cm high with a creeping rhizome. The leaves are narrowly ovatehastate-sagittate, mid-green adaxially, pale green abaxially, 22 cm long and 10 cm wide, with acuminate to cuspidate apex. The spathe is erect, pale green, not constricted, slightly oblong, about 6.5 cm long, with caudate tip about 1.5 cm long. Spadix is stipitate for about 40 cm, sub-equal to spathe. This longer description was made by Yuzammi (2000). The species was described by Schott in 1863 based on a type specimen from Mount Halimun (Yuzammi, 2000). Homalomena bellula is endemic to Java with limited distribution in West and Central Java (Yuzammi, 2000). A survey conducted in Mount Halimun by Bogor Botanic Garden team in 1997 and 1999, resulted in no records of the species, suggesting that the population may no longer exist in that area. A recent record of this species from an

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expedition in Mount Slamet done by Yuzammi and colleagues (Yuzammi, 2000). They found a single population of H. bellula in Baturraden, southern slope of Mount Slamet in Central Java, suggesting the existence of other H. bellula population in other parts of the area. According to Yuzammi (2000), H. bellula is distributed within a narrow range of altitude of around 750 m asl. Thus, it also has a specific altitude beside its limited geographic distribution. A species with a confined distribution and considered as rare is vulnerable to environment changes, and consequently more vulnerable to extinction (Sรถderstrรถm et al., 2007). This study was aimed to estimate the current population of H. bellula and to describe its population structure and microhabitat characteristics. This information is very important in understanding the ecology of this species, and will also provide ecological data useful for deciding future conservation policy.

MATERIAL AND METHODS Study sites Mount Slamet, approximately 3,432 m asl, is the highest peak in Central Java. It is surrounded by five districts: Purbalingga, Banyumas, Pemalang, Tegal, and Brebes. It covers an area of about 50,000 ha of which about (10,000) ha has been allocated as

protected forest. The forests are under the authority of the state company Perhutani (Indonesian State Forest Enterprise) as production forest, limited production forest and protected forest. The protected forests consist of three vegetation zones: sub montane (1,500-2,000 m asl), montane (2,000-2,400 m asl) and alpine (above 2,500 m asl), which providing a high biodiversity. Field work for this research was conducted in September, 2007 in Pancuran Tujuh Area, the southern slope of Mount Slamet, a tourism area managed by Perhutani, the private holding company inside the forest area (Figure 1). The altitude of the study area was between 750 to 1,500 m asl (S 0 0 07 18'590'' and E 109 13'088''). Total annual precipitation of this area is more than 5,000 mm/year, 0 0 with temperature ranging between 20 and 30 C. The soil type of the area is dominated by clay with dark reddish brown color. Species composition in the protected forest is comprised of several taxa such as Lauraceae, Myrtaceae, Annonaceae, Leeaceae, Sterculiaceae, Sapindaceae, Euphorbiaceae, and Rubiaceae. In the understorey, species composition dominated by herbs and small trees. The understorey species that are also suspected to be associated with H. Bellula as their coexistence are Schismatoglottis calyptrata, Elatostema rostratum, Cyrtandra sp., Curculigo sp., and Clidemia hirta.

Figure 1. Map of research area in southern slope of Mount Slamet.

KUSUMA & ASTUTI – Homalomena bellula Schott in Mount Slamet

Methods The populations of H. bellula were determined by random transect survey. All individuals were recorded and mapped by GPS (Global Positioning System). All groups of plant with at least 500 m distance apart were considered as a different population or regarded as subpopulations (Osunkoya, 1999). Natural or arbitrary boundaries such as a large stream or a cultivation area were also used to differentiate the populations. Density of each population was determined by 2 using quadrant plots (2 x 2 m ) in which the presence and abundance of H. bellula were recorded and counted. Population density was determined by dividing the number of all individuals within the total plot sample by the total area of plot sampled in each population. In recording and counting all individuals within the sample plots, three different growth stages of the plant were distinguished, i.e. juvenile (including seedling), vegetative, and reproductive stage. The dispersion pattern of each population was also calculated using Morisita’s Index with an assumption that the population consisted of clumps or patches of individuals of different densities, and that within a clump individuals are randomly spaced (Poole, 1974). As an addition data on microhabitat characteristics within sample plots for each population was also collected. These included temperature, humidity, soil temperature, soil humidity, light intensity, and canopy covering percentage. The microhabitat characteristics were used for estimating microhabitat preference of H. bellula. Data were subsequently analyzed descriptively, except for comparison of population density and microhabitat characteristics between populations. This comparison were analyzed using nonparametric Kruskal-Wallis tests because the underlying distributions of these variables were unknown (Potvin and Roff, 1993). The statistics analysis was carried out by SPSS version 15.0 (SPSS Inc.)

RESULTS AND DISCUSSION Population and density Based on random transect survey two groups of H. bellula with different altitude level were identified. One group in the area below 1,000 m asl. (colline zone) was identified as Pancuran Tujuh population (PTP) and another group in the area above 1000 m asl (submontane zone) was identified as Hutan Rimba Population (HRP). The Garmin mapsource program was used to determine the distance between two groups and giving a result of 1.2 km, approximately. According to Osunkoya (1999) and Yakimowski and Eckert (2007) those groups were considered as two separate populations based on long distance between groups (more than 1 km) and also in difference of

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topography which included altitudinal and arbitrary boundary, a cultivated pine and Agathis forest among both groups. The comparison of two populations using KruskalWallis test shows that there was no significant difference in population density of PTP and HRP (ρ≈0.09; U=59.5). Even though, from the table it was clearly shown that there were differences in the frequency of occurrence and the population density, which was PTP relatively denser than HRP (table 1). Many factors, biotic and abiotic can affects the abundance of plant populations. At the local scale, however, biotic interactions often determine the abundance of plants (Medel-Narvaez et al., 2006) Table 1. The population density of each population of H. bellula.

Site Pancuran Tujuh (PTP) Hutan Rimba (HRP)

Population Frequency of Altitude Habitat Plot density (mean occurrences (m asl) Type numbers ± SE) (%) (individual/m2) < Mixed 1000 Forest

61.90

0.86 ± 0.28

> Mixed 1000 Forest

22.22

0.25 ± 0.16

In Araceae, the abundance can also be greatly affected by vegetative reproduction (stolons, offshoots, etc.). In Sarawak, Schismatoglottis motleyana often dominates the herbaceous layer of entire valleys and yet seldom flowers and fruits. Such super-clones appear to have almost entirely given up sexual reproduction. A similar situation is found with the helophytes Homalomena rostrata and H. expedita (P. Boyce, pers. comm., 2009). In accordance with our field observation there were several traits that we predicted to be possibly affecting the differences of abundance and population density between both populations: the pollination, population patterns and habitat characteristics. Homalomena bellula, as the family of Araceae in general, has a unique inflorescence and required pollinator agent due to its protogynous anthesis. Araceae are generally pollinated by insects, principally beetles and flies but also bees and thrips (Gibernau et al., 1999). We suspected that pollination may play a crucial part in determining population density and relative abundance. Population structure and dispersion pattern The populations of H. bellula were quite different in the percentage number of each growths stage except in fruiting adult stage (Figure 1). About ten percent of both population members were at fruiting adult stage and eighty percent were in the other different stage classes. In contrast, the pattern of population

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structure of both populations was similar; both fitting the log-normal distribution. It revealed that the populations are expected for young growing populations under constant recruitment. 90 PTP

HRP

60 50 40 30 20 10 0 Juvenile

Non flowering adult plant

Flowering adult plant

Fruiting adult plant

Grow th stage

Number of individuals

Percentage (%)

sometimes by observing the dispersion patterns of the individuals some insight into the biological characteristics of the species and the reasons behind the changes in density of the population can be gained. Using Morisitha Index dispersion, both H. bellula populations are have the same type of dispersion, which is clump; the calculation yielded 4 and 2,853 for PTP and HTP, respectively.

25 20 15 10 5 0 0

200

400

600

800

1000

1200

1400

Altitude (m a.s.l)

Figure 2. Population characteristic of H. bellula.

Figure 3. The distribution of H. bellula based on altitude level.

The difference in amount of flowering and fruiting individuals was not obvious (Figure 2). Both populations bear individuals producing fruits. However, further observation is needed to study the reproductive biology; because we suggest that the pollinator play an important rule in the successful pollination. It is in accordance with our observation of H. bellula collection in Bogor Botanic Garden in which mature fruit of H. bellula was not successfully produced. In spite of producing fruit and seed, the species is likely favorable to use vegetative (clonal) reproduction in order to reproduce. Their creeping rhizomes are able to produce new shoots and develop to new individuals. Apparently reduced sexual reproduction and improved asexual reproduction is particularly common to plant in which sexual reproduction cannot occur (Eckert, 2002). The altitudinal distribution of this species in this study area varied between 767 to 1287 m asl. Yuzammi (2000) recorded the altitude level of H. bellula as far as 750 m asl., but in this study we discovered individuals at an altitude of more than 1000 m asl (Figure 3). Compared to Araceae in general that have a broad ecological range (Leimbeck et al., 2004), this distribution was regarded as a narrow distribution in altitudinal level. Density of plant populations can also be used to represent a function of the spacing between neighboring individuals (Kunin, 1997). This means that close distance of each individual within the population can increase the population density by stimulating accretion of population size. Spacing between neighboring individuals could be estimated using dispersion pattern of the population. Poole (1974) mentions that in field populations it is difficult to define sharply the population interactions, but

Habitat characteristics The microhabitat characteristics in both populations were compared using Kruskal-wallis test and revealed not significantly different, except for slope degree (Table 2). This means that both populations tend to occupy similar microhabitat. Degree of slope was the only significant habitat parameter that differs in both populations, leading to a difference in population density (Table 2). According to Boyce (2004), the aroids are overwhelmingly plants of humid and shaded forest which is easily found on steep slopes of stream valleys at low to mid-elevation (up to 850 m asl). The area in Pancuran Tujuh has more valleys than in Hutan Rimba, even though the valleys were steeper. Thus, the result confirm that steep slope within a certain degree, seems to be favored by H. bellula.

Table 2. The comparison of habitat characteristic in two H. bellula populations. Site Parameter

PTP HRP Asymp. Sig. (mean ± SE) (mean ± SE) (2-tailed)

Soil pH 6.8±0.04 6.9 .050 Soil humidity (% RH) 54.4±5.6 50 .566 0 Temperature ( C) 24.3±0.6 24.5±0.5 .469 Humidity (% RH) 85±3.6 85±5 1.000 Light Intensity (Lux) 842.3±156 107.5±22.5 .145 Topography Hilly Hilly o Slope ( ) 54.2±2.7 31±11 .024* Canopy covering (%) 55.6±8 70±10 .247 Note: PTP (Pancuran Tujuh Population); HRP (Hutan Rimba Population). Data were automatic recoded to fill the assumptions. * significant in α=5%

KUSUMA & ASTUTI – Homalomena bellula Schott in Mount Slamet

CONCLUSIONS There were two populations of H. bellula in Southern Slope of Mount Slamet; one occupy colline zone and the other occupy submontane zone. Both populations were similar in population density, population structure, dispersion pattern and microhabitat characteristics. The population seems to be in good condition due to its log-normal size distribution that expected for young, growing populations with constant recruitment, supported by many numbers of juveniles for population maintenance. Future study should focus on monitoring the population and estimating the population growth rate to address the best management system of the species.

ACKNOWLEDGMENTS We would like to thank Sutrisno, Annisa Satyanti and Peter Boyce for their insightful remarks and constructive suggestions, which led to a substantial improvement in the paper. We grateful thank to Yuzammi for her helpful discussion during the paper writing. We also would like to thank Subroto Widyatmoko (Perhutani) for the permission and Kartam for help and enjoyable discussion during the field work.

REFERENCES Boyce, P. 2004. The aroids of Borneo. Gardenwise. 23: 11-13. Singapore Botanic Garden. Eckert, C.G. 2002. The loss of sex in clonal plants. Evolutionary Ecology 15: 501-520. Gauthier, M.P.L., D. Barabe, and A. Bruneau. 2008. Molecular phylogeny of the genus Philodendron (Araceae): delimitation and infrageneric classification. Botanical Journal of the Linnean Society 156: 13-27.

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Gibernau, M., D. Barabe, P. Cerdan and A. Dejean. 1999. Beetle pollination of Philodendron solimoensense (Araceae) in Frecnh Guiana. International Journal of Plant Sciences 160 (6): 1135-1143. Govaerts, R. and D. G. Frodin. 2000. World Checklist and Bibliography of Araceae (and Acoraceae) Royal Botanic Gardens, Kew. Published in the Internet; http: //www.kew.org/wcsp/ accessed October 11, 2007. Hay, A., P. C. Boyce, W. L. A. Hetterscheid, N. Jacobsen, J. Murata and J. Bogner. 1995. Checklist and botanical bibliography of the Araceae of Malesia, Australia and The Tropical Western, Pacific Region. Blumea Supplement: 1-161. Hay, A.1999. Revision of Homalomena (Araceae-Homalomeneae) in New Guinea, The Bismarck Archipelago and Solomon Islands. Blumea 44: 41-71 IUCN. 2007. 2007 IUCN Red List of Threatened Species. www.iucnredlist.org. Accessed October 23, 2007. Kunin, W.E. 1997. Population size and density effects in pollination: pollinator foraging and plant reproductive success in experimental arrays of Brassica kaber. Journal of Ecology 85: 225-134. Leimbeck, R.F., T. Valencia and H. Balslev. 2004. Landscape diversity patterns and endemism of Araceae in Ecuador. Biodiersity and Conservation 13: 1755-1779. Medel-Narvaez, A., J. L. L. de la Luz, F. Freaner-Martinez and F. Molina-Freaner. 2006. Patterns of abundance and population structure of Pachycereus pringlei (Cactaceae), a columnar cactus of the Sonoran Desert. Plant Ecology 187: 1-14. Osunkoya, O.O.,1999. Population structure and breeding biology in relation to conservation in the Gardenia actinocarpa (Rubiaceae) - a rare shrub of North Queensland rainforest. Biological Conservation 88: 347-359. Poole, W.P. 1974. An Introduction to Quantitative Ecology. Tokyo: McGraw-Hill Kogakhusa. Potvin, C., and D. A. Roff. 1993. Distribution-free and robust statistical methods: viable alternatives to parametric statistics. Ecology 74: 1617-1628. Söderström, L., A. Séneca, and N. Santos. 2007. Rarity patterns in members of the Lophoziaceae/Scapaniaceae complex occurring North of the Tropics - Implications for conservation. Biological Conservation 135: 352- 359. Yakimowski, S.B., and C.G. Eckert. 2007. Threatened peripheral populations in context: geographical variation in population frequency and size and sexual reproduction in a clonal woody shrub. Conservation Biology 21 (3): 811-822. Yuzammi. 2000. A Taxonomic Revision of the Terrestrial and Aquatic Aroids (Araceae) in Java. [M.Sc. thesis]. School of Biological Sciences Faculty of Life Science. University of New South Wales.

BIODIVERSITAS Volume 10, Number 4, October 2009 Pages: 206-209

ISSN: 1412-033X (printed edition) ISSN: 2085-4722 (electronic) DOI: 10.13057/biodiv/d100408

The Escherichia coli Growth Inhibition Activity of Some Fermented Medicinal Plant Leaf Extract from the Karo Highland, North Sumatra â&#x2122;Ľ

EVI TRIANA , NOVIK NURHIDAYAT Microbiology Division, Research Center for Biology, Indonesian Institute of Sciences (LIPI), Cibinong-Bogor 16911 Received: 5th December 2008. Accepted: 3rd July 2009.

ABSTRACT A lot of traditional medicinal plant has antibacterial activities. Most of these plants are freshly chewed or grounded and used directly to treat infectious bacterial diseases. However, some practices employ a traditionally spontaneous fermentation on boiled extracted leaf, root or other parts of the plant. This work reports a laboratory stimulated spontaneous fermentation of leaf extracts from selected medicinal plants collected from the Karo Higland. The spontaneous fermentation was stimulated to be carried out by the Acetobacter xylinum and Saccharomyces cerevisiae. The anti-infectious agent activity was assayed on the Escherichia coli growth inhibition. A complementary non fermented leaf extract was also made and assayed as a comparative measure. Indeed, the fermented leaf extract of bitter bush (Eupatorium pallescens), cacao (Theobroma cacao), avocado (Persea gratissima), passion fruit (Passiflora edulis), cassava (Cassava utilissima), diamond flower (Hedyotis corymbosa), periwinkle (Catharanthus roseus), and gandarussa (Justicia gendarussa) have relatively higher anti-E. coli activity than those of non fermented ones. However, there were no anti-E. coli activity was detected in both fermented and non fermented leaf extract of the guava (Psidium guajava) and common betel (Piper nigrum). ÂŠ 2009 Biodiversitas, Journal of Biological Diversity Key words: medicinal plant, anti-bacteria, Escherichia coli, fermentation, Acetobacter xylinum, Saccharomyces cerevisiae.

INTRODUCTION Indonesia is one of the two mega-centers of biodiversity, alongside Brazil, with around 140 million hectares of rain forest. Therefore nature is deeply rooted in the life of people socially, economically and culturally. The flora diversity has been taken advantages for traditional remedies since ancient time (Ministry of Trade, 2006). It has been proved that many plants have healing and disease prevention properties as well as maintaining health or curing ailments. The plants known or believed having such pharmacological effects were called medicinal plants (Maiti, 2007). Medicinal plants are categorized into three groups: traditional medicinal plants are plants which known or believed having medicinal properties and have been used as traditional medicines; modern medicinal plants are plants which proved scientifically to produce medicinal lead compound and its application can be verified medically, and; potential medicinal plants are plants which have been proved scientifically to produce medicinal bioactive compound, nevertheless medically invalidated or its application as traditional medicines are difficult to ď&#x201A;Š Corresponding address: Jl. Raya Bogor Km. 46 Cibinong-Bogor 16911 Tel. +62-21-8765066. Fax. +62-21-8765063/62 e-mail: evitriana03@yahoo.com

trace (Zuhud et al., 1994). Most of traditionally Indonesia people have known and applied medicinal plants as one of the effort to deal with healthy issue before modern drugs were developed. At present days, they are still used as one of the alternative medication, and so-called traditional medicine. Traditional medicine may be seen as a product of the twofold wealth of Indonesia: its biodiversity and its cultural diversity. About 2500 medicinal plant species are presently used as traditional medicines. It relied on bioactive compound produced by the plants (Ministry of Trade, 2006). The diversity of bioactive compound produced by the plants rise efforts to further develop of drugs for many diseases, such as antibacterial agent, antifungal agent, antiparasites, anticancer, antivirus, etc (Slikkerveer, 2006). Most of the plants live in volcanic areas rich in selenium and sulphur, such as the Karo highlands may bear selenium based active compounds. In fact, selenium base bioactive compounds are one of the important assets from Indonesia microbial diversity. Previous studies have discovered spectrum of biological functions of those bioactive compound, such as antioxidant of lipid membrane peroxidation which play an important role in binding lipid radical in atherosclerosis prevention (Bulger and Maier, 2001); lowering cholesterol, anti cancer (Arteel and Sies, 2001; Dong et al., 2002), controlling hormonal balance, reproduction and normal growth of baby and

TRIANA & NURHIDAYAT â&#x20AC;&#x201C; Anti bacterial plant to E. coli of Karo Highland

children (Maehira et al., 2002), slowing aging, increasing fertility (Ellis et al., 2004; Kiefer, 2004) and anti microbes, including antibacteria which have significant importance in immune systems (Bulger and Maier 2001; Stanner 2001). Human body cannot synthesize selenoprotein. Therefore selenium intake depends on nutrient consumption. One way to fulfill the need of selenoprotein is bio-extraction of selenium from medicinal plant leaves and selenoprotein synthesis using a mixed acid fermentation of Acetobacter xylinum and Saccharomyces cerevisiae. This system will decrease pH to very low state in order to hydrolyze selenium that bind with methionine and cysteine to provide amino acid binding selenium that available to be absorbed and synthesized into selenoprotein by the microorganisms (Puspitasari, 2001). This preliminary research was conducted to reveal the antibacterial properties as one of the selenium base bioactive compound activities produced in a mixed acid fermentation of selected medicinal plant leaves. The extract was subjected to assay on the growth inhibition of Escherichia coli as causal agent of diarrhea. MATERIAL AND METHODS Bacterial culture. The microorganisms that were employed were A. xylinum and S. cerevisiae. These bacteria and yeast isolates were isolated from Indonesian traditional fermented food, i.e. from the Karo Highland, North Sumatra by Genetics Laboratory, Department of Microbiology, Research Center for Biology, LIPI Cibinong-Bogor, West Java. The E. coli JM109 was used in the antibacterial assay. Preparation of assay bacteria. The assay bacteria, E. coli, was streaked on Nutrient Agar (NA) media that prepared from 28 g NA (ready to use) in o 1000 mL distilled water, then incubated at 37 C for 3 days. Two loops of good growth E. coli were inoculated in 100 mL Nutrient Broth (8 g in 1000 mL o distilled water), then incubated at 37 C for 24 hours for further used. Medicinal plants material. The medicinal plants were selected based on their etnobotanical uses in infectious disease prevention and therapy as revealed by survey and interview some saman and people in the Karo Highland areas. Those plants were bitter bush (Eupatorium odoratum L), cacao (Theobroma cacao L), avocado (Persia gratissima Gaerth), guava (Psidium guajava L), betel (Piper betle L), passion fruit (Passiflora edulis Sims), cassava (Manihot utilissima Pohl), diamond flower (Hedyotis corymbosa [L] Lamk), periwinkle (Catharanthus roseus [L] G.Don), and gandarussa (Justicia gendarussa Burn). The plant leaves were grounded and then oven dried o at 40 C for two days. Preparing starter A. xylinum-S. cerevisiae. Starter was prepared from pineapples juice added with distilled water at composition 30:70 (v/v) and

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sugar at 5% (w/v) of total volume. The mixture was aliquoted into several little jars then covered with cheese cloth, plastics, and tied with rubber, then o autoclaved at 121 C, 15 psi for 15 minutes. After cooling, media were inoculated with A. xylinum-S. cerevisiae (10% total volume), then incubated at room temperature for 3-4 days to grow nata layer, for further inoculation to the leaf extract of medicinal plants. Leaf extract fermentation. One half gram of dried leaves was added with 10 g sucrose and 50 mL distilled water, then covered with cheese cloth, paper, plastics, and tied with rubber, then autoclaved at o 121 C, 15 psi for 15 minutes. Each sample consisted of 2 repeats and 1 control. All samples but controls were inoculated with 5 mL starter A. xylinum-S. cerevisiae. Fermentation process was performed for 9 days. Fermented leaf suspension preparation. After 9 days fermentation, pH of extracts was measured. As much as 1.5 mL extract was centrifuged at 3.000 rpm for 10 minutes. Supernathan was used for anti E. coli test. Assay of anti-E. coli activity of leaf extract. The E. coli were swabbed on the NA agar surface using soft tip to evenly distributed and dried. Paper discs were dipped and saturated in each leaf extract for 5 minutes. It was then put onto the surface of the media o inoculated with E. coli, then incubated at 30 C for 3 days to allow the bacterial growth. Anti-E. coli activity of the extract were then determined by measuring the respective zone of inhibition.

RESULTS AND DISCUSSION The mixed acid fermented leaf extract suspension had lower pH that that of the non fermented extract suspension (Table 1). It probably due to sugar fermentation by the S. cerevisiae producing ethanol and carbondioxide (CO2). The ethanol was then oxidized by A. xylinum to form acetic acid that in turn acidified the extracts suspension. The acid facilitated the selenium extraction and selenoprotein biosynthesis by the mixed acid fermentative microorganisms (Puspitasari, 2001). Tabel 1. pH of control and the mean of treatment (fermented leaf extracts) Plants Bitter bush Cacao Avocado Guava Betel Passion fruit Cassava Diamond flower Periwinkle Gandarussa

Mean of pH of treatment 2,992 3,103 2,868 3,106 2,976 3,021 2,902 3,101 3,255 3,544

pH of control 5,705 6,055 6,083 5,000 6,450 5,853 5,318 5,364 5,598 8,081

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Table 2 shows anti-E. coli growth of the selected fermented leaf suspension. This might indicates that some fermented leaf extracts contained active compound of selenium. Selenium is bioactive compound that has antibacterial activity (Stanner 2001). Tabel 2. Diameter of inhibition zone of fermented leaf extracts and control against assay bacteria E. coli. Diameter of inhibition zone (cm) Fermented leaf Control (without extract* fermentation) Bitter bush 2,53 2,90 Cacao 1,60 1,36 Avocado 1,22 Guava Betel Passion fruit 1,53 Cassava 1,51 Diamond flower 1,84 Periwinkle 1,58 Gandarussa 1,51 1,65 Note:-not detected, * fermented by A. xylinum and S. cerevisiae Plants

The medicinal plants having high potential anti-E. coli were bitter bush, cacao, avocado, passion fruit, cassava, diamond flower, periwinkle, and gandarussa. Their potency could be predicted from their leaf extracts activity to inhibit the growth of E. coli as indicated by the clear zone of no observed growth around the paper discs contained leaf extracts. In this study, leaf extract of passion fruit exhibited anti-E. coli activity. This result confirmed Ripaâ&#x20AC;&#x2122;s statement (2009) that such activity of chloroform extract of passion fruit leaf was exhibited. Probably the anti-E. coli activity of diamond flower and periwinkle is the first report since no available report yet. Both plants were known well for their activity against cancer cells (Sadasivan et al., 2006; Sudarsono, 1999; Taha et al., 2008). There is no available report yet also on the anti-E. coli activity of the gandarussa (Panthi and Chaudhary, 2006). However, there is a phenomenon that the anti-E. coli was detected after fermentation in some plant such as avocado, passion fruit, cassava, diamond flower, and periwinkle. That means the bioactive compounds need to be activated. No active compound activation is required in the case of plants bitter bush, cacao, and gandarussa. It is probably the nature of the active compound that could be extracted well with hot water and acidification during the fermentation did not play any comparative role. Table 2. showed that potential anti E. coli of unfermented leaf extracts of bitter bush, cacao, and gandarussa were relatively higher than fermented ones. One of the explanations is that selenium concentration in the beginning of

fermentation process might be used in the microbial metabolisms such as selenoprotein biosinthesis that transform the anti-E. coli into other functional molecules. In this case, instead of using fermented leaf extracts, direct use of unfermented ones to treat infectious disease caused by E. coli thought to be more effective, because it showed higher anti-E. coli activity. This result confirmed Heyneâ&#x20AC;&#x2122;s statement (1987) that bitter bush leaves have been long ago known and applied as traditional medicine to treat chronically diarrhea. In application, those leaves were used directly without fermentation process. Unfortunately, the potential anti-E. coli of cacao and gandarussa leaves has not been reported. Nevertheless, gandarussa is used to cure stomachache in Aceh (Ministry of Trade, 2006). Leaf extracts of guava and betel showed no anti-E. coli activity. Therefore hot water extract of betel and guava leaves could not be used as anti-E. coli medicine. However Syamsuhidayat et al. (1991) reported that guava and betel leaves have been long ago known and used as folklore medicine to cure diarrhea caused by E. coli. It suggests that the antidiarrhea activity of the guava leaf is not anti-E. coli in term of growth inhibition, probably the prevention of water excretion or inactivation of the causative microbial toxins (Hoque, 2007). On the other hand, anti-E. coli activity was exhibited by ethanol extract of betel leaf (Arambewala et al., 2005). In addition to leaves of bitter bush and guava which were long ago believed as anti diarrhea in traditional medicine (Morton, 1987; Heyne, 1987), the anti-E. coli activity of leaves of cacao, avocado, passion fruit, cassava, diamond flower, periwinkle, and gandarussa may enrich the etnobotanical information since those plants were not commonly employed as anti diarrhea or anti-E. coli. Based on the diameter of inhibition zone of the anti-E. coli of fermented leaf extracts, it could be list from the highest to the lowest were bitter bush, diamond flower, cacao, periwinkle, passion fruit; followed by gandarussa and cassava which have same activity; and avocado, respectively. The highest anti-E. coli of unfermented leaf extracts was bitter bush, followed by gandarussa, and cacao, respectively. Meanwhile, the leaf extracts of guava and betel exhibited no anti-E. coli activity.

CONCLUSSION The fermentation facilitated the leaf extracts of diamond flower, periwinkle, passion fruit, cassava, and avocado to demonstrate anti-E. coli activity. The leaf extracts of bitter bush, gandarussa, and cacao exhibited anti-E. coli activity with or without fermentation, therefore they could be used directly as anti-E. coli medicine. On the other hand, the leaf extracts of guava and betel exhibit no antibacterial activity with or without fermentation process.

TRIANA & NURHIDAYAT â&#x20AC;&#x201C; Anti bacterial plant to E. coli of Karo Highland

REFERENCE Arambewala, L., K. Kumaratungga, and K. Dias. 2005. Studies on Piper betel on Srilangka. Journal national of Science Foundation 33 (2): 133-139. Arteel, G.E. and H. Sies. 2001. The biochemistry of selenium and the gluthathione system. Environmental Toxicology and Pharmacology 10: 153-158. Bulger, E.M. and R.V. Maier. 2001. Antioxidants in critical illness. American Medical Association Arch Surgery 136: 1201-1207. Dong, Y., H.E. Gauther, C. Stewart and C. Ip. 2002. Identification of molecular target associated with selenium-induced growth inhibitor in human breast cells using cDNA microarrays. Cancer Research 62: 708-714. Ellis, D.R., T.G. Sors, D.G. Brunk, C. Albrecht, and C. Orser. 2004. Production of Se-methylselenocystein in transgenic plants expressing selenocysteine methyltransferase. BMC Plant Biology 4 (1): 1-11. Heyne, K. 1987. Tumbuhan Berguna Indonesia. Jilid II. Jakarta: Badan Penelitian dan Pengembangan Kehutanan, Departemen Kehutanan. Hoque, M., M.L. Bari, Y. Inatsu, V.K. Juneja, and S. Kawamoto. 2007. Antibacterial activity of guava (Psidium guajava L.) and Neem (Azadirachta indica A. Juss) extracts agains foodborne pathogens and spoilage bacteria. Foodborne Pathogens and Disease. 4 (4): 481-487. Kiefer, D. 2004. Getting serious about selenium. Life Extension Magazine Report . Desember 2004: 1-8. Maehira, F., G.A. Luyo, I. Miyagi. M. Oshiro, N. Yamane, M. Kuba, and Y. Nakazato. 2002. Alteration of serum selenium concentration in the acute phase of pathological conditions. Clinica Acta 316: 137-146. Maiti, S. and K.A. Geetha. 2007. Horticulture: Medicinal and Aromatic Plants in India. Gujarat: National Research Center for Medicinal and Aromatic Plants. Ministry of Trade. 2006. Biodiversity, traditional medicine and sustainable use of indigenous medicinal plants in Indonesia. Indonesia Export News. August: 3-7.

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Morton, J.F. 1987. Guava. In: Morton, J.F. (ed.). Fruit of Warm Climates. Lafayette: Purdue University. Panthi, M.P. and R.P. Chaudhary. 2006. Antibacterial activity of some selected folklore medicinal plants from west Nepal. Scientific World 4 (4): 16-21. Puspitasari, T.I. 2001. Analisis Kandungan Selenium pada Tanaman yang Potensial untuk Dimanfaatkan sebagai Antiketombe. [Skripsi] Bogor: IPB. Ripa, A.F., M. Haque, L. Nahar, Md. Monirul Islam. 2009. Antibacterial, cytotoxic and antioxidant activity of Passiflora edulis Sims. European Journal of Scientific Research 31 (4): 592-598. Sadasivan, S., P.G. Latha, J.M. Sasikumar, S. Rajathekaran, S. Shyamal, V.J. Shine. 2006. Hepatoprotective studies on Hedyotis corymbosa (L.) Lam. Journal of Ethnopharmacology 106 (2): 245-249. Slikkerveer, L.J. 2006. The challenge of non-experimental validation of Mac Plants. In: Bogers, R.J., L.E. Cracker, and D. Lange (eds). 2006. Medicinal and Aromatic Plants. Berlin: Springer Verlag. Stanner, S. 2001. A way to fight flu? British Nutrition Fundation. Nutrition Bulletin 26: 273-274. Sudarsono. 1999. Asperulosid, senyawa iridoid Hedyotis corymbosa (L.) Lamk. Suku Rubiaceae. Indonesian journal of Pharmacy 10 (3): 43-55. Syamsuhidayat, S. Sugati dan J.R. Hutapea. 1991. Inventaris Tanaman Obat Indonesia. Jilid I. Jakarta: Departemen Kesehatan RI. Taha, H.S., M.K. El-Bahr and M.M. Seif-El Nash. 2008. In vitro studies on Egyptian Catharanthus roseus (L.) G. Don.: 1-Calli production, direct shootlets regeneration and alkaloids determination. Journal of Applied Sciences Research 4 (8): 1017-1022. Zuhud, E.A.M., Ekarelawan dan S. Raswan. 1994. Hutan Tropika Indonesia Sebagai Sumber Keanekaragaman Plasma Nutfah Tumbuhan Obat dalam Pelestarian Konservasi Sumberdaya Hutan. Bogor: IPB dan Lembaga Alam Tropika Indonesia.

BIODIVERSITAS Volume 10, Number 4, October 2009 Pages: 210-214

ISSN: 1412-033X (printed edition) ISSN: 2085-4722 (electronic) DOI: 10.13057/biodiv/d100409

Review: Current Advances in Gloriosa superba L. RAVINDRA ADE, MAHENDRA K. RAI

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Department of Biotechnology, Sant Gadge Baba Amravati University, Amravati 444602, Maharashtra, India Received: 2nd June 2009. Accepted: 7th July 2009.

ABSTRACT Gloriosa superba L. is an important medicinal plant of Asia and Africa. It is used in diseases, like cancer, gout, scrofula and act as antipyretic, antihelmintic, purgative and antiabortive. It is a source of colchicines and colchicocides, which are very costly, being highly demanded by pharma industries. Due to excessive use of the plant for diverse medicinal purposes the species is on the verge of extinction and included in Red Data Book. The strenuous efforts of botanists, biotechnologists, policy makers and conservationists are required. It is a matter of great concern to conserve this plant otherwise we will be loosing it by 2020.The present review is focused on current status of the genus, source of alkaloids, poisonous nature, the strategies for its conservation and future perspectives of G. superba. © 2009 Biodiversitas, Journal of Biological Diversity Key words: Gloriosa superba L., colchicine, endangered plant, red data book, conservation.

INTRODUCTION From the beginning of human civilization the plants and their products particularly ethnomedicinal plants plays a great role. The antique traditional medicines and prescriptions at a halt exist and largely practiced. The ethnobotanists are trying to ascertain new medicines from the forests by the help of tribal people. In India, the population of tribal people is around fifty three million along with 555 tribal groups or communities, which are reside in forest and srounding. These people have enormous indigenous knowledge which is a possible tool to explore for novel cost-effective plants for food as well as medicine. Several medicinal plants were originally identified and developed through indigenous knowledge, thus ethnomedicines have played key role in the development of drugs used in modern system of medicine. The plant drugs which are originated and developed are cocaine, morphine, quinine, colchicine, atropine, ephedrine, codeine, emetine, caffeine, resurpine, vinablastin, vincristin, and guguline etc. The medicinal plants play a major role in remedial now a days. The entire planet is in

 Corresponding address: Dr. Mahendra K. Rai, Professor and Head Department of Biotechnology, S.G.B. Amravati University, Amravati 444602 Maharashtra, India Tel.: +91-721-2662208-9, ext. 267 Fax.: +91-721-2662135, 2660949 e-mail: mkrai123@rediffmail.com; pmkrai@hotmail.com

search of renovating herbal medicinal plants and its application for the betterment of mankind. World Health Organization (WHO) has previously recognized to re-establish the traditional knowledge of medicine among our conventional theaters. Traditional knowledge since 200 B.C. in Ayurveda is very well recognized especially in India among tribal people. Gloriosa superba L. (Colchicaceae) also known as Malabar glory lily or “kembang telang” (Java, Indonesia) is a perennial tuberous climbing herb, extensively scattered in the tropical and sub-tropical parts of the India, including the foothills of Himalayas. It is called as ‘Mauve beauty’, ‘Purple prince’, ‘Modest’, ‘Orange gem’, ‘Salman glow’ and ‘Orange glow’ (Bose and Yadav, 1989). It is adapted to different soil texture and climatic variation. The plant grows in sandy-loam soil in the mixed deciduous forest in sunny positions. It is very tolerant of nutrientpoor soils. It occurs in thickets, forest edges and boundaries of cultivated areas in warm countries upto height of 2530 m. (Neuwinger, 1994). G. superba is a inhabitant of tropical Africa and now found growing naturally in many countries of tropical Asia including Bangladesh, India, Sri Lanka, Malaysia and Myanmar. In India, it occurs commonly in tropical forests of Bengal and Karnataka (Sivakumar and Krishnamurthy, 2002). The plant thrive from Bundelkhand to humid Assam valley. It is known by different names in India such as ‘Kalihari’, ‘Agnishikha’, ‘Languliata’, and ‘Nangulika’. There are several associated species of Gloriosa including G. superba, G. simplex, G. grandiflora, G. lutea, G.

ADE & RAI â&#x20AC;&#x201C; Current advances in Gloriosa superba L.

plantii, G. lipolidii, G. longifolia, G. rothschildiana, G. virescens, G. sudanica, etc. These species are distributed mainly in Africa. Besides many hybrids popular garden cultivars like the purple prince, African chief, orange gem, orange gift, lavender lady, and orange ball are grown for ample range of flowers and color combination of the perianth, which bloom for long during the autumn. G. superba is also known as the national flower of Zimbabwe. Except diverse pharmaceutical products and other therapeutic preparations, it is also a popular plant for providing color in greenhouse and conservatories even immature flowers are gorgeous to behold (Kranse, 1986; Ghani, 2000). G. superba is a semi-woody herbaceous branched climber, reaching just about five meters in height. One to four stems arise from a single V-shaped fleshy cylindrical tuber. Gloriosa superba is an imperative medicinal plant, all parts are used in the medicine, which contains two important alkaloid, colchicine and colchicoside, leaves are used to treat cancer related diseases, also in ulcer, piles, and scrofula (Evans et al., 1981).

THE CURRENT STATUS The leaf juice of Gloriosa is used to kill-lice in hair, tubers contain the bitter principles, superbine and gloriosine, which in large doses are fatal; however, in small doses they are used as tonic, antiabortive, and purgatives. The white flour prepared from the tubers is bitter and used as stimulant. It is given with honey in gonorrhea, leprosy, colic and intestinal worms and for promoting labor pains, a paste of tubers is applied over the suprapubic region and vagina. Its warmpoultice is locally applied in rheumatism and neuralgic pains (Samy et al., 2008). Medicinally, the tuber is used as abortifacient, and in small dose it acts as a tonic, stomachic and anthelmintic. It is also used in the treatment of gout because it contains colchicine. Paste of the tuber is externally applied for parasitic skin diseases. Samy et al. (2008) conducted ethnobotanical survey of folk plants for the treatment of snakebites in southern part of Tamilnadu, India, in which traditional approach was evaluated methodically with some selected plant extracts which showed potent neutralizing effect against the venom. The conventional method of propagation is through corms, since poor seed germination restricts their use in multiplication. Therefore, propagation by tissue culture technique is necessary (Finnie and van Staden, 1989, 1991). G. superba contains about 0.10.8% colchicine in bulb which is used in plant breeding to induce mutation and polyploidy and also can solve an important problem in fuchsia breeding. G. superba also produces another alkaloid gloriosine. Colchicines affect cell membrane structure indirectly by inhibiting the synthesis of membrane constituent. It binds to tubelin preventing its polymerization into

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microtubules. This anti-mitotic property disrupts the spindle apparatus that separate chromosomes throughout metaphase, cells with high metabolic rates are most implicated by the arrest of mitosis. Kumar (1953) studied doubling of chromosomes induced by gloriosine isolated from G. superba. Jitpakdi et al. (1999) screened ten plant species including G. superba for metaphase chromosome preparation in adult mosquitoes (Diptera: Culicidae) using an inoculation technique for colchicine like substances using a mosquito cytogenetic assay have shown the increased metaphase chromosome. Due to overexploitation for its diverse medicinal applications G. superba has been endangered, therefore, there is urgent need to conserve the plant by biotechnological approaches like tissue culture. (Rajgopalan and Khader, 1994). This approach has been very important because it provides complete sterile and virus-free plants by rapid multiplication. G. superba is now promising as an industrial medicinal crop in Asia particularly in South India for its high colchicine content. For commercial production of colchicine and its derivatives, natural production from in vitro methods of the source plant are thus of great attention. In the past two decades, focus has been on plant biotechnology as a potential alternative production method, using cultured cells rather than plants.

SOURCE OF PRECIOUS ALKALOIDS Gloriosa superba produces two important alkaloid colchicine and gloriosine, which are present in seeds and tubers while the other compounds such as lumicolchicine, 3-demethyl-N-deformyl-N-deacetylcolchicine, 3-demethylcolchicine, N-formyl deacetylcolchcine have been isolated from the plant (Sugandhi, 2000; Suri et al., 2001). Suri et al. (2001) reported new colchicine glycoside, 3-O-demethylcolchicine-3-Oalpha-D-glucopyranoside in G. superba seeds. Kaur et al. (2007) studied purification of 3-monomeric monocot mannose-binding lectins and their evaluation for antipoxviral activity isolated from G. superba. Alkaloids are structurally heterogeneous class of secondary biomolecules derived from basically five amino acids ornithine, lysine, phenylalanine, tyrosine and tryptophan (Thakur et al., 1975). Thakur et al. (1975) reported the substances from plant of the subfamily Wurmbaeoideae and their derivatives along with alkaloids from the G. superba.

COLCHICINE It is conventional drug for gout obtained from corms of G. superba and Colchicum autumnale (Thakur et al., 1975; Sivakumar and Krishnamurthy, 2002). The term â&#x20AC;&#x153;colchicineâ&#x20AC;? is derived from area

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known as Colchis near black sea. C. autumnale grows wild in Europe and Africa. Thomson was the first who proposed early idea of action of colchicines in gout treatment. Gout and uric acid metabolism is same way linked and colchicines might act on this and it is caused by deposition of microcrystals of uric acid in joints and may be due to defective regulatory mechanism for endogenous purine synthesis but contradictory result for the action of colchicine on synthesis and extraction of urates have been recorded, colchicines interrupt, the cycle of new deposition which seem to be indispensable for the persistence of acute gout. Distressing side effect has also been recorded sporadically but colchicines remain the drug for acute gout. Modification of the side chain of rings does not abolish anti-gout activity as long as the configuration of C-ring confirms to that of colchicines. It also acts as anti-mitotic and anti-gout agent. It blocks or suppresses cell division by inhibiting mitosis. It inhibits the development of spindles as the nuclei are dividing (spindles are formed by the polymerization of tubuline) from a pool of subunit during a detached phase of cell-cycle and then depolymerized during other phase. It is also used to induce polyploidy initiation, occasionally other mutations also occur like chlorophyll mutations, but frequency is low. It can solve an important problem of fuchsia breeding. Most of the fuchsia species are diploid or tetraploid, a crossing between diploid and tetraploid result often in a triploid, which is mostly sterile because the process of meiosis (cell division for reproduction) requires the coupling of similar chromosomes and there is no mechanism allowing for the alignment of three similar chromosomes, triploid plants are not able to produce fertile reproductive cells. They are, therefore, sterile and unusable as parents. A special problem of colchicines induced ploidy, particularly in vegetatively propagated crops, is the chimerism caused by the instantaneous presence of tissue of different ploidy levels in one plant or plant parts. Colchicine is mostly used in its freshly prepared aqueous form. The range of concentration of colchicine applied varies from 0.0063%, concentration of about 0.05% is the most commonly used (Milne and Meek, 1998). Kannan et al. (2007) studied optimization of solvents for efficient isolation of colchicines from G. superba. The maximum yield of colchicine was obtained when it is extracted with water and alcohol in the ratio of 50:50. Bellet and Gaignault (1985) reported the production of colchicinic substance from G. superba. The colchicines-like activity of G. superba-extracted for mosquito (Diptera: Culicide) in which four fractions i.e. hexane fractions, dichloromethane fraction-1, dichloromethane fraction-2 and methanol fraction were investigated. The latter three fractions yielded hopefully high colchicine like activity, whereas hexane fraction yielded very low activity.

Ghosh et al. (2002) studied the root culture of G. superba by using direct and indirect precursor of the biosynthetic pathway for the enhancement of colchicine production. They successfully used aluminium chloride as an elicitor, in which they have used root cultures of G. superba treated with 5 mM methyl jasmonate and 125 ÂľM AlCl3. The enhancement of intracellular colchicine content was observed in the roots by 50-fold and 63-fold respectively. Ghosh et al. (2006) reported that colchicine can also be applied in the lanolin paste or as a solution, for instance, on a cotton dot, placed in a leaf axil. Khan et al. (2007) evaluated the enzyme inhibition activities of G. superba rhizomes extract against lipoxygenase, actylcholinesterase, butyrycholinesterase and urease in which wonderful inhibition was observed on lipoxygenease. Further, Khan et al. (2008) reported antimicrobial potential of G. superba extracts in which excellent antifungal activity was confirmed against Candida albicans, C. glabrata, Trichophyton longifusus, Microsporum canis and Staphylococcus aureus. Colchicine is synthesized using mainly aromatic amino acids such as tryptophan, phenylalanine and tyrosine. Key enzymes involved in colchicine metabolism are tyrosine ammonia lyase (TAL) and phenylalanine ammonia lyase (PAL). Sivakumar and Krishnamurthy (2004) reported the biosynthesis of colchicine, the in vitro supply of exogenous precursor using B5 medium from G. superba calluses. The maximum amount of colchicine i.e. 9.0 mg was detected in the medium fed with 30 ÎźM tyrosine. The activity of TAL was higher than that of PAL and a low frequency of tracheary elements was observed.

POISONING NATURE OF PLANT The colchicine which is a major component of Gloriosa is mainly responsible for toxic effect (Vishwanathan and Joshi, 1983). The toxins present have an inhibitory action on cell division, and depressant action on the bone marrow. Just after ingestion of tubers, the symptom develops within two hours; vomiting, numbness and severe effects on throat as well as diarrhea leading to dehydration. Alopecia and dermatitis are the major symptoms develop after two to three weeks after poisoning (Jayaweera, 1982). Colchicine poisoning following ingestion of G. superba tubers have been reported by various investigators (Nagratnam et al., 1973; Jose and Ravindran, 1988; Fernando and Fernando, 1990). Mendis (1989) reported the toxicity symptoms as gastroenteritis, acute renal failure, cordiotoxicity and hematological abnormalities. Moreover, there were eight deaths due to G. superba, while the gastrointestinal symptoms along with sweating, hypotension jaundice and convulsions were reported after the consumption (Aleem, 1992).

ADE & RAI – Current advances in Gloriosa superba L.

CONSERVATION BY MEANS OF IN VITRO PROPAGATION Gloriosa superba usually multiply by corm and seeds but due to low germination capability it restricts for the regeneration. Therefore, in order to safeguard and preserve this important plant biotechnological approaches would be very useful (Sivakumar and Krishnamurthy, 2002). The conventional method of propagation has many disadvantages as 50% of the yield has to be set aside for raising the next crop, transmittance of soil-borne diseases from one crop to the next, and from one location to another and during the 2-3 month storage period between harvest and the raising of next crop (Mrudul et al., 2001). Kala et al. (2004) studied the prioritization of medicinal plants on the basis of available knowledge, existing practices and use value status in Uttaranchal, India in order to understand the pattern of indigenous uses of medicinal plants available in the Uttaranchal state, India and documented 300 species including G. superba. Hassan and Roy (2005) reported 92% of the cultures of apical and axillary buds of young sprout from naturally grown G. superba plants regenerate four shoots per culture in MS basal medium fortified with 1.5 mg/L BA + 0.5 mg/L NAA. Custers and Bergervoet (1994) reported micropropagation of G. superba by shoot cuttings and explants from node, internode, leaves, flowers, pedicels and tubers. G. rothschildiana (duphur) vs. G. rothschildiana (new accession) and G. rothschildiana vs. G. superba were cultured on MS basal medium with 3% w/v sucrose, 0-10 mg/L Benzyl Adenine (BA) and 0.1 mg lndole Acetic Acid (IAA) and maintained at 24 days under 16 hours photoperiod. Addition of low level of Benzyl Adenine (BA) (1 mg/L) improved plant growth, whereas the high level of BA (10 mg/L) caused proliferation of multiple shoots, from rhizome meristem, by applying alternatively high and low BA level, a method of continued propagation was achieved which resulted in a 4-7 fold multiplication of qualitatively good plantlets every 18 week. The resulting shoots were incubated on MS medium, with 3% sucrose and 0-1 mg/L IAA or NAA. Transplantation into soil was only possible after the plants had formed. Samarajeewa et al. (1993) studied clonal propagation of G. superba from apical bud and node segment of shoot tip, cultured on solidified agar (0.8% w/v) Gamborg’s B5 medium containing BA, IAA, Kinetin, NAA, IBA or 2,4-D. The cultures were maintained under fluorescent light at 25-27ºC. Primary cultures were initiated in solid B5 medium containing 0.5 to 1 mg/L BA and 0.01-0.5 mg/L IAA, IBA, NAA when shoot tip of primary cultures were transferred to shoot multiplication media, shoot proliferation occurred via adventitious bud formation within 4-8 weeks. Somani et al. (1989) reported in vitro propagation and corm formation in G. superba. The fresh sprouts

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were excised from corms of G. superba and dissected propagules with shoot and root primordia were placed on MS basal medium (Murashige and Skoog, 1962) containing 3% sucrose and 0.6% agar. Explant germinated on the MS medium producing shoot and root, which formed new corm within one month. For shoot and cormlet regeneration, 1-4 mg/L kinetin was added to the medium. Cultures were maintained at 25ºC in white fluorescent light (2500 lux) with an 8h/day photoperiod. Sivakumar and Krishnamurthy (2002) reported in vitro organogenetic responses of G. superba. They used MS medium supplemented with ADS and BA, 98%. The callus induction occurred in non-dormant corm bud explants. The maximum number of multiple shoot (57%) was observed in corm-derived calluses. Gupta (1999) compared the production of different colchicinic substances from G. superba and C. autumnale. He reported extensive range of these colchicinic compounds like colchicines (0.9%), dimethyl-3-colchicine (0.19%), colchicoside (0.82%) and their formyl derivatives from G. superba. While these values were found to be less in case of C. autumnale which were reported as 0.62%, 0.9%, and 0.39% respectively. Sivakumar and Krishnamurthy (2002, 2004) studied induction of embryoids from leaf tissue of G. superba. The nodular calli were observed on S.H. medium supplemented with 2,4-D and 1 isopentyldene. Gupta et al. (1999) found hepatoprotective activity of G. superba. Jha et al. (2005) reported production of forskolin, withanolides, colchicine and tylophorine from plant source by using biotechnological approach.

CONCLUSION Gloriosa superba is a commercially imperative medicinal plant which has diverse medicinal applications and eventually due to over-exploitation this plant is facing local extinction. It has been affirmed as endangered plant by IUCN and hence there is a pressing need to conserve the plant by in situ and ex situ multiplication in general and micropropagation in particular so as to meet the everincreasing demand from the industries. Furthermore, responsiveness should be generated among the common people concerning the importance of G. superba and its overexploitation by the people. Peoples participation in conservation of rare and endangered medicinal plants like G. superba will also be very useful.

REFERENCES Aleem, H.M. 1992. Gloriosa superba poisoning. Journal of Association of Physicians of India 40: 541-2. Bellet, P., and J.C. Gaignanlt. 1985. Gloriosa superba L. and the production of colchicinic substances. Annales Pharmaceutiques Francaises 43: 345-347.

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Bose, T.K., and L.P. Yadav. 1989. Commercial Flowers. Kolkata: Nayaprakash. Custers, J.B.M. and J.H.W. Bergervoet. 1994. Micropropagation of Gloriosa: towards a practical protocol. Scientia Horticulturae. 57: 323-334. Evans, D.A., S.P. Tanis, and D.J. Hart. 1981. A convergent total synthesis of (and) (F) Desacetamido isocolchicine. Journal of American Chemical Society 103: 5813-5821. Fernando, R. and D.N. Fernando. 1990. Poisoning with plants and mushrooms in Sri Lanka: a retrospective hospital based study. Veterinary and Human Toxicology 32: 579-81. Finnie JF, van Staden J. 1989. In vitro propagation of Sandersonia and Gloriosa. Plant Cell, Tissue and Organ Culture 19: 151158. Finnie, J.F. and J. van Staden. 1991. Isolation of colchicine from Sudersania auranicacea and Gloriosa superba variation in the alkaloid level of plant grown in vivo. Journal of Plant Physiology 138: 691-695. Ghani, A. 2000. Medicinal plants for drug development: potentiality of medicinal plants of Bangladesh. 10th Asian Symposium on Medicinal Plants, Spices and other Natural products (ASOMPS X). Dhaka, Bangladesh, 18-23 November, 2000. Ghosh, B., S. Mukherjee, T.B. Jha, and S. Jha. 2002. Enhanced colchicine production in root cultures of Gloriosa superba by direct and indirect precursors of the biosynthetic pathways. Biotechnology Letters 24: 231-234. Ghosh, S., B. Ghosh, and S. Jha. 2006. Aluminium chloride enhances colchicine production in root cultures of Gloriosa superba. Biotechnology Letters 28: 497-503. Gupta, B.K. 1999. Production of colchicine from G. superba tubers in cultivation and Utilization of medicinal plants. CSIR Journal 270-278. Jayaweera, D.M.A. 1982. Medicinal Plants Used in Ceylon. Vol. 3. Colombo: National Science Council of Sri Lanka. Jha, S., M. Bandypadhyay, and K.N. Chaudhary. 2005. Biotechnological approaches for the production of farskolin, withanolides, colchicine and tylophorine. Plant Genetic Resource 3: 101-115. Jitpakdi, A., W. Choochote, and D. Insun. 1999. Screening of ten plant species for metaphase chromosome preparation in adult mosquitoes (Diptera: Culicidae) using an inoculation technique. Journal of Medical Entomology 36: 892-5. Jose, J. and M. Ravindran. 1988. A rare case of poisoning by Gloriosa superba. Journal of Association of Physicians of India 36: 451-452. Kala, C.P., N.A. Farooquee, and U. Dhar. 2004. Prioritization of medicinal plants on the basis of available knowledge, existing practices and use value status in Uttaranchal, India. Biodiversity and Conservation 13 (2): 453-469. Kannan, S., S.D. Wesley, and A. Ruba. 2007. Optimization of solvents for effective isolation of colchicine from Gloriosa superba L. seeds. Natural Product Research 5: 469-472. Kaur, A., S.S. Kamboj, and J. Singh. 2007. Purification of 3 monomeric monocot mannose-binding lectins and their evaluation for antipoxviral activity: potential application in multiple viral diseases caused by enveloped viruses. Biochemistry and Cell Biology 85: 88-95. Khan, H., M.A. Khan, and I. Hussan. 2007. Enzyme inhibition activities of the extracts from rhizomes of Gloriosa superba Linn (Colchicaceae). Journal of Enzyme Inhibition and Medicinal Chemistry 6: 722-725. Khan, H., M.A. Khan, and T. Mahmood. 2008. Antimicrobial activities of Gloriosa superba Linn extracts. Journal of Enzyme Inhibition and Medicinal Chemistry 6: 855-859.

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Authors Index Abbas, B. Ade, R. Agusta, A. Agustini, V. Akhdiya, A. Anggarwulan, E. Anwar, Y. Arobaya, A.Y.S. Astirin, O.P. Astuti, I.P. Bintoro, M.H. Budiarto, K. Campbell, S.J. Chairul Chairul, S.M. Chikmawati, T. Dona, S.E. Ehara, H. Elrahimi, S.A. Fadli, N. Fahrrurozi Guhardja, E. Gustafson, J.P. Handajani, N.S. Handayani, R. Hardiwinoto, S. Harini, M. Hartana, A. Hartati, S. Hendarwin, T. Hendra, M. Herdiana, Y. Hermawan, R. Hertina Hidayat, T Ilyas, M Indriyati Jamal, Y. Kanti, A. Kartawijaya, T. Kusdianti Kusuma, Y.W.C. Lapian, H.F.N. Larashati, S. Lestari, Y. Lukman, R. Meryandini, A. Miftahudin,

168 210 23 175 115 158 109 134 44 201 168 104 88 40 40 55, 120 6 168 88 181 115 98 55 44 151 195 44 120 6 115 98 88 63 23 19 23, 76 195 23 23 88 19 201 1 76 115 163 115 55

Mirmanto, E. Mulyaningsih, E.S. Nugraha, R.T.P. Nurhidayat, N. Pardede, S.T. Pattiselanno, F. Permana, Dj.R. Poerba, Y.S. Poerwanto, R. Praptiwi Priadi, D. Purwanto, Y. Rahayu, R.D. Rai, M.K. Rakhmawati, R. Retnaningtyas, E. Rismitasari, Rudi, E. Saprudin, D. Satyanti, A. Semiadi, G. Setiadi, D. Setiawan, F. Setyowati, F.M. Setyowati, N. Sinaga, S. Slamet-Loedin, I.H. Sobir, Sudarmonowati, E. Sufaati, S. Sugardjito, J Sugiyarto Suhadi Suharno Suharsono Sulistyo, J. Supyani Surahman, M. Susilo, F.X. Sutrisno, H. Tamelander, J Triana, E. Walujo, E.B. Wardah Widjaya, E.A. Widodo, P. Widodo, W. Widyastuti, U.

187 63 124 206 88 134 6 12 163 40 6 98 151 210 158 158 163 88 115 29 124 98 88 146 49 163 63 163 6 175 94 129 139 175 109, 168 151 70 168 195 34 88 206 98 146 12 120 81 109

Keywords Index [(E)-4-(2’,4’,5’-trimethoxyphenyl)but-3-en-1-ol] [(E)-4-(3’,4’,1-trimethoxy phenyl)but-3-en-1-ol] [(E)-4-(3’,4’-dimethoxyphenyl)but-3-en-1-ol] 35S-oshox4 abundance Acetobacter xylinum active fraction AFLP Agrobacterium tumefaciens agroforestry Alor anti-bacteria antibacterial Antipatharia apoptotic arabinofuranosidase Araceae Baluran National Park banteng beetle Bekol savannah Benuaq biodiversity black coral blood parameters BST buffalo Cacatua sulphurea parvula carnation (Dianthus carryophyllus L.) cassava cDNA cement base Central Kalimantan chemotaxonomy cloning coconut oil colchicine conservation coral coral reef CpMYRV1 Cryphonectria parasitica cultivar cysteine deer diversity Dyera costulata dynamic growth endangered plant endophytic fungi Escherichia coli EST estuary ethnobotany Euphorbiaceae feeding group fermentation Fisher’s α index free fruit size fused petal genetic diversity genetic relationships genotype germination Gloriosa superba L. grazing

40 40 40 63 76, 195 206 158 163 63 129 81 206 151 1 44 115 201 139 139 195 139 98 1, 34 1 124 158 139 81 104 6 109 181 187 23 109 151 210 34, 210 181 88 70 70 104 109 139 19, 76, 195 12 187 210 23 206 55 76 98, 146 19 94 151, 206 34 120 120 120 163 168 6 29 210 134

habitat HD-Zip oshox (oryza sativa homeobox) Homalomena bellula homologous hpt hyperhydricity immunostimulant in vitro conservation indigenous Indonesia inoculum intermediate isolation isozymes Java Javan warty pig Jayapura jelutong Kebar land use system lauric acid leaf leaves macro-moths Manado management type mangosteen Manihot esculenta Manokwari medicinal plant metallothionein microhabitat molecular characterization morphological character mortality mould Mt. Cycloops Nature Reserve mulching northern Acehnese reef orchid mycorrhiza p53 Pandanus conoideus Lamb. var. yellow fruit Pandanus uses parrot PCR PCR peat swamp forest petal fusion phenylbutenoid derivative phylogenetic analysis Picrasma javanica Bl. seed plantlet viability Pongo abelii population population density population structure primer screening Quercus gemelliflora RAPD Raphanus sativus L. rDNA ITS recruitment red data book reef fish rubble Rusa Saccharomyces cerevisiae

134 63 201 55 63 104 40 104 98 168 151 120 109 163 146 124 175 12 134 195 151 120 6 34 1 88 163 6 134 206 109 201 70 23 187 76 175 129 88 175 44 44 146 81 55 63 187 120 40 19 49 104 94 81, 168 201 201 12 29 12,168 158 1 187 210 88 181 134 206

sago palm Sebangau sediment seed weight seedling establishment seeds maturity selection sengon (Paraserianthes falcataria) social interaction soil macroinvertebrates soybean sperm Staphylococcus aureus stomata storage period Streptomyces Sumatra Sumatran orangutan

168 187 76 29 29 49 6 129 94 129 109 124 158 19 49 115 195 94

Sus verrucosus Syzygium Syzygium bankense T47D temperature terrestrial orchid transplantation travel-band Trichoglossus euteles tubers Uncaria gambier Roxb. upland rice vigor wheat xylanase xylooligosaccharide Zingiber cassumunar Roxb. Î˛-carotene

124 120 29 44 49 175 181 94 81 6 23 98 49 55 115 115 40 6

List of Peer Reviewers Abdel Fattah N. Abd Rabou, Dr. – Department of Biology, Faculty of Science, Islamic University of Gaza, Gaza Strip, Palestine Agung Budiarjo, M.Sc. – Department of Biology, Faculty of Mathematics and Natural Science, Sebelas Maret University, Solo, Indonesia Ahmad Dwi Setyawan, M.Sc. – Department of Biology, Faculty of Mathematics and Natural Science, Sebelas Maret University, Solo, Indonesia

Ali Saad Mohamed, Prof. Dr. – College of Veterinary Medicine, Sudan University of Science and Technology. Khartoum, North-Sudan Andi Salamah, Dr. – Department of Biology, Faculty of Mathematics and Natural Sciences, Indonesian University, Jakarta, Indonesia Ary Prihardhyanto Keim, Dr. – Botany Division, Research Centre for Biology, Indonesian Institute of Sciences (LIPI), Cibinong, Indonesia Dedi Darnaedi, Dr. – Botany Division, Research Centre for Biology, Indonesian Institute of Sciences (LIPI), Cibinong, Indonesia Dwi Nugroho Wibowo, Dr. – Faculty of Biology, General Soedirman University, Purwokerto, Indonesia Edi Rudi, Dr. – Department of Biology, Faculty of Mathematics and Natural Sciences, Syiah Kuala University, Banda Aceh, Indonesia Eko Baroto Walujo, Prof. Dr. – Botany Division, Research Centre for Biology, Indonesian Institute of Sciences (LIPI), Cibinong, Indonesia Elizabeth A. Widjaja, Prof. Dr. – Botany Division, Research Centre for Biology, Indonesian Institute of Sciences (LIPI), Cibinong, Indonesia Hamim, Dr. – Department of Biology, Faculty of Mathematics and Natural Sciences, Bogor Agricultural University, Bogor, Indonesia Hassan Poorbabaei, Dr. – Department of Forestry, Faculty of Natural Resources, University of Guilan, Somehsara, Islamic Republic of Iran Irawati, Dr. – Centre for Plant Conservation Bogor Botanic Gardens, Indonesian Institutes of Sciences (LIPI), Bogor, Indonesia J. Perry Gustafson, Prof. Dr. – Agronomy Department, University of Columbia, Columbia, MO, USA Joko Ridho Witono, Dr. – Centre for Plant Conservation Bogor Botanic Gardens, Indonesian Institutes of Sciences (LIPI), Bogor, Indonesia Katsuhiko Kondo, Prof. Dr. – Department of Agriculture, Faculty of Agriculture, Tokyo University of Agriculture, Atsugi City, Japan Mahendra K. Rai, Prof. Dr. – Department of Biotechnology, SGB Amravati University, Amravati, Maharashtra, India Marsusi, Dr. – Department of Biology, Faculty of Mathematics and Natural Science, Sebelas Maret University, Solo, Indonesia Nita Etikawati, M.Sc. – Department of Biology, Faculty of Mathematics and Natural Science, Sebelas Maret University, Solo, Indonesia Rita Rakhmawati, M.Sc. – Department of Biology, Faculty of Mathematics and Natural Science, Sebelas Maret University, Solo, Indonesia Roedhy Poerwanto, Prof. Dr. – Department of Agronomy, Faculty of Agriculture, Bogor Agricultural University, Bogor, Indonesia Sameer Ahmad Masoud, Ph.D. – Department of Biotechnology and Genetic Engineering, Faculty of Science, Philadelphia University, Amman, Jordan

Shahabuddin, Dr. – Department of Biology, Faculty of Mathematics and Natural Science, Tadulako University, Palu, Indonesia Siti M. Widiyastuti, Prof. Dr. – Faculty of Forestry, Gadjah Mada University, Yogyakarta, Indonesia Solichatun, M.Sc. – Department of Biology, Faculty of Mathematics and Natural Science, Sebelas Maret University, Solo, Indonesia Stuart J. Campbell, Dr. – Wildlife Conservation Society (WCS) Indonesian Program, Bogor, Indonesia Sugiyarto, Dr. – Department of Biology, Faculty of Mathematics and Natural Science, Sebelas Maret University, Solo, Indonesia Suranto, Prof. Dr. – Department of Biology, Faculty of Mathematics and Natural Science, Sebelas Maret University, Solo, Indonesia Surya Dewi Marlina, M.Sc. – Department of Chemistry, Faculty of Mathematics and Natural Science, Sebelas Maret University, Solo, Indonesia

Sutarno, Prof. Dr. – Department of Biology, Faculty of Mathematics and Natural Science, Sebelas Maret University, Solo, Indonesia Tjahjadi Purwoko, M.Sc. – Department of Biology, Faculty of Mathematics and Natural Science, Sebelas Maret University, Solo, Indonesia Widya Mudyantini, M.Sc. – Department of Biology, Faculty of Mathematics and Natural Science, Sebelas Maret University, Solo, Indonesia

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

MOLECULAR BIOLOGY AND GENETICS

Comparison Analysis of Genetic Diversity of Indonesian Mangosteens (Garcinia mangostana L.) 163-167 and Related Species by using on Isozymes and AFLP Markers SOBIR, SOALOON SINAGA, ROEDHY POERWANTO, RISMITASARI, RUDY LUKMAN TAXONOMY, BIOSYSTEMATICS, AND PHYLOGENETICS

168-174

Genetic Relationship of Sago Palm (Metroxylon sagu Rottb.) in Indonesia Based on RAPD Markers BARAHIMA ABBAS, MUHAMMAD HASIM BINTORO, SUDARSONO, MEMEN SURAHMAN, HIROSHI EHARA ECOLOGY AND BIOLOGICAL CONSERVATION

The Association of Mycorrhizal with Terrestrial Orchid Root in Mt. Cycloops Nature Reserve, Jayapura

175-180

VERENA AGUSTINI, SUPENI SUFAATI, SUHARNO

Growth Rate of Acropora formosa Fragments that Transplanted on Artificial Substrate Made from Coral Rubble

181-186

NUR FADLI

Forest Dynamics of Peat Swamp Forest in Sebangau, Central Kalimantan

187-194

EDI MIRMANTO

Diversity and Abundance of Beetle (Coleoptera) Functional Groups in a Range of Land Use Systems in Jambi, Sumatra

195-200

FRANCISCUS XAVERIUS SUSILO, INDRIYATI, SURYO HARDIWINOTO

Population and Microhabitat Characteristic of Homalomena bellula Schott in Mount Slamet, Central Java, Indonesia

201-205

YAYAN WAHYU CANDRA KUSUMA, INGGIT PUJI ASTUTI OTHERS

The Eschericia coli Growth Inhibition Activity of Some Fermented Medicinal Plant Leaf Extract from the Karo Highland, North Sumatra

206-209

EVI TRIANA, NOVIK NURHIDAYAT REVIEW

Review: Current Advances in Gloriosa superba L.

210-214

RAVINDRA ADE, MAHENDRA K. RAI

Front cover: Micrograph of Ceratorrhiza sp. (PHOTO: VERENA AGUSTINI)

Published four times in one year

PRINTED IN INDONESIA ISSN: 1412-033X (printed)

ISSN: 2085-4722 (electronic)