Nusantara Bioscience vol. 3, no. 1, March 2011

Jatropha curcas photo by A. Abdurrahman

| Nus Biosci | vol. 3 | no. 1 | pp. 1‐58 | March 2011 | ISSN 2087‐3940 (PRINT) | ISSN 2087‐3956 (ELECTRONIC)

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| Nus Biosci | vol. 3 | no. 1 | pp. 1‐58 | March 2011 | ISSN 2087‐3940 (PRINT) | ISSN 2087‐3956 (ELECTRONIC) I S E A J o u r n a l o f B i o l o g i c a l S c i e n c e s

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ISSN: 2087-3940 (print) ISSN: 2087-3956 (electronic)

Vol. 3, No. 1, Pp. 1-6 March 2011

Optimization of DNA extraction of physic nut (Jatropha curcas) by selecting the appropriate leaf EDI PRAYITNO, EINSTIVINA NURYANDANI♥ Open University, UPBJJ Semarang. Jl. Semarang-Kendal, Mangkang Wetan, Semarang 50156, Central Java, Indonesia, Tel. +62-24-8666044, Fax. +6224-8666045; ♥email: vina_ut@yahoo.co.id

Manuscript received: 11 November 2010. Revision accepted: 24 February 2011 (stay empty)

Abstract. Prayitno E, Nuryandani E. 2011. Optimization of DNA extraction of physic nut (Jatropha curcas) by selecting the appropriate leaf. Nusantara Bioscience 3: 1-6. Jatropha curcas L. has important roles as renewable source of bioenergy. The problem occurs on difficult of DNA extraction for its molecular breeding programs. The objectives of this research were to study which leaf best as source of DNA extraction. Four accession were used, namely J1 and J2 (Jawa Tengah), S1 (South Sumatra), and S2 (Bengkulu). First, third, fifth, seventh, and yellow leaves for each accession were extracted using modification of Doyle and Doyle (1987) method. Visualization and comparation with Lambda DNA, Spectrophotometer UV-Vis and cutting DNA with EcoRI enzyme were show quality and quantity of DNA. The result showed that third leaves have sufficient quality and quantity as source of DNA. Third leaves DNA quantity for J1 (19.33 µg/mL), J2 (26.21 µg/mL), S1 (31.20 µg/mL), dan S2 (61.03 µg/mL), and quality for each accession were 1.9063 (J1), 2.0162 (J2), 2.0116 (S1), and 2.0856 (S2).

Key words: Jatropha curcas, DNA extraction, appropriate, leaf.

Abstrak. Prayitno E, Nuryandani E. 2011. Optimalisasi ekstraksi DNA jarak pagar (Jatropha curcas) melalui pemilihan daun yang sesuai. Nusantara Bioscience. Nusantara Bioscience 3: 1-6. Jarak pagar (Jatropha curcas L.) mempunyai peran penting sebagai sumber bahan bakar nabati. Usaha pemuliaan tanaman ini secara molekuler sering terkendala sulitnya ekstraksi DNA. Penelitian ini bertujuan untuk mengetahui daun yang sesuai untuk digunakan sebagai sumber DNA. Penelitian ini dilakukan pada empat aksesi jarak pagar yaitu J1 dan J2 (Jawa Tengah), S1 (Sumatera Selatan), dan S2 (Bengkulu). Ekstraksi dilakukan pada daun pertama, ketiga, kelima, ketujuh, dan daun kuning dari setiap aksesi dengan metode Doyle and Doyle (1987) yang dimodifikasi. Kualitas dan kuantitas DNA hasil ekstraksi diketahui melalui visualisasi dengan pembanding DNA lambda, spektrofotometer UV-Vis pada panjang gelombang 260/280, dan pemotongan menggunakan enzim EcoRI. Hasil penelitian menunjukkan bahwa daun ketiga memadai untuk digunakan sebagai sumber DNA. Kuantitas DNA daun ketiga J1 (19,33 µg/mL), J2 (26,21 µg/mL), S1 (31,20 µg/mL), dan S2 (61,03 µg/mL). Sedangkan kemurniannya masing-masing yaitu 1,9063 (J1), 2,0162 (J2), 2,0116 (S1), dan 2,0856 (S2).

Kata kunci: Jatropha curcas, ekstraksi DNA, daun, sesuai.

INTRODUCTION Increased economic growth and spur the growth of population of high energy consumption. Energy source the world today is still dominated by fossil fuel that cannot be renewed (unrenewable). Various efforts have been made to solve energy problems (Raharjo 2007). Fuel from the plant has several advantages such as ease of storage and environmentally friendly, therefore biofuels were given priority for development. On January 25, 2006, the President of Indonesia issued Presidential Regulation No. 5/2006 regarding the national energy policy and Presidential Instruction No. 1/2006 concerning the provision and use of biofuels as alternative fules. Then on July 1, 2006, presidential and state officials conducting a retreat in the village of Losari, Grabag subdistric, Magelang district, and decided to develop a bioenergy or biofuel as an alternative energy.

Biofuel can be divided into two major categories, namely bioethanol and biodiesel. Bioethanol is ethanol derived from fermentation of raw materials that contain starch or sugar such as molasses and cassava. This fuel can be used to replace regular gasoline (gasoline). Ethanol can be used is alcohol-free pure water (anhydrous alcohol) and levels of more than 99.5%, or called with a fuel grade ethanol (FGE). Blend of premium and FGE is called gasohol. In Indonesia, Pertamina give biopremium trademark for the product. Biodiesel is a popular name for FAME (fatty acid methyl ester), is a biofuel that is used to power diesel engines as an alternative to diesel. This fuel derived from vegetable oils are converted through chemical and physical reactions, so that the nature of the chemical has changed from its original nature. Currently, Pertamina has issued such a product with trade name which is a blending FAME biodiesel with regular diesel (petrosolar) (Prihandana et al. 2007).

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Jatropha curcas is a native plant of Central America (Fairless 2007) and has been naturalized in tropical and subtropical regions, including Indonesia. This species is drought resistant and is commonly planted as a garden fence, but is also useful as an ornamental plant shrubs and herbs. Oil from the seeds is useful for medicine, insecticides, making soap and candles, as well as raw material for biodiesel (Gubitz et al. 1999). The use of castor oil as biodiesel ingredient is an ideal alternative, because it is a renewable oil resources (renewable fuels) and non-edible oil so it does not compete with human consumption requirements, such as palm oil, corn, soybeans and others (Dwimahyani 2005). In addition, Jatropha also contains secondary metabolites which are useful as protectant for plants and as an ingredient for human medicine (Debnath and Bisen 2008) Some of the obstacles encountered in developing castor oil, among others, lack of information about varieties that have beneficial properties such as high production, fast multiplication, high oil yield in seeds, as well as resistance to pests and diseases. This happens because so far the Jatropha plant is only regarded as hedgerows that have low economic value so that research and development of this plant is rarely done. To overcome this, plant breeding has a significant role.

Characterization of jatropha plant in Indonesia is carried out simply and not be universal. Often, the mention of Jatropha plant species is based solely on phenotypic appearance or region of origin. Characterization using morphological or phenotypic description has limitations because it is very influenced by the environment. Different morphological features can be caused by environmental stress, whereas the same genotype, whereas the same morphological features do not necessarily indicate that both types of plants are closely related, because the outer shape of a plant is the result of cooperation between the genotype by environment (Joshi et al. 1999; Karsinah 1999 .)

Therefore, it is necessary to develop universal genetic information. Molecular markers can provide information universally because it is not influenced by the environment (Azrai 2005), so that they can answer the problem in the characterization of physic nut plants.

Jatropha curcas is one of the many plants that contain latex, which is a true plant secondary metabolites. The presence of secondary metabolites such as polyphenols, tannins, and polysaccharides can inhibit the action of the enzyme (Porebski 1997; Pirtilla et al. 2001). Isolation of plant DNA at a distance often experienced problems due to high levels of secondary metabolites in the form of polysaccharides and polyphenols. According to Sharma et al. (2002) the presence of metabolites in several crops affect DNA isolation procedure, he was using a modified CTAB to isolate DNA from plant tissue containing high polysaccharide. In line with this Kiefer et al. (2000), Pirtilla et al. (2001) and Sanchez-Hernandes, C. and J.C. GaytanOyarzun (2006), states that the extraction of DNA and RNA from plants containing polysaccharides, polyphenols as well as sap and difficult.

Proper techniques of DNA extraction is needed in the plant breeding process to obtain DNA with a high quality

and quantity. To obtain pure DNA from plant sap, generally carried out repeated purification and modification of procedures (Kiefer et al. 2000), thus requiring additional cost and effort. For that, you can use parts of plants that contain little secondary metabolites. The content of secondary metabolites in plant tissues fluctuate in line with its development. Secondary metabolites may vary because of differences in age and plant part (Cirak et al. 2007a, b, 2008; Achakzai et al. 2009). Therefore, to simplify the DNA extraction process jatropha, have done research to learn the parts of plants containing secondary metabolites in small amounts and produce DNA with high quality and quantity.

This research aims to study the jatropha plant leaves at different levels of development that have the potential to produce the best quality and quantity of DNA in the DNA extraction process.

MATERIALS AND METHODS Time and place of study Research was conducted at the Open University UPBJJSemarang, Central Research Laboratory Tropical Fruit IPB, Bogor, West Java, and Laboratory of Structure and Function of Plant Diponegoro University in March to November 2009.

Plant material Jatropha plant materials used in this study are the three accessions of jatropha plants originated from areas of Klaten (Central Java) with the code J1 and J2, Palembang (South Sumatra) with codes S1, and Bengkulu, with the code S2.

Procedures Isolation of DNA. About 0.5 g of leaves from the first, third, fifth, seventh and yellow leaves from each sample was crushed in porcelain bowls by adding 0.1 grams of silica sand to be easily crushed. To prevent network browning by oxidation, polivinilpolipirilidon (PVPP) as much as 40 mg and added extraction buffer (2% CTAB, 100 mM Tris-HCl pH 8, 1.4 M NaCl, 20 mM EDTA) as much as 1 mL is added into a cup containing the sample which has added 1% merkaptoetanol. Samples that have been incorporated into the fine volume of 1.5 mL Eppendorf tube. Subsequently the mixture incubated at 65oC for 30 minutes while inverted, and then added 1 mL solution of chloroform: isoamilalchohol (24:1 = v/v) and divortek for 5 seconds. This solution was then separated using a centrifuge with a speed of 11,000 rpm for 10 minutes at a room temperature. Supernatant was separated from the pellet by putting it into a new Ependorf tube.

DNA in the supernatant was purified by adding 1 mL solution of chloroform: isoamilalkohol (24:1 = v/v) and disentrifuse at a speed of 11,000 rpm for 10 minutes at room temperature. Supernatant was transferred into a tube and added with 1 mL of cold isopropanol, shaken gently until white threads arise, which is DNA. Subsequently DNA was precipitated by incubation for 30 minutes at a

PRAYITNO & NURYANDANI – Optimization of DNA extraction of Jatropha curcas

present in yellow leaves, except on J1 where J1k (yellow) has a thicker ribbon smears compared J15 and J17 (Figure 1).

In genomic DNA extracted from young leaves, which are visible smear on the bottom of genomic DNA. Ribbon smear is a molecule with varying weights that can be derived from degraded DNA or other follow-up material that is not known (Herison 2003). Smears indicated that the isolated genomic DNA was not intact anymore, probably dismembered during the extraction takes place (Sisharmini et al. 2001). Genomic DNA damage can be caused by degradation of secondary compounds that are released when the cells were destroyed or damaged due to physical handling. The decline is likely influenced by the smear of secondary metabolites of plants and physical handling. In this case the physical handling for each sample the same can be said for using the same standard procedure, therefore, the greatest influences that cause differences in high and low smear is a secondary metabolite from the leaves of plants (Milligan 1992).

In certain plants, plant metabolites will be seen visually in the form of sap. Jatropha curcas is a plant sap, with pink latex (de Padua et al. 1999) or nodes in the young gradually turns cloudy/older if left in free air or dark brown when taken from the older plants (Heyne 1987). Young leaves contain more secondary metabolites than older leaves (Badawi 2006; Mulyani 2006). Young leaves generally contain secondary metabolites and enzymes that high because it requires in the process of growth, development, and division of cells’ leaf. In the development of plant secondary metabolite concentrations will gradually decline as the decline in leaf growth activity, and the leaves have yellowed, the concentration of enzymes and secondary metabolites in the leaves decreased significantly due to the ongoing process of senesensi (Salisbury and Ross 1995).

At this stage the plant will attract substances and enzymes that are still useful to the plant from old leaves for use in the process of development of the younger plants, so the possibility of plant secondary metabolites present in a very low level so that the DNA is not much degraded by the follow-up compound (Salisbury and Ross 1995; Herison 2003). Although the smear on the older leaves less and less, but the quantity of genomic DNA was also decreased, which lights up genomic DNA bands at the top of the wells that are running low on older leaves.

temperature of -20ºC. Solution containing the DNA that has been purified disentrifuse with speed 11 000 rpm for 10 minutes at room temperature and then the supernatant was discarded. DNA precipitate was washed with 70% alcohol and dried at room temperature. Further the DNA samples that was obtained was dissolved in 100 mL TE buffer (10 mM Tris-HCl pH 7.5, 10 mM EDTA) and incubated at 37° C for one hour and then mixed until uniform to further test its quality.

Test the quality and quantity of DNA. The quantity (concentration) and quality of DNA determined by UV-Vis spectrophotometer at wavelength 260 and 280 nm.

Determination of the total DNA quantity was calculated based on the value of absorbance at a wavelength of 260 nm. A at 260 = 1.0 equivalent amount of DNA is 50 ug/mL. λ DNA quality is considered good if the value of A260/280 approaching 1.8 to 2. To determine the concentration and quality of DNA, electrophoresis results were soaked in a solution of 1% EtBr and then observed under UV transluminator. The quantity of DNA is based on the thickness of the electrophoresis results of DNA samples are compared with the amount of lambda DNA of known concentration, ie 250 ug/mL. This study also tested the quality of DNA by cutting genomic DNA using EcoRI enzyme are visualized by electrophoresis on agarose gel.

RESULTS AND DISCUSSION Visualization of the extracted DNA The success of the isolation and extraction process of genomic DNA can be marked with resultant large DNA (high molecular weight DNA), that is not degraded during extraction and purification process, and can be cut by restriction enzymes that has been used (Herison 2003).

Results of isolation and extraction of jatropha’s DNS employed Doyle and Doyle method (1987) which has been modified to produce the desired genomic DNA bands, although relatively small quantity when compared to lambda DNA. Genomic DNA was seen as a ribbon that lights up at the top sinks electrophoresis results.

In general, smear on DNA extracted from young leaves (code J11, J21, S11, S21) and concentrated look taller than the smear on DNA extracted from the older leaves, then gradually decreasing concentration smear on leaves more old (leaves the third, fifth, and seventh), and smear the least L

J11

J13

J15

J17

J1K

J21

J23

J25

J27

J2K

S11

3

S13

S15

S17

S1K

L

L

S21

S23

S25

S27

S2K

L

Figure 1. Visualization of the extracted DNA from four accessions of Jatropha curcas Klaten (J1, J2), Palembang (S1) and Bengkulu (S2). L = LAMDA (ladder)

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The young leaves have a high cleavage activity. In the division process, DNA replication will experience, so the amount of DNA will double itself, thus DNA concentration is relatively high in young leaves. On older leaves, the division process could decrease, until finally stopped altogether. On the leaves that have yellowed, in addition to the absence of the division process, it also exacerbated the death of cells that were old, so the amount of DNA was also decreased dramatically (Salisbury and Ross 1995).

Test the quality and quantity of DNA with UV-Vis spectrophotometer The quantity (concentration) and quality of DNA determined by UV-Vis spectrophotometer at wavelength 260 and 280 nm. Determination of the total DNA quantity was calculated based on the value of absorbance at a wavelength of 260 nm. The highest DNA purity can be seen in the A260/280 ratio that produces the value of 1.8 to 2. According to Sambrook et al. (1989) DNA with a ratio in the range of figures have met the requirements of purity required in molecular analysis. Spectrophotometer results show relatively good purity DNA that has yet to reach 100% purity in some accessions. The concentration and purity of genomic DNA was analyzed using UV-Vis spectrophotometer can be seen in Table 1.

Genomic DNA which has a purity of 100% contained in the accession J1 was extracted from the third leaf with value ratio of 1.9063. Genomic DNA from the first leaf accession J1 has a value ratios approaching 100% purity with ratio of 2.0131. While the three other leaves, that leaves the fifth, seventh, and yellow leaves have a value ratio of less than 1.8 respectively, 1.7417, 1.2578, and 1.2356. Results DNA extraction leaves first, third, and fifth of the accession J2 has a value closer to purity ratio, respectively 2.0697, 2.0162, 2.0914, while the seventh leaves and yellow leaves have a ratio value that is still far from purity, namely 1.5873 and 1, 1940.

On the accession of S1, almost all of the extracted DNA purity approaching leaves, each leaf of the first, third, fifth, and seventh ratio is 2.0768, 2.0116, 2.0792, 2.0225, while the yellow leaves have value ratio far from the purity of 1.4434. DNA extracted first and third leaf from the accession of S2 close to the purity of the value ratio of 2.0611 and 2.0856. While leaf fifth, seventh, and yellow leaves have a ratio that is far from the purity of the respective ratios 2.2187, 2.1782, and 1.5177.

Besides the purity of genomic DNA samples, another consideration that must be considered is the quantity of

genomic DNA was generated from the DNA extraction process. Readings A260 = 1 means the concentration of DNA obtained at 50 ug/mL (Herison 2003). The concentration of genomic DNA was extracted was calculated by the formula: DNA concentration (ug/mL) = A260 x dilution factor x 50 ug/mL. DNA concentration resulting from the extraction process represents the amount of DNA contained in the leaf tissue used for the sample and treatment methods used in each sample is the same. Table 1 below is the concentration of DNA from samples of twenty leaves from four accessions of jatropha plant that is used. From Table 1, note the concentration ratio of genomic DNA from leaf tissue of each first, third, fifth, seventh, and yellow leaves, and comparison of genomic DNA concentration between sections. In general, genomic DNA concentration decreased with increasing age of leaves used as a sample.

Samples from the first leaf shows the quantity of genomic DNA is much larger than the sample leaves the third, fifth, seventh, and yellow leaves. Measurement of the quantity of genomic DNA samples from accessions J1 genomic DNA in Klaten produces relatively little compared to the accession of J2, S1, and S2, which is 27.69 ug/mL for the first leaf, 19.33 ug/mL for the third leaf, 3.68 tg/mL for the fifth leaf, 2.03 g/mL for the seventh leaf, and 4.51 ug/mL for yellow leaves. This is due to a smaller sample size compared to other accessions due to spill some of the samples by laboratory staff who worked on, so that DNA samples that were tested got reduced. While the accession J2, where accession was also derived from the same home with the accession of J1, which was from Klaten, Central Java, and comes from the same parent, the quantity of genomic DNA generated greater than J1, which is 62.06 ug/mL for the extraction of the first leaf, 26.21 ug/mL for the third leaf, 27.69 ug/mL for the fifth leaf, 5.37 g/mL for the seventh leaf, and 4.37 ug/mL for yellow leaves.

The concentration of genomic DNA for S1 accession on the first leaves produced 67.61 g/mL DNA, whereas the third leaf, the concentration of genomic DNA was 31.20 ug/mL, on the fifth leaves of 46.71 ug/mL, on the seventh leaf, 22, 90 ug/mL, and the yellow leaves of 7.59 g/mL. Accession S2 on the first leaves produced 101.35 g/mL genomic DNA, while the third leaf, the concentration of genomic DNA was 61.03 ug/mL, on the fifth leaves of 44.18 ug/mL, leaves the seventh, 26.27 ug/mL , and the yellow leaves of 5.37 g/mL. The Figure 1 shows the concentration of the extracted genomic DNA of

Table 1. Test the quality (purity) and quantity (concentration) of DNA using UV-Vis spectrophotometer in four accessions of jatropha from Klaten (J1, J2), Palembang (S1) dan Bengkulu (S2).

Leaves First Third Fifth Seventh Yellow

J1 2.0131 1.9063 1.7417 1.2578 1.2356

DNA purity J2 S1 2.0697 2.0768 2.0162 2.0116 2.0914 2.0792 1.5873 2.0225 1.1940 1.4434

S2 2.0611 2.0856 2.2187 2.1782 1.5177

J1 27.69 19.33 3.68 2.03 4.51

DNA concentration (Âľg/mL) J2 S1 62.06 67.61 26.21 31.20 27.69 46.71 5.37 22.90 4.37 7.59

S2 101.35 61.03 44.18 26.27 5.37

PRAYITNO & NURYANDANI – Optimization of DNA extraction of Jatropha curcas

as secondary metabolites, carbohydrates, proteins, and others, will hinder the work restriction enzymes.

Whether DNA can be cut with restriction enzymes is visible from at least smear results of electrophoresis bands after DNA cut with EcoRI enzyme (Herison 2003). EcoRI produce DNA bands when smears were electrophoresed because this restriction enzyme included in the frequent cutter (Vos et al. 1995). The result of cutting with EcoRI enzyme produces DNA fragments that appear as a smear on some samples, but most other samples can not be cut by this enzyme because of the high follow-up compounds that inhibit enzymes work. Smear only be observed in J13 and J15, while the other samples have not seen a clear smear as a result of enzyme EcoRI. Visible is the presence of minor compounds in the lower section sinks. possible follow-up material that inhibits this enzyme EcoRI work so as not to cut the genomic DNA tested jatropha.

The description above discussion shows that differences in leaf tissue age used influence the extraction of genomic DNA where the younger leaves will produce a quantity of genomic DNA was higher but also accompanied by the high follow-up material in the form of plant secondary metabolites that inhibit the work in the field of molecular further. Older leaves to produce the amount of genomic DNA are relatively fewer compared to young leaves, but the following secondary metabolites was also reduced in number. This study shows that the third leaf is better used as a source of genomic DNA since the DNA purity is better than the other leaves, and the quantity produced enough DNA to be used for further molecular analysis.

diminishing. This is related to the phase of leaf development that has been outlined above.

Results spectrophotometer for quantity of genomic DNA of the above shows that the largest quantity of genomic DNA from four accessions were found in the extraction of the first leaf. But considering the quality of the resulting DNA, the highest purity approaching 100% are found in the sample using the third leaf as a source of genomic DNA, although in terms of quantity, the number is lower than the samples originated from the first leaf.

Comparison of DNA extracted from five types of leaf samples from accessions used in J1 and J2 from Klaten, from the same parent tree can be seen in Table 1. From Table 1 it can be seen that the DNA genome of the first and second leaf (accession J1) and leaves the first, third, and fifth (accession J2) approached the purity, but purity is closest to the third leaf (accession J1 on the ratio of 1, 9063 (purity 100%) and the accession to the ratio of 2.0162 A2).

But in terms of quantity, J1 and J2 are not comparable although originating from the same parent because of the sample is not the same J1 J2 terms of number of samples tested for spill samples by the laboratory.

Some researches indicate that generally young leaves are used in DNA extraction because of the ease in getting the DNA with a high quantity. Mansyah et al. (2003) who conducted research on mangosteen states that extraction of DNA from old leaves is more difficult when compared with young leaves, so as to obtain DNA from old leaves with a sufficient quantity is required special treatment, namely with the addition of the extracted leaves up to 2 g and DNA purification with the addition of RNase. While Prana (2003) who perform DNA extraction on taro plants also use the young leaves (in this case the leaf shoots) as the source of DNA.

CONCLUSION The third leaf physic nut plants suitable for use as a source of DNA for molecular analysis of genomes, as in quantity and quality sufficient to produce genomic DNA for further molecular analysis such as PCR. Genomic DNA extracted from the third leaf is generally close to 100% purity and quantity of DNA produced is also large enough to be used for further molecular analysis.

Test the quality of DNA by using the enzyme EcoRI cuts The purity of DNA can be seen from the absence of a DNA sample can be cut by restriction enzyme such as EcoR1 (Figure 2). If a DNA sample has high purity, this DNA would be easy to cut by restriction enzymes. But if this is still contain DNA samples follow-up materials such

M

J11

J13

J15

J17

J1k

J21

J23

J25

J27

J2K

5

S11

S13

S15

S17

S1K

S21

S23

S25

S27

S2k

M

Figure 2. Visualization of results by the enzyme EcoRI cuts at the four accessions of jatropha from Klaten (J1, J2), Palembang (S1) and Bengkulu (S2).

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ISSN: 2087-3940 (print) ISSN: 2087-3956 (electronic)

Vol. 2, No. 1, Pp. 7-14 March 2010

Characterisation of taro (Colocasia esculenta) based on morphological and isozymic patterns markers TRIMANTO1,♥, SAJIDAN², SUGIYARTO² ¹ SMP Negeri 2 Gemolong, Sragen, Jl. Citro Sancakan No. 249, Sragen 57274, Central Java, Indonesia; Tel.: +92-0818754378 ² Bioscience Program, School of Graduates, Sebelas Maret University, Surakarta 57126, Central Java, Indonesia Manuscript received: 25 October 2009. Revision accepted: 15 February 2010.

Abstract. Trimanto, Sajidan, Sugiyarto. 2011. Characterization of taro (Colocasia esculenta) based on morphological and isozymic patterns markers. Nusantara Bioscience: 7-14. The aims of this research were to find out: (i) the variety of Colocasia esculenta based on the morphological characteristics; (ii) the variety of C. esculenta based on the isozymic banding pattern; and (iii) the correlation of genetic distance based on the morphological characteristics and isozymic banding pattern. Survey research conducted in the Karanganyar district, which include high, medium and low altitude. The sample was taken using random purposive sampling technique, including 9 sampling points. The morphological data was elaborated descriptively and then made dendogram. The data on isozymic banding pattern was analyzed quantitatively based on the presence or absence of bands appeared on the gel, and then made dendogram.

The correlation based on the morphological characteristics and isozymic banding pattern were analyzed based on the product-moment correlation coefficient with goodness of fit criterion. The result showed : (i) in Karanganyar was founded 10 variety of C. esculenta; (ii) morphological characteristics are not affected by altitude; (iii) isozymic banding pattern of peroxides forms 14 banding patterns, esterase forms 11 banding patterns and shikimic dehydrogenase forms 15 banding patterns; (iv) the correlation of morphological data and the isozymic banding pattern of peroxidase has good correlation (0.893542288) while esterase and shikimic dehydrogenase isozymes have very good correlation (0.917557716 and 0.9121985446); (v) isozymic banding pattern of data supports the morphological character data.

Key words: taro, Colocasia esculenta, morphology, isozyme.

Abstrak. Trimanto, Sajidan, Sugiyarto. 2011. Karakterisasi talas (Colocasia esculenta) berdasarkan penanda morfologi dan pola pita isozim. Nusantara Bioscience: 7-14. Tujuan penelitian ini adalah untuk mengetahui: (i) keragaman Colocasia esculenta berdasarkan karakter morfologi; (ii) keragaman C. esculenta berdasarkan pola pita isozim, dan (iii) hubungan jarak genetik berdasarkan karakter morfologi dan pola pita isozim. Survei penelitian dilakukan di Kabupaten Karanganyar, di ketinggian tinggi, sedang dan rendah. Sampel diambil menggunakan teknik random sampling purposif, mencakup 9 titik cuplikan. Data morfologi diuraikan secara deskriptif dan kemudian dibuat dendogram kekerabatan. Data pola pita isozim dianalisis secara kuantitatif berdasarkan ada atau tidaknya pita di gel, kemudian dibuat dendogramnya. Korelasi berdasarkan karakter morfologi dan pola pita isozim dianalisis berdasarkan korelasi koefisien momen-produk kriteria goodness of fit. Hasil penelitian menunjukkan: (i) di Karanganyar terdapat 10 varietas C. esculenta; (ii) karakter morfologi tidak terpengaruh oleh ketinggian; (iii) peroksidase membentuk 14 pola pita isozim, esterse membentuk 11 pola pita dan shikimate dehidrogenase membentuk 15 pola pita; (iv) data morfologi dengan isozim peroksidase memiliki korelasi yang baik (0,893542288), sementara data morfologi dengan isozim esterse dan shikimate dehidrogenase memiliki korelasi yang sangat baik (0,917557716 dan 0,9121985446); (v) data pola pita isozim mendukung data karakter morfologi.

Kata kunci: talas, Colocasia esculenta, morfologi, isozim

INTRODUCTION The diversity of food crops in Indonesia can be developed to overcome the food problem. Types of tubers that can be utilized more optimally as a staple food rice substitutes include cassava, sweet potato, taro, purse, arrowroot and canna. These tubers have a lot of the preeminent, among them having a high content of carbohydrates as energy sources (Liu et al. 2006), not containing gluten (Rekha and Padmaja 2002), containing angiotensin (Lee et al. 2003), antioxidative ( Nagai et al.

2006),which can be applied to various purposes (Aprianita 2009), and produce more energy per hectWEREthan rice and wheat. Tubers can be grown on marginal areas (Louwagie et al. 2006), where other plants cannot grow and

can be stored in the form of flour and starch (Aboubakar et al. 2008).

Taro has a good variety of morphological characters such as tubers, leaves and flowers as well as chemicals such as flavor, aroma and others (Xu et al. 2001).

Characterization of taro plants now has started to be developed through two approaches. The diversity among the varieties can be distinguished based on morphological and molecular markers. Diversity based on morphological marker has a weakness, because the morphological characteristics do not necessarily indicate genetic diversity.

Morphological diversity is influenced by the environment, because every environment has different conditions, so the plants do adapt to their home range.

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Molecular marker is an effective technique in genetic analysis of a plant variety. Molecular markers have been applied widely in plant breeding programs. Molecular marker that is often used to distinguish plant diversity is a marker of isozyme and DNA (Asains et al. 1995; Setyo 2001). Isozyme is a direct product of genes and relatively free from environmental factors. Isozyme can be used as a genetic trait to study and identify the diversity of individuals or a cultivar. Isozymes were enzymes that have active molecules and different chemical structure, but catalyze the same chemical reaction. Different forms of an enzyme molecule can be used as the basis of chemical separation, by electrophoresis method will result in banding patternsproduced by different distances (Purwanto et al. 2002).

Information about the genetic diversity of taro (Colocasia esculenta L.) is needed for plant breeding and improvement for the offsprings to obtain superior varieties.

Based on the background, the research was conducted on taro plants in different areas in a region that had high altitude, medium and low that included morphological characters and isozyme banding patter pita on different varieties of taro plants in Karanganyar, Central Java.

MATERIALS AND METHODS The experiment was conducted in March 2009 to August 2009. Taro plants (Colocasia esculenta L.) were collected from Karanganyar District, Central Java differentiated by differences in altitude, namely: (i) the highlands (> 1000 m asl), (ii) plain medium (500-1000 m asl), and (iii) Lowland (<500 m asl). Location of the study covers nine districts in the district Karanganyar (Table 1).

Characterization of isozyme of taro plants were conducted at the Faculty of Forestry Gadjah Mada University, Yogyakarta, using three enzyme systems namely esterase (EST), peroxidase (POD) and shikimate dehydrogenase (ShDH).

Characterization of morphology Characterization of morphology includes: range of plants, plant’s height, stolon’s number, stolon’s length, leaf shape of basalt, the dominant position of leaves, leaf edges, leaf color, leaf blade edge color, pattern Petiole junction, crossing the color, the color of the liquid at the tip of the leaf blade, the main color of the leaves of bone, bone leaf pattern, the ratio petiole length/leaf blade length, color Petiole upper third, middle third Petiole color, lower third Petiole color, color Petiole lines, color Petiole ring bottom, bottom Petiole transverse incision, midrib length ratio/display total Petiole, leaf midrib color, waxy coating on leaves. Manifestation cormus, cormus length, branch cormus, cormus shape, weight, cortex color, and the flesh color the middle, the color of the meat fibers, cormus skin surface, skin thickness cormus, cormus fiber levels, and color shoots.

Isozyme analysis The third leaf from top in was extracted with a mortal, by adding a solution of extract buffer ± 1 mL. Once

crushed and homogenized, the sample was inserted into the eppedorf, then played with the speed of 15,000 rpm for 20 minutes. Making the gel: Gel Poliacrilamide consists of two parts, ie running a gel that lies at the bottom with a concentration of 7.5% and spacer gel located on top of running gel with a concentration of 3.75%. Electrophoresis: electrophoresis tanks were filled with a solution of electrode buffer tanks as high as ± 2 cm. Mounted on gel electrophoresis, supernatant solution was filled into the hole 5 mL samples using injection equipment (stepper).

Electrophoresis process carried out by an electric current ± 100 mA for 180-200 min. Staining performed after gel electrophoresis, namely by putting that has been removed from the glass electrophoresis into a plastic tray, then soaked in dye solution of dye esterase (EST), peroxidase (POD) and shikimate dehydrogenase (ShDH). Observations gel performed after fixation with seeing a pattern emerging bands, and copy it in the form zimogram.

Data analysis Taro plant morphology data were described by descriptive method that covers all the observed variables in accordance with Kusumo et al. (2002). On isozyme data, the tape that emerged was given a value of 1 while the ones that did not arise given the value 0. Dendogram analysis performed with the method of grouping Average Linkage Cluster Method with DICE coefficient (Rohlf 2005). The grouping was done by UPGMA (Unweigthed Pair Group with arithmatic mean) is calculated by SHAN on NTSYS program (Numerical Taxonomy and Multivariate Analysis System) version of 2:02, while the dendogram analysis using the statistical program Minitab 14 Average linkage method with Euclidean distance measurement. The result made dendogram based isozimnya relationship. Results were analyzed by distance dendogram relationship more than 60% similarity (Cahyarini 2004). The correlation between genetic distance based on morphological characteristics and genetic similarity based on isozyme banding pattern was analyzed based on product-moment correlation coefficient with the criteria of goodness of fit based on the correlation according to Rohlf (2005).

RESULTS AND DISCUSSION Characterization of morphology of C. esculenta The results of characterization of taro plants performed on three different plains in the district Karanganyar ketinggianya obtained 11 variants showed that the plant C. esculenta were scattered in several districts, namely: Benthul, Lompongan, Laos, Mberek, Kladi, Plompong, Sarangan, Kladitem, Jabon, Japan, and Linjik. In this study 18 samples taken taro with research sites with environmental factors as listed in Table 1. The diversity seen in the type of plant, leaf and cormus (bulb). The characterization results show that there is a difference between 11 variants of taro. Description of the morphology of the leaf, midrib, and cormus (bulb) in each of the varieties of taro were as Table 2.

TRIMANTO et al. – Characterization of taro based on morphological and isozyme markers Tabel 1. Environmental conditions where the growth of taro in Karanganyar.

Locations/ Subdistricts Lowland Gondangrejo

Environmental factors Altitude Temp. Type of Rainfall CultiType of taro Shade (meter) (°C) soil (mm/y) vation Benthul Mberek Kladi Linjik Lompongan Plompong

150 150 98 98 320 95

29 29 29 29 30 29

Grumosol Grumosol Aluvial Aluvial Mediteran Mediteran

√ -

1537 1537 1680 1680 2012 2012

√ √ √ √

Plain medium Karangpandan Benthul Lompongan Sarangan Matesih Jabon Laos Linjik

650 600 650 700 750 700

28 28 28 28 27 28

Mediteran Mediteran Mediteran Litosol Litosol Litosol

√ √

2818 2818 2818 2480 2480 2480

√ √ √ √ -

Plateau Tawangmangu Kladitem Benthul Lompongan Ngargoyoso Laos Jatiyoso Sarangan Jepang

1500 1700 1500 1000 1300 1200

23 22 23 26 26 26

Andosol Andosol Andosol Andosol Andosol Andosol

√ √ √ -

3299 3299 3299 3182 3098 3098

√ √ √ √ √

Jaten Karanganyar Kebakkramat

In the dendrogram similarity coefficient of 60% was used to analyze the phylogenetic relationship of the 18 samples found in different locations with 11 different varieties. According Cahyarini (2004) said the similarity distance away if less than 0.60 or 60%, so that separate groups at a distance of less than 0.60 still has a close resemblance. In this dendogram analysis, the number 1 or 100% indicates that the group members have a perfect resemblance, while getting closer to the number 0 means the similarity distance farther.

Benthul Dendogram analysis results showed that the Benthul taro of different height have the same morphological characteristics and have a high relationship. This is evident in the coefficient of 0.60 which was still in one group. But there was a tendency that Benthul of different heights showes different sizes, ranging from leaf size, plant height, stem and tuber. Benthul is commonly grown as a crop population between the rice fields and gardens, and allowed to grow without special treatment. Environmental factors such as temperature at any altitude, soil and availability of different light and water, thought to cause the size of the plants experience the difference. According to Park et al. (1997) and Djukri (2006) each deal with environmental stress of plants continues to do the adaptation, including changes in morphological characteristics and physiology.

Benthul that grows in the highlands appear higher with habitus width, leaf midrib and stalk thin and big. This was observed in taro grown in ketinggianya more than 1500 m with high 22°C, and high rainfall reaches 2299 mm /±humidity, low temperature year. According to Basri

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(2002) plant growth is influenced by environmental factors. Altitude above 1500 m cause gas and water vapor content (humidity) and the number of clouds blocking sunlight to the plants, so plants were capturing light by raising levels of chlorophyll and surface area. Taro plants tend to have broad leaves because of the availability of adequate water due to high rainfall in the area still support the optimum process in photosynthesis.±Low temperature 22°C Benthul that grows in the lowlands tend to have narrower leaves and smaller and lighter bulbs. According to Menzel (1980) the temperature is too high may cause leaves to hinder the development of broad and narrow leaf photosynthetic rate high as a result reducing the weight of tuber. But when the temperature is too low to reach less than 10°C, the plant tissue can be damaged and an interruption of growth so the plants tend to be stunted.

Lompongan Dendogram Lompongan relationship found in three different heights showed only the size difference. Broadly speaking taro from the highlands, medium and low still have the same morphological characteristics. Lompongan plants grow wildly around the edge of rice fields and waterways.

Lompongan plants from the highlands have differences with the lowlands, such as: green leaf color is more concentrated, browner midrib color, and the size is larger.

Unlike Lompongan plants in the highlands that were often found on the outskirts of the river with shade trees around it, the ones in the lowlands were found in around the edges of fields full of water. Environmental factors in the form of light, temperature and humidity cause the plants to have different adaptations. According to Taiz and Zeiger (1991), leaf surface area increased because of the shade, and color changes due to the increased levels of chlorophyll a and b.

In the circumstances shaded light spectrum that is active in the process of photosynthesis (wavelength 400700 nm) get decreased. Plants will make adjustments to streamline the capture of light energy that is by increasing leaf area, plant height and chlorophyll a and b (Lambers et al. 1998).

Altitude causes humidity, light, temperature, and moisture content to vary. According to Fitter and Hay (1998) environmental factors were related one another so that the plant held a response to the environment. High water levels in the soil cause leaf’s cell turgor to increase which in turns causes leaf’s expansion. Reduced light causes the leaves to add the proportion of mesophyll tissue.

Temperatures that were too high (> 40 ° C) cause defective enzyme and respiration is rapid, so the plants have stunted growth. The temperature is too low (<1°C) causes decreased enzyme activity cause plant tissue damage and death. The optimum temperature for photosynthesis is 20-30°C.

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Table 2. 18 samples of C. esculenta in Karanganyar district with characteristics Characteristics

Varieties 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

A. Type of plant 1. Rentang tanaman 1. Sempit 2. Sedang 3. Lebar √ 2. Tinggi tanaman 1. Kerdil (< 50 cm) 2. Sedang (< 50 cm) 3. Tinggi (< 50 cm) √ 3. Jumlah stolon 1. 1-5 buah 2. 6- 10 buah √ 3. 11-20 buah 4. Panjang stolon 1. Pendek (<15 cm) √ 2. Panjang (>15 cm) B. Cormus (umbi) 1. Manifestasi cormus √ 1. Ada 2. Tidak ada 2. Panjang Cormus 1. Pendek (± 8 cm) 2. Sedang (± 12 cm) √ 3. Panjang (± 18 cm) 3. Cabang cormus 1. Bercabang 2. Tidak bercabang √ 4. Bentuk cormus √ 1. Kerucut 2. Membulat 3. Silindris 4. Memanjang 5. Datar dan terbuka 5. Berat cormus 1. Ringan (± 0.5 kg) 2. Sedang (± 2 kg) √ 3. Berat (± 4 kg) 6. Warna korteks cormus 1. Putih 2. Kuning-orange √ 7. Warna daging tengah 1. Putih 2. Kuning √ 3. Orange 8. Warna serat daging 1. Putih 2. Kuning muda 3. Kuning-orange √ 4. Merah 9. Permukaan kulit cormu 1. Berserabut 2. Bersisik 3. Berserabut dan bersisik√ 10. Ketebalan kulit √ 1. Tebal 2. Tipis 11. Tingkat serabut 1. Sedikit √ 2. Banyak 12. Warna tunas 1. Kuning hijau 2. Merah muda √ 3. Ungu -

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C. Daun 1. Posisi daun dominan 1. Mendatar 2. Mangkok √ 3. Tegak keatas 4. Tegak kebawah 2. Tepi daun 1. Penuh 2. Bergelombang 3. Berlekok-lekok √ 3. Warna Helai daun 1. Hijau 2. Hijau tua √ 3. Ungu 4. Warna tepi helai daun 1. Keputihan 2. Hijau 3. Merah muda 4. Ungu √ 5. Warna cairan ujung daun 1. Keputihan 2. Kuning 3. Merah muda √ 4. Merah tua 6. Warna utama tulang daun 1. Kuning 2. Hiaju 3. Merah muda √ 4. Ungu 7. Pola utama tulang daun 1. Bentuk Y √ 2. Bentuk Y meluas 8. Warna petiole Sepertiga atas 1. Kuning 2. Hijau muda √ 3. Cokelat 4. Ungu Sepwertiga bawah 1. Kuning 2. Hijau muda 3. Cokelat √ 4. Ungu 9. Warna garis petiole 1. Hijau 2. Ungu √ 10 Irisan melintang bawa 1. Terbuka √ 2. Tertutup 11. Warna cincin petiole 1. Putih 2. Kuning kehijauan √ 3. Merah muda 4. Ungu 12. Warna pelepah daun 1. Keputihan 2. Hijau muda 3. Merah Keunguan √ 13. Lapisan lilin 1. Tidak ada 2. Rendah √ 3. Sedang 4. Tinggi -

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TRIMANTO et al. â&#x20AC;&#x201C; Characterization of taro based on morphological and isozyme markers Note: 1. Benthul (plateau), 2. Benthul (plain medium), 3.Benthul (lowlands), 4. Lompongan (plateau), 5.Lompongan (plain medium), 6. Lompongan (lowlands), 7. Laos (plateau), 8. Laos (plain medium), 9. Linjik (plain medium), 10. Linjik (lowland), 11.

Sarangan (plateau), 12. Sarangan (plain medium), 13. Kladitem (plateau), 14. Plompong (lowland), 15. Kladi (lowland), 16. jabon (plain medium), 17. Mberek (lowland), 18. Japan (plateau).

Figure 1. Dendogram relationship 18 samples of C. esculenta from three different heights based on morphological characters.

Description: No. 1-18 same as Table 2.

Characterization of isozyme taro Peroxidase Results with the dye peroxidase isozyme analysis, shikimate dehydrogenase and esterase can be seen in Figure 2. Peroxidase in 18 samples of C. esculenta tested to form 14 different banding pattern. Banding pattern I with migration distance (Rf) 0586, 0630, 0717, 0761 and 0804 being owned by the sample 1. Banding pattern II with Rf 0586, 0717, 0761 and 0804 were owned by the sample 2 and 3. Banding pattern III is owned by sample 5 with Rf 0630, 0739, 0782 and 0826. Banding pattern IV with Rf 0630, 0739, 0782 owned by samples 4 and 6. Banding pattern V with Rf 0652, 0739, 0782 and 0874 were owned by the sample 7 and 8. Rf banding pattern VI 0652, 0739, 0782 and 0869 were owned by the sample 9 and 10.

Banding pattern VII with Rf 0565, 0717 0739 and 0847 held by the sample 11. Banding pattern VIII with Rf 0565, 0717 0739 owned by sample 12. IX banding pattern with a distance of 0630 and 0739 held by the sample 13. Banding pattern X with Rf 0630 and 0739 held by the sample 14. XI banding pattern with a distance of 0630, 0739, and 0804 is owned by the sample 15. XII banding pattern with a distance of 0630, 0739, and 0826 is owned by the sample 16. XIII banding pattern with a distance of 0630, 0717 and 0761 held by the sample 17. Banding pattern XIV with Rf 0607, 0652 and 0.761 were owned by the sample 18.

Shikimate dehydrogenase Isozyme analysis results with dye shikimate dehydrogenase (ShDH) on 18 samples of C. esculenta tested to form 15 different banding pattern. Banding pattern I with Rf 0523, 0568, and 0863 is owned by the sample 1.

Banding pattern II with Rf 0523, 0568, 0614 and 0863 were owned by the sample 2 and 3. Banding pattern III with Rf 0523, 0568, 0614 and 0840 were owned by the sample 4. Banding pattern IV with Rf 0500, 0523, 0568,

11

0614 and 0840 were owned by samples 5 and 6. Banding pattern V with Rf 0568 and 0840 were owned by the sample 7 and 8. Banding pattern VI with Rf 0523 and 0840 held by the sample 9. Banding pattern VII with Rf 0500, 0523 and 0840 were owned by the sample 10. Banding pattern VIII with Rf 0523 and 0818 held by the sample 11.

Banding pattern IX with Rf 0500, 0523 and 0818 were held by the sample 12. Banding pattern X with Rf 0416, 0432, 0523, 0795 owned by sample 13. Banding pattern of Rf 0500 XI, 0523, 0727 and 0750 were owned by the sample 14. XII banding pattern of Rf 0523, 0546, 0581 and 0818 held by the sample 15. Banding pattern XIII with Rf 0523, 0546, 0568 and 0795 held by the sample 16. Banding pattern XIV with Rf 0500, 0546, and 0795 was owned by the sample 17. Banding pattern XV with Rf 0546, 0568 and 0795 were held by the sample 18.

Esterase Results with the dye esterase isozyme analysis on 18 samples of C. esculenta were tested forming 11 different banding patterns. Banding pattern I with Rf the same but having different shapes, and shown at Rf 0.22, 12:26 and 12:32 were owned by the sample 1, 2 and 3 (quantitative and qualitative). Banding pattern II with Rf 0.20, 0:28, 0:32 and 0.36 were owned by the sample 4, 5 and 6 (quantitative and qualitative). Banding pattern III with Rf 0:30, 0:34, 0:38, 0:40 was owned by the sample 7 and 8 (quantitative and qualitative). Banding pattern IV with Rf 0.20, 0:30, 0:34, 0:38 and 0:44 is owned by the sample 9 and 10.

Banding pattern V with Rf 0.20, 12:26 and 12:38 were owned by the sample 11 and 12 (quantitative and qualitative). VI banding pattern was owned by the sample 13 with Rf 0.20, 0:28, 0:30, 0:46, 0:48. Banding pattern VII owned by the sample 14 with Rf 0.20, 0.26, 0:30, 0:34.

VIII banding pattern VIII was owned by the sample 15 with Rf 0.20, 0.26, 0:30, 0:36. Banding pattern IX was owned by the sample 16 with Rf 0.20, 0:22, 0:26, 0:32.

Banding pattern X was owned by the sample 17 with Rf 0.20, 0.24, 0:32 and banding pattern XI with Rf 0.20, 12:28 and 12:32 were owned by the sample 18.

Similarity on taro genetics based on isozyme markers Genetic similarity between samples can be tested using cluster analysis (group average analysis), which results in the form dendogram or tree diagram. The end result is a dendogram of relationship were tested by three different enzymes (peroxidase, shikimat dehydroginase, and esterase) (Figure 3).

Election peroxidase has advantages including: a broad spectrum and has a very important role in the process of plant physiology. This enzyme can be isolated and scattered in the cell or plant tissue, especially in plant tissues that had been developed (Butt 1980; Hartati 2001).

Shikimate dehydrogenase (ShDH) is an enzyme which spread to most living things. Shikimate dehydrogenase involved in oxidoreductase that catalyzes NADP + shikimate into three main products dehydroshikimate + NADPH+H+. At the plant, esterase is a hydrolytic enzyme that functions to withhold simple esters in organic acids, inorganic acids and phenols and alcohols have low molecular weight and easily soluble.

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A

B

C

Figure 2. The variation of 18 isozyme banding pattern of sample C. esculenta from three different heights. Description: a. Banding pattern of peroxidase, b. Shikimate dehydrogenase banding pattern, c. Esterase banding pattern. No. 1-18 same as Table 2.

A

B

C

Figure 3. Relationship dendogram 18 samples of C. esculenta from three different heights based on isozyme banding pattern. A. peroxidase, B. shikimate dehydrogenase, c. Esterase. No. 1-18 same as Table 2.

Results dendogram relationship between the use of peroxidase enzymes, shikimate dehydrogenase and esterase showed generally taro of the same variety have the same banding pattern, although from different locations ketinggianya, so that enzymatically still have a high relationship, since it is estimated the same parent. On a different taro varieties tend to have a different banding pattern. Formation of the group between the use of esterase, peroxidase and shikimate dehydrogenase gave different relationship relations, but in one variety is generally joined in one group at a distance of more than 60% similarity, although originating from different locationsâ&#x20AC;&#x2122; height.

Esterase formed seven groups which were of more than 60% similarity between one another, where there were taro who joined another group. Jabon formed a group with Plompong which were of 0.80 similarity. Kladi formed a group with Plompong at a distance of 0.75 similarities.

Lompongan joined with Japan at a distance of 0.70 similarities. Laos and Linjik form one group at a distance of 0.67 similarity. Even when they are different taro

varieties butwhen they form one group, they still have a high genetic relationship.

Peroxidase also formed seven groups. In general, in a variety of taro is still present in one group even though planted in different locations altitude place. Peroxidase formed a different group variation taro with esterase. In peroxidase, Laos and Linjik in one group that were of 0.75 similarity. Plompong and Kladi form one group, but Jabon joined at a distance of 0.70 similarity. Lompongan and Kladitem form one group at a distance of 0.75 similarity.

Peroxidase added information that was not the presence of new groups formed on the use of esterase.

Shikimate dehydrogenase provided the formation of different groups of taro with esterase and peroxidase. In shikimate dehydrogenase,formed three groups originating from different varieties, but it formed one group that was of more than 60% similarity. Lompongan and Benthul joined at 0.65 similarity distance. Kladi joined Sarangan at a distance of 0.62 similarity. Mberek and Japan formed one group that was of 0.67 similarity.

The use of different enzymes gave results in different groups, although there is formation of the same group with

TRIMANTO et al. â&#x20AC;&#x201C; Characterization of taro based on morphological and isozyme markers

a different enzymeâ&#x20AC;&#x2122;s use. The use of different enzyme will complement the formation of groups of different taro varieties. The genetic pattern of bands that formed in the use of enzymes is the expression of taro varieties in question. With a specific enzyme that cannot afford some taro that express ribbon patterns, but with other enzymes can express ribbon patterns. So that more types of enzymes used then it will complete the formation of groups on varieties of taro.

Results dendogram through morphological markers and isozyme banding pattern shows the difference. From the morphological marker of the 11 varieties, obtained taro formed 10 groups at a distance of 0.60 similarity. Different taro varieties, most will form a separate group means morphologically different taro varieties have different morphological characteristics. Talas who formed a group on the analysis of relationship is Kladi and Plompong.

When the isozyme was used, more groups were formed, this means that between the different taro varieties there is still a high relationship. If the different varieties of taro belong to one group with a distance of close to 1 it is possible that the similarity comes from the older of taro.

Environmental factors affect plant morphology, if the environmental factor is more dominant than genetic factors, the plant will experience a change in morphology (Suranto 1999, 2001). In the long term it is possible crop genetic trait changes in her body. Plants that were stressed environment would be possible to have mutations, so that in the long term can happen speciation.

New types were also possible as a result of hybridization, so having a close relationship with both of the parent species. The property of taro which has a close relationship is what can be used to search for a superior taro through crossbreeding. Some taros found in Karanganyar were a wild taro. Wild Taro and of likely no benefit are possibly to have genetic traits that superior, so that the hybridization process to obtain high yielding varieties can be applied.

Generative breeding of taro is naturally difficult to occur because the male and female flowersg et mature at different times and a new flowering occurs after more than 6 months of age. Many plants are not considered going through a flowering because the flowering process is too long. Many cultivated plants are harvested before adulthood, so many plants are difficult to perform in a generative breeding.

Characterization of taro plants through morphological marker is more easily done, by observing external nature, taro plants can be assumed to have superior properties. But genetic markers also play an important role because it is more fundamental and is not influenced environment. Data morphology and isozyme banding pattern on taro plants in Karanganyar can be used in addition to the identification of the food plant breeding efforts.

Characterization relations of morphology and isozyme The correlation between genetic distance based on morphological markers and similarity based on isozyme banding pattern were analyzed based on product-moment correlation coefficient with the criteria of goodness of fit

13

according to Rohlf (1993). Result of calculation correlation between genetic distance based on morphological markers and genetic similarity based on isozyme banding pattern showed that between morphology and isozyme has a good correlation and a very good (Table 4). Correlation between morphological data and isozyme banding pattern of peroxidase, esterase, and shikimate dehydrogenase, respectively, also were on the value of 0.893542288, 0.917557716, 0.9121985446.

This shows the characterization of taro based on morphological markers consistent with isozyme banding pattern, so that the isozyme data support the morphological data.

Diversity is difficult to observe the morphological marker would be more accurate if you have the genetic markers such as isozymes. Morphological characters that were equipped with the character of isozyme banding pattern adds accuracy of the data to identify plant diversity.

Isozyme has advantages because it requires little sample of the plant, were not inhibited during plant dormancy, can be used to perform characterization of the plant in very much.

Table 4. Relationships and morphological characterization characterization results based on isozyme banding pattern Characters that correlated

Level

Criteria

Morphology and POD Morphology and EST Morphology and ShDH

0.893542288 0.917557716 0.9121985446

good very good very good

The relationships of taro plants obtained from places of different heights can be made into a dendogram between morphology and marker pattern of the isozymeâ&#x20AC;&#x2122;s ribbon.

Dendogram based on morphological markers and isozyme banding pattern of peroxidase, shikimate dehydrogenase, and esterase showed that taro with the same type from a different altitude did not show any difference at a distance of 60% similarity. Of the eighteen samples were divided into 10 groups. Each taro with the same type, although located in different places still reflect the height of high relationship. This proved that taro plants of the same type belonged to a single group.

Figure 4. Dendogram relationship 18 samples of C. esculenta from three different heights based on morphological markers and isozyme banding pattern of peroxidase, esterase, and shikimate dehydrogenase. Description: No. 1-18 same as Table 2.

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Taro varieties which become one group is based on morphological markers and isozyme banding pattern, where the isozyme banding pattern supports the morphological data. This is evident in samples 1, 2, 3, ie Bentul from three different height locations which join one group. Other evidence were sample 4, 5 and 6, which were from three different altitude sites that also formed one group. This indicated that the isozyme data support the morphological data, so as to identify the plant in addition to morphological data, isozyme data is also needed to increase the accuracy of the data. There were varieties of taro which have a a close relationship that are Kladi and Plompong that have a high relationship when viewed from the merger with its isozyme morphological characteristics, both were at the coefficient of 0.68. Allegedly the two taro plants have elders who have a high kindship, because almost the same its relation of morphology and isozyme almost the same. From the characterization results obtained that has a relationship Kladi and Plompong highest compared with other varieties of taro. Taro with different varieties formed their own groups at a distance of 60% similarity. This means that at a distance of 60% of all varieties of taro had different characters.

CONCLUSION There is a diversity of morphological characters in 18 samples of taro plants (Colocasia esculenta L.) that grow in Karanganyar. Taro is still in one variety that is at different height diversity appears only on the size of the vegetative plant. The results showed isozyme banding pattern of the variability in isozyme banding pattern of peroxidase, esterase and shikimate dehydrogenase in taro varieties found in different locations. Characterization of taro based on morphological markers is consistent with the characterization based on isozymes. Isozyme data support the morphological character data.

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ISSN: 2087-3940 (print) ISSN: 2087-3956 (electronic)

Vol. 3, No. 1, Pp. 15-22 March 2011

Study on floristic and plant species diversity in the Lebanon oak (Quercus libani) site, Chenareh, Marivan, Kordestan Province, western Iran HASSAN POURBABAEI♥, SHIVA ZANDI NAVGRAN Determent of Forestry, Faculty of Natural Resources, University of Guilan, Somehsara, P.O.Box 1144, Tel.: +98-182-3220895, Fax.: +98-182-3223600, ♥ E-mail: h_pourbabaei@guilan.ac.ir Manuscript received: 28 Augustus 2010. Revision accepted: 4 October 2010.

Abstract. Pourbabaei H, Navgran SZ. 2011. Study on floristic and plant species diversity of the Lebanon oak site (Quercus libani) in Chenareh, Marivan, Kordestan Province, western Iran. Nusantara Bioscience 3: 15-22. In order to study floristic and plant species diversity, approximately 450 ha of oak forests were selected in Chenareh, Marivan of Kordestan province in western Iran. Inventory was selectively carried out in 50 m elevation range in the different aspects. Vegetation was surveyed in the four layers including: tree (dbh >5 cm), regeneration (dbh <5 cm), shrub and herb. Diversity and richness indices were used to analyze data in the different vegetation layers. Results indicated that 82 plant species found in the studied site, comprise of 9 tree, 3 shrub and 70 herbaceous. The mean diversities and richness measures were found to be the highest in southwestern and lowest in southeastern and northern aspects for the tree layer. Whereas for the regeneration layer, the mean diversity measures were found the highest in northeastern (i.e., 1-D and H′) and southern (i.e., N2 and N1) and lowest in southwestern (i.e., 1-D, H′ and N1) and southeastern (i.e., N2). The mean diversities were found the highest in northern (i.e. N2 and H′) and northwestern (i.e. 1-D and N1) and lowest in northeastern aspect in the shrub layer. The mean diversities were also found the highest in western and lowest in northeastern aspect in the herbaceous layer. Moreover, Mean richness and diversity were found the highest in 1500 m asl and lowest in 1750 and 1800 m asl in the tree and shrub layers. Mean richness and diversity were found the highest in 1500 m asl and lowest in 1750 m asl in the regeneration layer. Also, the mean diversities were found the highest in elevation 1700 m asl and lowest in elevation 1800 m asl in the herbaceous layer.

Key words: floristic, plant diversity, aspect, elevation, western Iran.

Abstrak. Studi keanekaragaman floristic dan jenis tumbuhan pada situs Lebanon ek (Quercus libani) di Chenareh, Marivan, Provinsi Kordestan, Iran barat. Nusantara Bioscience 3: 15-22. Dalam rangka meneliti floristik dan jenis tumbuhan, sekitar 450 ha hutan kayu ek dipilih di Chenareh, Marivan, Provinsi Kordestan di Iran barat. Inventarisasi dilakukan dilakukan secara selektif pada kisaran elevasi 50 m dengan aspek yang berbeda-beda. Vegetasi disurvei dalam empat lapisan termasuk: pohon (dbh> 5 cm), regenerasi (dbh <5 cm), semak dan herbal. Indeks keanekaragaman dan kekayaan digunakan untuk menganalisis data dalam lapisan vegetasi yang berbeda. Hasil penelitian menunjukkan bahwa 82 jenis tumbuhan ditemukan di lokasi penelitian, terdiri dari 9 pohon, 3 semak dan 70 herba. Pada pengukuran lapisan pohon mean keanekaragaman dan kekayaan ditemukan bahwa yang tertinggi di barat daya dan yang terendah di tenggara dan utara. Pada pengukuran lapisan regenerasi mean keanekaragaman ditemukan yang tertinggi di timur laut (yaitu, 1-D dan H ') dan selatan (yaitu, N2 dan N1) dan yang terendah di barat daya (yaitu, 1-D, H' dan N1) dan tenggara (yaitu, N2). Pada lapisan semak keanekaragaman ditemukan mean tertinggi di utara (N2 yaitu dan H ') dan barat laut (yaitu 1-D dan N1) dan terendah di timur laut. Pada keanekaragaman lapisan herba ditemukan mean tertinggi di barat dan terendah di timur laut. Selain itu, mean kekayaan dan keanekaragaman lapisan pohon dan semak ditemukan tertinggi pada ketinggian 1500 m dpl dan terendah pada 1750 dan 1800 m dpl.

Mean kekayaan dan keragaman lapisan regenerasi ditemukan tertinggi pada ketinggian 1500 m dpl dan terendah pada 1750 m dpl. Juga, ditemukan mean keragaman lapisan herba tertinggi ditemukan pada ketinggian 1700 m dpl dan terendah pada 1800 m dpl.

Kata kunci: flora, keanekaragaman tanaman, aspek, ketinggian, Iran barat.

INTRODUCTION The Zogros forests of western Iran extend from Piranshahr city in western Azarbayejan province in the Zagros and Bakhtiary mountains to around the Jahroum and Fasa cities in the Fars province. These forests cover approximately 5 million ha area, and because of dominancy of species of oak genus, these forests are called as western oak forests (Mohadjer 2005). The western oak forests have remarkable significance in regard to ecological services including water and soil conservation. Therefore, these forests play an important role in preventing of soil erosion.

About 40% of surface waters of our country run from Zogros mountains basin in which seven rivers exist with fresh water. In the other hand, these forests produce by products through their woody species, and also maintain sustainable agriculture in the lowlands.

In general, forest communities in these forests from lowland to highland as follows: Amygdaletum scopariae, Pistacio-Amygdaletum, Quercetum persicae (Q.brantiae), Juniperetum polycarpae. Juniperetum community has a large extent from Khorasan to Azarbayejan, Zagros, Bandar Abbas and Baluchestan (Sabeti 1994). In the Pistacio-Amygdaletum community, Amygdalus species

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affect on the natural regeneration of Pistacia species, that is, the seedlings of Pistacia species would be protected under the spiny bushes of Amygdalus species.

The Zogros mountains are divided into two parts: northern and southern. The northern Zagros is consisted of the growing site of Quercus infectoria Oliv. and also Q.libani Oliv. and Q.persica J. & Sp. (Q.brantii Lindl.) species are found in this part. However, the southern Zagros is included Q.persica site which it extended to Fars province (i.e., 29º 5´ N). The northern Zagros is wetter and cooler than the southern one. The dispersion areas of Lebanon oak (Q.libani) are mostly restricted to central and eastern mountains of Tavrous and Amanous of Anatolia in Turkey, the mountains of northeastern of Iraq and northwestern of Syria and western part of Iran (i.e., Kordestan province) (Browicz 1994). In addition, this species is found over 1000 m asl elevation and the best conditions range from 1200 to 1600 even to 1800 m asl to growing it, and also this species is found higher than 2000 m asl elevation in Ahir dagi and Herakol dagi mountains in southern Anatolia, Turkey. Western borderline of this species is located in the Goniah province in Anatolia and northern borderline restricted to latitude 40ºN in Erzincon province in Turkey (Davis 1982). In flora of Iraq, the distribution area of this species was cited in central regions of Iraq forests, north of Syria, Palestinian, Turkey and Iran on hillsides on the metamorphic and igneous rocks and on loam soils, and elevation ranges from 1800 to 2000 (occasionally 2100) m asl (Townsend and Guest 1980) . In Iran, the distribution of this species is restricted to highlands of Sardasht in Kordestan and Euromiah provinces, and horizontal distribution is from north of Sardasht to south of Marivan in Kordestan province and vertical distribution is from 1400 to 2150 m asl elevation (Fattahi 1994). This species is situated in latitude from 25º to 36º N and longitude from 45º to 46º E and is grown cold humid, cold sub humid or humid climates. The pure type of

this species is found in highlands and the mixed one mostly found associated with Q.infectoria and Q.brantii species.

This species is in relation to soil and climatic conditions.

The forests of this species are found as high and coppice forms, and the species covers 106316 ha area in western Iran of which 83844 is located in the Kordestsn province (Fattahi 1994).

There are numerous studies in relation to floristic composition all over the world (e.g., Andel 2001; Nebel et al. 2001; Ipor et al. 2002; Blanckaert et al. 2004; WardellJohnson et al. 2004; Ramírez et al. 2007; Cayuela et al.

2008; Gole et al. 2008; Macía 2008; El-Ghanim et al. , 2010; Figueroa et al. , 2011). In addition, plant species diversity has been assessed in forest ecosystems in recent decades (e.g., Brockway 1998; Pitkänen 1998; Khera et al.

2001; Ashton and Macintosh 2002; Aubert et al. 2003; Nagaike 2003; Jobidon et al. 2004; Chiarucci and Bonini 2005; Pant and Samant 2007; Aparicio et al. 2008; Macía 2008; Pe´rez-Ramos 2008; Hayat et al. , 2010). Whereas there is less studies about plant species diversity in Zagros forest ecosystems (Mirzaei et al. 2008; Pourbabaei et al. , 2010).

The aim of this study was to determine floristic composition and plant species diversity in the Lebanon oak site in Kordestan province of Iran.

MATERIALS AND METHODS Study area The study area is located in Marivan city of Kordestan province in western Iran, and Chenareh is situated 25 km from northwestern Marivan city (35° 29′ to 35° 45′ N latitude, 46° 14′ to 46° 29′ W longitude). Mean annual precipitation is 909.5 mm, ranging from 590.8 to 1422.2 mm (Figure 1).

Kordestan

Chenareh forest Marivan City

Figure 1. Study site maps Chenareh’s forests (blank circle) in Marivan District, Kordestan Province, IR Iran.

POURBABAEI & NAVGRAN – Plant species diversity of Chenareh forest

Mean annual temperature is 13.3º C, and the length of dry season is 4 month (based on embrothermic curve) from June to August. Type of climate is sub humid with cold winters in the basis of Emberger’s formula (Department of Forestry 2002). Edaphically, soils consist of developed brown (calciferous and eutroph), deep and semi deep, and young soils consisting of litho sol and colluvium which often are less deepness and shallow. Quercus brantii community are predominantly found on calcico brown soils, and Q.libani community often found on eutroph brown soils.

The research was conducted in 450 ha of Chenareh’s forests where included Lebanon oak and altitude ranges from 1500 to 1800 m asl These forests are located steep areas, and slope is more than 50% in the most area. Main aspects of these forests are northern and southern. These forests have been under anthropogenic disturbances in the past, therefore they are considered as manipulated forests now.

Sampling At first, oak site was quantified on the map with 1:50000 scale with surveying forests. Inventory was selectively carried out in 50 m elevation range from 1500 m to 1800 m asl in the different aspects in the basis of distribution of Lebanon oak population. Sampling plot area was 1000 m2 in size and circular (Zobeiri 1994). In total, 42 sampling plots were made. At each plot, diameter at 1.3 m (DBH) of tree ≥ 5 cm was measured and identified (high and coppice origin), and crown diameters (i.e., large and small) of regeneration with DBH < 5 cm were measured.

For shrub species, the number of individuals were recorded and identified. To collect herbaceous data, nested plot sampling was performed at center the plot (MullerDombois 1974), and minimal area ranged from 32 to 1000 m2 in the basis of different altitudes. Cover percentage was visually estimated, as accurately as possible, for each herbaceous species in the nested plots, and type of species was identified in the Herbarium of Faculty of Natural Resources, University of Guilan.

Data analysis Species richness (total number of species present) and evenness (the manner in which abundance is distributed among species) are the two principal components of diversity. Species richness is frequently characterized by the number of species present (S), Margalef species richness (R1) and Menhinick species richness (R2) (Ludwig and Reynolds 1988). In this study, Smith and Wilson’s evenness index (Evar) was applied to calculate evenness measures (Krebs 1999). Diversity indices combine species richness and evenness components into a single numeric value. The most commonly used indices of diversity, Simpson (1-D) and Shannon-Wiener (H′) were used in this study (Magurran 2004). Moreover, Hill’s N2 and McArthur’s N1 were calculated in the basis of these indices (Krebs 1999). Vegetation data were analyzed in four layers (i.e., tree, regeneration, shrub and herb) using richness, evenness and diversity indices. In tree layer, DBH was converted to basal area (m2) for each individual tree and summed for each species, and then substituted for the

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number of individuals in the diversity formula.

Furthermore, crown cover area (m2) was computed for each regeneration species and applied the formula. Data analyses were performed using Ecological Methodology and SPSS 13.0 software (Krebs 1999; Kinnear 2001).

RESULTS AND DISCUSSION Floristic composition A total of 82 plant species were found in the studied area, of which 12 woody species (9 trees, 3 shrubs) and 70 herbaceous species existed (Table 1) while 4 trees, 3 shrubs, one bush and 78 herbaceous species were identified in Ilam forests of Zagros (Pourbabaei et al. 2010).

Therefore, it is concluded that tree richness is high in the studied area. Also, it can be deduced from Table 1 that Rosaceae and Fagaceae families play an important role in among woody species. Moreover, Asteraceae and Poaceae families were most abundant amongst herbaceous species.

In addition, results were revealed that the Asteraceae family was dominant in Ilam forests of Zagros (Pourbabaei et al. 2010).

The number of plant species was considerable in the studied area when compare with northern Zagros mountains since there is 165 woody species (tree and shrub) in Zagros and 182 bush and herbaceous species only in northern Zagros (Jazirehi and Rostaghi 2003). The highest richness of woody species belong to Fagaceae and Rosaceae and the highest richness of herbaceous species belong to Asteraceae and Poaceae families in the studied area, these results were confirmed in the Zagros zone (Jazirehi and Rostaghi 2003).

Plant diversity based on different aspects Plant species diversity of four growth layers was obtained in terms of different aspects. The highest and lowest population of Lebanon oak was found in eastern (32%) and northwestern (24%) aspect, respectively. Figure 1 displays mean tree (high and coppice forms) diversity in the basis of different aspects.

The mean diversities were highest in southwestern and lowest in southeastern and northern aspects in the tree layer. The ANOVA test indicated that there were no significant differences amongst mean diversity measures in the different aspects (P > 0.05). Figure 3. displays mean tree richness, Margalef (R1) and Menhinick (R2) and evenness measures in the different aspects.

The mean richness, R1 and R2 measures were highest in southwestern, and lowest in southeastern and northern aspects, respectively. The mean Evar was the highest in southeastern and lowest in northeastern aspect. The Kruskal-Wallis test showed that there were no significant differences amongst mean richness values in the different aspects. Whereas, the ANOVA test indicated that there were significant differences amongst mean R1 and R2 in the different aspects (P < 0.05), and Tukey test showed that there was significant difference between southwestern and southeastern aspects in view of mean R1. Also, there was significant difference between southwestern and other aspects except southern in view of mean R2.

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Table 1. Plant species list based on growth layers Layer

Species

Tree

Acer monspessulanum L. (Aceraceae), Amygdalus communis L. (Rosaceae), Cerasus mahaleb L. (Rosaceae), Crataegus pontica C.Koch. (Rosaceae), Pistacia atlantica (Anacardiaceae), Pyrus syriaca Boiss. (Rosaceae), Quercus brantii Lindl.

(Fagaceae), Q.infectoria Oliv. (Fagaceae), Q.libani Oliv. (Fagaceae).

Shrub

Cerasus microcarpa (C.A.Mey) Boiss. (Rosaceae), Cotoneaster nummularia Fisch & Mey. (Rosaceae), Lonicera nummularifolia Jaub & Spach. (Caprifoliaceae).

Herbaceous

Acanthus dioscoridus L. (Acantaceae), Achillea filipendula L. (Asteraceae), A.millefolium L. (Asteraceae), Aegilops triuncialis L. (Poaceae), A.triuncialis L. (Poaceae), Alopecurus myosuroides Ovcz. (Poaceae), Antemis tinctoria L. (Asteraceae), Astragalus curvirstris Boiss. (Papilionaceae), A. michauxianus Boiss. (Papilionaceae), A. (tragacantha ) sp. (Papilionaceae), Aristolochia bottae Jaub & Spach. (Aristolochiaceae), Boissiera squarrosa Hochst. (Poaceae), Bromus tectorum L. (Poaceae), Buchingera axillaris Boiss. (Cruciferae), Bunium elegans (Fenzl.) Freyn. (Umbelliferae), Callipeltis cucularia Stev. (Rubiaceae), Centaurea virgata Lam. (Asteraceae), Cephalaria syriaca (L)Schrad.

(Dipsaceae), Chaerophyllum macropodum Boiss. (Umbelliferae), Cornilla varia L. (Papilionaceae), Dactylis glomerata L. (Poaceae), Dianthus tabrizianus Adams. (Caryophylaceae), Echinops orientalis Trauth. (Asteraceae), E.ritrodes Bunge. (Asteraceae), Eremopoa persica (Trin.) Roshev (Poaceae), Eryngium thyrsoides F.Delaroche. (Umbelliferae), Euphorbia macroclada Boiss (Euphorbiaceae), Ferula orientalis L. (Umbelliferae), Fibijia macrocarpa Boiss.

(Cruciferae), Galium aparine L. (Rubiaceae), Grammosciadium platycarpum Boiss. (Umbelliferae), Gundelia tournefortii L. (Asteraceae), Helianthemum ledifolium (L.) Miller. (Cistaceae), Heteranthelium piliferum (Banks & Soland) (Poaceae), Hordeum bulbosum L. (Poaceae), Hypericum scabrum L. (Hypericaceae), Inula britanica L. (Asteraceae), Lamium album L. (Labiatae), Marrubium vulgare L. (Labiatae), Mesostemma kotschyanum Wed. (Caryophylaceae), Milium pedicellare Bornm. (Poaceae), Onopordon kurdicum Bornm& Beauv (Asteraceae), Onosma elwendicum L. (Boraginaceae), O. microcarpa DC. (Boraginaceae), Phlomis olivieri Benth. (Labiatae), P.rigida Labill.

(Labiatae), Picnomon acarna L. (Asteraceae), Poa bulbosa L. (Poaceae), Potentila kurdica Boiss & Hohen. (Rosaceae), Prangos ferulaceae L. (Umbelliferae), Rhaponticum insigne Boiss. (Asteraceae), Rhabdoscidium aucheri Boiss.

(Umbelliferae), Salvia bracteata Banks & Soland. (Labiatae), Sanguisorba minor Scop. (Rosaceae), Scabiosa calocephala Boiss.(Dipsacaceae), S. leucactis Patzak. .(Dipsacaceae), Scutellaria pinnatifida A.Hamilt. (Labiatae), Serratula grandifolia Boiss. (Asteraceae), Smyrnium aucheri Boiss. (Umbelliferae), Stachys inflata Benth. (Labiatae), Taeniatherum crinitum (Schreb).Nevski (Poaceae), Teucrium polium L. (Labiatae), Trifolium campestre Schreb.

(Papilionaceae), T.pratens L. (Papilionaceae), Turginia latifolia L. (Umbelliferae), Valerianella dactylophylla Boiss & Hohen. (Valerianaceae), Veronica kurdica Benth. (Scrophulariaceae), Vicia variabilis Freyn & Sint. (Papilionaceae), Xeranthemum inaepertum Boiss. (Asteraceae), Zoegea leptaurea L. (Asteraceae).

The mean diversity measures were highest in northeastern (i.e., 1-D and H′) and southern (i.e., N2 and N1) and lowest in southwestern (i.e., 1-D, H′ and N1) and southeastern (i.e., N2) in the regeneration layer (Figure 4).

The ANOVA test showed that there were significant differences amongst mean 1-D measures in the different aspects, but no significant differences amongst other diversity indices. In addition, Tukey test showed that there was significant difference between northeastern and southwestern aspects.

The mean richness, R1 and R2 measures were highest in northern, southern and lowest in southwestern and southeastern aspects, and mean Evar was the highest in southwestern and lowest in northern aspect (Figure 5).

There were significant differences amongst mean richness, R1 and R2 measures. Tukey test showed that there was significant difference between northern and southwestern aspect in view of richness.

The mean diversities were highest in northern (i.e. N2 and H′) and northwestern (i.e. 1-D and N1) and lowest in northeastern aspect in the shrub layer (Figure 6). The ANOVA test showed that there were no significant differences amongst mean diversities measures in the different aspects.

The mean richness and R1 measures were highest in northern, while R2 was the highest in eastern aspect. The mean richness was lowest in other aspects and also the mean of R1 and R2 were found the lowest in northwestern

aspect, and the Evar were found the highest in northwestern and lowest in northeastern (Figure 7). There were no significant differences amongst mean richness, R1, R2 and Evar measures in the different aspects.

The mean diversities were highest in western and lowest in northeastern aspect in the herbaceous layer (Figure 8). The ANOVA test showed that there were significant differences amongst mean diversities measures in the different aspects. The differences among means were detected using Tukey’s test which are characterized by different letters on the histogram of Figure 8.

The mean richness and Evar measures were highest in western and lowest in northern aspect in the herbaceous layer and there were significant differences amongst mean measures in different aspects (Figure 9).

The Lebanon oak was found in all aspects, but it had the most abundant in eastern and the least in northwestern aspect since it requires plenty of sunlight in eastern aspect (Maroufi 2000). This species is preferred northern and eastern aspects and ecological needs of Q.libani is higher than Q.infectoria and Q.brantii (Jazirehi and Rostaghi 2003).

The tree species diversity was found the highest in southwestern and lowest in southeastern and northern aspects, because richness and richness indices had the highest and lowest values the mentioned aspects, and evenness had the highest and lowest values in southeastern and northeastern aspects, respectively.

POURBABAEI & NAVGRAN â&#x20AC;&#x201C; Plant species diversity of Chenareh forest

4.5

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Figure 1. Mean diversity measures and their standard errors based on different aspects in the tree layer (1. northern, 2. northeastern, 3. northwestern, 4. eastern, 5. southern, 6. southwestern, 7. southeastern, 8. western).

S

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Richness and evenness indices

Richness and evenness indices

Figure 6. Mean diversity measures and their standard errors based on different aspects in the shrub layer.

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Figure 3. Mean richness, R1, R2 and Evar measures and their standard errors based on different aspects in the tree layer.

Figure 7. Mean richness, R1, R2 and Evar measures and their standard errors based on different aspects in the shrub layer.

1-D

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12 Diversity indices

3 Diversity indices

cd

1-D N2 H' N1

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Figure 4. Mean diversity measures and their standard errors based on different aspects in the regeneration layer.

Figure 8. Mean diversity measures and their standard errors based on different aspects in the herbaceous layer (The same letters on the histogram indicate that there are no significant differences amongst mean values).

S

4.5

R1

4

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Evar

3 2.5 2 1.5 1 0.5 0

14 Richness and evenness indices

5 Richness and evenness indices

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bcd

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Figure 5. Mean richness, R1, R2 and Evar measures and their standard errors based on different aspects in the regeneration layer.

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Figure 9. Mean richness and Evar measures and their standard errors based on different aspects in the herbaceous layer.

Â

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3 (1): 15-22, March 2011

3.5

1-D

3

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N1 2 1.5 1

Diversity indices

Diversity indices

2

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1700

Elevation (m a.s.l.)

Figure 10. Mean diversity measures and their standard errors based on elevation classes in the tree layer.

Richness and evenness indices

R1

4.5

R2

4

Evar

3.5 3 2.5 2 1.5 1 0.5 0 1500

1550

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1800 Elevation (m a.s.l.)

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1-D N2 H' N1

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Figure 15. Mean richness, R1, R2 and Evar measures and their standard errors based on elevation classes in the shrub layer.

0.5 0 1500

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10 9 8 7 6 5 4 3 2 1 0

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Figure 11. Mean richness, R1, R2 and Evar measures and their standard errors based on elevation classes in the tree layer.

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Figure 16. Mean diversity measures and their standard errors based on elevation classes in the herbaceous layer.

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Richness and evenness indices

5 4.5 4 3.5 3 2.5 2 1.5 1 0.5 0

1750

Elevation (m a.s.l.)

Elevation (m a.s.l.)

Figure 12. Mean diversity measures and their standard errors based on elevation classes in the regeneration layer.

1800

Figure 14. Mean diversity measures and their standard errors based on elevation classes in the shrub layer

Richness and evenness indices

S

5

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Elevation (m a.s.l.)

S

10

Evar

8 6 4 2 0

1500

1550

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Figure 13. Mean richness, R1, R2 and Evar measures and their standard errors based on elevation classes in the regeneration layer.

1500

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1750

1800 Elevation (m a.s .l.)

Figure 17. Mean richness and Evar measures and their standard errors based on elevation classes in the herbaceous layer.

POURBABAEI & NAVGRAN – Plant species diversity of Chenareh forest

These results are to be confirmed with obtained results from Zagros forests in Ilam (Mirzaei et al. 2008; Pourbabaei et al. 2010). The Lebanon oak trees have been overexploited in southwestern aspect and as a result, population of other species such as Amygdalus communis and Crataegus pontica have increased in this aspect and also caused to increase tree species diversity.

The regeneration diversity of woody species was found the highest in northeastern (i.e., 1-D and H´) and southern (i.e., N1 and N2) and the lowest in southwestern (i.e., 1-D and H´) and southeastern (i.e., N2). The highest value of richness, R1 and R2 were found in northern and southern aspects and the lowest in southwestern and southeastern aspects. The highest value of evenness was found in southwestern and the lowest in northern aspect.

The shrub diversity was found the highest in northern (i.e., H´ and N2) and northwestern (i.e., 1-D and N1) and the lowest in northeastern aspect. The highest value of richness and R1 was found in northern and R2 in eastern aspect. The lowest value of richness was found in other aspects, and R1 and R2 in northwestern aspect. The highest value of evenness was found in northwestern and the lowest in northeastern.

The herbaceous diversity was highest in western and the lowest in northeastern aspect. The highest value of richness and evenness were found in western and the lowest were in northern aspect. The number of tree individuals per hectare and its crown cover were low in western aspect and as a result the herbaceous diversity was the highest in this aspect. The population of tree species was more in northeastern aspect and crown coverage was 60 to 80 percent and as a result the herbaceous diversity was lower in this aspect.

Plant diversity based on elevation classes The elevation distribution of Lebanon oak species stretch from 1500 to 1800 m asl in the studied area. The highest and lowest Lebanon oak population was found from 1600 to 1750 m asl (18%) and from 1500 to 1600 m asl (8%), respectively. The mean diversities were found the highest in elevation 1500 m asl and lowest in elevation 1800 m asl in the tree layer (Figure 10). There were no significant differences amongst mean diversity measures in the different elevations (P > 0.05). These results are to be confirmed with gained results of Zagros forests in Ilam (Mizaei et al. 2008).

The mean richness, R1 and R2 measures were found the highest and lowest in elevation 1500 and 1800 m asl, respectively in the tree layer, while the highest and lowest of mean Evar was found in elevation 1650 and 1600 m asl, respectively (Figure 11). There were no significant differences amongst mean these parameters in the different elevations.

The mean diversities were found the highest in elevation 1500 m asl and lowest in elevation 1800 m asl in the regeneration layer (Figure 12). The mean richness and R1 measures were found the highest in elevation 1500 m asl, and the highest value of R2 was in elevation 1700 m asl and these parameters were lowest in elevation 1650 and 1800 m asl, respectively in the regeneration layer. The highest and lowest of Evar were found in elevation 1700 and 1800 m asl, respectively (Figure 13). There were no

21

significant differences amongst mean diversity, richness and evenness measures in elevation classes in the regeneration layer.

The mean diversities were found the highest in elevation 1500 m asl and lowest in elevation 1750 m asl in the shrub layer (Figure 14). The mean richness and R1 measures were also found the highest in elevation 1500 m asl, and the highest value of R2 was in elevation 1750 m asl and these parameters were lowest in elevation 1600 m asl in the shrub layer. The highest and lowest of Evar were found in elevation 1600 and 1750 m asl, respectively (Figure 15). There were no significant differences amongst mean diversity, richness and evenness measures in elevation classes in the shrub layer.

The mean diversities were found the highest in elevation 1700 m asl and lowest in elevation 1800 m asl in the herbaceous layer (Figure 16). The mean richness was found the highest in elevation 1500 m asl, and lowest in elevation 1800 m asl, and the highest and lowest of Evar were found in elevation 1600 and 1800 m asl, respectively, in the this layer (Figure 17). There were no significant differences amongst mean diversity, richness and evenness measures in elevation classes in the herbaceous layer.

The most population of Lebanon oak was found from 1600 to 1750 m asl elevation. Maroufi (2000) indicated that this tree was distributed upper 1400 m asl elevation and it formed pure stands in elevation from 1600 to 1700 m asl The Quercus brantii, Q.infectoria and Q.libani species were observed with each other in elevation from 1500 to 1600 m asl, and in elevation of 1600 to 1650 m asl Q.infectoria and Q.libani species found with together, and from 1650 to 1800 m asl just Q.libani was found (Tabatabaei and Geisarani 1992).The Q.libani species is distributed from 1500 to 2100 m asl and the best elevational range of this species was characterized from 1600 to 1800 m asl (Jazirehi and Rostaghi 2003).

The herbaceous species of Vicia variabilis Fren & Sint. has more population in sites where Q.libani population is plentiful. With increasing elevation up to 1700 m asl, V.variabilis population is also increased. The Q.libani forms pure stands in higher elevations (1650 to 1800 m asl) and population of Mesostemma kotschyanum is increased in comparing with V.variabilis since ecological needs of Mesostemma kotschyanum lower than is Vicia variabilis.

The herbaceous coverage is to be increased in western and eastern aspects due to decreasing crown cover of oak species, and Turginia latifolia L. species is formed the most coverage percent since it has less ecological needs and it also is a thorny species.

CONCLUSION The Zogros are divided into two parts: northern and southern. Northern Zagros is determined in the basis of distribution of Quercus infectoria Oliv. and Q. libani Oliv.

Southern Zagros is also determined based on distribution of Quercus brantii Lindl. The Lebanon oak was found in all aspects, but it had the most population in eastern aspect and also this species was preferred northern aspect due to high

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3 (1): 15-22, March 2011

ecological needs. The most population of Lebanon oak was found from 1600 to 1750 m asl elevation because of suitable humidity and edaphically conditions. In fact, elevational distribution of Lebanon oak is as spindle shape, that is, population of this species is increasing when the elevation is increasing and the population is decreasing in higher elevation. The disturbance is approximately high in elevation of 1500 m asl, as a result herbaceous and other woody species have been dominated and Lebanon oak decreased. Therefore, in order to rehabilitate the northern Zagros it is recommended that plantation of Lebanon oak is greatly conducted in the mentioned aspects and elevations.

Regarding that plant species diversity and richness are considerable in studied area, it is better that this site is considered as genetic reservoir.

ACKNOWLEDGEMENTS We would like to thank to Hosein Maroufi who helped us in identification of plant species specimens. Also, we wish to acknowledge our field assistants that had helped us during the data collection.

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Brockway DG (1998) Forest plant diversity at local and landscape scales in the Cascade Mountains of southwestern Washington. For Ecol Manag 109: 323-341.

Browicz K (1994) Chorology of trees and shrubs in south-west Asia.

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Department of Forestry (2002) Forestry plan, Ghablianeh district, Dow viseh region, Marivan. University of Kordestan. Sanandaj. [Persian] El-Ghanim WM, Hassan LM, Galal TM, Badr A (2010) Floristic composition and vegetation analysis in Hail region north of central Saudi Arabia. Saudi J Biol Sci, 17: 119-128.

Fattahi, M (1994) Study on Zagros oak forests and the most important their destruction causes. Institute of Forests and Rangelands Research press. Sanandaj. [Persian] Figueroa JA, Teillier S, Castro SA (2011) Diversity patterns and composition of native and exotic floras in central Chile. Acta Oecologica 37 (2): 103-109.

Gole TW, Borsch T, Denich M., Teketay D (2008) Floristic composition and environmental factors characterizing coffee forests in southwest Ethiopia. For Ecol Manag 255: 2138-2150.

Hayat MSA, Kudus KA, Faridah-Hanum I, Noor AGA, Nazre M (2010) Assessment of Plant Species Diversity at Pasir Tengkorak Forest Reserve, Langkawi Island, Malaysia. J Agric Sci 2(1): 31-38.

Ipor I, Sani H, Tawan C (2002) Floristic composition of forest formation at Mahua, Crocker range national park, Sabah. ASEAN Review of Biodiversity and Environmental Conservation (ARBEC), JulySeptember, 1-8.

Jazirehi MH, Rostaghi EM (2003) Silviculture in Zagros. University of Tehran Press. Tehran. [Persian] Jobidon R, Cyr G, Thiffault N (2004) Plant species diversity and composition along an experimental gradient of northern hardwood abundance in Picea mariana plantations. For Ecol Manag 198: 209-221.

Khera N, Kumar A, Ram J, Tewari A (2001) Plant biodiversity assessment in relation to disturbances in mid- elevational forest of Central Himalaya, India. Tropical Ecol 42 (1): 83-95 Kinnear PR, Gray CO (2001) SPSS for windows made simple release 10 (translated by Ardakani AF). Psychology Press. Tehran. [Persian] Krebs JC (1999) Ecological methodology. Harper and Row. New York.

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ISSN: 2087-3940 (print) ISSN: 2087-3956 (electronic)

Vol. 3, No. 1, Pp.: 23-27 March 2011

Evaluation structural diversity of Carpinus betulus stand in Golestan Province, North of Iran VAHAB SOHRABI1,♥, RAMIN RAHMANI1, SHAHROKH JABBARI2, HADI MOAYERI1 Faculty of Forestry, Gorgan University of Agricultural Science and Natural Resources. PO Box 386, Shahid Beheshti Street, Gorgan, Golestan, Islamic Republic of Iran, Tel. +98 (171) 222 0028, Fax. +98 (171) 222 598, ♥Email: vahabsohrabi61@yahoo.com 2 Super Council of Forests, Range, Watershade Management Organization, Islamic Republic of Iran.

Manuscript received: 19 February 2011 Revision accepted: 3 March 2011.

Abstract. Sohrabi V, Rahmani R, Jabbari S, Moayeri H. 2011. Evaluation structural diversity of Carpinus betulus stand in Golestan Province, Northern Iran. Nusantara Bioscience 3: 23-27. In order to investigate structural diversity of Carpinus betulus type in Golestan province 30 modified Whittaker plots by systematic random system were located. Per plot the characteristic of trees and shrubs species (Species name, diameter and height of trees) are recorded. The heterogenity indices of Simpson, Shannon–Wiener, Simpson’s reciprocal and number of equally common species were used for the quantitative data. Toward better understand from diversity condition in horizontal and vertical composition of stand, the diameter divided in 10 cm classes and Method of Mohajer and the height divided in 10 m height classes and dominant height, then number of diversity of each class extracted by Ecological Methodology software V.7. The results showed with increase of diameter and height classes, decrease species diversity. Also regeneration layers diversity has significant difference with trees layers. Thus, the study of biodiversity changes in different diameter and height category cause ecologically precise perspective in management of forest stands.

Key words: structure diversity, indices diversity, diameter and height classes.

Abstrak. Sohrabi V, Rahmani R, Jabbari S, Moayeri H. 2011. Evaluasi keragaman struktur tegakan Carpinus betulus di Provinsi Golestan, Iran bagian utara. Nusantara Bioscience 3: 23-27. Dalam rangka untuk menyelidiki struktur keragaman tipe Carpinus betulus di provinsi Golestan, 30 plot Whittaker yang telah dimodifikasi dibuat secara sistem random sistematis. Pada setiap plot, karakteristik spesies pepohonan dan semak (nama spesies, diameter dan tinggi pohon) dicatat. Indeks heterogenitas dari beberapa macam indeks Simpson, Shannon-Wiener, Simpson’s reciprocal dan jumlah spesies yang umum ditemukan digunakan untuk data kuantitatif. Untuk lebih memahami kondisi keanekaragaman dalam tegakan horizontal dan vertikal, maka dikelompokkan ke dalam diameter dalam kelas 10 cm, metode Mohajer, tinggi dalam kelas 10 m, dan ketinggian yang dominan, kemudian jumlah keragaman setiap kelas ditentukan dengan software Ecological Methodology v.7.0. Hasil penelitian menunjukkan bahwa peningkatan kelas diameter dan tinggi, menyebabkan penurunan keragaman spesies. Keragaman lapisan regenerasi memiliki perbedaan signifikan dengan lapisan pohon. Studi perubahan keanekaragaman hayati dengan kategori diameter dan tinggi yang berbeda memerlukan perspektif ekologis yang tepat dalam pengelolaan tegakan hutan.

Kata kunci: keanekaragaman struktur, indeks diversitas, kelas diameter dan tinggi.

INTRODUCTION Human knows the concept and the importance of biodiversity from earlier century. Plato frequently point out the diversity and believe that if there is more diversity in the world, the world will be better (Beasapour 2000, Ejtehadi et al. 2009). Today, the word of biodiversity applies by various science experts, such as ecologists. The convention of biodiversity of USA, describe biodiversity as: there is a difference in all the life type all sources such as marine, ground and ecological complex combination and include the diversity within species, between species and ecosystems (Markandya et al. 2008). One of the constant keys of management of uneven age forest is the true understanding about spatial structure of forest (Costanza et al. 2007). Forest structure is the important feature in

management of forest ecosystems (Zenner and Hibbs 2000). Structural features are used to determine the species neech heterogeneous experiment and plant dynamic time, management of regeneration patterns and fragmentation dynamic, description of microclimate diversity and predicting the wood production (Youngblooda et al. 2004).

Management of forest stands performs by stands structure control (age, size and tree density) and forest structure (size and spatial order of tree) because the concept of forest structure is more important than species combination (Oheimb et al. 2005). The study of natural forests structure defined the way of desired structure that the use of appropriate silviculture operation and stimulation of natural structure in under management stands considered as the way to keep the biological diversity and forest dynamic and stability (Markandya et al. 2003). The study of forest

Â

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structure especially in virgin forests is very important and gives us comprehensive information about the condition in forest for programming. The diversity of a forest stand may not be sufficiently described by tree species diversity alone.

Structural diversity, resulting from recruitment of trees of different sizes into multilayered canopies, should also be taken into account (Liang et al. 2007). This characteristic, which can be approximated by the diversity of tree size, affects the amount of light and precipitation received by subordinate trees and understory plants (Anderson et al.

1969), and may thus influence the productivity of forest ecosystems. Thus manipulating tree-size diversity is a practical tool for forestmanagers who strive for greater biodiversity and/or greater productivity (Varga et al.

2005).Varus studing done about in forest structure.

Ahani et al (2006) do the research about species diversity of tree based on the diameter class in Acer sites in Shafarud forests. So, rhombus plots in half hectare study in forest according to Acer (34 plots). First the feature within each plot, its slope, aspect, height from sea level and then total diameter of trees up to more than 10 cm

measured.Biodiversity accounted in four diameter alasses (10-30, 35-50, 55-80, 80-120 cm). The result showed that the Shanon and N1 Mac Arthur indices in diameter class of 35-50 cm, have greatest amount, while the index of Simpson and N2 hill shows the greatest amount in diameter class of 10-30 cm. The purpose of this paper is the evaluation of structural diversity in diameter and height classes and their changing process with changing of diameter classes and height category in Carpinus betulus (Persian: Mamarz) type in Golestan province, IR Iran.

MATERIALS AND METHODS The regions of study Kohmian forestry plan is located in 98 wateshade domain which is limited in north is village of Kohmian, Fazel Abad, Khanduz Sadat and Marzbone, in south and west to Naeem s forestry plan and in east to vatan forestry plan. Its east longitude is 55-14-49 to 55-10-30 and its north width is 37-65-15 to 37-00-00 degrees (Figure 1).

GolestanÂ

6

4

5

3 2 1

Figure 1. Map of the site study in in Golestan Province, North of Iran. 1. Shastkalateh, 2. Tavir, 3. Kohmian, 4. Takht, 5. Loveh, 6.

Farsian.

SOHRABI et al. – Diversity of Carpinus betulus stand in Golestan, Iran

25

Table 1. Indices used in this paper (Ejtehadi et al. 2009) Equation

1 − D = 1 − ∑ ( pi )

Index 2

Simpson

H ′ = ∑ ( Pi )( Log 2Pi )

Shannon–Wiener

1

Simpson’s reciprocal

s

i =1

D

=

1

∑ pi

2

N1 = eH ′

Number of equally common species

Description of equation (1-D) = Simson’s index of diversity p1 = proportion of individual species I in the community H’ = information content of sample (bits/individual) = index of species diversity s = number of species p1 = proportion of total sample belonging to i-th species 1/D = Simson’s reciprocal index (= Hill’s N2) p1 = proportion of individual species i in the community H’ = information content of sample (bits/individual) = index of species diversity s = number of species p1 = proportion of total sample belonging to i-th species

Research method This research is basee on sampling by systematic random system and the center of plots in forest is determined. To study and investigation, 30 modified Whittaker plots in range of 850-950 m altitude from the sea level in north aspect were located. In this 20x50 meter frame, the characteristic of trees and shrubs species (species name, diameter and height of trees) are recorded.

The heterogenity indices of Simpson, Shannon–Wiener, Simpson’s reciprocal and number of equally common species and evenness indices of Simpson, Camargo, SmithWilson and modified nee were used for the quantitative data (Table 1). Then aforesaid characteristics saved as information bank in Excell 2010. Then indices account by Ecological Methodology software v.7.0 (Krebs 1999).

Analyze of data was done by analyze of variance (ANOVA) and Duncan’s multiple range test (DMRT).

Diversity indices in 10 cm diameter classes The under study diversity indices in this paper shows the decrease in the diameter classes of 10cm with increase of classes. The most diversity number is in diameter class of 0-10 cm and the least diversity number is in diameter class of 90-100 cm. other than Simpson diversity index that shows the least diversity number in diameter class of more than 100cm, the significant different is between diameter classes in 1% level (Figure 2).

Diversity indices in diameter classes by method of Mohajer Diversity indices shows decrease process with the increase of diameter classes but it increase again in last class (dbh>80). The most diversity number is in the class of 0-10 cm and the least diversity number is in class of 60_80 cm. The diameter classas (20-30, 30-60, dbh>80) are not significant different. The significant different is between diameter classes in 1% level (Figure 3).

RESULTS AND DISCUSSION Next of survey recorded number of 10 trees species dependent of 8 families and 3 shrubs species dependent of 2 families that show notable statistics (Table 2).

Table 2. Composition of trees and shrubs species.

Scientific name Quercus castanefolia Carpinus betulus Parrotia persica Tilia begunda Acer insigne Ulmus glabra Acer cappadocicum Alnus glutinosa Crataegus monogyna Mespilus germanica Prunus avium Sorbus torminalis Diospyros lotus

Family Fagaceae Betulaceae Hamameliadaceae Tiliaceae Acearaceae Ulmaceae Acearaceae Betulaceae Rosaceae Rosaceae Rosaceae Rosaceae Ebenaceae

Trees/Shrubs T T T T T T T T S S S T S

Diversity indices in 10m height classes Diversity indices have orderly decrease process. The most diversity number is in height class of 0-10m and the least diversity number is in height class of 40-50m. , the significant different is between height classes in 1% level (Figure 4).

Diversity indices in dominant height of height classes The most diversity number in all indices is for h<1/3hm class and the least diversity number is for 1/3h<h>2/3hm height class. Diversity indices, first, decrease then increase in third class. First height class has significant difference with other classes in level of 1% (Figure 5).

Discussion Forest structure is the important feature in management of forest ecosystems (Zenner and Hibbs 2000). The study of natural forests structure defined the way of desired structure that the use of appropriate silviculture operation and stimulation of natural structure in under management stands considered as the way to keep the biological diversity and forest dynamic and stability (Markandya et al.

2003). Whereas, structure characterize the building (vertical and horizontal), composition and diversity of

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Figure 2. The comparison of diversity indices in 10cm diameter classes. A. Simpson, B. Simpson’s reciprocal, C. Shannon– Wiener, D. Number of equally common species.

Figure 3. The comparison of diversity indices by method of Mohajer (2005). A. Simpson, B. Simpson’s reciprocal, C. Shannon–Wiener, D. Number of equally common species.

Figure 4. The comparison of diversity indices in 10m height classes. A. Simpson, B. Simpson’s reciprocal, C. Shannon– Wiener, D. Number of equally common species

Figure 5. The comparison of diversity indices in height classes by dominant height. A. Simpson, B. Simpson’s reciprocal, C. Shannon–Wiener, D. Number of equally common species.

forest stands. Forest stands have different structure in various sections (linear and phenomenal) like a building.

For recognition, study and precise programming of forest stands, its features need to consider according to different sections. Various profiles (linear and phenomenal) could be dividing for forest stands. The study of forest stand profile especially in virgin forests is very important and gives us comprehensive information about structure of these forests (Mohajer 2005). For better understanding of the structure of forest stand, we analyzed it according to the vertical and horizontal structure. Species diversity of tree and shrub in this type have significant different in low diameter and height classes with up diameter and height classes classes.

Diameter and height classes below of 10 cm, account as 10 regeneration layer, so diversity of regeneration layer is more than the diversity of tree layers (Pourbabaei et al.

2006; Sohrabi 2010). This is due to the decrease of canopy of small saplings and it need low light than higher age process in this classes. By the increase of diametrical and height classes, the diversity decrease. It is obvious that the structure diversity naturally in the virgin forest decrease depend on site condition and with increase of stand age and its move toward climax, because gradually increase of trees age dominant species dominant against the under species.

Trees are the main elements in forest ecosystems that other living thing life of this ecosystem depends on the life of them. Therefore removing of the tree threatened the life of the existent in this ecosystem. The main role of forest engineer is the marketing of forest (Mohajer 2005). In this step choosing of trees perform by considering of target diameter from defined species and gradually the number of trees in defined diameter decreased and so the repeating act might remove some class of trees. It is threatened the structure diversity and the species diversity. Trees diversity in higher diametrical and altitudinal categories is part of the lower diametrical category diversity. Any changes in above level might change the ground cover. Tree dimension diversity has an effect on the amount of light and raining by small plant and trees (Anderson et al.1969). This has influence on the produce of forest ecosystems.

SOHRABI et al. – Diversity of Carpinus betulus stand in Golestan, Iran

CONCUSION The increasing of diameter and height classes, decrease species diversity. Regeneration layers diversity has significant difference with trees layers. Thus, the study of biodiversity changes in different diameter and height category cause ecologically precise perspective in management of forest stands.

ACKNOWLEDGEMENTS Therefore I express gratitude to any ones who is useful in my life. By the way, I thank Ezazi to give us translation of this paper.

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ISSN: 2087-3940 (print) ISSN: 2087-3956 (electronic)

Vol. 3, No. 1, Pp.: 28-35 March 2011

Microanatomy alteration of gills and kidneys in freshwater mussel (Anodonta woodiana) due to cadmium exposure FUAD FITRIAWAN1,♥, SUTARNO², SUNARTO² ¹ Open University, UPBJJ Bandar Lampung. Jl. Soekarno-Hatta No. 108 B Rajabasa, Bandar Lampung 35144, Lampung, Indonesia. Tel.: +92-721704772. Fak.: +92-721-709026. E-mail: ut-bandarlampung@upbjj.ut.ac.id, maz_afid@yahoo.co.id ² Bioscience Program, School of Graduates, Sebelas Maret University, Surakarta 57126, Central Java, Indonesia Manuscript received: 15 December 2010. Revision accepted: 26 February 2011.

Abstract. Fitriawan F, Sutarno, Sunarto. 2011. Microanatomy alteration of gills and kidneys in freshwater mussel (Anodonta woodiana) due to cadmium exposure. Nusantara Bioscience 3: 28-35. The purpose of this study were to determine the level of Cd accumulation in the gills and kidneys, to khow the changes in microanatomic structure of A. woodiana after the various treatments of heavy metals. Completely randomized design pattern of 5 x 3 as used in this laboratory experiment. The amount of exposure of heavy metals Cd were (0 ppm, 0.5 ppm, 1 ppm, 5 ppm, 10 ppm), while the variation of length of exprosure time to Cd were (7 days, 14 days, and 30 days). The parameters of Cd accumulation in the gills and kidney was analyzed by using AAS method, while abnormalities of gills and kidney were detected by microanatomy structure. Data collected were then analyzed using the analysis of variance (ANOVA) and continued with further test the DMRT. The results indicated that there is a significant effect in 475.3 > 0.000 and 60150.3 >0.000 with 5% significance level (P<0.05) of Cd treatment on gill and kidney microanatomy of A. woodiana. The changes in microanatomy structure of those organs are including edema, hyperplasia, fusion of lamella, necrosis and atrophy.

Key words: gills, kidneys, Anodonta woodiana, cadmium.

Abstrak. Fitriawan F, Sutarno, Sunarto. 2011. Perubahan mikroanatomi pada insang dan ginjal kerang air tawar (Anodonta woodiana) terhadap paparan kadmium. Nusantara Bioscience 3: 28-35. Tujuan penelitian ini untuk mengetahui tingkat akumulasi, perubahan struktur mikroanatomi setelah perlakuan logam berat Cd pada insang dan ginjal A. woodiana. Jenis penelitian yang digunakan yaitu eksperimental laboratorium dengan rancangan acak lengkap (5 x 3) berupa besarnya paparan Cd (0 ppm, 0,5 ppm, 1 ppm, 5 ppm, 10 ppm) dan waktu pemaparan Cd (setelah 7 hari, 14 hari, dan 30 hari). Parameter pengujian mencakup uji akumulasi Cd pada insang dan ginjal dengan metode AAS, dan abnormalitas insang dan ginjal akibat akumulasi Cd dengan metode preparasi. Analisis akumulasi Cd pada insang dan ginjal menggunakan analisis varian (ANAVA) dan dilanjutkan dengan uji lanjut jarak berganda Duncan (DMRT). Hasil penelitian menunjukkan pengaruh pemberian beberapa perlakuan Cd terhadap kontrol insang dan ginjal A. woodiana signifikan sebesar 475,3 > 0,000 dan 60150,3 > 0,000 dengan taraf signifikansi rata-rata 5% (P<0,05) yang ditandai dengan perubahan struktur mikroanatomi pada insang berupa edema, hiperplasia, fusi lamella, nekrosis hingga atropi. Sedangkan pada ginjal berupa edema, hiperplasia dan nekrosis pada tubulus, glomerulus, dan mineralisasi pada sel darah hingga mengalami pendarahan.

Kata kunci: insang, ginjal, Anodonta woodiana, kadmium.

INTRODUCTION Cadmium (Cd) is one type of heavy metals that are useful in several industries. For example in the textile batteries industry and electroplating, as coloring matters in ink. Cd also exisst naturally in foods even if only in small amounts absorbed by the intestine (5-8%) (Palar 1994). But on the other hand, a heavy metal can cause problems; problems can occur more severe if waste management is not done properly, so that it will have an impact on the environment as a micro-pollutants (Soegianto et al. 2004).

Incoming cadmium in fresh water will be joined with a metal ion cofactor so that the shape of Cd2+ causes the toxicity of the water. Cd2+ ‘s toxicity levels in water depend on salinity. The toxicity of Cd2+ in the water will rise if the salinity is low.

To determine the level of pollution in a region we can use a particular bioindicator organism typical of one that can be used, namely A. woodiana. The advantages of this animal is that they settle in one place, and have a slow movement, so that if an environment is exposed to heavy metal waste Cd then indirectly affects the lives of these biota. Cd accumulation in an organism other than cause exposure to the organ, it will also cause interference on enzyme activity. The nature of the toxic metal is due to its very effectiveness in binding itself withh the group of sulfuhidril (SH) in the enzyme system of cells that form bonds and metaloprotein metaloenzim so that enzyme activity for cell life processes cannot take place (Connell and Miller 1995).

Gills and kidneys are vital organs. Gills play a role in the process of respiration, acid-base balance, ionic and osmotic regulation because of the branchial epithelium

FITRIAWAN et al. â&#x20AC;&#x201C; Effect of Cd on freshwater mussels A. woodiana

tissue which became the meeting place of active transport between organisms and the environment (Soegianto et al.

2004). Renal function begins in the glomerular ultrafilter that is formed from the plasma. Ultrafilter will enter the Bowman's capsule and into the lumen of the tubule.

Filtering through the various segments of the tubules causes changes in the volume and composition of fluid filtration as a result of the process of reabsorption and secretion along the tubules (Tresnati et al. 2007).

Glomerulus is composed of blood capillaries to function as a selective filter from the blood mainly in the normal blood screening (Takashima and Hibiya 1995). Following through on glomerular filtration and being re-absorbed in the tubular, it produces urine as a result of secretion in normal circumstances (Tresnati et al. 2007).

The purpose of this study are: (i) to know the content of Cd accumulation, (ii) changes in the microanatomy structure, and (iii) in gill and kidney of A. woodiana after treatment.

MATERIALS AND METHODS Time and place The research of the Cd treatment on A. woodiana conducted at the Laboratory of Pharmacy and Food Academy Analyst Sunan Giri, Roxburgh. Analysis of heavy metal content by AAS method was carried out in sub lab Chemistry Laboratory of Mathematics and Science Center UNS Surakarta, while the preparation for the analysis was conducted in the laboratory animal anatomy Faculty of Veterinary Medicine, Gadjah Mada University in Yogyakarta. The experiment was conducted in OctoberNovember 2009.

Materials Freshwater mussels (A. woodiana) were obtained from farms in the fishing village of the tourist site of Janti, Polanharjo Subdistrict, Klaten District, Central Java.

Procedures Freshwater bivalve A. woodiana was selected based onthe maximum growth and uniform size. The shellfish was acclimatized for 15 days, after which it was examined by using the compound of Cd for 30 days with repeated 3 times at day 7, 14 and 30. Physical-chemical parameters measured mencakuip pH, DO and water temperature where the experiment. Cd content of the examination conducted on the gill and kidney A. woodiana with AAS method.

Preparation of gill and kidney preparations performed with haematoxylin-eosin (HE) method with treatment stages, ie trimming, dehydration, embedding, cutting, stainning, mounting and reading the results.

Data analysis Environmental chemistry parameters (pH, DO, temperature) were described with a descriptive method.

Effects of Cd exposure on the gill and kidney A. woodiana were analyzed by ANOVA one-way significance level of 5% (P> 0.05), followed by a further test of significant

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difference or Duncan's multiple range test (DMRT).

Abnormality in the microanatomy structure of gills and kidneys of A. woodiana was directly observed and described with a descriptive method.

RESULTS AND DISCUSSION Water environmental parameters Examination of physical and chemical parameters of water quality used in this study include the degree of acidity (pH), dissolved oxygen (DO), and water temperature.

Degree of acidity (pH) The degree of acidity or pH is a value that shows the activity of hydrogen ions in water. The pH of a water reflects the balance between acid and base in these waters.

The pH range 1-14, pH 7 is the boundary halfway between the acid and alkaline (neutral). The higher the pH of the water, the greater the base nature will be, and the lower the pH the more acidic the water. PH value is influenced by several parameters, including biological activity, temperature, oxygen content and the ions. From the biological activity, CO2 gas is generated as a result of respiration. This gas will form a buffer or buffer ions to maintain the pH range in the waters in order to remain stable (Erland 2007).

In this study, the pH is very important as water quality parameters, for controlling the type and rate of speed of reaction some materials in the water. In addition, A. woodiana live at a certain pH interval, so that by knowing the value of pH, it can be known whether or not the water supports their lives. Based on Figure 1A, it is known that the higher Cd concentration, the higher the value of the range of pH waters. On day 7, pH values ranged from 7.34 to 8.44, on day 14 ranged from 7.37 to 8.40, and the dayto-30 range from 7.31 to 8.68. According to Erland (2007), pH to function as an index of environmental conditions and limiting factors, where each organism has a different tolerance to pH maximum, minimum and optimal.

According to Erland (2007) pH value of water has a special characteristic, the hydrogen ion concentration be measured by the balance between acids and bases. Acidfree mineral acid and carbonic acid will lower pH value (acid), while the carbonate (CO3), hydroxide (OH-) and bicarbonate to raise pH (alkaline). Rochyatun et al. (2006) states, that at a relatively high metal content will be alkaline pH values (pH 7.40 to 8.59), where the metal is difficult to dissolve and settle to the bottom of the water.

When in the treatment, pH values from 0.5 to 10 ppm, in the study it ranged from 7.92 to 8.68, indicating water has been polluted quite heavily, with the level of alkalinity in excess of tolerance. According to Connell and Miller (1995) increase in pH in the waters will be followed by decreasing the solubility of heavy metals that tend to settle.

Deposition can occur in sediments and food; the food will enter and accumulate in the body of A. woodiana. Given the Cd is a non-essential metal that cannot be degraded so that it will cause interference with the organs, such as the gill and kidney.

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increasing the rate of respiration and dissolved CO2 increases, so the toxins more and more absorbed in the body through the gills. The higher the level of aquatic toxicity, the higher the rate of breathing will be (Budiono 2003).

Dissolved oxygen is essential for respiratory zoobentos and other aquatic organisms (Odum 1993). In addition, the solubility of oxygen is also affected by temperature, at high temperature and low oxygen solubility at low temperatures the high oxygen solubility. Each species of aquatic biota have a range of different tolerances to the concentration of dissolved oxygen in the water. Species with wide tolerance range and wide distribution of species with narrow tolerance range only live in certain places. Budiono (2003) stated that the excessive presence of heavy metals in the waters will affect the respiratory system of aquatic organisms, causing low dissolved oxygen levels, which disturb the life of aquatic organisms.

Water solubility of oxygen (DO) Oxygen is one of the gases dissolved in natural waters with varying levels are influenced by temperature, salinity, water turbulence, and atmospheric pressure. Besides necessary for the survival of aquatic organisms, oxygen is also needed in the process of decomposition of organic compounds. Sources of dissolved oxygen are mainly derived from the diffusion of oxygen from the atmosphere.

This diffusion occurs directly on stagnant conditions (silent), or because of agitation (water mass unrest) caused by waves or wind.

Figure 1B shows that the higher concentration of Cd treatment, then progressively decreasing levels of dissolved oxygen (DO) in water. Ardi (2002) classified water quality based on the DO into four types namely; not contaminated (> 6.5 mg/L), lightly polluted (4.5 to 6.5 mg/L), being contaminated (2.0 to 4, 4 mg/L) and heavily polluted (<2.0 mg/L). In this study, the DO in the first test of 10.10 ppm decreased to 3.19 ppm, in the second test from 10.11 ppm to 3.25 ppm, and the third repeat of 10.26 ppm to 3.76 ppm. From the above results the pollutuion can still be considered moderate.

Decreased levels of oxygen in the water is inversely proportional to the high Cd treatment in these waters.

Cadmium is an inorganic contaminants/minerals that can accumulate in water or in food. In general, the Cd that entered the waters will be Cd2+ which causes the toxicity waters, and the presence of sediment in the diet will be very easy to be consumed by aquatic biota, including A. woodiana. Solubility of oxygen is essential for the sustainability of aquatic life, oxygen is used as a tool of biota metabolism so it can carry out their duties as pendekomposisi and degrading organic materials in order to more easily broken down by bacteria (Warlina 2004; Ardi 2002). If an aquatic tecemar by a heavy metal inorganic, then A. woodiana not able to decompose organic materials, so that the decomposition process is highly dependent aerobic bacteria that need oxygen is very high, and can cause a deficit of oxygen in these waters.

According to Destiany (2007) with increasing concentrations of heavy metals, the dissolved oxygen content will decrease, and the CO2 will rose, due to low oxygen levels that require aquatic biota such as A. woodiana to pump water through their gills, thereby

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Temperature Each of aquatic organisms has different tolerance limits to changes in water temperature to the life and growth of aquatic organisms. Therefore, the temperature is one factor that physically is very important for aquatic organisms or aquatic life. In general, the temperature directly affects the aquatic biota of enzymatic reactions in the organism and does not directly influence the structure of organs and the spread of aquatic animals (Nontji 1984).

From Figure 1C, it is known that the temperature of different water looks increasingly high Cd treatment at each treatment, which ranged from 25.8 to 26.6°C on the first test, and the second test ranged from 26.2 to 27°C, the third test ranged from 26.8 to 27.4°C. This is influenced metal accumulation in each treatment with the higher concentration, thus causing the water temperature value is also higher. It is inversely proportional to the solubility of oxygen in water, ie at high temperature low oxygen solubility, and low solubility of oxygen at high temperature (Odum 1993).

The Relationship between the temperature rise of heavy metal accumulation in the water is strong. Cd is an inorganic non-essential metal that cannot be in the degradation of benthos organisms and microorganism. The presence of metal causes the metabolic rate of aquatic biota

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Figure 1. The physical-chemical parameters condition of waters in the experimental location after administration of Cd. A. the degree of acidity (pH), B. DO, C. temperature.

FITRIAWAN et al. â&#x20AC;&#x201C; Effect of Cd on freshwater mussels A. woodiana

increased in order to defend themselves, so that automatically the oxygen demand is very much while on the other hand, given the concentration of heavy metals higher, thus increasing the concentration of heavy metals that enter the more carbon dioxide (CO2) are released that cause the oxygen content dwindling waters so that the rising water temperature.

According to Connell and Miller (1995) the role of water temperature is very important to help the body's metabolism of aquatic animals. The increase in water temperature can cause the immune system of aquatic biota to decrease. So if a toxic Cd2+ enters the body of A. woodiana the biota will be very difficult to retain yourself from the poison.

Accumulation of Cd in the gills of A. woodiana The result of the content of Cd in the gill and kidney A. woodiana with AAS method are shown in Table 1. From the results it is known that the increasing Cd treatment, the increase of Cd accumulatd in the gill of A. woodiana. In the control (0 ppm), Cd accumulation in the gills of A. woodiana was 0.12 ppm, the accumulation in the control is still below the maximum tolerance limit Cd accumulation in organs, as specified by the FAO (1972) and MOH (1989), namely a maximum accumulation of Cd in organs of 1 ppm. It is also in accordance with preliminary studies that have been made to the content of Cd in water samples, with the result that the content of Cd in Janti aquaculture that was still in the normal state that was 0.0028 ppm (IGR No. 82/2001; EPA 1986).

After the examination after 7 days the average values obtained Cd accumulation in gill A. woodiana in the treatment of 0.5 ppm was 0.58 ppm, treatment of 1 ppm was 0.87 ppm, 5 ppm was 1.00 ppm and 10 ppm was 2.15 ppm. Meanwhile, after the examination on day 14, obtained an average value of accumulated Cd at 0.5 ppm treatment was 0.78, treatment of 1 ppm 0.93 ppm, 5 ppm treatment at 1.24 ppm, and on treatment 10 ppm at 2.34 ppm. After the examination on day-30, it was obtained an average value of Cd accumulation in the treatment of 0.5 ppm 1.43 ppm, 1 ppm treatment at 1.01, 5 ppm treatment at 2.58, and the treatment of 10 ppm 3.49 ppm.

Darmono (1995) states that the relationship between the amount of metal absorption and metal content in water is usually in proportion, the increase in metal content in the network in accordance with the increase of metal content in water. According Sunarto (2007) Cd will also experience the process of biotransformation and bioaccumulation in aquatic biota. Cadmium enters the body along the water or food consumed, but water or food has been contaminated

by Cd. The amount of metal that accumulates in the gills will continue to increase, even very likely continue to enter through the accumulation of Cd digestive tract to the kidney; in addition to increasing levels of pollutants in the presence of Cd may also biomagnification process in the water body. If the amount of Cd that enters the body and has exceeded the threshold value, it will experience death and even extinction.

Cd treatment against gill A. woodiana with a concentration of 0 ppm, 0.5 ppm, 1 ppm, 5 ppm and 10

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ppm for 7 days, 14 days and 30 days to yield significant results (P <0.05). This is in accordance with the opinion Darmono (1995) which states, that the relationship between the amount of metal absorption and metal content in water is usually in proportion, the increase in metal content in the network in accordance with the increase of metal content in water.

ANOVA test and Duncan's range test showed no association of Cd accumulation in gill of A. woodiana with the concentration of Cd (Table 1). The higher the concentration of Cd is given, the higher the exposure to Cd on the gills of A. woodiana will be. The most obvious difference is indicated by the treatment concentration of 10 ppm, which is the highest concentration, thus giving an average value of accumulated most Cd concentrations higher than below it. As for the concentration of 0.5 ppm and 1 ppm, obtained test results are less tangible difference, possibly because the treatment they are not too much difference compared with other treatments, so that the results of cadmium exposure on A. woodiana not too apparent.

Table 1. Treatment test results in the accumulated Cd concentration of Cd in gill and kidney A. woodiana.

Treatment of Cd Average Cd Average Cd concentration accumulation in accumulation in (ppm) gill (ppm) kidneys (ppm) 0 0.12 a 0.018933 a 0.5 0.93 b 0.045200 b 1 0.94 b 0.042956 b 5 1.61 c 0.082844 c 10 2.66 d 0.660111 d Note: Value having the same letter notation means that no effect significantly different

ANOVA test and Duncan's range test that also shows a relationship between the length of the treatments with the level of Cd accumulation in the gill of A. woodiana (Table 2). The longer treatment time means the accumulation of Cd in the gills. In Table 2, It can be seen that a 30-day treatment gave the highest average of the Cd accumulation on the gills of of A. woodiana. Then further test the distance from Duncan to get the illustration relations and exposure levels of Cd accumulation in the gill of A. woodiana to the long treatment, where the old high Cd treatment is given, the higher accumulation of Cd in the gills of A. woodiana.

Table 2. Old test results the treatment of Cd accumulation in gill and kidney A. woodiana Average Cd Average Cd accumulation in gill accumulation in (ppm) the kidney (ppm) 7 0.940313 a 0.048420 a 14 1.081420 a 0.063740 a 30 1.728753 b 0.397867 b Note: Value having the same letter notation means that no effect significantly different Length of treatment (days)

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Analysis of treatment outcome ofthe renal Cd of A. woodiana Test results on the Cd content of the kidney A. woodiana after treatment is shown in Table 1. The average value of the kidney of A. woodiana in the control group amounted to 0.019 ppm (0.018933 ppm), the value accumulated in the control was still below the maximum tolerance limit Cd accumulation in organs, as specified by the FAO (1972) and MOH (1989) that is equal to 1 ppm. It is also in accordance with preliminary studies that have been made to the content of Cd in the water, that the content of Cd in the water of Janti aquaculture is still in the normal state is 0.0028 ppm (IGR No. 82/2001; EPA 1986).

Later in the treatment of 0.5 ppm Cd in the kidneys after examination, AAS average accumulation after 7 days was at 0.020 ppm, after 14 days was at 0.029 ppm, after 30 days was at 0.086 ppm. Later in the treatment of 1 ppm after 7 days accumulation of 0.030 ppm, 0.031 ppm after 14 days, and after 30 days at 0.066 ppm. Later in the treatment of 5 ppm Cd accumulation after 7 days at 0.057 ppm, after 14 days at 0.085 ppm, and after 30 days at 0.107 ppm. And in the treatment of 10 ppm Cd accumulation in the kidney after 7 days at 0.116 ppm, after 14 days at 0.150 ppm and after 30 days showed the exposure of 1.717 ppm.

From the above data, it is known that the higher the concentration of Cd treatment A. woodiana, the higher the value of exposure to cadmium in the kidneys of A. woodiana. This is similar to what has been mentioned by Sunarto (2007) that Cd will also experience the process of biotransformation and bioaccumulation in aquatic biota.

Cadmium enters the body along the water or food consumed, where water or food has been contaminated by Cd. The amount of metal that accumulates in the gills will continue to increase, even very likely continue to enter through the accumulation of Cd digestive tract to the kidney.

Based on ANOVA statistical test, it is known that Cd treatment on the kidney of A. woodiana with a concentration of 0 ppm, 0.5 ppm, 1 ppm, 5 ppm and 10 ppm for 7 days, 14 days and 30 days, yield significant results (P <0.05). This is in line with the significance indicated on the gills of A. woodiana. Furthermore, Duncan range test indicated that the higher the concentration of Cd treatment, the higher the accumulation of Cd in the kidneys of A. woodiana. Treatment of Cd concentration of 10 ppm gave the highest cumulative impact with the most obvious significance level, of the treatment concentration underneath. Later the treatment of 0.5 ppm and 1 ppm did not show significant differences, it might be that the range of Cd concentrations that had been given is not far adrift, so that the value of exposure and accumulation of Cd in the kidneys was directly proportional to the treatment given.

Cd concentration of 10 ppm gave the most obvious difference.

To know the effect of long exposure to the treatment of kidney due to accumulation of Cd in A. woodiana, it was also performed ANOVA test followed by a significantly different test (Duncan), where the longer treatment time means the higher the impact on exposure accumulation of Cd in the kidneys of A. woodiana, where this long a period of 30 days of treatment gave the greatest average rating of

accumulation.

According Destiany (2007) said that, the process of accumulation of chemicals in living things is described as follows: foods that accumulate heavy metals such as cadmium, will be eaten by aquatic biota, including the types of bivalves and will enter into the digestive tract.

From within the digestive (gastrointestinal) through the walls will go to the circulatory fluid, then after the circulatory fluid would most foodstuffs in metabolism and partly met with several networks, so it will be in storage in fat tissue. Then chemicals such as cadmium in the fluid circulatory oxidized to Cd2+ that cause toxicity and will accumulate in the liver, because the nature of the Cd is a material non esesial, its presence in the liver cannot be inactivated by the enzyme, so it continues to settle the kidneys and and create sediment there.

Microanatomical changes in the gill of A. woodiana after exposure to Cd Accumulation of Cd has caused a variety of physiological damage to the organs of A. woodiana, because the nature of the toxicity of Cd accumulated in the body has exceeded the maximum threshold of 1 ppm (FAO 1972), where the metal LC50 at 3 ppm occurs 48-72 hours after treatment (Kraak et al. 1992). In this experiment, some damaged organ/tissue shrinkage as siphon, foot, gill and kidney. According to Palar (1994) Cd can damage aquatic biota in the physiological system urinary system, gill, kidney and blood circulation. Damage caused by Cd contacts continuously through the cell membrane results in the degeneration of the membrane. If Cd enters through the gill, the gill will cause the deficiency so that the body's metabolic function gets disrupted.

Result analysis of changes in cellular of the microanatomical structure of the gill and kidney of A. woodiana is shown in Table 3. From the data results of the transverse slice preparations of the gill of A. woodiana after accumulation of Cd, it is known that the symptoms of cell damage on the gill was known at a concentration of 0.5 ppm with marked with edema in the lamella branchialis, so that on day 14 and day 30 the hiperflasia was more visible impact. The worst damage at the cellular level of gills occured on thetreatment of 10 ppm, where the gills showed symptoms of edema that was accompanied by hyperplasia, and eventually the entire network of experienced fusion up to each lamella having atrhopi (Figure 2).

In the normal gill at the concentrations of 0 ppm, with a concentration of 0.1004 to 0.1321 ppm Cd accumulation (Figure 2A), it can be seen all the parts of cells from epithelial cells, basement membrane, Lacuna, the blood cells until the cell pillar that were still in normal circumstances. The accumulation of these metals has been carried by each of biota samples from the sampling site that is in the area of aquaculture of Janti. So to make a sample without the accumulated metal is extremely difficult. In addition, according to Rahman (2006) in general, heavy metal content in a body of water is different form the one with heavy metals that have been dissolved in the aquatic sediments especially heavy metals in the organ. A heavy metal when the waters would go down and settles to form sedimentation, it will cause the organisms that eat at the

FITRIAWAN et al. â&#x20AC;&#x201C; Effect of Cd on freshwater mussels A. woodiana

were also seen pillar cells began to separate from the bottom of epithelial cells (middle lamella). When experiencing edema,l Cd accumulation in gills occurred at the accumulation of 0.5111 ppm.

Gills in Figure 2C has undergone thorough hyperplasia and the fusion is taking place in two parts of the middle lamella, with a marked by the epithelial cells started to scarp, accompanied by the loss widened Lacuna red blood cells and pillar cells apart. Laksman (2003) says that hyperplasia is a process of formation of excessive tissue due to the increase in cell volume. Hyperplasia caused by excessive edema so that red blood cells out of kapilernya and separated from the backers. In the event this hyperplasia Cd accumulation began at 0.6829 ppm exposure level.

Condition of cells and gill tissue had fused to lamella (Figure 2D), and began to show marked necrosis with epithelial cells in each lamella started together with epithelial cells on the other lamella, Lacuna also began to rupture causing respiratory function failure which affects the metabolism of A. woodiana. Secondary lamella fusion caused by the swelling in the cells of the gills (edema). The occurrence of secondary lamella fusion resulting in impaired function of the secondary lamella in the case of oxygen-making process and therefore contributes to the death of A. woodiana (Susilowati 2005). At a concentration of 5 ppm after 30 days A. woodiana experience death. In this incident the gills accumulate heavy metals at a concentration of 0.9280 ppm.

At that last stage of a gill would experience the highest levels of damage, this damage can lead A. woodiana to experience the death of the level of necrosis and atrophy.

Condition of cells and gill tissue necrosis and atrophy experienced (Figure 2E), characterized by the merging of each cell in lamella and lamella with bone loss starting institutions. Atrophy is a reduction (shrinking) the size of a cell, tissue, organ or body part (Harjono 1996). In this study occurred atrophy in primary lamella. Atrophy occurs due to experimental animals exposed to cadmium at high concentrations and in a long exposure time. Cells in primary lamella shrinkage (atrophy).

Laksman (2003) states that the necrosis is cellâ&#x20AC;&#x2122;s death that occurrs due to hyperplasia and excessive fusion of secondary lamella, so that the gill tissue is no longer intact form or in other words necrosis occurs accompanied with the death of a biota. In the event necrosis and atrophy of the accumulated Cd in the gills of A. woodiana started at 2.1279 ppm exposure and atrophy starting at the level of accumulation of 2.337 ppm.

bottom waters, such as A. woodiana (bivalves) will have a great opportunity for exposure to heavy metals that have been bound and form sediment.

Table 3. Changes in cellular structure mikroanatomi A. Gill woodiana after exposure to heavy metals cadmium with HE staining preparation.

Time of Fusion Concentration Hypersurgery Edema of Necrosis Atrophy (ppm) plasia (days) lamella 0 7 14 30 0,5 7 + 14 ++ + 30 +++ ++ 1 7 ++ + 14 ++ ++ ++ 30 ++++ +++ +++ 5 7 ++++ +++ + 14 ++++ ++++ +++ (dead) 30 ++++ ++++ ++++ +++ 10 7 ++++ +++ + 14 ++++ ++++ ++++ ++++ ++ (mati) 30 ++++ ++++ ++++ ++++ +++ Note: -: no change in the microanatomical structure (0%); +: there was a slight change in the microanatomical structure (1% -25%); + +: there are changes in the microanatomical structure (26% 50%); + + +: occurred many changes in the microanatomical structure (51% -75%); + + + +: there are very many changes in the microanatomical structure (76% -100%).

Gill cellular conditions experiencing edema (Figure 2B), visible basement membrane began to stretch out, the field narrowing Lacuna cell deficiency causes gill function and difficulty in breathing process, so that the metabolism of the body began to fail. Edema is swelling of the cell or excessive accumulation of fluid in body tissues (Laksman 2003). The presence of edema can cause fusion of secondary lamella of the lamella. In this study the occurrence of edema caused by the influx of Cd into the gills of A. woodiana resultied in the cell cell irritating so that the cell would swell.

The process of entry of Cd into the gills by Palar (1994), together with other metal ions and the food that has been accumulated Cd, and will form ions that can dissolve in fat. Ions were able to penetrate the gill cell membrane, so it can get into the gills, and then there will be a process of loss of volume regulation in the cell. In this treatment

A

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Figure 2. Structural changes in gill cells A. woodiana. Note: A. Tues normal gills, B. Tues gill edema, C. Tues gill hyperplasia, D. Tues fusion gill lamella, E. Tues gill necrosis.

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Microanatomical changes in kidney of A. woodiana after exposure to Cd Changes in the microanatomical structure of the kidney of A. woodiana after administration of Cd are shown in Table 4. From this table, it is known that changes in cellular structure mikroanatomi kidneys began to occur at a concentration of 0.5 ppm for 7 days, edema of the tubules begin to appear and be perfect edema at 30 days and began to show more than 25% hyperplasia. Perfect hyperplasia is shown at a concentration of 1 ppm after 14 days of inspection, then the fusion epithelium of the kidney evenly shown at concentrations of 5 ppm after 30 days of inspection.

Table 4. Changes in cellular structure of kidney mikroanatomi A. woodiana after exposure to heavy metals cadmium with haematoxylin-eosin staining preparation.

Time of surgery (Days)

Concentration (ppm) 0

7 14 30 0.5 7 14 30 1 7 14 30 5 7 14 (dead) 30 10 7 14 (dead) 30 Note: same as Table 9.

Edema

Hyperplasia

+ +++ ++++ +++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++

++ + ++++ ++++ +++ ++++ ++++ +++ ++++ ++++

Fusion of Necrosis lamella ++ +++ ++ +++ + ++++ +++ +++ ++++ +++ ++++ ++++

The kidney cells have shown complete necrosis at a concentration of 10 ppm after 30 days, while the concentration of 5 ppm to 10 ppm starting from day 14 and day of the 30th state of A. woodiana have been many who experienced the death (LC50), so the kidneys and gills partially preserved in a freezer with a temperature of -4 ° C for further examination. In Figure 2A, are shown in the picture kidney that is still in normal circumstances from the control A. woodiana.

Situation normal kidney cells and tissues in the control A. woodiana or the treatment of 0 ppm (Figure 3A), visible layer between cells in glomeruli and tubules and blood cells are still visible above and below normal. The metal accumulation in the kidney ranged from 0.0095 to 0.0242

A

B

ppm. Cd pollution levels, according to FAO (1972) still in the normal category below the threshold of fishery water quality (1 ppm), so it can be said that the content of Cd in the kidneys of A. woodiana the control is still normal. State accumulation is still at normal levels it may also occur due to A. woodiana, located diantrara aduktor posterior kidney, heart and pericardium (Suwignyo et al. 2005). Kidney position located on the inside and is relatively protected from the environment cause the accumulation of Cd is relatively small when compared to the accumulation of Cd in the gills.

In Figure 3B, indicated changes in cell structure that has undergone kidney mikroanatomi edema in all parts of the tubules to glomeruli (indicated by black color), and seems to bleed blood cells due to accumulated Cd logan continuously. In clinical edema in kidney cells caused by erasifikasi proteins in the renal tubules in the network, so that the urine comes out containing excessive protein (Anonymous 2008). In these conditions, the accumulation of Cd to the kidney began to be exposed at a concentration of 0.0200 ppm.

Then on further changes, where the higher Cd exposure then suffered kidney cell hyperplasia (Figure 3C), which is marked by the outbreak of the tubules, and the resulting mixing of intra cell with extra fluid cell, and then also in the glomerulus looks very black, because the glomerulus has accumulated more Cd long, which will result in epitelnya cells will rupture at any time. Then the blood cells were also seen indicating blackish blood has been contaminated with Cd. The range of Cd accumulation in kidney condition hyperplasia began to occur on exposure of 0.0849 ppm.

Highest level of damage to the kidney, necrosis of kidney cells have shown in Figure 3D, which has entered the stage of renal cell necrosis seen any broken tubules, glomeruli also broken so that mixed the cells with extra fluid cells, and whole blood cells were blackened due to Acute accumulation of Cd. The content of Cd in kidney like this occured at the exposure of 0.0786 ppm.

According to Atdjas (2008) accumulated Cd at the highest level will cause some kidney disorder that is poisoning the nephrons of the kidney (nephrotoxicity), proteinuria or protein in the form contained in the urine, diabetes where there is the content of glucose in the urine (glikosuria), and aminoasidiuria or amino acid content in the urine accompanied by a decline in kidney filtration rate glumerolus.

C

D

Figure 3. Structural changes in renal cell woodiana. Notes: A. Tues normal gills, B. Tues gill edema, C. Tues fusion gill lamella, D. Tues gill necrosis

FITRIAWAN et al. â&#x20AC;&#x201C; Effect of Cd on freshwater mussels A. woodiana

CONCLUSION There is significant heavy metal accumulation of Cd in each treatment against the gill and kidney A. woodiana as evidenced by the Anova test data for 475.3> 60150.3 0.000 and> 0.000 with an average significance level of 5% (P <0.05). There are structural changes in the kidneys marked by microanatomical forms which were edema, hyperplasia, fusion of lamella and necrosis, whereas in the kidney in proved by the occurrence of edema, hyperplasia and necrosis of the tubules, glomeruli and mineralization in the blood cells to bleed.

REFERENCES Ardi 2002. Utilization of macrozoobenthos as an indicator of the quality of coastal waters. Science philosophy paper. School of Graduates BAU. Bogor. [Indonesia] Atdjas D. 2008. Impact of cadmium (Cd) levels in green mussel body (Perna viridis) in Muara Karang estuary pond area Jakarta on human health. Graduate Program, ITS. Surabaya. [Indonesia] Budiono A. 2003. Effect of mercury pollution on water biota. Science philosophy paper. School of Graduates, Bogor Agricultural University. Bogor. [Indonesia] Connel DW, Miller GJ. 1995. Chemistry and ecotoxicology pollution. UI Press. Jakarta. [Indonesia] Darmono S. 1995. Metals in biological systems of living things. UI Press.

Jakarta. [Indonesia] Destiany M. 2007. The effect of mercury chloride on the microanatomical structure of carp liver. [Thesis S1]. Biology Department, FMNS, State University of Semarang. Semarang. [Indonesia] EPA. 1986. Quality criteria for water. EPA Standart 440/5-86-001.

Washington DC. FAO 1972. Food compotition table for use in East Asia; food policy and nutrition division. FAO. Rome.

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Harjono RM, Hartono A, Surya S. 1996. Dorlandâ&#x20AC;&#x2122;s medical dictionary.

EGC. Jakarta. [Indonesia] Kraak MH, Lavy D, Peeters WH, David C. 1992. Chronic ecotoxicity of copper and cadmium to the zebra mussel Dreissena polymorpha. Arc Environ Contam Toxicol 23 (3): 363-369.

Nontji A. 1984. Biomass and productivity of phytoplankton in the waters of Jakarta Bay and its relation to environmental factors.

[Dissertation]. Graduate Faculty, Bogor Agricultural University.

Bogor. [Indonesia] Odum EP. 1993. Fundamentals of ecology. GMU Press. Yogyakarta.

[Indonesia] Palar H. 1994. Contamination and toxicology of heavy metals. Rineka Cipta. Jakarta. [Indonesia] Indonesian Government Regulation No. 82 of 2001 (PP No. 82/2001) on Water Quality Management and Control of Water Pollution.

[Indonesia] Rahman A. 2006. The content of heavy metals lead (Pb) and cadmium (Cd) on some species of crustacean on the beach Batakan and Takisung, Tanah Laut district, South Kalimantan. Bioscientiae 2 (3): 93-101.

Soegianto A, Primarastri NA, Winarni D. 2004. The effect of cadmium on the survival rates and damage to gill structure and hepatopankreas on regang shrimp [Macrobrachium sintangense (de Man)]. Berk Penel Hayati 10: 59-66. [Indonesia] Sunarto 2007. Bioindicator of heavy metal pollutant cadmium (Cd) with microanatomical structural analysis, the efficiency of gill function, morphology and condition of shells of freshwater mussels (Anodonta woodiana Lea). [Dissertation]. Airlangga University. Surabaya.

[Indonesia] Susilowati E. 2005. Acute effect of administration of cadmium to milkfish gills microanatomical structure. [Thesis S1]. State University of Semarang. Semarang. [Indonesia] Takashima F, Hibiya T. 1995. An atlas of fish histology normal and pathological features. 2nd ed. Kodansha. Tokyo.

Tresnati J, Djawad MI, Bulqys AS. 2007. Kidney damage of ikan pari kembang (Dasyatis kuhlii) caused by lead heavy metals (Pb). J Sains Teknol 7 (3): 153-160. [Indonesia] Warlina L. 2004. Water pollution: sources, impacts and mitigation.

Science philosophy paper. School of Graduates, Bogor Agricultural University. Bogor. [Indonesia]

ISSN: 2087-3940 (print) ISSN: 2087-3956 (electronic)

Vol. 3, No. 1, Pp. 36-43 March 2011

Site suitability to tourist use or management programs South Marsa Alam, Red Sea, Egypt

1

MOHAMMED SHOKRY AHMED AMMAR1,♥, MOHAMMED HASSANEIN2, HASHEM ABBAS MADKOUR1, AMRO ABD-ELHAMID ABD-ELGAWAD2

National Institute of Oceanography and Fisheries (NIOF), Suez, P.O. Box 182, Egypt. Tel. (Inst.) 0020 62 3360015. Fax. (Inst.) 0020 62 3360016. ♥ Email: shokry_1@yahoo.com 2 Tourism Development Authority, Cairo, Egypt Manuscript received: 3 February 2011. Revision accepted: 3 March 2011.

Abstract. Ammar MSA, Hassanein M, Madkour HA, Abd-Elgawad AE. 2011. Site suitability to tourist use or management programs South Marsa Alam, Red Sea, Egypt. Nusantara Bioscience 3: 36-43. Twenty sites in the southern Egyptian Red Sea (Marsa Alam-Ras Banas sector) were surveyed principally for sensitivity significance throughout the periode 2002-2003. Sensitivity of the study area was derived from internationally known criteria, the key words of each criterion and a brief description of its use was described. The present study assigned for the first time a numerical total environmental significance score that gives a full sensitivity significance evaluation for any site to decide to select either for tourist use or management purposes. However, the results of the study still have the availability to arrange sites with respect to one criterion or only two or many of the used criteria whichever needed. Sites selected for protection are categorized as belonging to the following protected area categories: sites 7, 10 (category vi), site 18 (category ib), site 5 (category iv), sites 16, 17 (category ii). Sites selected for tourist uses are suggested to be classified into 2 categories: first category sites (sites 1, 3, 8, 11, 13, 15) which are recommended as tourist use sites with management of the sensitive resources beside non consumptive recreational activities like swimming, diving, boating, surfing, wind-surfing, jet skiing, bird watching, snorkelling, etc.; second category sites (sites 2, 4, 6, 9, 12, 14, 19, 20) which are recommended as tourist use sites with both non consumptive and managed consumptive recreational activities like fishing.

Key words: sensitivity significance, selection criteria, tourist use, management programs, Marsa Alam, Red Sea, Egypt

Abstrak. Ammar MSA, Hassanein M, Abd-Elmegid AE. 2011. Kesesuaian untuk lokasi wisata atau program manajemen Marsa Alam Selatan, Laut Merah, Mesir. Nusantara Bioscience 3: 36-43. Dua puluh situs di Laut Merah bagian selatan Mesir (sektor Marsa AlamRas Banas) disurvei terutama untuk signifikansi sensitivitas sepanjang periode 2002-2003. Sensitivitas suatu daerah penelitian merupakan kriteria yang dikenal secara internasional, kata kunci setiap kriteria dan deskripsi singkat tentang penggunaannya dijelaskan.

Penelitian ini dilakukan untuk pertama kalinya berupa skor nilai total signifikansi lingkungan yang memberikan arti evaluasi sensitivitas penuh untuk situs apapun untuk memutuskan memilih baik untuk tujuan wisata atau tujuan pengelolaan lainnya. Namun, hasil penelitian ini masih memiliki ketersediaan untuk mengatur situs-situs yang berkaitan dengan satu kriteria, hanya dua atau banyak dari kriteria yang digunakan mana yang diperlukan. Situs dipilih untuk perlindungan dikategorikan sebagai milik kategori kawasan lindung sebagai berikut: situs 7, 10 (kategori vi), situs 18 (kategori ib), situs 5 (kategori iv), situs 16, 17 (kategori ii). Situs yang dipilih untuk keperluan wisatawan disarankan harus diklasifikasikan menjadi dua kategori: situs kategori pertama (situs 1, 3, 8, 11, 13, 15) yang direkomendasikan sebagai situs menggunakan wisata dengan manajemen sumber daya sensitif di samping kegiatan rekreasi non konsumtif seperti berenang, menyelam, berperahu, berselancar, wind-surfing, jet ski, mengamati burung, snorkeling, dan lain-lain; situs kategori kedua (situs 2, 4, 6, 9, 12, 14, 19, 20) yang direkomendasikan sebagai tempat wisata baik kegiatan non konsumtif atau kegiatan rekreasi non konsumtif yang dikelola seperti memancing.

Kata kunci: signifikansi sensitivitas, kriteria seleksi, kegunaan wisata, program manajemen, Marsa Alam Selatan, Laut Merah

INTRODUCTION South Marsa Alam’s diverse coastal and marine environments are valuable community resource which may be good sites providing recreation and pleasure for visitors and tourists or scientific materials for scientists to do monitoring and conservation programs. There is no getting around the fact that tourism is huge, already categorized as the world’s largest industry and will continue to be the dominant developing force in the 21st century (Hill 1998).

As environmental conservation and protection is critically

important in some sites, sustainable tourism is critically important as well since it may provide source of finance for parks and conservation, serve as an economic justification for park protection, offer local people economically sound and sustainable alternatives to natural resource depletion or destruction, promote conservation and build support with commercial constituencies (Hawkins 1998).

Tourist uses includes a diversity of activities that take place in both coastal zone and coastal waters (Watson et al.

2000), which involve the development of tourism capacities (hotels, resorts, second homes, restaurants, etc.)

AMMAR et al. â&#x20AC;&#x201C; Tourist and management of South Marsa Alam, Egypt

and support infrastructures (ports, marinas, fishing, diving shops and other facilities). Coastal recreation activities include two main types: consumptive and non-consumptive ones: Activities such as fishing, shell fishing and shell collection, etc. belong to the consumptive recreational uses while the non consumptive activities include swimming, diving, boating, surfing, wind-surfing, jet skiing, bird watching, snorkelling, etc (Porter and Bright 2003). Tourist uses is based on a unique resource combination at the interface of land and sea offering amenities such as water, beaches, scenic beauty, rich terrestrial and marine biodiversity, diversified cultural and historic heritage, healthy food and good infrastructure.

Management programs are the programs that are used for preserving an area to provide lasting protection for part or all of the natural marine environments therein. IUCN (1994) defined the protected area as an area of land and/or sea especially dedicated to the protection and maintenance of biological diversity, and of natural and associated cultural resources, and managed through legal or other effective means. To help improve understanding and promote awareness of protected area purposes, IUCN has developed a six category system of protected areas identified by their primary management objective (IUCN 1994) as follows: I. Strict Nature Reserve/Wilderness Area: Protected area managed mainly for science or wilderness protection.

Ia. Strict Nature Reserve: Protected area managed mainly for science.

Ib. Wilderness Area: Protected area managed mainly for wilderness protection.

II. National Park: Protected area managed mainly for ecosystem protection and recreation.

III. Natural Monument: Protected area managed mainly for conservation of specific natural features.

IV. Habitat/Species Management Area: Protected area managed mainly for conservation through management intervention.

V. Protected Landscape/Seascape: Protected area managed mainly for landscape/seascape conservation and recreation.

VI. Managed Resource Protected Area: Protected area managed mainly for the sustainable use of natural ecosystem Carrying capacity is important to discuss on dealing with coastal sustainable tourism. The term "carrying capacity" is the number of organisms the resources of a given area can support over a given time period (MPA NEWS 2004). Adapted to tourism management, it has a similar meaning: the number of people who can use a given area without an unacceptable alteration in the physical environment. Carrying capacity can differ from site to site.

Dixon et al. (1994), on analyzing coral cover, they estimated that the diver carrying capacity threshold for the Bonaire Marine Park is between 4000 and 6000 dives per site per year. Surveying the percent of damaged coral colonies in the Red Sea Ras Mohammed National Park, Hawkins and Roberts (1997) suggest 5000 to 6000 dives per site per year in the absence of a site specific data.

Sampling a suite of invertebrates (hard corals, soft corals,

37

sea fans, branching hydrocorals, and erect sponges), Chadwick-Furman (1996) found the threshold for diving sites in the US Virgin Islands to be only 500 dives per site per year and attributed this significantly lower estimate to the fragility of the various reef organisms in the study area.

However, effective diver education programs can allow coral reef managers to increase carrying capacities (Medio et al. 1997). Mooring buoys and the management of the number of vessels using mooring buoys with respect to time and location are other effective tools coral reef managers use in reducing the anchor and diver damage to coral reefs.

The purpose of this study is not to replace existing criteria with a new set, but to use existing frameworks for site selection to classify south Marsa Alam sites either for tourist use or management programs in order to assign sites either to EEAA (Egyptian Environmental Affairs Agency) for management purposes or to TDA (Tourism Development Authority) for tourist uses. It is also aimed to develop a total numerical value of sensitivity significance (by scoring and summing techniques) that can be used for site selection (tourist use or management programs), then using single criteria scoring for a particular management or tourist use.

MATERIALS AND METHODS Twenty sites in the southern Egyptian Red Sea (between Marsa Alam and Ras Banas) were surveyed principally for sensitivity significance. The survey was conducted throughout the periode 2002-2003. The sites were determined by fixing more or less equal distances between them, however determining the position of sites was done during the preliminary survey. The area of study is shown in Figure 1.

The ecological survey was performed using Scuba diving. For corals and other benthic fauna and flora, the transect line method applied by Rogers et al. (1983) was used by using a 30 m long tape for surveying the percent cover. The intercepted lengths of every individual coral and any other benthic organism or habitat were measured; these lengths are then used to calculate the percent cover using the formula: % cover = (intercepted length/transect length) * 100 Three transects were used per depth zone and the average was calculated for all transects.

For fishes, the stationary fish census applied by Bohnsack and Bannerot (1986) was used by using a 50 m long transect for the survey. Transects were laid parallel to the shore at 4 m depth in the deep reefs or just above the reef patch in case of the patchy reefs. The survey was basically done at 4m depth since it is the area of maximum fish abundance.

Sensitivity significance of the study area is derived from internationally known criteria, however the key words of each criterion and a brief description of its use can be described as follow: Diversity (Ratcliffe 1977; IEEM 2006): large numbers of species, particularly when represented by large popu-

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3 (1): 36-43, March 2011

lations are to be valued. A high species diversity is usually also reflected by a high diversity of different communities which show variation in environmental conditions.

Rarity (Tubbs and Blackwood 1971; Wittig et al. 1983; Edwards-Jones et al. 2000): Applied to habitats or species where areas are limited, population numbers low or the habitat or species limited in distribution.

Fragility (Ratcliffe 1977; IEEM 2006): Habitats or species vulnerable to disturbance and loss because of small area, low population or reliance on a single key resource.

Ecological functions (IEEM 2006): Loss of ecological function of the physical conditions can be measured by calculating the area of vegetation that is removed or the area of nearshore habitat that is covered by the pier structure.

Typicalness (Fandiño 1996; Edwards-Jones et al.

2000): A measure of how well a site reflects all the habitats that are expected to occur in that geographical region.The more representative a site is of a region, the better.

Naturalness (Ratcliffe 1977; IEEM 2006): Habitats largely unmodified by human activity (e.g. salt marsh, blanket bog).

Scientific value (Wright 1977, Edwards-Jones et al.

2000): The degree of interest of a natural area in terms of current or potential research. It may also be related to the extent to which a site has been used for past research. Sites with good histories (e.g., description of ecosystems’ dynamics in the past 50 years) are more valuable to science because they enhance our understanding of ecology Environmental significance (IEEM 2006): Significance of the site to the environment where that significance is global, natural or local Scenic value (Ratcliffe 1977): The combination of landforms and habitats is identified as having high scenic value in the context of surrounding landscape Size (Ratcliffe 1977; IEEM 2006): In general, nature conservation value increases with size. Large sites in general contain more species and larger populations of animals and plants than small ones. Chance extinction of species, either as a result of natural or man-made factors, is reduced if a species is present in large numbers.

10, so 0.2% rare biota or habitats = an estimated score of 2 (0.2*10) and so on.

Fragility: Each 1% fragile habitats (nesting, feeding, breeding), relative to the total cover, was given an optimal score of 10, so each 0.3% fragile habitats = an estimated score of 3 (0.3*10) and so on.

Ecological function: Each 6.66% vital ecological function (vegetation or habitats not removed by physical conditions) was assigned a score of 1 (6.66/6.66), thus a vital ecological function of 26.64% will have an estimated score of 26.64/6.66 = an estimated score of 4 and so on.

Typicalness: A site representing 80% of the number of the characteristic ecosystems of a geographical area was assigned a score of 10% (80/8), thus a site having 24% characteristic ecosystems will have an estimated score of 24/8=3% and so on.

Naturalness: A 10% virgin area (with no human caused alteration) was assigned a score of 1 (=10/10), thus a 30% virgin area has an estimated score of 30/10=3 and a virgin area of 50% has an estimated score of 50/10=5 and so on.

Scientific value: A site used for scientific research for the past 10 years was assigned a score of 1 (=10/10), thus a site used for the past 30 years will have an estimated score of 3 (=30/10), a site used for the past 50 years will have an estimated score of 5 (=50/10) and so on.

Environmental significance: Global significance was assigned a score of 3, each of national and local significance was given a score of 1.

Scenic value: Scenic value of the landscape depends on the value of the following dimensions: 1-visual dimension 2-geology 3-topography 4-soils 5-ecology 6-landscape history 7-Anthropology 8-architecture 9-culture associations 10-public places. A site that fulfil the scenic value with respect to those 10 items was assigned a score of 5 (=10/2), thus a site that fulfil 4 items will have an estimated score of 4/2=2, a site that fulfil 2 items will have an estimated score of 2/2=1 and so on.

Size: Each 5000m2 habitats was assigned a score of 1 (=5000/5000), so a size of 10000m2 will have an estimated score of 2 (10000/5000) and so on.

Estimating sensitivity significance (developed by the author) An optimal sensitivity score (the optimal score) was supposed for each criterion; this was the score at which the site could be optimal. In addition, an estimated score was assigned to each crierion depending on how much the site meets the conditions of the optimal score, then all sensitivity scores for each site were summed to get the total sensitivity significance. Methods of how values have been assigned to each site per each criterion is described as follows (developed by the author) (Table 1).

Diversity: Diversity value of 1 (according to ShannonWiener 1948 formula) was assigned a sensitivity significance score of 5, so .diverrsity value of 1.2 = estimated score of 6 (1.2*5) and so on.

Rarity: Each 1% of rare biota, relative to the total abundance, was assigned a sensitivity significance score of

List of sites and their positions Site 1. Marsa Nakry: 24o55`35.476”N, 34o57`40.993”E Site 2. between Marsa Nakry and Gabal Dorry: 24o54`36.428”N, 34o58`25.453”E Site 3. 1 km south of Gabal Dorry: 24o47`33.942”N, 34o59` 14.139”E Site 4. South Host Mark: 24o 47`33.942”N, 35o01`58.197”E Site 5. Northern Sharmel Fokairy: Transect 1: 24o45`16.192”N, 35o03`55.792”E Transect 2: 24o45`22.126”N, 35o03`50.218” E Site 6. Southern Sharmel Fokairy: 24o38`20”N, 35o04`51” E Site 7. Sha’b North Ras Baghdadi:24o40`25``N,35 o05`38``E Site 8. Northern Ras Baghdadi: 24o40`05.900”N, 35o05`52.625”E Site 9. Southern Ras Baghdadi: 24o39`16.800”N, 35o05`54.200”E Site 10. North Sharmel Loly: 24o36`50.460”N, 35o06`59.248”E

AMMAR et al. – Tourist and management of South Marsa Alam, Egypt

Site 11. Southern Sharmel Loly: 24o36`39.2666”N, 35o07`08.795”E Site 12. North Hankourab: 24o34`49.624”N, 35o08`40.185”E Site13. South Hankourab: 24o33`23.20”N, 35o09`02.405”E Site 14. North Ummel Abas: 24o30`44.200”N, 35o08`16.927”E Site 15. Middle Ummel Abas: 24o30`46.024”N, 35o08`16.300”E Site 16. South Ummel Abas: 24o30`24.642”N, 35o08`31.717”E Site 17. Wadi El-Mahara: 24o24`27.674”N, 35o13` 41.471” E Site 18. a mangroove area: 24o16`32.400”N, 35o3`15.815”E Site 19. South Hamata city: 24o 16` 32.400” N, 35o23` 15.815” E Site 20: Lahmy; South El-Gharabawy: 24o12`09.494”N, 35o25`37.744”E

39

RESULTS AND DISCUSSION Site priorities for management and protection Dealing with the total assigned value of sensitivity significance and considering sensitivity significance score ≥ 50 to be suitable for management purposes, the following site priorities are suggested for management purposes: sites 10, 7, 18, 17, 5 and 16 having significant scores of 86, 77, 73, 61, 57 and 54 respectively. However, dealing with each criterion separately, site 10 has first priority for managing diversity and rarity; site 18 for fragility; sites 7, 10, 18 for ecological functions, scientific value, and environmental significance; sites 7, 10 for typicalness and size; site 10 for naturalness; site 18 for scenic value. Moreover, if we used many few of the used criteria, we'll have different site priorities according to the criteria selected for comparison.

1 23 45 67 8 9 10 11 12 13 14 15 16 17 18 19 20

Figure 1. Location of the study area South Marsa Alam (from Marsa Alam to Ras Banas) on the Egyptian Red Sea.

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3 (1): 36-43, March 2011

Also, choosing a higher number of criteria used for comparison gives rise to shefting the priority into the site that is appropriate to most of the used criteria.

Site priorities for tourist uses Acoording to the total assigned sensitivity significance and considering a sensitivity significance score < 50 to be suitable for touristic use, 16 sites were selected are suggested for touristc uses. As many of these sites have some sensitive resources, these sites are suggested to be divided into two categories: first category sites includes sites with some sensitive resources and with 30 â&#x2030;¤ sensitivity significance < 50, second category sites include sites without sensitive resources and with sensitivity significance < 30. First category sites are sites 1, 3, 8, 11, 13, 15. Second category sites are sites 2, 4, 6, 9, 12, 14, 19, 20. Sensitive habitats for first category sites are rarity for site 1, diversity and rarity for site 3, fragility for site 8, diversity for site 11 and typicalness for site 13.

Table 1. Environmental sensitivity of each of the studied sites Zone Site 1 Site 2 Site 3 Site 4 Site 5 Site 6 Site 7 Site 8

Site description Site 1 (Marsa Nakry) is characterized by a 95% degraded reef flat, 38% dead corals with increase in the hydrocoral Millepora dichotoma at 1-5m zone. However, one threatened manta ray (Taenura lymma) was observed. Site 2 is very poor and far from the shore with increased algae, sands and dead corals. Site 3 has 45% dead corals and one threatened stingray (Taenura lymma). Site 4 is dominated with algae while site 5 is characterized by two main ecosystems: a coral reef ecosystem and a seagrass ecosystem, 38.5% live corals, 66.5% dead corals and one threatened threatened Manta Ray (Himantura uarnak). Site 6 has the shoreline heavily condensed with quite a lot amount of plastic bags, glass and plastic bottles, wood pieces, steel pieces, robes, old shoes, small and big canes with a very poor marine life. Site 7 has 65% live corals, 14% dead corals and one endangered reptile, (the green turtle Chelonia mydas. Site 8 has 20% live corals and 55% dead corals, site 9 has 46.5% live corals and 50% dead corals while site 10 has 91% live corals and 5% dead corals. Sites 11 and 12 have a poor marine life except some algae and spots of corals while site 13 is a clean sandy beach with few small coral patches having 47% dead corals and 35% live corals. Site 14 is mostly sand with few patches of algae, sea grasses and a well developed reef. Site 15 has a seagrass patch, a small reef patch suffering from old dynamite fishing and a lot of deep crab niches on the shoreline while site 16 has a fringing reef, a patch reef and a barrier reef with a seagrass bed in between. Site 17 has a a

degraded reef flat, with 40% live corals, 30% dead corals with juveniles of the threatened organ pipe coral Tubipora musica attaching rocks, dead corals and rubble on the reef crest, fishes were mostly of large sizes. while site 18 has heavily condensed mangrove trees on both land and water

Site 9 Site 10 Site 11 Site 12 Site 13 Site 14 Site 15 Site 16 Site 17 Site 18 Site 19 Site 20

Div (15) 1 5 1.2 6 1.6 8 0.4 2 1.2 6 1 5 2.4 12 1.4 7 1 5 3 15 1.4 7 0.6 3 1 5 0.6 3 1.2 6 1.6 8 1.4 7 2.6 13 1.6 8 0.8 4

Rar (15) 1 10 0.2 2 1 10 0 0 0.9 9 0.2 2 1 10 0 0 0 0 1.4 14 0.4 4 0 0 0.6 6 0.1 1 0.3 3 0.3 3 1.3 13 0.4 4 0 0 0 0

Frag (15) 0.3 3 0.2 2 0.3 3 0 0 1 10 0.2 2 0.9 9 0.9 9 0 0 1 10 0.4 4 0 0 0.5 5 0.3 3 0.9 9 1 10 0.9 9 1.3 13 0 0 0 0

EcFu (15) 26.64 4 13.32 2 19.98 3 6.66 1 59.94 9 6.66 1 86.58 13 39.96 6 33.3 5 86.58 13 26.64 4 0 0 39.96 6 19.98 3 59.94 9 79.92 12 66.6 10 86.58 13 0 0 0 0

Typ (10) 24 3 24 3 24 3 0 0 64 8 0 0 80 10 48 6 32 4 80 10 24 3 16 2 40 5 24 3 24 3 48 6 48 6 64 8 24 3 16 2

Nat (10) 30 3 30 3 30 3 0 0 50 5 30 3 70 7 30 3 20 2 80 8 30 3 30 3 30 3 10 1 30 3 20 2 40 4 60 6 40 4 30 3

SciVa (5) 30 3 20 2 20 2 10 1 30 3 20 2 40 4 30 3 10 1 40 4 30 3 20 2 20 2 10 1 20 2 30 3 30 3 40 4 40 4 20 2

EnSi SceVa Size (5) (5) (5) 4 3 2 2 4 2 2 4 4 2 2 4 4 2 2 1 4 2 2 3 2 1 1 2 6 4 3 5 4 3 2 3 2 1 1 3 6 4 3 5 4 3 2 3 6 2 3 3 4 2 2 2 2 1 1 1 4 2 2 2 6 3 3 4 4 3 2 4 8 4 4 4 4 2 2 3 2 2 1 2

Tot (100) 38 28 40 9 57 19 77 42 22 86 36 18 38 18 41 54 61 73 26 16

Note: Div = diversity, Rar = rarity, Frag = fragility, EcFu = Ecological function, Typ = typicalness, Nat = naturalness, SciVa = scientific value, EnSi = environmental significance, SceVa = scenic value, Tot = total. Site 2 = between Marsa Nakry and Dorry. Values in parenthesis are the optimum score for each criterion. Diversity: upper value in the table is the Shannon estimate of diversity, lower value is the estimated score. Rarity: upper value in the table is the percent rare biota or habitats, lower value is the estimated score. Fragility: upper value is the percent fragile habitats, lower value is the estimated score. Ecological function: upper value is the percent non removed vegetation or habitats, lower value is the estimated score. Typicalness: upper value is the percent of characteristic ecosystems of a geographical area, lower value is the estimated score. Naturalness: upper value is the percent of area of no human caused alteration, lower value is the estimated score. Scientific value: upper value is the number of past years the site has been used for scientific researches, lower value is the estimated score. Environmental significance: values in the table are the estimated scores. Scenic value: upper value is the number of items the site fulfil for scenic value, lower value is the estimated score. Size: values in the table are the estimated scores.

AMMAR et al. â&#x20AC;&#x201C; Tourist and management of South Marsa Alam, Egypt

beside having seagrasses (10% of the bottom cover). Site 19 is characterized by a dirty shoreline full of plastic bags, robs, bottles and canes with a very poor reef while site 20 is a typical example of sandy beach having also a trace of an old reef completely degraded and buried with sand.

Approaching a total mathematical sensitivity significance score Evaluation of sensitivity significance criteria in the previous studies dealt just phonetically with each criterion separately like for example Ratcliffe (1977), IEEM (2006) for evaluation of diversity as high, medium or low, fragility as reversible or irreversible, naturalness as virgin, semivirgin or altered, size as large, medium or small. Other criteria were phonetically evaluated like Tubbs and Blackwood for evaluation of rarity; IEEM (2006) for ecological functions; FandiĂąo (1996) and Edwards-Jones et al. (2000) for typicalness; Wright (1977) and EdwardsJones et al. (2000) for scientific value; IEEM (2006) for environmental significance; Ratcliffe (1977) for scenic value. Such phonetic evaluation can only deal with each criterion separately making it difficult to compare several sites for a group of criteria together, in turn making it difficult to arrange those group of sites according to their importance with respect to several criteria. The present study solved that problem by assigning for the first time a numerical score for each criterion (explained in the material and methods section), then summing all mathematical scores to give a total sensitivity significance score. However, the study still has the availability to arrange the sites with respect to one criterion or only two or many of the used criteria whichever needed according to the management purpose. Although Croom and Crosby (1998) mentioned that scoring and summing techniques was used to minimize the personal bias, he used scoring and summing techniques with respect to only one separate criterion e.g. rarity. Approaching a total sensitivity significance score in the present study is important to select a site that is much appropriate with most of the used criteria. Salm and Clark (1984) and Ray and Legates (1998) expected that extremely complicated scoring and summing techniques may seem the most objective and defensible way to choose a priority site. They further related the reason of using a simple assessment system to the fact that it is easier to use, requires fewer resources and can be evaluated by a diverse group of individuals with varying levels of expertise.

Site priorities for management purposes Since priorities for site selection with respect to a single criterion differ from those given on using another criterion and from those given on using the total sensitivity significance; it is important, after selecting sites for management purposes, to use the appropriate criterion for selecting the appropriate site for the appropriate management. Parkes (1990) favoured the rating of individual assets, but differed in how multiple values at a site should be reconciled. He suggested that, where a site has several assets of varying levels of biological significance, the site rating should be based on the value of

41

the dominant asset at the site, or the majority of assets at the site. Selection criteria can be used to order candidate sites according to priority in the selection process (Nilsson 1998). However, the present study has been directed mainly to solve the struggle between EEAA (Egyptian Environmental Affairs Agency) and TDA (Tourist Development Authority) for attaining as many sites as possible to EEAA for management purposes or to TDA for tourist uses. Therefore, it was important to think in developing a numerical total environmental significance score by which we can decide either to assign the site for EEAA or for TDA. Latimer (2009) stated that the use of precise numerical criteria, or indices for the evaluation of size, diversity or rarity could provide a guideline reference scale, he further mentioned that professional judgement is also important. According to the purpose of the study and considering a total sensitivity significance â&#x2030;Ľ50 to be significant and appropriate for assigning the site for management (protection) purposes, priorities of site selection assigned for management purposes are site 10, site 7, site 18, site 17, site 5 and site 16, other sites are assigned for touristic uses.

Categorization, carrying capacity and management objectives of sites selected for management purposes Although sites 7 and 10 have high sensitivity significance with respect to all criteria, they are recommended as managed resource protected areas (category VI) since they contain fishing communities and fishing activities. It is important to sustain fishery resources by restricting fishing activities seasonally or temporarily to let the areas recover. Areas managed to sustain fisheries are very rarely promoted to MPAs, but there are exceptions like the fish habitat reserves in Australia. Site 18 having the highest sensitivity significance with respect to fragility and ecological functions, and being inhabited with mangrove trees, is recommended as wilderness area (category Ib) which is managed mainly for wilderness protection. Sites 7, 10 and 18 having fragile habitats should have a diver carrying capacity threshold of 500 dives per site per year according to Chadwick-Furman (1996). However, site 5 has considerable sensitivity significance with respect to fragility and ecological functions, being inhabited with the fragile seagrasses, it is recommended as habitat/species management area (protected area, category IV). Similar to sites 7, 10, 18; site 5 should have a diver carrying capacity of 500 dives per site per year. Sites 16 and 17 though having considerable sensitivity significance with respect to diversity, rarity, fragility, ecological functions and typicalness, they are recommended as national park (protected areas, category II) since they have a significant size which will increase their diver carrying capacity so as to tolerate recreation. According to Dixon et al. (1994) in Bonaire Marine Park and Hawkins and Roberts (1997) in Ras Mohammed National Park, sites 16 and 17 should have a diver carrying capacity of 4000-6000 dives per site per year. A matrix of management objectives in the sites assigned as protected areas are explained (Table 2) according to IUCN (1994).

42

3 (1): 36-43, March 2011

Table 2. Management objectives of sites selected for management purposes Sites 7, 10 Site 18 Site 5 Category VI Category Ib Category IV Scientific research 3 3 2 Wilderness protection 2 1 3 Preservation of species and genetic diversity (biodiversity) 1 2 1 Maintenance of environmental services 1 1 1 Protection of specific natural / cultural features 3 – 3 Tourism and recreation 3 2 3 Education 3 – 2 Sustainable use of resources from natural ecosystems 1 3 2 Maintenance of cultural/traditional attributes 2 – – Note: 1 = Primary objective; 2 = Secondary objective; 3 = Potentially applicable objective; – = not applicable.

Management objective

Site priorities for tourist uses Sites classified as first category sites (sites 1, 3, 8, 11, 13 and 15) are recommended as tourist use sites with management of the sensitive resources and non consumptive recreational activities like swimming, diving, boating, surfing, wind-surfing, jet skiing, bird watching, snorkelling, etc. Locations of recreational activities could have a carrying capacity of up to 6000 dives per site per year (Roberts 1997) while in the sensitive locations, it should not exceed 500 dives per site per year (ChadwickFurman 1996). However, effective diver education programs can allow coral reef managers to increase carrying capacities (Medio et al. 1997), also mooring buoys and the management of the number of vessels using mooring buoys with respect to time and location are other effective tools coral reef managers use in reducing the anchor and diver damage to coral reefs. Management of sensitive habitats in first category of tourist use sites includes protection of rarity for sites 1, diversity and rarity for site 3, fragility for site 8, diversity for site 11 and typicalness for site 13. Second category sites (sites 2, 4, 6, 9, 12, 14, 19 and 20) are recommended as tourist use sites with non consumptive and managed consumptive recreational activities like fishing. Diver carrying capacity of these sites could approach 6000 dives per site per year.

Site 4 having the lowest sensitivity significance and most minimum values with respect to every sensitivity criterion is suggested to allocate a part of it for building an artificial reef to restore the damaged ones (Ammar 2009a).

Site description Damaged reef flat in site 1 is due to the absence of reef access points to deep water. Ammar (2009b) indicated the importance of reef access points in his assessment of some coral reef sites along the Gulf of Aqaba, Egypt. Increased algae and sands in site 2 with increased dead corals agree with Pearson (1981) and Nezali et al. (1998) that algae are among the most important factors which can influence coral recolonization. The high percentage cover of the hydrocoral Millepora dichotoma at 1-5m depth in Marsa Nakry as well as in other sites having that species, agrees with the finding of Ammar (2004) that, Millepora sp. (a hydrocoral) prefers high illumination and has a strong skeletal density to tolerate strong waves. The relatively low sensitivity significance in spite of the presence of the

Sites 16, 17 Category II 2 2 1 1 2 1 2 3 –

threatened species (the blue spotted stingray Taenura lymma) in sites 1 and 3, indicates the importance of using a particular criterion when dealing with a particular management purpose. The green turtle Chelonia mydas found in site 7 is categorized as a taxon having an observed, estimated, inferred or suspected reduction of at least 80% over the last 10 years or three generations, whichever is the longer (IUCN 2002). The lower recorded amount of dead corals in site 10 (Sharm El Loly) though it is highly used by fishing boats, is due to the fact that these boats anchor on the inlet terminal, away from the reef and go to open water through the middle of the inlet. Reporting juveniles of the vulnerable organ pipe coral Tubipora musica in site 17 (Wadi El-Mahara) is the reason of increased sensitivity significance with respect to rarity in that site. Ammar (2005) categorized the organ pipe coral Tubipora musica as vulnerable according to IUCN (2001), as there is an estimated population size reduction of ≥ 50% over the last 10 years, based on the index of abundance and the decline in area of occupancy. Site 18 having a mangrove ecosystem, a seagrass ecosystem and a coral reef ecosystem integrating together helped to increase most of the selection criteria, in turn increasing the overall sensitivity significance. Broody (1998) stated that selection criteria help to provide a rational basis for choosing among potential sites.

CONCLUSIONS The present study approached for the first time a numerical total sensitivity significance score for each site to select a site that is much appropriate with most of the used criteria. This is important to classify a group of sites to be suitable either for tourist use or management purposes. Since priorities for site selection differ from one sensitivity criterion to the other and from the total sensitivity significance, it is important, after selecting a site for management (using the total sensitivity significance), to specify the appropriate criterion for deciding the appropriate management purpose per site. Sites selected for management (protection) purposes are categorized as belonging to the following protected area categories: sites 7, 10 (category vi), site 18 (category ib), site 5 (category iv), sites 16, 17 (category ii). Sites selected for tourist uses are classified into 2 categories: 1- First category sites (sites

AMMAR et al. – Tourist and management of South Marsa Alam, Egypt

1, 3, 8, 11, 13, 15) which are recommended as tourist use sites with management of the sensitive resources and non consumptive recreational activities like swimming, diving, boating, surfing, wind-surfing, jet skiing, bird watching, snorkelling, etc. 2- Second category sites (sites 2, 4, 6, 9, 12, 14, 19, 20) which are recommended as tourist use sites with non consumptive and managed consumptive recreational activities like fishing.

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ISSN: 2087-3940 (print) ISSN: 2087-3956 (electronic)

Vol. 3, No. 1, Pp.: 44-58 March 2011

Review: Natural products from Genus Selaginella (Selaginellaceae) AHMAD DWI SETYAWANâ&#x2122;Ľ Department of Biology, Faculty of Mathematics and Natural Sciences, Sebelas Maret University, Surakarta 57126. Jl. Ir. Sutami 36A Surakarta 57126, Tel./fax. +62-271-663375, email: volatileoils@gmail.com Manuscript received: 28 Augustus 2010. Revision accepted: 4 October 2010.

Abstract. Setyawan AD. 2011. Natural products from Genus Selaginella (Selaginellaceae). Nusantara Bioscience 3: 44-58. Selaginella is a potent medicinal-stuff, which contains diverse of natural products such as alkaloid, phenolic (flavonoid), and terpenoid. This species is traditionally used to cure several diseases especially for wound, after childbirth, and menstrual disorder. Biflavonoid, a dimeric form of flavonoids, is the most valuable natural products of Selaginella, which constituted at least 13 compounds, namely amentoflavone, 2',8''-biapigenin, delicaflavone, ginkgetin, heveaflavone, hinokiflavone, isocryptomerin, kayaflavone, ochnaflavone, podocarpusflavone A, robustaflavone, sumaflavone, and taiwaniaflavone. Ecologically, plants use biflavonoid to response environmental condition such as defense against pests, diseases, herbivory, and competitions; while human medically use biflavonoid especially for antioxidant, antiinflammatory, and anti carcinogenic. Selaginella also contains valuable disaccharide, namely trehalose that has long been known for protecting from desiccation and allows surviving severe environmental stress. The compound has very prospects as molecular stabilizer in the industries based bioresources.

Key words: natural products, biflavonoid, trehalose, Selaginella.

Abstrak. Setyawan AD. 2011. Bahan alam dari Genus Selaginella (Selaginellaceae). Nusantara Bioscience 3: 44-58. Selaginella adalah bahan baku obat yang potensial, yang mengandung beragam metabolit sekunder seperti alkaloid, fenolik (flavonoid), dan terpenoid.

Spesies ini secara tradisional digunakan untuk menyembuhkan beberapa penyakit terutama untuk luka, nifas, dan gangguan haid.

Biflavonoid, suatu bentuk dimer dari flavonoid, adalah salah satu produk alam yang paling berharga dari Selaginella, yang meliputi sekurang-kurangnya 13 senyawa, yaitu amentoflavone, 2',8''-biapigenin, delicaflavone, ginkgetin, heveaflavone, hinokiflavone, isocryptomerin, kayaflavone, ochnaflavone, podocarpusflavone A, robustaflavone, sumaflavone, dan taiwaniaflavone. Secara ekologis, tumbuhan menggunakan biflavonoid untuk merespon kondisi lingkungan seperti pertahanan terhadap hama, penyakit, herbivora, dan kompetisi, sedangkan manusia menggunakan biflavonoid secara medis terutama untuk antioksidan, anti-inflamasi, dan anti karsinogenik. Selaginella juga mengandung trehalosa suatu disakarida yang telah lama dikenal untuk melindungi dari pengeringan dan memungkinkan bertahan terhadap tekanan lingkungan hidup yang keras. Senyawa ini sangat berpotensi sebagai stabilizer molekul dalam industri berbasis sumberdaya hayati.

Kata kunci: produk alami, biflavonoid, trehalosa, Selaginella.

INTRODUCTION Medicinal plant is plant containing substance which can be used for the medication or become precursor of drug synthesis (Sofowora 1982). Medicinal plant has been source of human health since ancient time, whereas about 60-75% of world populations require plant for carrying health (Farnsworth 1994; Joy et al. 1998; Harvey 2000).

Plants and microbes are the main source of natural products (Hayashi et al. 1997; Armaka et al. 1999; Lin et al.

1999a,b; Basso et al. 2005), and consistently become main source of the newest drugs (Harvey 2000). The drug development from natural sources are based on the bioassay-guided isolation of natural products, due to the traditional uses of local plants (ethnobotanical and ethanopharmacological applications) (Atta-ur-Rahman and Choudhary 1999).

Traditional medication system by using plant medicines has been developed during thousands of year especially by Chinese (Wu-Hsing) and India (Ayurveda, Unani and

Siddha) (Peter 2004; Ahmad et al. 2006), while the most advanced, widespread and oldest traditional medication system in Nusantara or Malay Archipelago (Malesia) is jamu which developed by Javanese. Jamu contains several recipes that compiled by about 30 plant species. Relief at Borobudur temple about making jamu indicates that jamu has been widely recognized since early 9th century (Jansen 1993). This system has been documented for centuries in many serat and primbon, Javanese literary (Soedibjo 1989, 1990; Sutarjadi 1990); and spreaded by trading, migration and expansion of several kingdoms such as Mataram Hindu (Sanjaya), Srivijaya (Saylendra) and Majapahit.

Selaginella Pal. Beauv. (Selaginellaceae Reichb.) has been used as complementary and alternative medicines in several traditional medication. This matter is traditionally used to cure wound, after childbirth, menstrual disorder, skin disease, headache, fever, infection of exhalation channel, infection of urethra, cirrhosis, cancer, rheumatism, bone fracture, etc. Part to be used is entire plant, though only referred as leaves or herbs (Setyawan 2009; Setyawan

SETYAWAN â&#x20AC;&#x201C; Natural products of Selaginella

and Darusman 2008). The usage can be conducted single or combination, fresh or dried, direct eaten or boiled (Dalimartha 1999; Wijayakusuma 2004). This plant has sweet taste and gives warm effect on the body (Bensky et al. 2004). The use of Selaginella as medicinal matter is occurred in the entire world. The largest usage is conducted

by Chinese, especially for S. tamariscina, S. doederleinii, S. moellendorffii, S. uncinata, and S. involvens (Lin et al.

1991; Chang et al. 2000; Wang and Wang 2001).

Unfortunately, Selaginella is rarely exploited in Nusantara.

Traditional jamu of Java use more cultivated spices and rhizomes than wild herbs or grasses.

Plant medicinal properties are contributed by natural products or secondary metabolites, such as phenolic (flavonoid), alkaloid, terpenoid, as well as non protein amino acid (Smith 1976). Natural products are chemical compounds or substances produced by a living organism and found in nature that usually has a biological activity for use in pharmaceutical drug discovery and drug design (Cutler and Cutler 2000). In this following discourse, the authors studied diversity of natural products from Selaginella, especially biflavonoid and trehalose compounds; and biological activity of Selaginellaâ&#x20AC;&#x2122;s bifavonoid in modern medication.

NATURAL PRODUCTS DIVERSITY Previous phytochemical studies on the constituents of genus Selaginella leds to the discovery of many compounds, including biflavonoids, the main secondary metabolite of Selaginella (Sun et al. 1997; Silva et al. 1995; Chen et al. 2005b; Lin et al. 1994; 2000). Biflavonoid has also distributed to Selaginellales, Psilotales, and Gymnosperms (Seigler 1998), several Bryophytes and about 15 families of Angiosperms (DNP 1992). The other compounds are including lignin (White and Towers 1967); lignan (Lin et al. 1994), lignanoside (Lin et al. 1990; Zheng et al. 2004, 2008b), alkaloid (Zheng et al. 2004; Lin et al.

1997), selaginellin (Zhang et al. 2007; Cheng et al. 2008), glycosides (Man and Takahashi 2002; Zhu et al. 2008), glucosides (Dai et al. 2006; Yuan et al. 2008), Cglycosylflavones (Richardson et al. 1989), etc. Selaginella species of Java contains alkaloid, phenolic (flavonoid, tannin, saponin), and terpenoid (triterpene, steroid) (Chikmawati and Miftahudin. 2008; Chikmawati et al.

2008). Some species of Japan consist of a steroid type namely ekdisteroid (Takemoto et al. 1967, Hikino et al.

1973; Yen et al. 1974). The diversity and content of other compound is relatively lower than biflavonoid, nevertheless they have also certain bioactivities.

Water extracts of S. tamariscina also has several natural products such as ferulic acid, caffeic acid, vanillic acid, syringic acid, umbelliferone (Bi et al. 2004b); tamariscinoside A, tamariscinoside B, adenosine, guanosine, arbutin (Bi el al. 2004a); tamariscinoside C, tyrosine, D-mannitol, and shikimic acid (Zheng et al.

2004). The EtOH extract of the whole herbs of S. tamariscina that fractionated by chloroform and ethyl acetate contains selaginellin A and selaginellin B (Cheng et al. 2008). The main constituen of S. tamariscina

45

subsequently is amentoflavone, robustaflavone, bilobetin, hinokiflavone, isocryptomerin and an apigenin-diglucoside (Yuan et al. 2008). S. tamariscina has also many sterols that inhibit the growth of human leukemia HL-60 cells indicating anti cancer property (Gao et al. 2007). The aerial parts of S. pulvinata has steroid constituent (Zheng et al.

2007), and several Selaginella has also sterol (Chiu et al.

1988). Steroid compound namely ekdisteroid has been found in Japanese species of S. deliculata, S. doederleinii, S. moellendorffii, S. nipponica, S. involvens (= S. pachystachys), S. stauntoniana (= S. pseudo-involvens), S. remotifolia var. japonica, S. tamariscina, and S. uncinata (Takemoto et al. 1967; Hikino et al. 1973; Yen et al. 1974).

Methanolic extract of S. lepidophylla contains 3methylenhydroxy-5-methoxy-2,4-dihydroxy tetrahydrofurane, which can a slight inhibitory effect on the uterus contraction (Perez et al. 1994). S. lepidophylla is also reported contain volatile oils (Andrade-Cetto and Heinrich 2005). The acetone extract of S. sinensis contains selaginellin A, an unusual flavonoid pigment (Zhang et al.

2007). S. sinensis has a glucoside, namely selaginoside (Dai et al. 2006), a sesquilignan, namely sinensiol A (Wang et al. 2007), secolignans, namely styraxlignolide D and neolloydosin (Feng et al. 2009), and (+)-pinoresinol (Umezawa 2003a,b). S. uncinata also has chromone glycosides, namely uncinoside A and uncinoside B (Man and Takahashi 2002), which shows antiviral activities against RSV and PIV-3 (Ma et al. 2003). Ethanol extract of S. uncinata also contains flavonoids that possessing a benzoic acid substituent (Zheng et al. 2008a).

S. doederleinii contains several phenolic compounds such as (+)-matairesinol, (-)-lirioresinol A, (-)-lirioresinol B, (-)-nortracheloside (Lin et al. 1994), and (-)matairesinol, (+)-syringaresinol, (+)-wikstromol, (+)nortrachelogenin (Umezawa 2003a,b). The (-)-matairesinol has inhibitory activity against cAMP and acts as an insecticide synergist, while (+)-syringaresinol has cytotoxic effect (Harborne et al. 1999). S. doederleinii also contains a glycosidic hordenine (Markham et al. 1992), which increases hypertension (Lin et al. 1991).

S. caulescens, S. involvens, and S. uncinata contain about 0.2% silicon, higher than the most of other club mosses and true ferns (Ma and Takahashi 2002), which may improve plant tolerant to disease, drought, and metal toxicities (Epstein 1999; Richmond and Sussman 2003; Ma 2004). S. labordei contains 4'-methylether robustaflavone, robustaflavone, eriodictyol and amentoflavone (Tan et al 2009). S. apoda yields substantial amounts of 3-O-methylD-galactose (Popper et al. 2001). S. moellendorfii contains several pyrrolidinoindoline alkaloids (Wang et al. 2009).

Other natural products, beside biflavonoid and trehalose, also have several molecular properties that can increase human health and have economical values; and need for further observation.

Natural products of Selaginella can vary depend on climate, location, and soil factors; as well as harvesting and extraction procedure (Nahrstedt and Butterweck 1997); and also plant species or variety, parts to be extracted and age.

The different species of Selaginella shows different HPLC fingerprint characteristic. The samples of the similar

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3 (1): 44-58, March 2011

species, collected in different period, different environment or different locations shows certain difference in fingerprints. However, it also generate main fingerprint peaks, which can be used to evaluate and distinguish the different species or infra species (Fan et al. 2007).

Phenol

BIFLAVONOID Selaginella species have a large number of bioactive compounds, the most important being biflavonoids (Silva et al. 1995; Lin et al. 1999). Biflavonoids are naturally occurring compounds that are ubiquitous in all vascular plants and have many favorable biological and pharmacological effects (Lee et al. 1996; Baureithel et al.

1997; Lobstein-Guth et al. 1998). One of flavonoid structure that has high medicinal valuable is biflavonoid; a dimeric form of flavonoid which formed by binding of two flavone units or mixture between flavone and flavanon or aurone (Geiger and Quinn 1976; DNP 1992; Ferreira et al.

2006).

Flavonoid (or flavanoid) is widespread plant natural products (5-10%); its chemical structure and biological role are very diverse (Macheix et al. 1990). This compound is formed by shikimate and phenylpropanoid pathways (Harborne 1989), with a few alternative biosynthesis (Robards and Antolovich 1997). Flavonoid is derived from phenols having basic structure of phenylbenzopiron (tocopherol) (Middleton et al. 2000); distinguished by 15 carbon skeletons (C6-C3-C6) consisted of one oxygenated ring and two aromatic rings (Figure 1). Substitution of chemical group at flavonoid is generally hydroxylation, methoxylation, methylation and glycosilation (Harborne 1980). Flavonoid is classified diversely; among them are flavone, flavonone, isoflavone, flavanol, flavanon, anthocyanin, and chalchone (Porter 1994; Ferreira and Bekker 1996; Ferreira et al. 1999a,b). More than 6467 flavonoid compounds have been identified and amount of new discovery is consistently increasing (Harborne and Baxter 1999). This compound is playing important role in determining color, favor, aroma, and quality of nutritional food (Macheix et al. 1990). Flavonoid is mostly monomeric form, but there is also dimer (biflavonoid), trimer, tetramer, and polymer (Perruchon 2004).

Biflavonoid (or biflavonil, flavandiol) is a dimeric form of flavonoid which formed by bonding of two flavone units or mixture between flavone and flavanon or aurone (Geiger and Quinn 1976; DNP 1992; Ferreira et al. 2006). Basic structure of biflavonoid is 2,3-dihydroapigeninil-(I-3’,II3’)-apigenin (Figure 1.). This compound has interflavanil C-C bond between carbon C-3’ at each flavone group.

There is also some biflavonoid with interflavanil C-O-C bonding (Bennie et al. 2000, 2001, 2002; Ferreira et al.

2006). Locksley (1973) suggest generic term ‘biflavanoid’ to replace ‘biflavonil’ which is early used. Term ‘biflavanoid’ is assumed more accurate than ‘biflavonoid’ because indicating saturated in nature. Suffix ‘oid’ indicates homogeneous dimeric type, including biflavanon, biflavon, biflavan, etc. However, term ‘biflavonoid’ is more regularly used because articulated easier.

B C Flavonoid

Biflavonoid

Figure 1. Basic structure of phenol, flavanoid and biflavanoid.

Bicyclic ring system is named A and C rings, while unicyclic ring is named B ring. The two unit of monomeric biflavonoid is marked by Roman number I and II. Position number at each monomer is started from containing oxygen atom ring, position of C-9 and C-10 indicate unification of them (Rahman et al. 2007; ).

Biflavonoid is found at fruit, vegetable, and other part of plant. This compound is originally found by Furukawa in 1929 (Lin et al. 1997) from leaf extract of G. biloba in form of yellow colored compound, later named ginkgetin (I-4’, I-7-dimetoxy, II-4’, I-5, II-5, II-7-tetrahydroxy I-3’, II-8 biflavone) (Baker and Simmonds 1940). Nowdays, amount of biflavonoid which isolated and characterized from nature continually increase (Oliveira et al. 2002; Ariyasena et al. 2004; Chen et al. 2005a), but learning to bioactivity is still limited. The most observed biflavonoid is ginkgetin, isoginkgetin, amentoflavone, morelloflavone, robustaflavone, hinokiflavone, and ochnaflavone. Those compounds have similar basic structure, i.e. 5,7,4’trihydroxy flavonoid, but differing at nature and position of flavonoid bond (Rahman et al. 2007).

Biflavonoid has several namenclaturing systems, such as Loksley, IUPAC, and vernacular name. The first of two systems is the most systematic, but the most used is vernacular name.

Locksley (1973) standardize nomenclature and position number of biflavonil ring skeleton. Every monomer unit is marked by Roman numerals I and II that indicate bonding between monomer, followed by Arabic numerals indicate that bonding position. The two numeral from two monomer unit compiled dimeric, than paired with hyphen to show bonding position of two monomer. Number of substitution group at monomer unit follow IUPAC system for flavone.

In Locksley system, amentoflavone named I-4’, II-4’, I-5, II-5, I-7, II-7-hexahydroxy I-3’, II-8 biflavone, while hinokiflavone which its flavone unit bonded with an oxygen is named by II-4’, I-5, II-5, I-7, II-7-pentahydroxy I-4’-O-II-6 biflavone. This system is intuitive, logical, and depicts the chemical structure. In IUPAC, amentoflavone is named by 8-5-(5,7-dihydroxy-4-oxo-4H-chromen-2-il)-2hydroxyphenyl-5,7-dihydroxy2-(4-hydroxy-phenyl)chromen-4-on, while hinokiflavone is 6-4-(5,7-dihydroxy4-oxo-4H-chromen-2-il)-phenoxy5,7-dihydroxy-2-(4-

SETYAWAN – Natural products of Selaginella

hydroxyphenyl)- chromen-4-on. Basic difference between two systems is reference of structural skeleton. Locksley use flavanoid structure, while IUPAC use chromen structure that more complex (Rahman et al. 2007). The above two nomenclature is rarely used because its complication. Vernacular name that given by each inventor is often used because simpler and easier, though it is not systematic and does not depict chemical structure, such as amentoflavone, hinokiflavone, ginkgetin, etc.

In vivo biosynthesis of flavonoid in nature is relatively mysterious, but there are some approaches by in vitro to explain biosynthesis. According to Rahman et al. (2007) there are nine pathways of biflavonoid synthesis, namely: (i) Ullmann coupling halogenated flavones; (ii) synthesis of biflavones via 1,1’-biphenyls; (iii) metal catalyzed cross coupling of flavones; (iv) Wessely-Moser rearrangements; (v) phenol oxidative coupling of flavones; (vi) Ullmann condensation with flavone salts; (vii) nucleophilic substitution; (viii) dehydrogenation of biflavanones into biflavones; and (ix) dehydrogenation of biflavone into biflavanone.

In East Asia, biflavonoid is usually produced from leaf of Ginkgo biloba which main constituent is ginkgetin (Krauze-Baranowska and Wiart 2002; Dubber 2005). In sub Sahara-Africa, it is especially produced from seed of Garcinia cola which main constituent is kolaviron (Iwu and Igboko 1982; Iwu 1985, 1999; Iwu et al. 1987, 1990; Braide 1989, 1993; Han et al. 2006; Farombi et al. 2005; Adaramoye and Medeiros 2009). The biflavanones are the most dominant in the most Garcinia species (Waterman and Hussain 1983), pericarp of Javanese mangosteen (G. mangoestana) contains amentoflavone and other flavonoids (ADS 2008, data not be shown). In Europe, biflavonoid is commonly produced from herbs of Hypericum perforatum which main constituent is amentoflavone (Berghofer and Holzl 1987, 1989; Nahrstedt and Butterweck 1997; Borlis et al. 1998; Tolonen 2003; Kraus 2005). Selaginella has potent as source of biflavonoid, which can yield various biflavonoid compounds depending on species. It has cosmopolitanly distributed and able to cultivate almost all the words depending on species.

DIVERSITY OF BIFLAVONOID Selaginella is one of the potential medicinal plants as a source biflavonoid in Nusantara, where 200 of the 700-750 species from the entire world are found (Setyawan 2008).

A total of 13 biflavonoid compounds have been isolated from Selaginella, including amentoflavone (3',8”biapigenin), 2',8''-biapigenin, delicaflavone, ginkgetin, heveaflavone, hinokiflavone, isocryptomerin, kayaflavone, ochnaflavone, podocarpusflavone A, robustaflavone, sumaflavone, and taiwaniaflavone (Figure 2). In Setyawan and Darusman (2008) mentioned that the number is only 12 biflavonoid compounds. Some biflavonoid are easily found at various species of Selaginella, but the other is only found at certain species. Amentoflavone and ginkgetin is biflavonoid compound of the most Selaginella, while sumaflavone is only reported from S. tamariscina (Yang et

47

al. 2006; Lee et al. 2008) and delicaflavone is only reported from S. delcatula (Andersen and Markham 2006). At least 11 species of Selaginella have been tested by amentoflavone content (Sun et al. 2006). There are also biflavonoid which is rarely found at Selaginella but it is commonly found at other species. Preliminary study shows that amentoflavone is found in high content (> 20%) at two of about 35 species of Malesian Selaginella, namely S. subalpina and S. involvens (ADS 2008, data not be shown).

In Selaginella, taiwaniaflavone is only reported from S. tamariscina (Pokharel et al. 2006), while this is also found at other plant such as Taiwania cryptomerioides (Kamil et al. 1981).

Selaginella is generally extracted from whole plant, though it is only conceived as herbs or leaves. Extraction can be conducted by various solvent, i.e. polar, semi-polar and non polar. For example: boiling in water, extraction by using methanol, ethanol, buthanol, ethyl acetate, chloroform, or extraction by using solvent mixture such as alcohol-water, ethanol-ethyl acetate, and ethanolchloroform. Methanol and ethanol are the most solvent used for biflavonoid extraction. Solvent types and extraction procedure can influence obtaining chemical structure and bioactivity of extract. Disease which is most treated by Selaginella extract is cancer. Besides, Selaginella extract also has many other usefulness, namely antioxidant, anti-inflammatory, antimicrobial (virus, bacterium, fungi, and protozoa), anti UV irradiation, anti allergy, vasorelaxation, anti diabetes, blood pressure stability, anti hemorrhagic, and antinociceptive.

Biflavonoid needs evaluation for its medical and nutritional value (Harborne and Williams 2000). Selaginella contains various biflavonoid with difference medical properties (Table 2).

Amentoflavone. Amentoflavone, the most common biflavonoid of Selaginella, has various biological and pharmacological effects, including antioxidant (Mora et al.

1990; Cholbi et al. 1991; Shi et al. 2008), anti cancer (Silva et al. 1995; Lee et al. 1996; Lin et al. 2000; Guruvayoorappan and Kuttan 2007), anti-inflammatory (Gambhir et al. 1978; Baureithel et al. 1997; Gil et al.

1997; Kim et al. 1998; Lin et al.. 2000; Woo et al.. 2005), antimicrobial (Woo et al. 2005; Jung et al. 2007), antivirus such as influenza (A, B), hepatitis (B), human immunodeficiency virus (HIV-1), herpes (HSV-1, HSV-2), herpes zoster (VZV), measles (Lin et al. 1998, 1999a,b, 2002; Flavin et al. 2001, 2002), and respiratory syncytial virus (RSV) (Lin et al. 1999a,b; Ma et al. 2001), vasorelaxation (Kang et al. 2004), anti-urcerogenic (Gambhir et al. 1987), anti stomachic-ache (Kim et al.

1998), anti depressant (Baureithel et al. 1997), anxiolytic (Cassels et al. 1998, 1999), analgesic (Silva et al. 2001), and anti-angiogenesis agent (Lee et al. 2009c).

2',8''-biapigenin. 2',8''-biapigenin is an anticancer, which inhibit transactivation of iNOS gene and cyclooxigenase-2 (COX-2) through inactivate nuclear factor-κB (NF-κB) and prevent translocation of p65 (Chen et al. 2005b; Woo et al. 2006); and anti-inflammatory (Grijalva et al. 2004; Woo et al. 2005 2006; Pokharel et al.

2006).

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3 (1): 44-58, March 2011 OH OH HO

OH

O HO

HO

OH

2’

8”

O

O OH

HO

O

Delicaflavone

2’,8”-biapigenin Amentoflavone (3',8”-biapigenin)

OCH3

OH

OCH3 OH CH3O HO

CH3O

Hinokiflavon

CH3O OH

O

Hinokiflavone

OH

Heveaflavone

Ginkgetin

OCH3

OCH3 CH3O

HO CH3O

Ochnaflavone

Isocryptomerin Kayaflavone

OCH3

HO HO

Podocarpusflavone A

Sumaflavone

OH

OH

HO

OH OH

Robustaflavone

HO

Taiwaniaflavone

Figure 2. Structure of biflavonoid from Selaginella, namely: amentoflavone, 2',8''-biapigenin, delicaflavone, ginkgetin, heveaflavone, hinokiflavone, Isocryptomerin, kayaflavone, ochnaflavone, podocarpusflavone A, robustaflavone, sumaflavone, and taiwaniaflavone.

SETYAWAN â&#x20AC;&#x201C; Natural products of Selaginella

Delicaflavone. Its bioactivity is not observed yet from Selaginella.

Ginkgetin. This compound is the second most studied biflavonoid of Selaginella beside amentoflavone. It has several properties including antioxidant (Su et al. 2000; Sah et al. 2005; Shi et al. 2008), anti-inflammatory (Grijalva et al. 2004; Woo et al. 2005, 2006; Pokharel et al.

2006), anti viral such as herpes and cytomegalovirus (Hayashi et al. 1992); anti protozoan such as Trypanosoma cruzi (Weniger et al. 2006); anti cancer (Sun et al. 1997; Kim and Park 2002; Yang et al. 2007), such as such as ovarian adenocarcinoma (OVCAR-3), cervical carcinoma (HeLa) and foreskin fibroblast (FS-5) (Su et al. 2000).

Ginkgetin is the strongest biflavonoid that inhibit cancer (Kim and Park 2002). Besides, this matter increase activity of neuroprotective against cytotoxic stress, and has potent for curing neurodegenerative disease such as stroke and Alzheimer (Kang et al. 2004; Han et al. 2006). Ginkgetin can also replace caffeine in food-stuff and medicines without generating addiction (Zhou 2002).

Heveaflavone. Heveaflavone has cytotoxic activity against cancer cell of murine L 929 (Lin et al. 1994).

Hinokiflavone. Hinokiflavone has antioxidant, antiviral and anti protozoan effect. This matter assists cell growth and protect from free radical cased by hydrogen peroxide (H2O2) (Sah et al. 2005). It also inhibit sialidase influenza virus (Yamada et al. 2007; Miki et al. 2008); has high resistance to HIV-1 by in vivo and to polymerase HIV-1 RTASE by in vitro (Lin et al. 1997). Lin et al. (1998, 1999a,b, 2002) and Flavin et al. (2001, 2002) is patenting antiviral effect of hinokiflavone and others to influenza virus (A, B), hepatitis (B), human immunodeficiency virus (HIV-1), herpes (HSV-1, HSV-2), herpes zoster (VZV), and measles. It has antiprotozoan activity by in vitro against Plasmodium falciparum, Leishmania donovani and Trypanosoma sp. (Kunert et al. 2008).

Isocryptomerin. Isocryptomerin has anti cancer property as well as anti-inflammatory, immunosuppressant and analgesic (Kang et al. 1998, 2001). It has cytotoxic activity against various cancer cells (Silva et al. 1995), including P-388 and HT-29 (Chen et al. 2005b). It has antibacterial activity against Gram-positive and Gramnegative bacteria (Lee et al. 2009b); and also has antifungal properties, which can depolarize fungal plasma membrane of Candida albicans (Lee et al. 2009a).

Kayaflavone. Kayaflavone has moderately anti cancer property (Sun et al. 1997; Yang et al. 2007) and antioxidant, such as depleting H2O2 (Su et al. 2000).

Ochnaflavone. Ochnaflavone derivatives may have antioxidant activity that inhibits expression of gene COX-2 at colon cancer cell (Chen et al. 2005b).

Podocarpusflavone A. It has moderately anti cancer (Sun et al. 1997; Yang et al. 2007) and antioxidant properties (Su et al. 2000; Shi et al. 2008).

Robustaflavone. Robustaflavone has anti cancer and anti virus properties. This matter significantly cytotoxic to various cancer cells (Silva et al. 1995) and significantly inhibits tumor cell of Raji and Calu-1 (Lin et al. 2000), cancer cell of P-388 and HT-29 (Chen et al. 2005b). It has also antiviral properties, which indicates high resistance to

49

polymerase HIV-1 RTASE by in vitro (Lin et al. 1997) and also influenza virus (A, B), hepatitis (B), human immunodeficiency virus (HIV-1), herpes (HSV-1, HSV-2), herpes zoster (VZV), and measles (Lin et al. 1998, 1999a,b 2002; Flavin et al. 2001, 2002).

Sumaflavone. Sumaflavone has anti-inflammatory property that able to inhibit production of NO, by mean blocking lipopolysaccharide formation that induces iNOS gene expression (Yang et al. 2006). It can also significantly inhibit ability of UV irradiation to induce matrix metalloprotease-1 and -2 (MMP-1 and -2) activities at fibroblast of primary human skin (Lee et al. 2008).

Taiwaniaflavone. It has anti-inflammatory, such as induce iNOS and COX-2 at macrophage of RAW 264.7 (Pokharel, et al. 2006).

MOLECULAR BIOACTIVITIES Selaginella is traditionally treated to cure several disease depending on species, such as cancer or tumor (uterus, nasopharyngeal, lung, etc), wound, after childbirth, menstrual disorder, female reproduction disease, expulsion of the placenta, tonic (for after childbirth, increase body endurance, anti ageing, etc), pneumonia, respiratory infection, exhalation channel infection, inflamed lung, cough, tonsil inflammation, asthma, urethra infection, bladder infection, kidney stone, cirrhosis, hepatitis, cystisis, bone fracture, rheumatism, headache, fever, skin diseases, eczema, depurative, vertigo, toothache, backache, blood purify, blood coagulation, amenorrhea, hemorrhage (resulting menstrual/obstetrical hemorrhage, stomachic, pile or prolepses of the rectum), diarrhea, stomach-ache, sedative, gastric ulcers, gastro-intestinal disorder, rectocele, itches, ringworm, bacterial disease, bellyache, neutralize poison caused by snakebite or sprained, bruise, paralysis, fatigue, dyspepsia, spleen disease (diabetic mellitus), emmenagogue, diuretic, and to refuse black magic (Martinez 1961; Bouquet et al. 1971; Dixit and Bhatt 1974; Ahmad and Raji 1992; Bourdy and Lee et al. 1992; Bourdy and Walter 1992; Nasution 1993; Lin et al. 1994; Kambuou 1996; Caniago and Siebert 1998; Sequiera 1998; Dalimartha 1999; Mathew et al. 1999; Abu-Shamah et al.

2000; van Andel 2000; Uluk et al. 2001; Harada et al.

2002; Man and Takahashi 2002; Warintek 2002; Winter and Jansen 2003; ARCBC 2004; Batugal et al. 2004; de Almeida-Agra and Dantas 2004; DeFilipps et al. 2004; Wijayakusuma 2004; Mamedov 2005; Khare 2007; Pam. 2008; Setyawan and Darusman 2008) (Table 1). This plant has sweet taste, and gives warm effect on the body (Bensky et al. 2004).

Plants ecologically use biflavonoid to response environmental condition such as defense against pests, diseases, herbivory, and competitions; while human medically use as antioxidant, anti-inflammatory, anti cancer, anti allergy, antimicrobial, antifungal, antibacterial, antivirus, antiprotozoan, protection to UV irradiation, vasorelaxation (vasorelaxant), heart strengthener, anti hypertension, anti blood coagulation, and influence enzyme

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3 (1): 44-58, March 2011

metabolism (Havsteen 1983, 2002; Kandaswami and Middleton 1993, 1994; Lale et al. 1996; Bisnack et al.

2001; Duarte et al. 2001; Kromhout 2001; Kang et al.

2004; Moltke et al. 2004; Arts and Hollman 2005; Martens and Mithofer 2005; Yamaguchi et al. 2005). The antioxidant, anti cancer and anti-inflammatory are the most important bioactivities of this secondary metabolite.

Selaginella is known possess various molecular bioactivities depending on species, but only a few species has been detailed observe in the advanced research. Several species that also distributed in Nusantara are observed, such as S. tamariscina, S. doederleinii, S. involvens, S. moellendorffii, S. uncinata, and S. willdenowii; while the most distributed Selaginella in Nusantara namely S. plana has not been investigated yet (Table 2).

S. tamariscina is the most powerful and the most useful plant Selaginella in the world. This herb is widely used as anti cancer, antioxidant and anti-inflammatory; and also used as anti UV irradiation, anti allergy, vasorelaxation, anti diabetic, immunosuppressant, analgesic, neuro protectant, antibacteria, antifungal, and possess estrogenic activity. As anti cancer, S. tamariscina can decrease expression of MMP-2 and -9, urokinase plasminogen activator, and inhibits growth of metastasis A549 cell and Lewis lung carcinoma (LLC) (Yang et al. 2007); inhibits proliferation of mesangial cell which activated by IL-1β and IL-6 (Kuo et al. 1998); inhibits leukemia cancer cell of HL-60 cell (Lee et al. 1999); induces expression of tumor suppressor gene of p53 (Lee et al. 1996); degrades leukemia cancer cell of U937 (Lee et al. 1996; Yang et al.

2007); reduces proliferation nucleus antigen cell from stomach epithelium (Lee et al. 1999); chemopreventive for gastric cancer (Lee et al. 1999); induces apoptosis of cancer cell trough DNA fragmentation and nucleus clotting (Ahn et al. 2006); and induces breast cancer apoptosis through blockade of fatty acid synthesis (Lee et al. 2009c). This property is mostly given by amentoflavone and isocryptomerin (Kang et al. 1998, 2001; Lee et al. 2009c), while ginkgetin is also acted as anti cancer to OVCAR-3 (Sun et al. 1997). As antioxidant, amentoflavone from S. tamariscina inhibits production of NO, which inactivates NF-κB, while sumaflavone blocks lipopolysaccharide formation that induces iNOS gene expression (Yang et al.

2006).

As anti-inflammatory, amentoflavone, taiwaniaflavone and ginkgetin from S. tamariscina inhibit inflammation that induce iNOS and COX-2 at macrophage RAW 264.7 which stimulated by lipopolysaccharide (Grijalva et al. 2004; Woo et al. 2005; Pokharel et al.

2006). Amentoflavone inhibits activity of phospholipase Cγ1 (Lee et al. 1996); phospholipase A-2 (PLA-2) and COX-2 (Kim et al. 1998), while 2',8''-biapigenin inhibits transactivation of iNOS gene and COX-2 through inactivate NF-κB and prevent translocation of p65 (Woo et al. 2006).

Amentoflavone from S. tamariscina inhibits fungi (Junk et al. 2006), anti influenza and resist to HSV-1 and -2 (Rayne and Mazza 2007); hinokiflavone inhibits sialidase influenza virus (Yamada et al. 2007; Miki et al. 2008) and resists to HIV-1 (Lin et al. 1997); robustaflavone and hinokiflavone resist to polymerase HIV-1 RTASE (Lin et

al. 1997); ginkgetin inhibits herpes and cytomegalovirus (Hayashi et al. 1992), by degrading protein synthesis of virus and depress gene transcription (Middleton et al.

2000). Isocryptomerin from S. tamariscina shows potent antibacterial activity against Gram-positive and Gramnegative (Lee et al. 2009b). Amentoflavone from S. tamariscina inhibits several pathogenic fungi (Woo et al.

2005; Jung et al. 2007). Isocryptomerin from S. tamariscina can depolarize fungal plasma membrane of C. albicans (Lee et al. 2009a).

S. tamariscina is effective ingredient to prevent and cure acute brain degenerative disease, such as stroke and dementia (Han et al. 2006). Capability to prevent brain damage is especially given by amentoflavone (Kang et al.

1998). S. tamariscina can elastic vascular smooth muscle through endothelium related to nitric oxide (NO) activity (Yin et al. 2005). Amentoflavone from S. tamariscina induces relaxation of phenylephrin which responsible to aorta contraction (Kang et al. 2004; Yin et al. 2005). S. tamariscina containing sumaflavone and amentoflavone inhibit ability of UV irradiation to induce MMP-1 and -2 at fibroblast (Lee et al. 2008). S. tamariscina reduces histamine from peritoneal mast cell causing allergic reaction (Dai et al. 2005). S. tamariscina decreases sugar blood and lipid peroxide, and also increases insulin concentration (Miao et al. 1996). Amentoflavone from S. tamariscina inhibits activity of tyrosine phosphatase 1B to maintain type-2 diabetic and obesity (Na et al. 2007).

S. articulate is treated as anti hemorrhagic. Water extract of this matter can moderately neutralize hemorrhagic effect and inhibits proteolysis of casein by venom (Otero et al. 2000; Winter and Jansen 2003).

S. bryopteris acts as antioxidant, anti-inflammatory, antiprotozoan, anti UV-irradiation and anti spasmodic.

Water extract of S. bryopteris increases endurance to oxidative stress; and assists cell growth and protects from free radical stress caused by H2O2 (Sah et al. 2005). S. bryopteris is treated as anti-inflammatory and cures veneral disease (Agarwal and Singh 1999). Amentoflavone and hinokiflavone from S. bryopteris have antiprotozoan activity against P. falciparum, L. donovani and Trypanosoma sp (Kunert et al. 2008). Water extract of S. bryopteris also significantly reduces potent cell dying caused by UV irradiation (Sah et al. 2005), while ethanolic extract can cure stomachic (Pandey et al. 1993).

S. delicatula acts as anti cancer and antioxidant. Water extract of S. delicatula has antioxidant characteristic and degrades blood cholesterol (Gayathri et al. 2005). Extract of S. delicatula that contained by robustaflavone and amentoflavone or its derivatives is cytotoxic against cancer cell of P-388, HT-29 (Chen et al. 2005b), Raji, Calu-1, lymphoma and leukemia (Lin et al. 2000) S. doederleinii is usually treated as anti cancer, but also acts as antiviral and anti-inflammatory. Water extract of S. doederleinii has antimutagenic against both picrolonic acid- and benzo[α]pyrene-induced mutation to cancer cell (Lee and Lin 1988). Ethanolic extract of S. doederleinii that is amentoflavone and heveaflavone has cytotoxic activity against cancer cell of murine L 929 (Lin et al.

1994). Extract of S. doederleinii also has cytotoxic against

SETYAWAN – Natural products of Selaginella

the three human cancer cell lines, HCT, NCI-H358, and K562 (Lee et al. 2008), and has anti mutagenic effect against cholangiocarcinoma cancer, but my cause bone marrow depression (Pan et al. 2001). Amentoflavone from S. doederleinii has potent as antiviral and antiinflammatory agents (Lin et al. 2000). However, hordenine that isolated from S. doederleinii increases hypertension (Lin et al. 1991).

S. involvens has characteristics as antioxidant, antiinflammatory and anti bacteria. Extract of S. involvens can inhibit production and effect of free radicals of NO and expression of iNOS/IL-1β (Joo et al. 2007). Water extract of S. involvens has significantly antioxidant effect to lipid peroxides (EC50 = 2 ug/mL). This extract is non toxic and degrades blood cholesterol (Gayathri et al. 2005). Water extract of S. involvens kills the various Leptospira strains, which causes infectious of leptospirosis diseases (Wang et al. 1963). Extract of S. involvens depresses activity of Propionibacterium acnes (> 100 ug/mL), which responses to acne inflammation; although has no antibiotic property (Joo et al. 2007). Beside, water extract of S. involvens may have analgesic activity (ECMM 1997; Ko et al. 2007).

S. labordei indicates antioxidant, anti cancer, and anti virus characteristics. S. labordei can inhibit activity of xanthine oxidase (XOD) and lipoxygenase (LOX), and absorb free radical (Chen et al. 2005b; Tan et al. 2009). It also down-regulate COX-2 gene expression in human colon adenocarcinoma CaCo-2 cells (Chen et al. 2005b).

Robustaflavone of S. labordei can inhibit hepatitis B virus (Tan et al. 2009) S. lepidophylla has hypoglycemic property (AndradeCetto and Heinrich 2005); while non-biflavonoid compound from methanolic extract of S. lepidophylla, 3methylenhydroxy-5-methoxy-2,4-dihydroxy tetrahydrofuran, has moderate resistance to uterus contraction (Perez et al. 1994).

S. moellendorffii contains antioxidant and anti cancer properties. Ethyl acetate extract of S. moellendorffii contains amentoflavone, hinokiflavone, podocarpusflavone A, and ginkgetin that has antioxidant properties (Shi et al.

2008). Ginkgetin that extracted by ethanol or ethyl acetate from S. moellendorffii can inhibit cancer cell growth of OVCAR-3, HeLa, and FS-5 (Sun et al. 1997; Su et al.

2000). It also act as anti-metastasis at lung cancer cell of A549 and LLC (Yang et al. 2007); and apoptosis resulting caspase activation by H2O2 (Su et al. 2000); while amentoflavone and its derivatives, kayaflavone, and podocarpusflavone A, have no this bioactivity (Sun et al.

1997).

S. pallescens has moderately antimicrobials and anti spasmodic activities. S. pallescens contains an endophytic Fusarium sp. that produce pentaketide anti fungal agent, CR377 (Brady and Clardy 2000). Chloroform-methanolic extract of S. pallescens can inhibit spontaneously contraction of ileum muscle (Rojas et al. 1999).

S. rupestris contains amentoflavone which has antispasmodic effect to ileum; and strengthening heart in case of normodinamic and hypodinamic (Chakravarthy et al. 1981)

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S. sinensis contains amentoflavone which has antiviral actifity against RSV (Ma et al. 2001) S. uncinata has activity as anti virus but generated by non biflavonoid compounds. S. uncinata has chromone glycosides, namely uncinoside A and B (Man and Takahashi 2002), which showed antiviral activities against RSV and PIV-3 (Ma et al. 2003).

S. willdenowii contains isocryptomerin and derivatives of amentoflavone and robustaflavone which significantly cytotoxic against various cancer cell (Silva et al. 1995).

TREHALOSE Trehalose is formed by α,α-1,1-glycosidic linkage of two low energy hexose moieties (Paiva and Panek 1996; Elbein et al 2003; Grennan 2007). This matter is a unique simple sugar which non reactive, very stable, colorless, odor-free, non-reducing disaccharide, and capable to protect biomolecules against environmental stress (Schiraldi et al. 2002). Therefore, this compound is a natural product, although not as commonly secondary metabolites of natural products. It works as osmoprotectant during desiccation stress (Adams et al. 1990); such as compatible solute in the stabilization of biological structures under abiotic stress (Garg et al. 2002); serves as a source of energy and carbon (Elbein et al 2003; Schluepmann et al. 2003); serves as signaling molecule to control certain metabolic pathways (Muller et al. 2001; Elbein et al 2003; Avonce et al 2005); protects proteins and cellular membranes from inactivation or denaturation caused by harsh environmental stress, such as desiccation, dehydration (drought), thermal heat, cold freezing, oxidation, nutrient starvation, and salt (Avigad 1982; Elbein et al. 2003; Wu et al. 2006). Trehalose acts as a global protectant against abiotic stress (Jang et al. 2003).

This matter is proved to be an active stabilizer of enzymes, proteins, biomasses, pharmaceutical preparations and even organs for transplantation (Schiraldi et al. 2002), and very prospects as molecular bio stabilizer in cosmetic, pharmacy and food (Roser 1991; Kidd and Devorak 1994). These multiple effects of trehalose on protein stability and folding suggest promising applications (Singer and Lindquist 1998).

Trehalose has long been known for protecting certain organisms from desiccation. The accumulation of the disaccharide trehalose in anhydrobiotic organisms allows them to survive severe environmental stress (Zentella et al.

1999). Trehalose also promotes survival under extreme heat conditions, by enabling proteins to retain their native conformation at elevated temperatures and suppressing the aggregation of denatured proteins (Singer and Lindquist 1998). Desiccation can reduce the lipid component in thylakoid membranes (Guschina et al. 2002). However, in desiccation-tolerant plants, membrane integrity appears not to be affected during drought-stress. S. lepidophylla retain their structural organization as intact bilayers (Platt et al.

1994) and often referred as resurrection plant because able to live on long drought and recovery through rehydration

Â

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process (Crowe et al. 1992), even when the most water body (99%) is evaporated (Schiraldi et al. 2002; van Dijck et al. 2002). Another species, S. tamariscina, can also remain alive in a desiccated state and resurrect when water becomes available (Liu et al. 2008). The drought can change fluorescence and pigmentation, but can not cause dying (Casper et al. 1993).

Trehalose exists in a wide variety of organisms, including bacteria, yeast, fungi, insects, invertebrates, and lower and higher plants (Elbein 1974; Crowe et al. 1984; Elbein et al. 2003), but rarely find in Angiosperms (Muller et al. 1995) and does not find in mammals (Teramoto et al 2008), and it is not accumulated to detectable levels in the most plants (Garg et al. 2002). This sugar plays important roles in cryptobiosis of Selaginella and other organisms, which revive with water from a state of suspended animation induced by desiccation (Teramoto et al 2008).

Trehalose is the major sugar formed in photosynthesis of Selaginella (White and Towers 1967). Some Selaginella contains high concentration of trehalose, such as S. lepidophylla (Adams et al. 1990; Mueller et al. 1995; Zentella et al. 1995), S. sartorii (Iturriaga et al. 2000), S. martensii (Roberts and Tovey 1969), S. densa, and S. wallacei (White and Towers 1967). Trehalose can reach 10-15% of cell dry weight (Grba et al. 1975).

Trehalose is not merely chemical compounds that responsible to resurrection ability of Selaginella. The protective effect of trehalose is correlated with a trapping of the protein in a harmonic potential, even at relatively high temperature (Cordone et al. 1999). Deeba et al (2009) suggest that S. bryopteris, one kind of resurrection plants, has about 250 proteins that expressed in response to dehydration and rehydration, and involved in transport, targeting and degradation in the desiccated fronds. Harten and Eickmeier (1986) suggest that several conservationed enzymes are beneficial for rapid resumption of metabolic activity of S. lepidophyla. Furthermore, Eickmeier (1979; 1982) suggests that both organelle- and cytoplasm-directed protein syntheses are necessary for full photosynthetic recovery during rehydration of S. lepidophyla.

FUTURE RESEARCH Research on Selaginella is still widely challenging. In the most elementary study of plant taxonomy, the high morphological variation of Selaginella causes several misidentification of this taxon. In ecology, global warming, habitat fragmentation and degradation that affected on sustainability of this resource need to be observed. In physiology, changes of fluorescens and pigmentation caused by environmental factor and age need to be explained. In biochemistry, several natural products are not exploited yet. One of non-biflavonoid compound from Selaginella that needs to be further investigated is trehalose. Molecular study is also required clarifying certain identity and phylogenetics relationship.

In Indonesia, several authors often misidentify Selaginella species, especially on popular article. This

matter is often identified as S. doederleinii, including Javanese wild species. The most authors agree that S. doederleinii is recognized as non native plant of Indonesia, which natural distribution is India, Burma, Thailand, Laos, Cambodia, Vietnam, Malaya, Chinese, Hong Kong, Taiwan, and Japan (Huang 2006; USDA 2008). Java has no species of S. doederleinii according to Alston (1935a) and observation on Selaginella collection of Herbarium Bogoriense, through several Kalimantan collection is suspected and has morphological similarity to this species (ADS 2007, data is not shown). This matter is possibility caused by referring to Dalimartha (1999), which include S. doederleinii in Indonesian plant medicines. Harada et al.

(2002) conduct similar misidentification, which cite S. plana as one of plant medicine in Mount Halimun NP (nowadays Mount Halimun-Salak NP), but the main picture presented is S. willdenowii. Field survey indicate that S. willdenowii is easily found in road side to Cikaniki Research Station of Mount Halimun-Salak NP, at rice field, shrubs land, primary and secondary forest, while S. plana is easier to be found in countrifield at lower height (ADS 2008, personal observation).

Species misidentification impacts on drug properties, because each species differ chemical constituent. Natural products content of Selaginella highly vary depending on species, although does not always congruent with traditional medical recipes. Sundanese people of Mount Halimun-Salak NP complementarily or substitutionally uses several Selaginella for treatment of after childbirth including S. ornata, S. willdenowii, S. involvens, and S. intermedia, but for similar recipe Sundanese around Bogor only uses S. plana (ADS 2008, personal observation).

Morphological diversity at infra specific level, and changes on pigmentation caused by age, drought and other environmental factors able to entangle identify base on morphological characteristics. It needs identification base on molecular characteristic, such as Korall et al. (1999) and Korall and Kenrick (2002, 2004). Beside, taxonomy of Malesian Selaginella needs to revise, because still based on old literature namely Alderwereld van Rosenburgh (1915a,b; 1916, 1917, 1918, 1920, 1922) and Alston (1934, 1935a,b; 1937, 1940). In a research brief about the traditional utilization of Selaginella in Indonesia, Setyawan (2009) collect at least 40 species of which half are estimated to new species or new records.

Completely research on variability of biflavonoid compounds of various Selaginella species with various solvent have not conducted yet. This matter is only conducted to certain species, compounds, and solvents.

Natural products of certain plant determine economical value that required in industrial scale of modern pharmacy.

Species with various low content of natural products less value than species with restricted high content, because modern pharmacy exploits natural products at molecular level. However, this matter is not always become consideration in traditional medication, because it generally uses simplicia that can be easily substituted by each others.

In phytochemistry and chemotaxonomy, high variety of natural products can assist identification, though each has no high content. However, a very low compound is not

SETYAWAN – Natural products of Selaginella

significantly important for identification, because often influenced by environmental factors, not merely to genetic factor. Bioactivity of each biflavonoid also requires to be observed because nowadays only bioactivity of amentoflavone and ginkgetin has been completely studied.

HPLC is potent method for analyzing of natural products of Selaginella (Fan et al. 2007).

Besides, trehalose observation on Selaginella is still restricted on a few species, and need to be conducted to amount of other species caused by potent economic value that can be generated. It can preliminary indicated by species that incurling leaves in hot weather or drough condition.

Biflavonoid study of Selaginella is still require attention such as: (i) the importance of assuring species identity caused by height morphological variety including by using molecular method; (ii) the importance of extending research coverage most of biflavonoid type, species, and extraction method; and also (iii) the importance of extending investigation on bioactivity, including nonbiflavonoid compound, which also have high economic potent such as trehalose.

CONCLUSION Selaginella is a potent medicinal matter, which mostly contains phenolic (flavonoid), alkaloid, and terpenoid. This matter is traditionally used to cure several diseases especially for wound, after childbirth, and menstrual disorder. Biflavonoid, a dimeric form of flavonoids, is one of the most valuable natural products of Selaginella, which constituted at least 13 compounds, namely amentoflavone, 2',8''-biapigenin, delicaflavone, ginkgetin, heveaflavone, hinokiflavone, isocryptomerin, kayaflavone, ochnaflavone, podocarpusflavone A, robustaflavone, sumaflavone, and taiwaniaflavone. Human medically use biflavonoid especially for antioxidant, anti-inflammatory, and anti cancer. Selaginella also contains several natural products, such as trehalose which valuable for bioindustry.

Selaginella research exhaustively needs to be conducted to explore all natural products constituents and their bioactivities.

ACKNOWLEDGEMENTS I would like to thank Prof. Dr. Keon Wook Kang from Chosun University, Gwangju, South Korea which delivered a number of valuable articles on chemistry of biflavonoid. I also thank Prof. Dr. Umesh R. Desai from Commonwealth University, Virginia, USA for permitting to cite a very interesting article on biosynthesis of biflavonoid. Grateful thank to Prof. Dr. Raphael Ghogomu-Tih from University of Yaoundé, Cameron for suggesting to write this article in international language. Sincere thanks are expressed to the late Dr. Muhammad Ahkam Soebroto from Research Center for Biotechnology, Indonesian Institute of Science,

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Cibinong-Bogor and anonymous peer reviewer of this article for their criticism. I also would like to thank Prof.

Dr. Wasmen Manalu from SEAMEO-BIOTROP Bogor which had invited me for training in writing scientific articles.

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| Nus Biosci | vol. 3 | no. 1 | pp. 1‐58 | March 2011 | ISSN 2087‐3940 (PRINT) | ISSN 2087‐3956 (ELECTRONIC) I S E A J o u r n a l o f B i o l o g i c a l S c i e n c e s

Optimization of DNA extraction of physic nut (Jatropha curcas) by selecting the appropriate leaf EDI PRAYITNO, EINSTIVINA NURYANDANI

1‐6

Characterisation of taro (Colocasia esculenta) based on morphological and isozymic patterns markers TRIMANTO, SAJIDAN, SUGIYARTO

7‐14

Study on floristic and plant species diversity of the Lebanon oak site (Quercus libani) in Chenareh, Marivan, Kordestan Province, western Iran HASSAN POURBABAEI, SHIVA ZANDI NAVGRAN

15‐22

Evaluation structural diversity of Carpinus betulus stand in Golestan Province, North of Iran VAHAB SOHRABI, RAMIN RAHMANI, SHAHROKH JABBARI, HADI MOAYERI

23‐27

Microanatomy alteration of gills and kidneys in freshwater mussel (Anodonta woodiana) due to cadmium exposure FUAD FITRIAWAN, SUTARNO, SUNARTO

28‐35

Site suitability to tourist use or management programs South Marsa Alam, Red Sea, Egypt MOHAMMED SHOKRY AHMED AMMAR, MOHAMMED HASSANEIN, HASHEM ABBAS MADKOUR, AMRO ABD‐ELHAMID ABD‐ELGAWAD

36‐43

Review: Natural products from Genus Selaginella (Selaginellaceae) AHMAD DWI SETYAWAN

44‐58

Published three times in one year PRINTED IN INDONESIA

ISSN 2087‐3940 (print)

ISSN 2087‐3956 (electronic)