STUDY ON THE LEVEL OF GENETIC DIVERSITY OF Diospyros celebica

ISBN 978-602-8964-17-3

STUDY ON THE LEVEL OF GENETIC DIVERSITY OF

Diospyros celebica, Eusideroxylon zwageri AND

Michelia spp. USING RAPD MARKERS

CENTER FOR CONSERVATION AND REHABILITATIONRESEARCH AND DEVELOPMENT,

FORESTRY RESEARCH AND DEVELOPMENT AGENCYMINISTRY OF FORESTRY

BOGOR – INDONESIA2011

Anthonius YPBC WidyatmokoILG Nurtjahjaningsih

Prastyono

STUDY ON THE LEVEL OF GENETIC DIVERSITY OF

Diospyros celebica, Eusideroxylon zwageri AND Michelia spp. USING RAPD MARKERS

Anthonius YPBC Widyatmoko ILG Nurtjahjaningsih

Prastyono

ITTO PROJECT PD 539/09 REV.1 (F)

IN COOPERATION WITH

CENTER FOR CONSERVATION AND REHABILITATION RESEARCH AND DEVELOPMENT,

FORESTRY RESEARCH AND DEVELOPMENT AGENCY MINISTRY OF FORESTRY

BOGOR – INDONESIA February 2011

Study on the Level of Genetic Diversity of Diospyros celebica, Eusideroxylon zwageri and Michelia spp. Using RAPD Markers Copyright©2011 By: Anthonius YPBC Widyatmoko, ILG Nurtjahjaningsih and Prastyono This report is a part of program ITTO Project PD 539/09 Rev.1 (F) “Promoting Conservation of Selected Tree Species Currently Threatened by Habitat Disturbance and Population Depletion” in cooperation with Center for Conservation and Rehabilitation Research and Development, Forestry Research and Development Agency, Ministry of Forestry. ISBN 978-602-8964-17-3 Published by Center for Conservation and Rehabilitation Research and Development, Forestry Research and Development Agency, Ministry of Forestry, Indonesia Jl. Gunung Batu No. 5 Bogor, Indonesia 16610 Phone : 62-251-8315222, 7520067 Facs. : 62-251-8638111 e-mail : [email protected] Printed by CV. Biografika, Bogor

mailto:[email protected]

PREFACE

This Technical Report is result of Activity 1.2.1, “to observe the level of genetic diversity and vulnerability of selected species to determine the conservation strategy of the selected species”. The activity is part of ITTO Project PD 539/09 Rev.1 (F), “Promoting Conservation of Selected Tree Species Currently Threatened by Habitat Disturbance and Population Depletion”. Sincere thanks and appreciation go to the Project Coordinator, Dr. Ir. Murniati, M.Si for support and invaluable advice. I would like also to express my gratitude thanks to all staff of ITTO Project PD 539/09 Rev.1 (F) for administration support. Colleagues at Molecular Genetic Laboratory, Center for Forest Biotechnology and Tree Improvement Research, are gratefully thanks for their support on laboratory works. Finally, we hope the result of this activity can support genetic conservation activities and strategy of the selected species in Indonesia. Bogor, February 2011 Authors

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TABLE OF CONTENT

PREFACE …………………………...…………………………………. iii

TABLE OF CONTENT ………………………………………………... v

LIST OF TABLES ……..………………………………………………… vi

LIST OF FIGURES ……………………………………………………… vii

ABSTRACT ……………………………………………………………... viii

INTRODUCTION ……………………………………………………….. 1

APPLIED METHODOLOGY …………………………………………... DNA extraction ……………………………………………………… RAPD analysis ………………………………………………………. Screening of RAPD primers …………………………………………

2 2 2 3

PRESENTATION OF THE DATA ………………………………………. 4

ANALYSIS AND INTERPRETATION OF THE DATA AND RESULTS Screening of RAPD Primers ………………………………………… Genetic Diversity and Population Relationship of Diospyros celebica Genetic Diversity and Provenances Relationship of Eusideroxylon zwageri ………………………………………………………………. Genetic Diversity and Provenances Relationship of Michelia spp …..

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CONCLUSIONS ………………………………………………………… 13

RECOMMENDATIONS ………………………………………………… 14

IMPLICATION FOR PRACTICE ………………………………………. 15

BIBLIOGRAPHY ……………………………………………………….. 17

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LIST OF TABLES

Table 1. List of used samples …………………………………………... 4 Table 2. Number of screened RAPD primer, selected primers, total

number of loci and means of loci per primer for the three species …………………………………………………………

5 Table 3. Genetic diversity within (diagonal) and between (below

diagonal) provenances for three provenances of Ebony from South Sulawesi and Central Sulawesi …………………………


diagonal) group for six sample groups of Ebony from South Sulawesi and Centre Sulawesi …………………………………


diagonal) groups for six sample groups of Ulin from Jambi and South Sumatera ………………………………………………...


diagonal) groups for seven sample groups of Michelia spp. from South Sumatera and East Java …………………………...

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LIST OF FIGURES

Figure 1. Dendrogram of genetic relationship between three provenances of Ebony from South Sulawesi (SS) and Centre Sulawesi (CS) based on UPGMA (Nei, 1978) ..........................

6 Figure 2. Dendrogram of genetic relationship between six sample

groups of Ebony from South Sulawesi (SS) and Centre Sulawesi (CS) based on UPGMA (Nei, 1978) ..........................

8 Figure 3. Dendrogram of genetic relationship between six sample

groups of Ulin from Jambi (Jb) and South Sumatera (SS) based on UPGMA (Nei, 1978) ………………………………..

10 Figure 4. Dendrogram of genetic relationship between seven groups of

Michelia spp. from South Sumatera (SS) and East Java (EJ) based on UPGMA (Nei, 1978) ..................................................

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ABSTRACT

Diospyros celebica, Eusideroxylon zwageri and Michelia spp are involed in threatened species according to their potential in the natural forest. Thus conservation of these species becomes a very crucial activity to be carried out. In order to conserve the species effective and efficient, information of genetic diversity, its distribution and genetic relationship between populations is very important. Information on genetic erosion in within population is also important to decide conservation strategy of each species. Analysis of genetic diversity and genetic erosion of each species were carried out using 5 selected RAPD primers. Total number of loci obtained from the primers has variation between 22 to 25. Mean genetic diversity of three provenances of Diospyros celebica (Ebony) was 0.2886. Mean genetic distance between provenances of D. celebica was 0.3029. Mean genetic diversity of two provenances of Eusideroxylon zwageri (Ulin) was 0.3678. Mean genetic distance between provenances of E. zwageri was 0.2572. Mean genetic diversity of two provenances of Michelia spp was 0.1878. Mean genetic distance between provenances of Michelia spp. was 0.6648. Based on cluster analysis, provenances of the three species were divided into two clusters. The clusters were correlated with geographic originality of the samples. Basically, different province will be clustered into different group. Genetic erosion from trees into poles and or wildling was not revealed in this study for D. celebica and E. zwageri. However, for M. champaca, genetic diversity was tend to decrease from trees to poles and wildlings. Key words: Diospyros celebica, Eusideroxylon zwageri, Michelia, RAPD,

genetic diversity, genetic erosion.

Study on the Level of Genetic Diversity of Diospyros celebica, Eusideroxylon zwageri and Michelia spp. Using RAPD Markers

INTRODUCTION

In Activity 2.1, study on the level of genetic diversity of three species (Diospyros celebica, Eusideroxylon zwageri and Michelia spp) was carried out in order to provide genetic information of the species for a purpose of establishing ex situ conservation plots. This was achieved by employing DNA markers.

The level of genetic diversity of the species is limited. Ideally, any genetic

conservation works should have information on genetic diversity of the species before hand. However, the genetic diversity information for most tropical rain forest species is still limited. Several studies on genetic diversity have been reported for Alstonia scholaris (Hartati et al., 2007), Instia bijuga (Rimbawanto and Widyatmoko, 2006), Santalum album (Rimbawanto et al., 2006) and Gyrinops verstigii (Widyatmoko et al., 2009). Genetic diversity of E. zwageri has been reported by Sulistyowati et al. (2005) and Rimbawanto et al. (2006a).

The availability of information on genetic diversity of these species is

essential for designing an appropriate sampling strategy for genetic conservation purposes. Genetic markers are needed to study many aspects of forest trees such as reproduction system, genetic diversity and gene flow.

RAPD (Random Amplified Polymorphic DNA) analysis (Wiliams et al.,

1990; Welsh and McClelland, 1991) is one of the most effective tools of DNA based fingerprinting techniques applied to analyze genetic diversity. RAPD analysis that is based on PCR (polymerase chain reaction) with 10-mer random oligonucleotide primer is relative easier than any DNA markers and could be carried out in a simple instrument.

The aim of this study is to investigate genetic diversity, its distribution and

genetic relationship between populations of Diospyros celebica (ebony), Eusideroxylon zwageri (ulin) and Michelia spp (cempaka) using RAPD markers.

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APPLIED METHODOLOGY

DNA extraction

Total genomic DNA was extracted using a modified Cetyl Trimethyl Ammonium Bromide (CTAB) protocol reported by Shiraishi and Watanabe (1995).

RAPD analysis RAPD analysis was performed in a reaction containing 10 mM Tris-HCl (pH

8.3), 10 mM KCl, 3.0 mM MgCl2, 0.2 mM each of dNTPs, 0.5 unit/10µl AmpliTaq DNA polymerase, a Stoffel Fragment (Applied Biosystem), 0.25 µM each of primers (Operon Technologies), and 10 ng/10µl template DNA. The condition of amplification was 94°C for 1 min., 45 cycles of 30 s at 94°C, 30 s at 37°C, and 90 s at 72°C, followed by 7 min. at 72°C. The amplification products were separated by electrophoresis in 1% agarose gel with ethidium bromide and detected with a 302-nm UV transilluminator.

Twenty RAPD primers were tested for screening polymorphic RAPD

primers which will be used in the study of genetic diversity for the three species. All the RAPD primers were supplied by Operon Technologies. Some criteria have been used to select polymorphic RAPD primers in the screening. Those criteria were: number of polymorphic loci, clear bands between 200 - 800 bps and reproducibility of the locus.

The presence (1) or absence (0) of the polymorphic fragments attained from

electrophoresis was noted as 1/0 data. Based on this data, the genetic similarity (S) and genetic distance (D = 1 - S) among all individuals were calculated using a simple matching coefficient (Sokal and Michener, 1958). Parameter genetic i.e. level of genetic diversity (h) was analyzed within populations. A dendrogram was constructed using the UPGMA method from the matrix of genetic distance among individuals. These parameters genetic were calculated using Popgene 1.32 computer program (Yeh et al., 1999).

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Screening of RAPD primers

Three criteria were used to select polymorphic RAPD primers in the screening of RAPD primers. The first criterion was number of polymorphic loci. This criterion is important to obtain as many information as possible with less number of primers. This method is used not only because of its time efficiency, but also the relatively low cost for analysis. The second criterion was clearance of RAPD band. RAPD analysis normally produces a lot of bands/loci, however because of competition among the bands some of them are weak band. The weak band can also be caused by mis-annealing of RAPD primers. Thus, the weak band normally has low reproducibility. Only the clear RAPD bands were selected since the clear bands are always high reproducibility. Some loci have clear and weak bands, in which clear band maybe homozygote and the weak band is heterozygote. Another reason is the loci contain two different loci. Thus, this type of loci was not selected in order to avoid mislabeling. The clear band is all bands which appear in one locus. Thus, reproducibility of the locus should be checked for all bands which appear in the locus. Reproducibility of the locus was the third criterion for screening.

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PRESENTATION OF THE DATA

Plant Materials collection

Leaf sample of individual trees was collected from the tree species. Diospyros celebica leaf samples were collected from South Sulawesi and Central Sulawesi. Eusideroxylon zwageri leaf samples were also collected from two provenances, Jambi and South Sumatera. Michelia spp leaf samples were collected from South Sumatera and East Java. Details of collected samples of the three species are shown in Table 1. Table 1. List of used samples

No. Provenance Materials No. of samples

DBH (cm)

Diospyros celebica Trees 8 35 – 45

1 Wasupoda District Luwu Timur, South Sulawesi S 2o29.191’, E 121o06.474’, 401 m asl

Wildlings 8

Trees 8 35 – 45 2

Mangkutana District Luwu Timur, South Sulawesi S 2o27.087’, E 120o48.891’, 66 m asl

Wildlings 8

Trees 8 35 – 45 3

Parigi Moutong, Central Sulawesi S 01o03’45’’ - 01o04’25’’ E 121o31’12” - 121o31’55”, 150 m asl

Wildlings 8

Eusideroxylon zwageri Trees 8 35 – 120 Poles 8 15 – 30 1 Batanghari, Jambi Wildlings 8 Trees 8 83 – 95 Poles 8 5 – 15 2 Musi Rawas, South Sumatera Wildlings 8

Michelia champaca Trees 8 15 – 32.5 Arjuno Mountain, Purwodadi Sub-

District, Pasuruan District Poles 2 4 1 Wilis Mountain, Ngawi Poles 6 1 – 7.6

Trees 6 75 – 90 Poles 6 15 – 40 Lahat1, South Sumatera Wildlings 8 2

Lahat2, South Sumatera Trees 4 50 – 70 Michelia alba Arjuno Mountain, Purwodadi Sub-

District, Pasuruan District Trees 4 16 – 37,7

Bumiaji Arboretum, Malang Trees 2 21.3 – 56.3

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ANALYSIS AND INTERPRETATION OF THE DATA AND RESULTS

Screening of RAPD Primers

Total of twenty Operon RAPD primers were screened for each species to find

polymorphic RAPD primers for a genetic diversity analysis. Screening of RAPD primers were carried out using three criteria as mentioned above. By using the three criteria, number of selected RAPD primers, total loci and mean of loci per primer are presented in Table 2.

Table 2. Number of screened RAPD primer, selected primers, total number of loci

and mean of loci per primer for the three species

Criteria D. celebica E. zwageri Michelia spp Number of screened primer 20 20 20 Selected RAPD primers 5 5 5 Total number of loci 23 25 22 Mean of loci per primer 4,6 5 4,4

Genetic Diversity and Population Relationship of Diospyros celebica

Based on selected RAPD markers, genetic variation of populations of ebony

ranged from 0.2476 to 0.3217 (Table 3). The highest genetic diversity was shown by Parigi population (0.3217) and followed by Mangkutana population (0.2966). The lowest genetic diversity was shown by Wasupoda population (0.2476). Details of genetic diversity of each population are presented in Table 3. Mean of genetic diversity of the three populations of ebony was 0.2886, which is higher than coniferous species i.e. Pinus attenuata (0,011), P. radiata (0.08), P. sylvestris (0.022), P. menziesii (0.050), even another broadleaf species i.e. Alstonia scholaris (0.247).

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Table 3. Genetic diversity within (diagonal) and between (below diagonal) provenances for three provenances of ebony from South Sulawesi and Central Sulawesi

Provenance Parigi Wasupoda Mangkutana

Parigi 0.3217 Wasupoda 0.4542 0.2476 Mangkutana 0.4191 0.0354 0.2966

Eventhough number of individual trees of the species in several provenances

have been decreased, the populations still have high genetic diversity. Reasons of this condition are due to some following posibilities: genetic diversity base of the provenances was initially high, there is high cross pollination between individual trees within provenance, and the exploitation of the trees in the provenance was carried out in the recent year.

In order to clarify the relationship of the species among the provenances, a

UPGMA dendrogram based on Nei’s standard genetic distance was constructed from the genetic distances (Fig. 1). The highest mean genetic distance between provenances was recognized between Parigi and Wasupoda (0.4542), and the close relation between two provenances was shown by Wasupoda and Mangkutana (0.0354).

Parigi (CS)

Wasupoda (SS)

Mangkutana (SS) Fig 1. Dendrogram of genetic relationship between three provenances of ebony

from South Sulawesi (SS) and Centre Sulawesi (CS) based on UPGMA (Nei, 1978).

The UPGMA cluster analysis reflected two main clusters. The first cluster

comprised population from Central Sulawesi (Parigi), and the second cluster consisted of populations from South Sulawesi (Wasupoda and Mangkutana). The

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genetic distance between the two clusters was 0.4366. These results indicate that the two clusters are genetically different according to geographic position.

The proportion of ± 70% of genetic diversity was distributed within

provenance, while the remaining 30% was distributed between provenances. This condition is likely due to evolution and adaptation process of the species and its provenances. The high genetic distance between the two clusters (province) might be influenced by high exploitation of the species.

In order to reveal genetic degradation within population, two sample groups

were collected from each provenance i.e. trees and wildlings. Genetic diversity within and between samples groups is shown in Table 4. Table 4. Genetic diversity within (diagonal) and between (below diagonal) group

for six sample groups of ebony from South Sulawesi and Centre Sulawesi

Groups P-t P-w W-t W-w M-t M-w

Parigi-trees (P-t) 0.2527

Parigi-wildlings (P-w) 0.1062 0.3048

Wasupoda-trees (W-t) 0.5186 0.3664 0.1848

Wasupoda-wildlings (W-w) 0.4029 0.3648 0.1499 0.3533

Mangkutana-trees (M-t) 0.5729 0.5536 0.1397 0.1110 0.2080

Mangkutana-wildlings (M-w) 0.5270 0.4862 0.0886 0.0762 0.0508 0.2290

The highest genetic diversity was shown by Wasupoda-widlings (0.3533), yet Wasupoda-trees had the lowest genetic diversity. Whilst, there was no significant different of genetic diversity between trees and wildings groups of the other two provenances (Parigi and Mangkutana). Random mating system in these two provenances and/or small samples using in this study may be reasonable for this condition. If the number of samples is increased, genetic diversity between trees and wildlings may be similar.

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Wildlings of Parigi and Mangkutana showed slightly higher genetic diversity than their parent trees. Random mating system in the provenance and small number of sample used in this study may account for this condition. If the number of samples is increased, genetic diversity between trees and wildlings may be similar. Significant difference of genetic diversity between Wasupoda-trees and Wasupoda-wildling may be due to related to following factors, such as random mating system, small number of trees and gene flow from other provenances.

Parigi-trees

Parigi-widlings

Wasupoda-trees

Wasupoda-wildlings

Mangkutana-trees

Mangkutana-wildlings

Fig 2. Dendrogram of genetic relationship between six sample groups of ebony from South Sulawesi (SS) and Centre Sulawesi (CS) based on UPGMA (Nei, 1978).

Fig. 2 shows a UPGMA dendrogram of the ebony when each provenance

was divided into two groups (trees and wildings). There are distinct groups of ebony taken from Central Sulawesi and South Sulawesi. Generally, wildling of each provenance has a close relationship with their parent trees, however in this case, wildling of Wasupoda group was in a group of Mangkutana population. This condition is most likely due to the high genetic diversity of Wasupoda-wildlings and low genetic diversity of Wasupoda-trees.

Genetic Diversity and Provenances Relationship of Eusideroxylon zwageri

Genetic diversity of provenances of ulin based on selected RAPD markers

ranged from 0.2461 to 0.4258 (Table 5). The highest genetic diversity was shown by Musi Rawas-wildlings (0.4258) and followed by Musi Rawas-poles (0.4102).

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The lowest genetic diversity was shown by Batanghari-big trees (0.2461). Detail of genetic diversity of each provenance is presented in Table 5. Mean genetic diversity of two populations of ulin was 0.3678, which is higher than ebony’s mean genetic diversity obtained in this study (0.2886).

Table 5. Genetic diversity within (diagonal) and between (below diagonal) groups

for six sample groups of ulin from Jambi and South Sumatera

Groups B-t B-p B-w M-t M-p M-w

Batanghari- trees (B-t) 0.2461

Batanghari-poles (B-p) 0.0226 0.3516

Batanghari-wildlings (B-w) 0.0276 0.0060 0.3672

Musi Rawas-trees (M-t) 0.3897 0.2396 0.2195 0.4062

Musi Rawas-poles (M-p) 0.3609 0.2204 0.1926 0.0234 0.4102

Musi Rawas-wildlings (M-w) 0.3144 0.1992 0.1792 0.0167 0.0202 0.4258

In order to reveal the relationship among six groups of Eusideroxylon zwageri (ulin), a UPGMA dendrogram was constructed from the genetic distances (Fig. 3). The highest mean genetic distance between groups was 0.3897 which was between Batanghari-trees and Musi Rawas-trees , and the closest relation between two groups was shown by Batanghari-poles and Batanghari-wildlings (0.0060).

There were two distinct clusters identified. The first cluster comprised

groups from Jambi (Batanghari-trees, Batanghari-poles and Batanghari-wildlings), and the second cluster consisted of groups from South Sumatera (Musi Rawas-trees, Musi Rawas-poles and Musi Rawas-widlings). A very close relationship was shown among three groups in each cluster.

Genetic degradation from trees to poles, and from poles to wildlings almost

did not reveal in both provenances of ulin, otherwise, it tends to increase. Trees group of Batanghari population in particular has the lowest genetic diversity to compare with poles and wildlings. There was a clear evidence that number of trees in the provenances was decreased significantly caused by over exploitation.

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Batanghari- trees (Jb)

Musi Rawas-trees (SS)

Batanghari- poles (Jb)

Batanghari- wildlings (Jb)

Musi Rawas-poles (SS)

Musi Rawas-wildlings (SS)

Fig 3. Dendrogram of genetic relationship between six sample groups of ulin

from Jambi (Jb) and South Sumatera (SS) based on UPGMA (Nei, 1978). Genetic Diversity and Provenances Relationship of Michelia spp

Based on selected RAPD markers, genetic variation of provenances of

Michelia spp ranged from 0.1000 to 0.2560 (Table 6). The highest genetic diversity was shown by Lahat1-poles (0.2560) and followed by Pasuruan-trees (0.2966). The lowest genetic diversity was shown by Lahat1-seedling (0.1000). Detail of genetic diversity of each provenance is presented in Table 6. Mean genetic diversity of six groups of Michelia champaca was 0.1878, which is lower than ebony (0.2886) and ulin (0.3678) obtained in this study. Table 6. Genetic diversity within (diagonal) and between (below diagonal) groups

for seven sample groups of Michelia spp. from South Sumatera and East Java

Groups L1-t L1-p L1-s L2-t P-t P-p M.alba

Lahat1-trees (L1-t) 0.1731 Lahat1-poles (L1-p) 0.0256 0.2560 Lahat1-seedlings (L1-s) 0.0473 0.0428 0.1000 Lahat2-trees (L2-t) 0.1565 0.1573 0.2292 0.1512 Pasuruan-trees (P-t) 0.5847 0.5141 0.6470 0.6790 0.2419 Pasuruan-poles (P-p) 0.7282 0.6145 0.8405 0.7103 0.0888 0.2050 M. alba 0.7399 0.6458 0.8623 0.8116 0.2382 0.0754 0.1368

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In order to clarify the relationship among six groups of Michelia champaca and one population of M. alba, a UPGMA dendrogram was constructed from these genetic distances (Fig. 4). The highest mean genetic distance between groups was 0.8405 which was between Lahat1-seedling and Pasuruan poles (0.8405), and the closest relation between two groups was shown by Lahat1-trees and Lahat1-poles (0.0256).

Lahat1-trees (SS)

Lahat1-poles (SS)

Lahat1-seedlings (SS)

Lahat2-trees (SS)

Pasuruan-trees

Pasuruan-poles

M. alba (EJ)

Fig 4. Dendrogram of genetic relationship between seven groups of Michelia spp. from South Sumatera (SS) and East Java (EJ) based on UPGMA (Nei, 1978).

Two distinct clusters were clearly identified. The first cluster comprised groups from South Sumatera (Lahat1-trees, Lahat1-poles, Lahat1-seedlings and Lahat2-trees), and the second cluster consisted of groups from East Java (Pasuruan-trees, Pasuruan-poles, M. alba). There were four groups included in the first cluster which were clustered into two groups, namely Lahat 1 samples and Lahat 2 samples. The grouping revealed the origin of the groups. Lahat 1 was owned by one farmer, and Lahat 2 was belonged to another farmer. Each farmer established the plantation/seed orchard from different population, provenance or mother trees.

Group of samples from East Java was also divided into two small clusters.

However, Pasuruan-poles close to M. Alba rather than their parents. This condition is most likely due to two factors, namely inter-species hybrid between M. champaca and M. alba occurred in the same area and small number of samples of M. alba used in this study.

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Genetic diversity of seedling in Lahat1 (South Sumatera) and poles in Pasuruan (East Java) tended to decrease comparing to their parents trees which is different to the fact in ebony as described above. High inbreeding caused by a non-random mating system of mother trees may account for the genetic degradation of Michelia spp.

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CONCLUSIONS

1. Mean genetic diversity of three provenances of Diospyros celebica (ebony) was 0.2886. Mean genetic distance between the provenances of D. celebica was 0.3029. Genetic distance between provenances within province was 0.0354.

2. Mean genetic diversity of two provenances of Eusideroxylon zwageri (ulin) was 0.3678. Mean genetic distance between the provenances of E. zwageri was 0.2572.

3. Mean genetic diversity of two provenances of Michelia champaca was 0.1878. Mean genetic distance between the provenances of Michelia spp. was 0.6648.

4. Based on cluster analysis, provenances of the three species were divided into two clusters. The clusters were related to geographic originality of the samples. Basically, different province will be clustered into different group.

5. Genetic degradation from trees to poles and or wildling was not revealed in this study for D. celebica and E. zwageri. On the other hand genetic diversity of M. champaca tended to decrease from trees to poles and wildlings.

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RECOMMENDATIONS

Study on the level of genetic diversity of Diospyros celebica, Eusideroxylon zwageri and Michelia spp. has been carried out in this project. Based on the results obtained in this study, three recommendations are proposed as follows:

1. Diospyros celebica and Eusideroxylon zwageri have been recognized as threatened species due to their remaining populations or provenances and number of remaining individual in their natural distribution. According to analysis of genetic diversity of the two species in this project, however, they still have high genetic diversities indicating that both species will be survived and exist providing the genetic diversity of both species are well maintained.. It is therefore, in-situ and ex-situ conservation program are required.

2. Michelia spp has different condition to compare with the other two species (Diospyros celebica and Eusideroxylon zwageri). Although natural distributions of Michelia spp are as limited as the others, many plantations have been established, especially for M. champaca. Therefore, natural distribution survey and mapping for Michelia spp are of critically important to decide in-situ conservation plan of the species. Ex-situ conservations of M. champaca can be carried in parallel with tree breeding programs or plantations establishment.

3. A number of information on genetic diversity of the three species (Diospyros celebica, Eusideroxylon zwageri and Michelia spp) have been obtained. However, information of mating system of natural population, include inbreeding coefficient and gene flow of D. celebica and E. zwageri is still required to design a conservation plan for both species.

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IMPLICATION FOR PRACTICE

The potential applications of molecular markers to facilitate gene conservation in the tropics could be divided in to two steps. Firstly, they can be used to evaluate the status of genetic background of ex situ plantations and in situ sites of any forest tree species which are already established based on conventional silviculture practice whether they contain correct clones and ramets and have sufficient genetic diversity for the conservation as the representative of the species gene pool.

Secondly, they can be applied to evaluate the status of genetic resources of

those which have never been established but planned to establish conservation programs by determining genetic variation within and among population or provenance and mating system as well as gene flow. Generally speaking, they can be used as a guideline of how and where to collect samples for ex-situ gene conservation and which sites are suitable as in-situ gene conservation. To maximize the latter application, it should be combined with an eco-geographic survey and adaptive traits measurement.

In this project, the second step is appropriate to the three species because ex-

situ and in-situ conservations of the species have never been established in Indonesia but planned to be established. Information on genetic variation within and between populations or provenances, and genetic relationship between populations or provenances has been obtained in this study. Genetic diversity of ebony and ulin is still high. Genetic variation within provenance is higher than that between populations. However, genetic distance between clusters was also high. Based on this information, collection of genetic materials for an ex-situ conservation should be focused both on individual trees within populations and each cluster. Each province should be represented by one population.

Genetic degradation was not revealed for both D. celebica and E. zwageri. It

is indicating that the genetic diversity of both species can be kept in this recent level when the remaining populations are well maintained and conserved. Genetic materials for establishing an ex-situ conservation can be treated as individual mother tree where information of every single tree is recorded or can be bulked during the collection. It is critically important, however, to ensure that the genetic materials were collected from several individual mother trees.

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In case of M. champaca, genetic diversity tended to decrease from trees to poles, and from poles to wildlings/seedlings. Therefore, in order to establish an ex-situ conservation plot of the species, there are two important factors to be concerned during genetic materials collection. The first is that genetic materials should be collected from as many as individual trees (collected separately). The second, the individual mother trees should represent all populations in the area. For example in Lahat area, mother trees should be selected from several individual trees from each farmer area.

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BIBLIOGRAPHY

Hartati, D., Rimbawanto, A., Taryono, Sulistyaningsih, E. dan Widyatmoko, AYPBC. 2007. Pendugaan keragaman genetik di dalam dan antar provenan Pulai menggunakan penanda RAPD. Jurnal Pemuliaan Tanaman Hutan 1(2): 51-98.

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STUDY ON THE LEVEL OF GENETIC DIVERSITY OF Diospyros celebica