INTRODUCTION OBJECTIVE REGIONAL ANALYSIS ON STOCK IDENTIFICATION OF GREEN AND HAWKSBILL TURTLES IN THE SOUTHEAST ASIAN REGION

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1 The Third Technical Consultation Meeting (3rd TCM) Research for Stock Enhancement of Sea Turtles (Japanese Trust Fund IV Program) 7 October 2008 REGIONAL ANALYSIS ON STOCK IDENTIFICATION OF GREEN AND HAWKSBILL TURTLES IN THE SOUTHEAST ASIAN REGION Wahidah Mohd Arshaad, Syed Abdullah Syed Abdul Kadir 2 Marine Fishery Resources Development and Management Department, Taman Perikanan Chendering,2080 Kuala Terengganu, Terengganu. 2 Turtle and Marine Ecosysten Centre, 200 Dungun, Terengganu. (wahidah@seafdec.org.my) INTRODUCTION Knowledge of population dynamics is largely obtained from long-term mark-recapture studies of females tagged while nesting on the beaches. These studies show that breeding female turtles display high fidelity to the same nesting beaches (Hendrickson 98; Carr & Ogren 960) and Carr (967) hypothesized that mature nesting female turtles were selecting their natal beach to deposit eggs. Studies in the southern Great Barrier Reef (sgbr) demonstrate that green turtles also display fidelity to resident feeding grounds throughout their adult lives (Limpus et al. 992) with females reaching sexual maturity at about 40 years (Limpus & Chaloupka 997). Detection of subpopulations of sea turtles is a very essential ecological aspect to promote their conservation and enhancement because the resources-protect schemes should be made to the each population unit. Mitochondrial DNA has proved particularly effective for detecting population structure in marine turtles. Analysis of mtdna structure in Atlantic green turtle population supported the natal homing hypothesis, as geographically distant were found to have heterogeneous mtdna frequencies (Bowen & Avise 996). ) To detect subpopulations of nesting green and hawksbill turtles in Southeast Asian region OBJECTIVE 2) To identify the genetic markers of different management units

2 Table : Number of samples green turtles collected for the Trust Fund IV. METHOD Country Location N Brunei Brunei Beach 4 Indonesia Derawan Sangalaki Panjang Malaysia Myanmar Philippines Thailand Vietnam Redang Sarawak Turtle s Sabah Turtle s Tameahla Coco Panikian APO Reef NP Baguan Huyong Khram Con Dao Minh Chau Total 332 Figure : Sampling sites of green turtle for Trust Fund IV study. Table 2: Number of samples hawksbill turtles collected for the Trust Fund IV. Thamee Hla Coco Huyong Minh Chau Khram Con Dao Redang Sarawak Turtle s APO Reef National Park Brunei Beach Philiphines Turtle s Panikian Sabah Turtle Derawan/Sangalaki/Panjang Country Location N Brunei Brunei Beach 4 Indonesia Kimar Belitung 38 Malaysia Melaka Sabah Turtle s 29 Myanmar Coco 4 Philipphine Dava0 Gulf APO Reef Natural Park Misamis Oriental Bataan Thailand Khram 9 Total 36 Figure 2: Sampling sites of hawksbill turtle for Trust Fund IV study. Coco Khram Melaka APO Reef National Park Brunei Beach Misamis Oriental Davao Gulf Sabah Turtle SAMPLES ANALYSIS Belitung 2

3 DNA was extracted from skin tissue PCR mtdna DNA control region - primer TCR and TCR6 (green), LTEi9 and H90 (hawksbill) - target an area of 389 bp and 740 bp of the control region Sequenced PCR product forward and reverse by automated sequencer Statistical analysis - Align sequences (Clustal X/W) - Blast with GenBank database for similar sequences - MEGA 4 (phlogenetic tree) - Arlequin 3. (estimate h and π, population differentiation & quantify genetic diversity) RESULT Figure 3: Neighbour-joining tree of Kimura 2-parameter distance for green turtle haplotypes based on mtdna control region sequences, using the hawksbill haplotype as an outgroup. GREEN SEA TURTLE Table 3: Frequencies of green turtles (Chelonia mydas) mtdna haplotypes. Clade I Clade II Clade III C CMP G G G G G Haplotypes M2 C Country Location N R T M C4 D2 C3 M3 C7 P2 B T2 T3 A2 A3 A6 P 8 4 G2 G3 G4 G G7 No. 3 Brunei Brunei Beach 4 2 Indonesia Derawan Sangalaki Panjang Pengumbahan Enu Redang Malaysia Paka 4 2 Pahang Perak Sarawak Sipadan Sabah Turtle s Tameahla 8 4 Myanmar 2 9 Coco Philippine Panikian 9 9 APO Reef NP 4 4 Phi Turtle s Huyong Thailand 8 Khram Con Dao Vietnam Minh Chau Total * Haplotypes in black () was published by Moritz et al. (2002) and haplotypes in red (9) are new. Figure 4: Neighbour-joining tree of Kimura 2-parameter distance for all sample rookeries of green turtle haplotypes based on mtdna control region sequences, using the hawksbill haplotype as an outgroup. 3

4 Table 4: Polymorphic sites in the 38 bp sequence Polymorphic sites (47) Haplo types () D2 G A G A C C A - T G T A T A C G G G G G G T G A A G C C C T T G G C A G T A C T G A A CMP A R A G C A G... M A G. T A C A A C A C A M3 A A C A C C.... T.. -. A M A C A A P A A.. G A G A.. G4. G C G C G G.. A G G G G A B A. A. T T G A. A. G.. T A.... A C. G G.. T T C T C T2 A. A. T T G A. A. G.. T A.... A C. G G.. T T C... G... T C T3 A. A. T T G A. A. G.. T A.... A C. G G.. T T C C. T C G4 A. A. T T T -... G.. T A.... A A. G G.. T T C T C A2.... T T G - C..... T A.... A C A G G. T. T C... T. T C G T G A3... G T T G - C A.... T A.... A C A G G. T T T C... T. A C G T G P... G T T G - C A.... T A.... A C A G G. T T T C... T. A C G T A6.... T T G - C A.... T A.... A C A G G. T T T C... T. A C G T G G7... G T T G - C..... T A.... A C A G G. T T..... T. A C G T Table : Haplotype (h) and nucleotide (π) diversities by nesting beaches. No. Country Nesting beach N h hd π Brunei Brunei Beach Indonesia Derawan Sangalaki Panjang Pengumbahan Enu Malaysia Redang Paka Pahang Perak Sarawak Sipadan Sabah Turtle s Myanmar Tamaehla Coco APO Reef Natural Philiphine Park 7 Panikian Philiphine Turtle s Thailand Huyong Khram Vietnam Minh Chau Con Dao Overall Table 6: Haplotype (h) and nucleotide (π) diversities by management units (Exact test of sample differentiation based on haplotype frequencies, Raymond M. and F. Rousset. 99). No. Country Nesting beach N h hd π Indonesia Derawan Sangalaki Pengumbahan Enu Malaysia/ Redang+Paka+Perak Philippine Pahang Sarawak Sipadan Sabah+Philippine TI Myanmar Tamaehla+Coco Thailand Huyong+Khram Vietnam Minh Chau+Con Dao Overall Figure : Location of 2 genetically distinct breeding stocks or management units (Exact test of sample differentiation based on haplotype frequencies Raymond M. and F. Rousset. 99). Thamee Hla Coco Huyong Khram Con Dao Redang Pahang Sarawak Turtle s Pengumbahan Minh Chau Sangalaki Philiphines Turtle s Sipadan Derawan Sabah Turtle Enu Table 7 : Frequencies of hawksbill turtles mtdna haplotypes. HAWKSBILL TURTLE Clade I Clade II Country Location N N 2 Ei_ Ei_ Ei_ Ei_ Ei_ Ei_ Ei_ Ei_ Ei_ Ei_ Ei_ Ei_ Ei_ Ei_ Ei_ H Brunei Brunei Beach Indonesia Kimar Belitung Malaysia Melaka Sabah Turtle s Myanmar Coco Philipphine Dava0 Gulf 2 7 APO Reef Natural Park 4 2 Misamis Oriental Bataan Thailand Khram Total *Note: N = No. of samples received, N 2 = No. of samples analyzed, H = No. of haplotypes. 4

5 Figure 6: Neighbour-joining tree of Kimura 2-parameter distance for all green turtle haplotypes based on mtdna control region sequences. Figure 7: Neighbour-joining tree of Kimura 2-parameter distance for all sample countries of hawksbill turtle haplotypes based on 6 mtdna control region sequences. Haplotypes () 6 Table 8: Polymorphic sites in the 740 bp sequence Polymorphic sites (43) Ei_ A - A G A G T T G A C T C A T T A A G A C A T A A A A T G A T A C G A G A A G T G A A G Ei_2. - G Ei_ C.... Ei_ G G.... G. Ei_ T G G.... G. Ei_ T... G G..... C Ei_ T... G G Ei_ G..... G Ei_9 G T. A C T C C A. T C T. C C. C A G T. C.. G C. G..... G A.. A C A. G A Ei_0 G T. A C T C C A. T C.. C C. C A G T. C... C. G..... G A.. A C A. G A Ei_ G T. A C T C C A. T C T G C C. C A G T G C... C G A.. A C A G G A Ei_2 G T. A C T C C A. T C T. C C. C A G T G C... C G A.. A C A. G A Ei_3 G T. A C T C C A. T C T. C C. C A G T. C G.. C G G A.. A C A. G A Ei_4 G T. A C T C C A. T C. G C C. C A G T. C... C G A.. A C A. G A Ei_ G T.. C T C. A G T C... C. C. G T. C... C.. G C A G..... A. A. G. 6 0 Table 9: Haplotype (h) and nucleotide (π) diversities by nesting beaches. No. Country Nesting Beach N H h π. Brunei Brunei Beach Indonesia Kimar Belitung Malaysia Melaka Sabah Turtle s Myanmar Coco Philiphine Dava0 Gulf APO Reef Natural Park Misamis Oriental Bataan 6. Thailand Khram Total 88 *Note: H = No. of haplotypes, h = haplotype diversity and π = nucleotide diversity. GREEN TURTLE haplotypes were detected which was published by Moritz et al and 9 are new. Dominant haplotypes are C3, found at all samples sites except Enu, Indonesia and Panikian, Philiphine. DISCUSSION C3 dominant haplotype for Pengumbahan (7/23), Redang (42/73), Paka (4/), Pahang (8/2), Perak (3/), Sipadan (24/40), Tameahla (8/), Coco (7/), Huyong (0/9), Khram (/), Vietnam (/3).

6 Dominant: C4, Sarawak (40/62), D2, Sabah (47/8) & Philippine Turtle (44/62), C4, Enu (27/28), A3, Panikian (9/9). haplotypes are share between nesting beach and 9 haplotypes are unique to individual nesting beach. HAWKSBILL TURTLE Due to the limited of samples number, the output of this study will not give a true figure of hawksbill population genetic. 3 haplotypes are share between nesting beach and 2 haplotypes are unique to individual rookeries. Melaka, no haplotype diversity. Table 0: Polymorphic sites of haplotypes found in Melaka (392 bp) Figure 6: Phylogenetic tree of haplotypes found in Melaka. Polymorphic sites (43) Haplo types () H2 C G G G G T C T A C A G T C G C A H3. A. A.. T C G H2... A A. T H3. A. A A G T Ei_9 T A A A A. T.. A G A C T A G T CONCLUSION This had study identified 2 genetically breeding aggregations of green turtle throughout Southeast Asia (Management Units). The population diversity overall for green turtle are still high except for Enu and Vietnam. More effort should be focused on these nesting populations to keep population size high and maintain this diversity. RECOMMENDATION 6

7 Research on feeding ground must done to know the feeding ground of haplotype C3 for green turtles. More work should do on identify the genetic of hawksbill turtles. AKNOWLEDGEMENT Japanese Trust Fund (Trust Fund IV, Research for stock enhancement of sea turtles) All Technical Members Countries (Brunei, Cambodia, Indonesia, Malaysia, Thailand,The Philiphines and Vietnam. Dr. Juanita Joseph (University Malaysia Terengganu) Nurul Azrin Yusof and Nik Zuraini Omar. Contract staffs, Ms. Zuhailah, Mr. Abdul Hadi and Ms. Tengku Noor Aminah. All practical students. 7