1 MITOCHONDRIAL DNA DIVERSITY OF THE LONG · PDF fileSequence Analysis, Genetic Distance and Phylogenetic Relationship Multiple alignments of the sequences were done by using the program

Journal of Wildlife and Parks (2014) 29 : 1-8 1

MITOCHONDRIAL DNA DIVERSITY OF THE LONG-TAILED MACAQUE (Macaca fascicularis) FROM THE NORTHERN REGION OF PENINSULAR MALAYSIA

Jeffrine J. Rovie-Ryan*1, 3, Abdullah, M.T.2, Frankie T. Sitam1, 3, Soon Guan Tan4, Misliah Mohamad Basir3, 5, Zaaba Zainol Abidin6, Charles Keliang3 & Azroie Denel3

1Wildlife Genetic Resource Bank (WGRB) Laboratory, Ex-Situ Conservation Division,Department of Wildlife and National Parks (DWNP) Peninsular Malaysia,

KM10, Jalan Cheras, 56100 Kuala Lumpur, Malaysia2Kenyir Ecosystem Research Centre, Universiti Malaysia Terengganu, 21030 Kuala Terengganu,

Terengganu, Malaysia3Outbreak Response Team (ORT), DWNP, KM 10, Jalan Cheras, 56100 Kuala Lumpur

4Department of Cell and Molecular Biology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia

5Deputy Director General I Office, DWNP, KM 10, Jalan Cheras, 56100 Kuala Lumpur6Deputy Director General II Office, DWNP, KM 10, Jalan Cheras, 56100 Kuala Lumpur

* Corresponding author: [email protected]; [email protected]

ABSTRACT

We examined the genetic diversity of 64 long-tailed macaques from the northern states of Peninsular Malaysia covering the states of Perlis and Kedah including the Langkawi Island using the complete control region (CR) segment of the mitochondrial DNA. Standard genetic diversity including nucleotide diversity, haplotype diversity and genetic divergence were calculated. Moderate nucleotide diversity (π = 0.021) was observed which is higher than a previous study on the Penang M. fascicularis population. Twenty-three haplotypes were detected with haplotype diversity, h of 0.936. Haplotype sharing was observed among Langkawi and Perlis macaques indicating historical connection between the island and the mainland. Phylogenetic trees constructed grouped the samples into 4 groups without any obvious populations structuring.

Keywords: Control region, Genetic diversity, Long-tailed macaque, mtDNA, Northern Peninsular Malaysia, Phylogenetic relationships

INTRODUCTION

The long-tailed macaque, Macaca fascicularis, also known as the crab-eating or cynomolgus macaques are the second most studied non-human primates after the rhesus macaque, M. mulatta. Distributed across vast areas of the mainland and insular Southeast Asia (Fooden, 1995), this species is very common and lives sympatrically with the other primate species including human. Commonly used as primate model in biomedical research, M. fascicularis are the prime candidate for the study of human diseases (Villano et al., 2009, Shiina et al., 2010).

In 2007, a Non Detrimental Findings (NDF) study was conducted by the Department of Wildlife and National Parks (DWNP) to estimate the M. fascicularis population in Peninsular Malaysia, indicating approximately 740,000 individuals (DWNP, unpublished report). The ban imposed by the Malaysian government on primate exportation and hunting in 1985 (DWNP, 1985) led to the population increase and since then had caused conflicts with human and considered as pest (DWNP, 2006). A recent field census conducted by the DWNP in 2011 revealed a total of 127,050 conflict macaques (DWNP, unpublished report).

Jeffrine J. Rovie-Ryan, Abdullah, M.T., Frankie T. Sitam, Soon Guan Tan, Misliah Mohamad Basir, Zaaba Zainol Abidin,

Charles Keliang & Azroie Denel

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Several studies on M. fascicularis using mitochondrial DNA (mtDNA) are available on the intraspecies variation (Smith et al., 2007), population genetics (Lawler et al., 1995; Harihara et al., 1988; Kawamoto et al., 2008; Shiina et al., 2010), phylogeography (Tosi et al., 2002; Blancher et al., 2008), and demography (Melnick & Hoelzer, 1992). In this pilot study, using the control region (CR) segment of the mtDNA, we attempt to assess the genetic diversity of the M. fascicularis from the northern region of Peninsular Malaysia.

METHODOLOGY

Sample Collection, DNA Extraction, PCR Amplification and Sequencing

Samples from conflict long-tailed macaques were collected from the northern states of Peninsular Malaysia: Perlis and Kedah (including the Langkawi Island; Figure 1). Appendix 1 summarizes the details of the samples used in this study.

Figure 1. The sampling locations (blue circles) in this study. Green circles represents sampling locations from a previous study at Penang by Rovie-Ryan et al. (2014).

Mitochondrial DNA Diversity of the Long-tailed Macaque (Macaca fascicularis) from the Northern Region of Peninsular Malaysia

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Total genomic DNA from 64 blood samples was extracted using the QIAamp DNA Kit (QIAGEN Ag., Germany) following the protocol provided by the manufacturer. A pair of primers; WGRB/MFCR/F15978 and WGRB/MFCR/R580 (Rovie-Ryan et al., 2014) were used to amplify the complete length of the CR. PCR amplifications were conducted using the following PCR profile: a preliminary denaturation at 98°C for 2 min followed by 30 cycles of 95°C for 30 sec, 69°C for 30 sec and 72°C for 40 sec, and later followed by a final extension period of 72°C for 3 min before the samples were cooled to 4°C. A 15µl reaction volume consisting of 0.5µl of DNA template (~15–20ng), 0.2 µl (0.13 µM) of each primers and 14.1 µl of GoTaq® Colorless Master Mix (Promega) was used. Cycle sequencing on both primers were done on the ABI PRISM®377 DNA Sequencer as provided by the 1st Base Laboratories Sdn. Bhd., Selangor, Malaysia.

Sequence Analysis, Genetic Distance and Phylogenetic Relationship

Multiple alignments of the sequences were done by using the program Geneious v5.6 (Drummond et al., 2012) and later manually checked. MEGA v5 (Tamura et al., 2011) was used to check for the sequence characterisation which includes the variable sites, conserved sites and parsimony-informative sites. DnaSP v5 (Librado et al., 2009) were later used to calculate the genetic diversity indices which includes the number of haplotypes, haplotype diversity (Hd) (Nei, 1987), and nucleotide diversity (Pi) (Nei, 1987).

The genetic distances among the samples were calculated by using the Kimura two-parameter model (Kimura, 1980) as calculated in MEGA v5. Phylogenetic trees were then constructed using the neighbour-joining (NJ), maximum parsimony (MP) and maximum likelihood (ML) methods as implemented in MEGA, and also the Bayesian inference (BI) by using MrBayes (Huelsenbeck et al., 2001) as a plug-in in the Geneious v5.6 program. Sequences of the other Macaca species, M. mulatta (AY612638), M. thibetana (NC_011519), and M. sylvanus (NC_002764), and the outgroup species of the Tribe Papionini, Papio hamdryas (NC_001992) were obtained from the GenBank database and included in the analysis. All trees were bootstrapped (Felsenstein, 1985) with 1,000 replicates.

RESULTS AND DISCUSSION

Variation and Genetic Distance in the mtDNA CR

PCR products with the length of 1,092 and 1,093 base-pairs (bp) were obtained from the 64 individual samples. A single deletion mutation was observed in two samples from Langkawi (WDSP/12/0117 and WDSP/12/0118). This deletion mutation is similar with the findings by Rovie-Ryan et al. (2014), where they discovered the same condition in some of the samples from Penang. The authors suggested that the condition were due to genetic stress experienced by the M. fascicularis during the Pleistocene era, which included intense climatic and vegetation changes, and coupled by volcanic activities in the region.

Within the 64 samples, 998 conserved sites (91.3%) and 95 variable sites (8.7%) were detected. A total of 82 parsimony informative sites (86.3%) were detected within the 95 variable sites. Overall, the northern region M. fascicularis (N = 64) exhibit a nucleotide diversity (π) of 0.021. This is comparatively higher than the π of M. fascicularis from Penang (0.012; N= 46) as calculated by Rovie-Ryan et al. (2014). DnaSP detected 23 haplotypes (h = 0.936) from the total samples with no haplotype sharing with the Penang population. Interestingly, haplotype sharing was observed among Langkawi and Perlis samples indicating historical connection between the island and the mainland. The overall



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genetic distance calculated was at 0.021. Within Kedah, Perlis and Langkawi Island, genetic distances were similar at 0.017, 0.015, and 0.016, respectively.

Phylogenetic Relationship

Phylogenetic tree constructed using all four methods (NJ, MP, ML, and BI) produced identical topologies, thus we represent them by the ML tree (-lnL= 4348.63, Figure 2). Generally, the topology separated the M. fascicularis into 4 major groups with no obvious structuring. Two samples from Langkawi Island were grouped with the Penang population, while the remaining samples grouped with the samples from Perlis. Samples from Kedah and Perlis formed two groups (Group 2 and 4).

Figure 2. Phylogenetic tree constructed showing the four major groupings. Numbers above the branches represents bootstrapping values of 1,000 replicates.


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In summary, the findings in this study revealed that the northern of Peninsular Malaysia M. fascicularis population exhibits moderate level of genetic diversity with no obvious genetic structuring observed. This finding could help the DWNP in the management of M. fascicularis population due to the human-macaque conflict. Since the current results have no obvious population structuring, thus translocation of conflict individuals could be conducted without fearing for genetic contamination. Several translocation have been conducted in the late 1980s, from the state of Penang to the adjacent mainland, and to the Taman Negara National Park in the state of Pahang (Elagupillay, S., pers. comm.). Finally, we conclude that the CR of the mtDNA is indeed a powerful segment to be used in addressing the genetic diversity of M. fascicularis.

ACKNOWLEDGEMENTS

This pilot study is part of Rovie-Ryan J.J. PhD research work on the genetics and evolution of the long-tailed macaques in Malaysia. Special thanks to the DWNP for providing the facilities, equipments, and personnel to conduct this pilot study and also to the State Directors of DWNP Kedah and Perlis, and the staffs for their assistance. This project is funded by the DWNP, and partly by the MoSTI-MGI-MGRC-UNIMAS Proboscis genome research grant led by MT Abdullah and colleagues. The following individuals are greatly influential in making this project a success: Abdullah Zawawi bin Yazid and Mohamad Nizam bin Rahammah.

REFERENCES

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Drummond, A.J., Ashton, B., Buxton, S., Cheung, M., Cooper, A., Duran, C., Field, M., Heled, J., Kearse, M., Markowitz, S., Moir, R., Stones-Havas, S., Sturrock, S., Thierer, T. & Wilson, A. (2012). Geneious v5.6. Available from http://www.geneious.com.

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Fooden, J. (1995). Systematic review of Southeast Asian long-tail macaques, Macaca fascicularis (Raffles, [1821]). Fieldiana Zoology, new series (81): 1–206.

Harihara, S., Saitou, N., Hirai, M., Aoto, N., Terao, K., Cho, F., Honjo, S. & Omoto, K. (1988) Differentiation of mitochondrial DNA types in Macaca fascicularis. Primates, 29: 117–127.

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Karimullah, & Shahrul, A. (2011). Condition and population size of Macaca fascicularis (long-tailed macaque). Journal of Cell & Animal Biology, 5(3): 41-46.



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Kawamoto, Y., Kawamoto, S., Matsubayashi, K., Nozawa, K., Watanabe, T., Stanley, M.A. & Perwitasari-Farajallah, D. (2008). Genetic diversity of longtail macaques (Macaca fascicularis) on the island of Mauritius: an assessment of nuclear and mitochondrial DNA polymorphisms. Journal of Medical Primatology, 37: 45–54.

Kimura, M. (1980). A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotides sequence. Journal of Molecular Evolution, 16: 111-120.

Lawler, S.H., Sussman, R.W. & Taylor, L.L. (1995). Mitochondrial DNA of the Mauritian macaques (Macaca fascicularis): an example of the founder effect. American Journal of Physical Anthropology, 96: 133–141.

Librado, P. & Rozas, J. (2009). DnaSP v5: A software for comprehensive analysis of DNA polymorphism data. Bioinformatics, 25: 1451-1452.

Melnick, D.J. & Hoelzer, G.A. (1992). Differences in male and female macaque dispersal lead to contrasting distributions of nuclear and mitochondrial DNA variation. International Journal of Primatology, 13: 379–393.

Nei, M. (1987). Molecular Evolutionary Genetics. New York: Columbia University Press.

Rovie-Ryan, J.J., Abdullah, M.T., Sitam, F.T., Tan, S.G., Zainuddin, Z.Z., Basir, M.M., Abidin, Z.Z., Keliang, C., Denel, A., Joeneh, E. & Ali, F.M. (2014). Genetic diversity of Macaca fascicularis (Cercopithecidae) from Penang, Malaysia as inferred from mitochondrial control region segment. Journal of Indonesian Natural History, 2(1): 14-25.

Shiina, T., Tanaka, K., Katsuyama, Y., Otabe, K., Sakamoto, K., Kurata, M., Nomura, M., Yamanaka, H., Nakagawa, H., Inoko, H. & Ota M. (2010). Mitochondrial DNA diversity among three subpopulations of cynomolgus macaques (Macaca fascicularis) originating from the Indochinese region. Experimental Animals, 59(5): 567-578.

Smith, D.G., McDonough, J.W. & George, D.A. (2007). Mitochondrial DNA variation within and among regional populations of longtail macaques (Macaca fascicularis) in relation to other species of the fascicularis group of macaques. American Journal of Primatology, 69: 182–198.

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Tosi, A.J., Morales, J.C. & Melnick, D.J. (2002). Y-chromosome and mitochondrial markers in Macaca fascicularis indicate introgression with indochinese M. mulatta and a biogeographic barrier in the Isthmus of Kra. International Journal of Primatology, 23: 161-178.

Villano, J.S., Ogden, B.E., Yong, P.P., Lood, N.M. & Sharp, P.E. (2009). Morphometrics and pelage characterization of longtailed macaques (Macaca fascicularis) from Pulau Bintan, Indonesia; Singapore; and Southern Vietnam. Journal of the American Association for Laboratory Animal Science, 48(6): 727-733.


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Appendix 1. Detailed information of the samples used in this study.

No Sample Label Location/GPS of capture

Loc

atio

n in

M

ap

Sex

Hap

. No.

1 WDSP/12/0078 Kg. Titi Kerbau, Canglun, Kedah (269200 714500) 1 M 12 WDSP/12/0079 Kg. Tengah, Derang, Kedah (276457 687221) 2 M 23 WDSP/12/0081 Kg. Tengah, Derang, Kedah (276457 687221) 2 F 34 WDSP/12/0082 Kg. Tengah, Derang, Kedah (276457 687221) 2 F 25 WDSP/12/0084 ABM, Sintok, Kedah (283213 714029) 3 M 46 WDSP/12/0085 ABM, Sintok, Kedah (283213 714029) 3 F 57 WDSP/12/0086 ABM, Sintok, Kedah (283213 714029) 3 F 48 WDSP/12/0087 ABM, Sintok, Kedah (283213 714029) 3 M 69 WDSP/12/0088 ABM, Sintok, Kedah (283213 714029) 3 M 7

10 WDSP/12/0089 ABM, Sintok, Kedah (283213 714029) 3 M 811 WDSP/12/0090 ABM, Sintok, Kedah (283213 714029) 3 F 912 WDSP/12/0091 ABM, Sintok, Kedah (283213 714029) 3 M 913 WDSP/12/0092 Tmn Putrajaya, Alor Setar, Kedah (266214 676377) 4 M 914 WDSP/12/0093 Tmn Putrajaya, Alor Setar, Kedah (266214 676377) 4 M 915 WDSP/12/0094 Tmn Putrajaya, Alor Setar, Kedah (266214 676377) 4 M 916 WDSP/12/0095 Tmn Putrajaya, Alor Setar, Kedah (266214 676377) 4 M 917 WDSP/12/0096 Tmn Putrajaya, Alor Setar, Kedah (266214 676377) 4 M 918 WDSP/12/0097 Kg. Sungai Layar Hujung, S/P, Kedah (276171 628148) 5 M 419 WDSP/12/0098 Kg. Sungai Layar Hujung, S/P, Kedah (276171 628148) 5 M 520 WDSP/12/0099 Kg. Sg. Rotan, Gurun, Kedah (278706 648805) 6 M 521 WDSP/12/0100 Kg. Jubai SC, Kulim, Kedah (296804 610770) 7 M 1022 WDSP/12/0101 Kg. Jubai SC, Kulim, Kedah (296804 610770) 7 M 1023 WDSP/12/0102 Kg. Jubai SC, Kulim, Kedah (296804 610770) 7 M 1124 WDSP/12/0103 Kg. Jubai SC, Kulim, Kedah (296804 610770) 7 M 1225 WDSP/12/0104 Kg. Jubai SC, Kulim, Kedah (296804 610770) 7 M 1026 WDSP/12/0105 Kg. Jubai SC, Kulim, Kedah (296804 610770) 7 M 1027 WDSP/12/0106 Kg. Jubai SC, Kulim, Kedah (296804 610770) 7 M 1028 WDSP/12/0107 Kg. Jubai SC, Kulim, Kedah (296804 610770) 7 M 1229 WDSP/12/0108 Kg. Jubai SC, Kulim, Kedah (296804 610770) 7 F 1230 WDSP/12/0110 Kg. Dato Kayaman, Perlis (252333 720791) 8 M 1331 WDSP/12/0111 Kg. Seri Kesinai, Perlis (255209 736975) 9 F 1432 WDSP/12/0112 Kg. Seri Kesinai, Perlis (255209 736975) 9 M 1533 WDSP/12/0120 Kg. Seri Kesinai, Perlis (255209 736975) 9 F 1934 WDSP/12/0113 Kg. Bukit Malut, Langkawi, Kedah (201141 698270) 10 M 1335 WDSP/12/0115 Kg. Bukit Malut, Langkawi, Kedah (201141 698270) 10 M 1336 WDSP/12/0117 Kg. Bukit Malut, Langkawi, Kedah (201141 698270) 10 F 1837 WDSP/12/0118 Kg. Bukit Malut, Langkawi, Kedah (201141 698270) 10 M 1838 WDSP/12/0119 Kg. Bukit Malut, Langkawi, Kedah (201141 698270) 10 M 1339 WDSP/12/0114 Kg. Kilim, Langkawi, Kedah (207797 699364) 11 F 1640 WDSP/12/0116 Kg. Kilim, Langkawi, Kedah (207797 699364) 11 M 1741 WDSP/12/0121 Kg. Titi Besi Chuping, Perlis (252884 719624) 12 M 2042 WDSP/12/0122 Kg. Titi Besi Chuping, Perlis (252884 719624) 12 F 2143 WDSP/12/0123 Kg. Titi Besi Chuping, Perlis (252884 719624) 12 F 2144 WDSP/12/0124 Kg. Melayu, Perlis (247716 734307) 13 F 2145 WDSP/12/0125 Kg. Melayu, Perlis (247716 734307) 13 M 20



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46 WDSP/12/0126 Kg. Melayu, Perlis (247716 734307) 13 M 1547 WDSP/12/0127 Kg. Melayu, Perlis (247716 734307) 13 M 1548 WDSP/12/0128 Kg. Melayu, Perlis (247716 734307) 13 M 1549 WDSP/12/0129 FRIM, Perlis (251413 736659) 14 F 1950 WDSP/12/0130 FRIM, Perlis (251413 736659) 14 M 2251 WDSP/12/0131 FRIM, Perlis (251413 736659) 14 F 2252 WDSP/12/0132 FRIM, Perlis (251413 736659) 14 M 2053 WDSP/12/0133 FRIM, Perlis (251413 736659) 14 M 2154 WDSP/12/0134 Simpang Tiga, Perlis (244234 720576) 15 M 1355 WDSP/12/0135 Simpang Tiga, Perlis (244234 720576) 15 F 2156 WDSP/12/0136 Simpang Tiga, Perlis (244234 720576) 15 F 1357 WDSP/12/0137 Simpang Tiga, Perlis (244234 720576) 15 M 2358 WDSP/12/0138 Simpang Tiga, Perlis (244234 720576) 15 F 1359 WDSP/12/0139 PKHL Sg. Bt. Pahat, Perlis (243353 722458) 16 M 2160 WDSP/12/0140 PKHL Sg. Bt. Pahat, Perlis (243353 722458) 16 F 2161 WDSP/12/0141 PKHL Sg. Bt. Pahat, Perlis (243353 722458) 16 F 2162 WDSP/12/0142 Bukit Lagi, Perlis (245693 712417) 17 F 2163 WDSP/12/0143 Bukit Lagi, Perlis (245693 712417) 17 F 2164 WDSP/12/0144 Bukit Lagi, Perlis (245693 712417) 17 F 21

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1 MITOCHONDRIAL DNA DIVERSITY OF THE LONG · PDF fileSequence Analysis, Genetic Distance and Phylogenetic Relationship Multiple alignments of the sequences were done by using the program