JOURNAL OF WILDLIFE AND PARKS Published by Department of Wildlife and National Parks (DWNP) Peninsular Malaysia Published 2014 MANAGEMENT BOARD Advisor Dato’ Abd. Rasid Samsudin Director of General, DWNP Chairperson Dato’ Misliah Mohamad Basir Deputy Director General I, DWNP Members Dato’ Dr. Zaaba Zainol Abidin Tn.Hj.Zainuddin Ab Shukor Abdul Kadir Abu Hashim Shabrina Mohd Shariff Salman Hj Saaban Mohd Taufik Abdul Rahman Nosrat Ravichandran DWNP Editor-In-Chief Dr. Sivananthan T.Elagupillay DWNP Editorial Board Prof. Emeritus Dr. Yong Hoi Sen, University Malaya Prof. Dr. Shukor Md. Nor, University Kebangsaan Malaysia Jeffrine Rovie Ryan Japning, DWNP Kayal Vizi Karuppannan, DWNP Secretariat Tan Poai Ean Frankie Thomas Sitam Hartini Ithnin Norsyamimi Rosli Noor Azleen Mohd Kulaimi Badmanathan Munisamy HONORARY ADVISOR Dr. Lim Boo Liat General Correspondence: Director General Department of Wildlife and National Parks (DWNP) / PERHILITAN, Peninsular Malaysia, KM 10, Jalan Cheras, 56100 Kuala Lumpur, Malaysia. Tel: 03-90866800 Fax: 03-90752873 Email: [email protected] Website: www.wildlife.gov.my Authorisation has been granted to the DWNP to reproduce these pages. Printed by Universal Iprint Sdn Bhd, Kuala Lumpur iii Journal of Wildlife and Parks Vol. XXIX (2014) ISSN 0121-8126 CONTENTS FULL PAPER PAGE Jeffrine J. Rovie-Ryan, Abdullah, M.T., Frankie T. Sitam, Soon Guan Tan, Misliah Mohamad Basir, Zaaba Zainol Abidin, Charles Keliang & Azroie Denel Mitochondrial DNA Diversity of the Long-Tailed Macaque (Macaca fascicularis) from the Northern Region of Peninsular Malaysia 1 - 8 Masrom, H., Omar, Y. & Mohd. Norfaizal, G. Flora Diversity of Tasek Bera Ramsar Site, Pahang, Malaysia 9- 11 Amirrudin B. Ahmad, M. Fahmi-Ahmad & Syed Ahmad Rizal Fish Diversity in Small Streams of Sungkai Wildlife Reserve, Perak, Malaysia 13 - 21 M. Izzat-Husna & Amirrudin A. Odonata (Class Insecta) of Sungkai Wildlife Reserve, Perak, Malaysia 23 - 30 Mohd Norfaizal, G. Masrom, H., Omar, Y. & Mohd Shukri, M.A. A Preliminary Flora Survey of Sungkai Wildlife Reserve, Perak, Malaysia 31 - 35 John Rasmussen Food Choice and Feeding Habits of the Flat-Headed Cat (Prionailurus planiceps) in Captivity 37 - 44 Thary Gazi Goh, Johannes Huijbregts, Hii Ning & Ahimsa Campos-Arceiz Beetles Recorded to Visit Elephant Dung in Temenggor Forest, Malaysia 45 - 48 Mohd. Sanusi, M., Traeholt, C., Khadijah-Ghani, S.A., Simson, B., Shukor M.N. & Rovie-Ryan, J. Jeffrine Evolusi Projek Konservasi Tapir Malaya 49 - 53 iv CONTENTS FULL PAPER PAGE Ang, L.H., Ho, W.M. & Tang, L.K. A Depository of Lowland Forest Tree Species Established on a Brown-Filled Site in Ara Damansara, Selangor, Malaysia 55 - 60 Ang, L.H., Ho, W.M. & Tang, L.K. A Model of Greened Ex-Tin Mine as a Lowland Biodiversity Depository in Malaysia 61 - 67 Magintan, D., Mohd. Aminurddin Ahmad, Adnan Ismail & Idlan Rasdi Observation of Dhole (Cuon alpinus) at Sungkai Wildlife Reserve, Perak, Malaysia 69 - 72 Ho, W.M., Tang, L.K., Ang, L.H., Ho, S.K. & Lee, D.K. Enrichment Planting in a Greened Slime Tailings in Peninsular Malaysia 73 - 76 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, Malaysia 2Kenyir Ecosystem Research Centre, Universiti Malaysia Terengganu, 21030 Kuala Terengganu, Terengganu, Malaysia 3Outbreak 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 Lumpur 6Deputy 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). 2 Jeffrine J. Rovie-Ryan, Abdullah, M.T., Frankie T. Sitam, Soon Guan Tan, Misliah Mohamad Basir, Zaaba Zainol Abidin, Charles Keliang & Azroie Denel 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) 3 from the Northern Region of Peninsular Malaysia 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.