Sains Malaysiana 53(7)(2024): 1509-1523

http://doi.org/10.17576/jsm-2024-5307-03

Molecular Phylogenetics and Phylogeography of Malaysian Mousedeer (Tragulus kanchil) Based on Mitochondrial DNA Sequences of the D-Loop Region

(Filogenetik Molekul dan Filogeografi Pelanduk Malaysia (Tragulus kanchil) Berdasarkan Jujukan DNA Mitokondria Kawasan D-Loop)

MOHAMAD AZAM AKMAL ABU-BAKAR^1,2, NOR RAHMAN AIFAT³, JEFFRINE JAPNING ROVIE-RYAN^4,5, MUHAMMAD ABU BAKAR ABDUL-LATIFF⁶, AHMAD AMPENG⁷, SHUKOR MD-NOR¹ & BADRUL MUNIR MD-ZAIN^1,*

¹Department of Biological Sciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor, Malaysia

²Faculty of Applied Science, Universiti Teknologi MARA Pahang, Jengka Campus, 26400 Bandar Tun Abdul Razak, Jengka, Pahang, Malaysia

³Faculty of Tropical Forestry, Universiti Malaysia Sabah (UMS), Jalan UMS, 88400 Kota Kinabalu, Sabah, Malaysia

⁴National Wildlife Forensic Laboratory, Ex-Situ Conservation Division, Department of Wildlife and National Park (DWNP) Peninsular Malaysia, KM 10 Jalan Cheras, 56100 Kuala Lumpur, Malaysia

⁵Institute of Biodiversity and Environmental Conservation (IBEC), Universiti Malaysia Sarawak, 94300 Kota Samarahan, Sarawak, Malaysia

⁶Faculty of Applied Sciences and Technology, Universiti Tun Hussein Onn Malaysia (Pagoh Campus), 84600 Muar, Johor, Malaysia

⁷Forest Department Sarawak, Bangunan Baitul Makmur II, Medan Raya, 93050 Petra Jaya, Kuching, Sarawak, Malaysia

Diserahkan: 22 Jun 2022/Diterima: 4 Jun 2024

Abstract

Two sympatric mousedeer species, Tragulus kanchil and Tragulus napu, inhabit the Malaysian tropical rainforests. Previous studies have established their phylogenetic relationships based on morphological variations; however, comprehensive genetic data have yet to be extensively used to relate the relationships especially from different populations. The main objectives of this study were to determine the phylogenetic relationships, population genetics, and phylogeography of mousedeer species based on DNA sequences of the mitochondrial D-loop region. DNA from 32 mousedeer samples, representing various populations in Malaysia, was sequenced and analyzed using Neighbor-Joining, Maximum Parsimony, and Bayesian Inference approaches. The phylogenetic analyses showed two main clades representing the populations of T. kanchil and T. napu. The results also showed that the T. kanchil populations in Borneo was separated from Peninsular Malaysia taxa in MP and BI phylogenetic tree. However, Borneo population was nested in east population of Peninsular Malaysia in NJ tree. In addition, the T. kanchil population in Peninsular Malaysia further separated into east and west coast populations of Titiwangsa Range. It was supported with finding in population genetic relation that showed relatively low levels among population. This is expected because some of the populations are isolated geographically. The divergence of these populations is likely due to the Titiwangsa Range which acts as a barrier separating the east and west Peninsular Malaysia populations, and the South China Sea separates the populations of Peninsular Malaysia and Borneo. Molecular clock tree reconstruction showed that the separation of T. kanchil and T. napu occurred around 17.11 million years ago (MYAs). Furthermore, the T. kanchil populations from the east and west Peninsular Malaysia showed a branching pattern from those of Borneo of about 11.04 and 9.14 MYAs, respectively. The results of this study increase our understanding of Malaysian mousedeer phylogeny and phylogeography.

Keywords: Mousedeer; phylogeography; Tragulus kanchil; Tragulus napu; ungulate

Abstrak

Dua spesies pelanduk Tragulus kanchil dan Tragulus napu, bersimpatrik menghuni hutan hujan tropika Malaysia. Kajian terdahulu telah mengesahkan pertalian filogenetik pelanduk berdasarkan variasi morfologi. Walau bagaimanapun, data genetik yang komprehensif masih belum digunakan sepenuhnya untuk mengaitkan hubungan terutamanya populasi yang berlainan. Objektif utama kajian ini adalah untuk menentukan pertalian filogenetik, genetik populasi dan filogeografi kedua-dua spesies pelanduk menggunakan jujukan DNA mitokondria kawasan D-loop. DNA daripada 32 sampel pelanduk yang mewakili populasi di Malaysia telah dijujuk dan dianalisis menggunakan kaedah Neighbor-Joining (NJ), Maximum Parsimony (MP) dan Bayesian Inference (BI). Analisis filogenetik menunjukkan terhasil dua klad utama mewakili populasi T. kanchil dan T. napu. Keputusan juga menunjukkan populasi T. kanchil di Borneo terpisah daripada populasi Semenanjung Malaysia berdasarkan pokok filogenetik MP dan BI. Walau bagaimanapun, populasi Borneo masih tergolong dalam satu kumpulan bersama populasi Timur Semenanjung Malaysia dalam pokok NJ. Selain itu, populasi T. kanchil di Semenanjung Malaysia terpisah kepada populasi barat dan timur Banjaran Titiwangsa. Ini juga disokong dengan hasil analisis populasi genetik menunjukkan tahap hubungan yang rendah antara populasi. Hal ini dijangka disebabkan populasi ini terpisah oleh halangan geografi. Pencapahan populasi ini berkemungkinan disebabkan Banjaran Titiwangsa bertindak sebagai penghalang yang memisahkan populasi Timur dan Barat Semenanjung Malaysia dan Laut China Selatan yang memisahkannya dengan populasi Borneo. Pohon jam molekul juga menunjukkan pemisahan spesies T. kancil dan T. napu berlaku sekitar 17.11 juta tahun dahulu (JTD). Tambahan lagi, populasi T. kancil Timur dan Barat Semenanjung Malaysia menunjukkan percambahan daripada populasi Borneo masing-masing sekitar 11.04 JTD dan 9.14 JTD. Hasil kajian ini dapat meningkatkan pemahaman tentang filogeni dan filogeografi spesies kancil di Malaysia.

Kata kunci: Filogeografi; pelanduk; Tragulus kanchil; Tragulus napu; ungulat

RUJUKAN

Abdul-Latiff, M.A.B. & Md-Zain, B.M. 2021. Taxonomy, evolutionary and dispersal events of pig-tailed macaque, Macaca nemestrina (Linnaeus, 1766) in Southeast Asia with description of a new subspecies, Macaca nemestrina perakensis in Malaysia. Zool. Stud. 60: 50.

Abdul-Latiff, M.A.B., Baharuddin, H., Abdul-Patah, P. & Md-Zain, B.M. 2019. Is Malaysia’s banded langur, Presbytis femoralis femoralis, actually Presbytis neglectus neglectus? Taxonomic revision with new insights on the radiation history of the Presbytis species group in Southeast Asia. Primates 60: 63-79.

Abdul-Latiff, M.A.B., Ruslin, F., Fui, V.V., Abu, M.H., Rovie-Ryan, J.J., Abdul-Patah, P., Lakim, M., Roos, C., Yaakop, S. & Md-Zain, B.M. 2014a. Phylogenetic relationships of Malaysia's long-tailed macaques, Macaca fascicularis, based on cytochrome b sequences. Zookeys 407: 121-140.

Abdul-Latiff, M.A.B., Ruslin, F., Faiq, H., Hairul, M.S., Rovie-Ryan, J.J., Abdul-Patah, P., Yaakop, S. & Md-Zain, B.M. 2014b. Continental monophyly and molecular divergence of Peninsular Malaysia’s Macaca fascicularis fascicularis. Biomed. Res. Int. 2014: 897682.

Abdul-Patah, P., Sasaki, H., Sekiguchi, T., Shukor, M.N., Mohd-Yusof, N.S., Abdul-Latiff, M.A.B. & Md-Zain, B.M. 2020. Molecular DNA-based spatial mapping technique predicting diversity and distribution of otters (Lutrinae) in Peninsular Malaysia using non-invasive fecal samples. Mammal Research 65: 691-700.

Aifat, N.R. & Md-Zain, B.M. 2021. Genetic identification of White-Handed Gibbons (Hylobates lar) in captivity.  Journal of Sustainability Science and Management 16: 316-326.

Aifat, N.R., Yaakop, S. & Md-Zain, B.M. 2016. Optimization of partial Cyt b gene sequence from selected ancient Presbytis museum skin specimens. Malays. Appl. Biol. 45: 93-96.

Aifat, N.R., Abdul-Latiff, M.A.B., Roos, C. & Md-Zain, B.M. 2020. Taxonomic revision and evolutionary phylogeography of dusky langur (Trachypithecus obscurus) in Peninsular Malaysia. Zool. Stud. 59: 64.

Brunke, J., Russo, IR.M., Orozco-terWengel, P., Zimmermann, E., Bruford, M.W., Goossens, B. & Radespel, U. 2020. Dispersal and genetic structure in tropical small mammal, the Bornean tree shrew (Tupaia longipes), in a fragmented landscape along the Kinabatangan River, Sabah, Malaysia. BMC Genet. 21: 43.

Chowdhury, S.M., Omar, A.R., Aini, I., Hair-Bejo, M., Jamaluddin, A.A., Md-Zain, B.M. & Kono, Y. 2003. Pathogenicity, sequence and phylogenetic analysis of Malaysian Chicken anaemia virus obtained after low and high passages in MSB-1 cells. Arch. Virol. 148: 2437-2448.

Corbet, G.B. & Hill, J.E. 1992. The Mammals of the Indomalayan Region: A Systematic Review. Oxford: Oxford University Press.

Drummond, A.J. & Rambaut, A. 2007. BEAST: Bayesian evolutionary analysis by sampling trees. BMC Evolutionary Biology 7: 214.

Endo, H., Fukuta, K., Kimura, J., Sasaki, M., Hayashi, Y. & Oshida, T. 2004. Phylogenetic relationships among populations of the mouse deer in the Southeast Asian Region from the nucleotide sequence of cytochrome b gene. Mammal Study 29: 119-123.

Farida, W.R., Semiada, G., Handayani, T.H. & Harun. 2006. Habitat distribution and diversity of plants of feed resources on mouse deer (Tragulus javanicus) and barking deer (Muntiacus muntjak) in Gunung Halimunan National Park. Tropics 15: 371-376.

Frankham, R., Ballou, J.D. & Briscoe, D.A. 2002. Introduction to Conservation Genetics. Cambridge: Cambridge University Press.

Halim, M., Aman-Zuki, A., Syed-Ahmad, S.Z., Muhaimin, A.M.D., Atikah, A.R., Masri, M.M., Md-Zain, B.M. & Yaakop, S. 2018. Exploring the abundance and DNA barcode information of eight parasitoid wasps species (Hymenoptera), the natural enemies of the important pest of oil palm, bagworm, Metisa plana (Lepidoptera: Psychidae) toward the biocontrol approach and it’s application in Malaysia. Journal of Asia-Pacific Entomology 21(4): 1359-1365.

Hall, R. 2013. The palaeogeography of Sundaland and Wallacea since the Late Jurassic. J. Limnol. 72: 1-17.

Hall, R. 2002. Cenozoic geological and plate tectonic evolution of SE Asia and the SW Pacific: Computer-based reconstruction, model and animations. J. Asian Earth Sci. 20: 353-431.

Hall, R. 2001. Cenozoic reconstruction of SE Asia and the SW pacific: Changing pattern of land and sea. In Faunal and Floral Migration and Evolution in SE Asia Australasia, edited by Metcalfe, I., Smith, J.M.B., Morwood, M. & Davidson, I. Lisse: Swets and Zeitlinger. pp. 35-56.

Harrison, M.E. & Chivers, D.J. 2007. The orang-utan mating system and the unflanged male: A product of increased food stress during the late Miocene and Pliocene? J. Hum. Evol. 52: 275-293.

Hassanin, A., Delsuc, F., Ropiquet, A., Hammer, C., Jansen van Vuuren, B., Matthee, C., Ruiz-Garcia, M., Catzeflis, F., Areskoug, V., Nguyen, T.T. & Couloux, A. 2012. Pattern and timing of diversification of Cetartiodactyla (Mammalia, Laurasiatheria), as revealed by a comprehensive analysis of mitochondrial genomes. C. R. Biol. 335: 32-50. 

Houben, A.J.P., van Mourik, C.A., Montanari, A., Coccioni, R. & Brinkhuis, H. 2012. The Eocene-Oligocene transition: Changes in sea level, temperature, or both? Palaeogeogr. Palaeoclimatol. Palaeoecol. 335-336: 75-83.

Huelsenbeck, J.P. & Ronquist, F. 2001. MRBAYES: Bayesian inference of phylogenetic trees. Bioinformatics 17: 754-755.

Hurzaid, A., Jaafar, I., Awang, Z. & Siti-Azizah, M.N. 2014. Genetic structure of the Asian Grass Frog, Fejevarya limnocharis (Amphibia: Anura: Dicroglossidae) of Peninsular Malaysia: A preliminary report. Zool. Stud.  53: 77.

Kamaruddin, K.R. & Esa, Y. 2009. Phylogeny and phylogeography of Barbonymus schwanenfeldii (Cyprinidae) from Malaysia inferred using partial cytochrome b mtDNA gene. Journal of Tropical Biology and Conservation 5: 1-13.

Khalil, A.R.A., Setiawan, A., Rustiati, E.L., Harianto, S.P. & Nurarifin, I. 2019. Keragaman dan kelimpahan Artiodactyla menggunakan kamera jebak di Kesatuan Pengelolaan Hutan I Pesisir Barat. Jurnal Sylva Lestari 7: 350-358.

Kim, K.S., Tanaka, K., Ismail, D.B., Maruyama, S., Matsubayashi, H., Endo, H., Fukuta, K. & Kimura, J. 2004. Cytogenetic comparison of the lesser mousedeer (Tragulus javanicus) and the greater mousedeer (T. napu). Caryologia 57: 229-243.

Kocher, T.D., Thomas, W.K., Meyer, A., Edwards, S.V., Paabo, S., Villablanca, F.X. & Wilson, A.C. 1989. Dynamics of mitochondrial DNA evolution in animals: Amplification and sequencing with conserved primers. Proc. Natl. Acad. Sci. 86: 6196-6200.

Kumar, S., Stecher, G., Li, M., Knyaz, C. & Tamura, K. 2018. MEGA X: Molecular evolutionary genetics analysis across computing platforms. Mol. Biol. Evol. 35: 1547-1549.

Kusuda, S., Adachi, I., Fujioka, K., Nakamura, M., Amano-Hanzawa, N., Goto, N., Furuhashi, S. & Doi, O. 2013. Reproductive characteristics of female lesser mouse deers (Tragulus javanicus) based on fecal progestagens and breeding records. Anim. Reprod. Sci. 137: 69-73.

Kuznetsov, G.V. & Borissenko, A.V. 2004. A new record of Tragulus versicolor (Artiodactyla, Tragulidae) from Vietnam, and its sympatric occurrence with T. kanchil. Russian J. Theriol. 3: 9-13.

Loo, A.H.B., Tan, H.T.W., Kumar, P.P. & Saw, L.G. 2001. Intraspecific variation in Licuala glabra Griff. (Palmae) in Peninsular Malaysia–a morphometric analysis. Biol. J. Linn Soc. 72: 115-128.

Matsubayashi, H. & Sukor, J.R.A. 2005. Activity and habitat use of lesser mousedeer and greater mouse-deer species, Tragulus javanicus dan Tragulus napu, in Sabah, Malaysia, Borneo. Malay. Nat. J. 57: 235-241.

Md-Zain, B.M., Lee, S.J., Lakim, M., Ampeng, A. & Mahani, M.C. 2010. Phylogenetic position of Tarsius bancanus based on partial Cytochrome b DNA sequences. Journal of Biological Sciences 10: 348-354.

Meijaard, E. & Groves, C.P. 2004. A taxonomic revision of the Tragulus mousedeer (Artiodactyla). Zool. J. Linn Soc. 140: 63-102.

Meijaard, E., Umilaela & de Wijeyeratne, G.S. 2010. Aquatic escape behaviour in mousedeer provides insight into tragulid evolution. Mamm. Biol. 75: 471-473.

Mètais, G., Chaimanee, Y., Jaeger, J.J. & Ducrocq, S. 2007. Eocene bunoselenodont Artiodactyla from southern Thailand and the early evolution of Ruminantia in South Asia. Naturwissens 94: 493-498.

Mètais, G., Chaimanee, Y., Jaeger, J.J. & Ducrocq, S. 2001. New remains of primitive ruminants from Thailand: Evidence of the early evolution of the Ruminantia in Asia. Zool. Scr. 30: 231-248.

Miller, K.G., Kominz, M.A., Browning, J.V., Wright, J.D., Mountain, G.S., Katz, M.E., Sugarman, P.J., Cramer, B.S., Christie-Blick, N. & Pekar, S.F. 2005. The Phanerozoic record of global sea-level change. Science 310: 1293-1298.

Mohd-Ridwan, A.R. & Abdullah, M.T. 2012. Population genetics of the cave-dwelling dusky fruit bat, Penthetor lucasi, based on four populations in Malaysia. Pertanika J. Trop. Agric. Sci. 3: 459-484.

Mohd-Yusof, N.S., Senawi, J., Nor, S.M. & Md-Zain, B.M. 2020. Haplotype and network analysis of Island Flying Fox (Pteropus hypomelanus) using D-Loop region of mitochondrial DNA to confirm subspecies designation. Mammal Research 65: 375-385.

Nur-Syuhada, N., Magintan, D., Siti-Hajar, A.R., Aisah, M.S. & Shukor, M.N. 2016. The wildlife research & rescue programme for mammals at Hulu Terengganu Hydroelectric Project (HTHEP), Terengganu, Peninsular Malaysia. In AIP Conference Proceedings 1784(1): 060036. AIP Publishing LLC.

Posada, D. & Crandall, K.A. 1998. Modeltest: Testing the model of DNA substitution. Bioinformatics 14: 817-818.

Rosli, M.K., Zamzuriada, A.S., Syed-Shabthar, S.M., Mahani, M.C., Abas-Mazni, O. & Md-Zain, B.M. 2011. Optimization of PCR conditions to amplify Cyt b, COI and 12S rRNA gene fragments of Malayan gaur (Bos gaurus hubbacki) mtDNA. Genet. Mol. Res. 10: 2554-2568. 

Rozas, J., Ferrer-Mata, A., Sanchez-DelBarrio, J.C., Guirao-Rico, S., Librado, P., Ramos-Onsins, S.E. & Sanchez-Gracia, A. 2017. DnaSP 6: DNA sequence polymorphism analysis of large data sets. Mol. Biol. Evol. 34: 3299-3302.

Rubinoff, D. & Hollad, B.S. 2005. Between two extremes:mitochondrial DNA is neither the panacea nor the nemesis of phylogenetic and taxonomic inference. Syst. Biol. 54: 952-961.

Sum, J.S., Lee, W.C., Amir, A., Braima, K.A., Jeffery, J., Abdul-Aziz, N.M., Mun-Yik, F. & Yee-Ling, L. 2014. Phylogenetic study of six species of Anopheles mosquitoes in Peninsular Malaysia based on inter-transcribed spacer region 2 (ITS2) of ribosomal DNA. Parasit Vectors 7: 309.

Syed-Shabthar, S.M., Rosli, M.K., Mohd-Zin, N.A., Romaino, S.M., Fazly-Ann, Z.A, Mahani, M.C., Abas-Mazni, O., Zainuddin, R., Yaakop, S. & Md-Zain, B.M. 2013. The molecular phylogenetic signature of Bali cattle revealed by maternal and paternal markers. Mol. Biol. Rep. 40: 5165-5176.

Timmins, R. & Duckworth, J.W. 2015. Tragulus kanchil. The IUCN Red List of Threatened Species 2015: e.T136297A61978576.

van Dort, M. 1988. Note on the skull size in the sympatric mouse deer species, Tragulus javanicus (Osbeck, 1765) and Tragulus napu (F. Cuvier, 1822). Zeitschrift für Säugetierkunde 53: 124-125.

Vislobokova, I.A. 2013. On the origin of Cetartiodactyla: Comparison of data on evolutionary morphology and molecular biology. Paleontol. J. 47: 321-334.

Voris, H.K. 2000. Maps of Pleistocene sea levels in Southeast Asia: Shoreline, river systems and time durations. Journal of Biogeogr. 27: 1153-1167.

Whitlock, M.C. & McCauley, D.E. 1999. Indirect measures of gene flow and migration: FST≠1/(4N_m+1). Heredity 82: 117-125.

Wirdateti, W. & Nugraha, T.P. 2016. Variasi dan filogeni Kancil dan Napu (Tragulus spp.) di Indonesia menggunakan gen 12s rRNA mitokondria. Jurnal Veteriner Maret 17: 22-29.

Wright, S. 1965. The interpretation of population structure by F-statistics with special regard to systems of mating. Evolution 19: 395-420.

*Pengarang untuk surat-menyurat; email: abgbadd@ukm.edu.my