, Volume 57, Issue 4, pp 449–453 | Cite as

No evidence for mitochondrial genetic variability in the largest population of critically endangered Tonkin snub-nosed monkeys in Vietnam

  • Andie AngEmail author
  • Amrita Srivathsan
  • Rudolf Meier
  • Tuong Bach Luu
  • Quyet Khac Le
  • Herbert Covert
News and Perspectives


The Tonkin snub-nosed monkey (Rhinopithecus avunculus) with a global population of <250 is listed as critically endangered. It is endemic to northeastern Vietnam and was feared extinct until its rediscovery in 1989. The largest single population of R. avunculus consists of 125–130 individuals in an area of forest called Khau Ca in Ha Giang Province. We used non-invasively collected fecal samples to establish the amount of genetic diversity in this population based on mitochondrial information. We amplified and sequenced a 467- to 650-bp section of the hypervariable region I (HVI) of the mitochondrial D-loop for 201 samples and reconstructed the full mitochondrial genomes for five samples based on metagenomic data. All 201 HVI sequences were identical and no variability was found in the five mitochondrial genomes. Our results highlight the immediate need for a comprehensive assessment of the genetic diversity of all populations of R. avunculus based on mitochondrial and nuclear markers. The latter need to be developed for this species.


Mitochondria D-loop Hypervariable region I Colobine Conservation 



We thank everyone for their help, particularly the management board of the Tonkin Snub-nosed Monkey Species Habitat Conservation Area at Khau Ca; Mr Hoang Van Tue, Vietnam Administration of Forestry, Southern Institute of Ecology; Mr Dan Van Khoan, his family and his team; and the Evolutionary Biology Lab (National University of Singapore). We would also like to thank AIT Biotech for facilitating the NGS sequencing.

Compliance with Ethical Standards

We followed the Code of Best Practices for Field Primatology (2014) and the ethical guidelines of the University of Colorado Boulder and the National University of Singapore. Research permits were obtained and we followed the rules and regulations of the Forest Protection Department of Ha Giang, the Protected Area Management Board of Khau Ca, and relevant Vietnamese authorities (permit no. 2039/TCLN-BTTN).


This research was funded by Wildlife Reserves Singapore, the Mohamed bin Zayed Species Conservation Fund, the Beverly Sears Graduate Student Research Grant, the Scott Ferris Graduate Research Award, the Department of Anthropology Pre-Dissertation Grant, SEABIG (NUS) R-154-000-648-646 and R-154-000-648-733, and Dr Vilma D’Rozario.

Conflict of interest

The authors declare that they have no conflict of interest and gave final approval for publication.

Supplementary material

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Supplementary material 1 (DOCX 15 kb)
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Supplementary material 2 (DOCX 31 kb)
10329_2016_571_MOESM3_ESM.docx (13 kb)
Supplementary material 3 (DOCX 13 kb)


  1. Ang A, Srivathsan A, Md-Zain BM, Ismail MRB, Meier R (2012) Low genetic variability in the recovering urban banded leaf monkey population of Singapore. Raffles B Zool 60:589–594Google Scholar
  2. Baillie JEM, Butcher ER (2012) Priceless or worthless? The world’s most threatened species. Zoological Society of London, UKGoogle Scholar
  3. Bogler AM, Lohse M, Usadel B (2014) Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30(15):2114–2120CrossRefGoogle Scholar
  4. Castro-Prieto A, Wachter B, Sommer S (2011) Cheetah paradigm revisited: MHC diversity in the world’s largest free-ranging population. Mol Biol Evol 28(4):1455–1468CrossRefPubMedGoogle Scholar
  5. Faircloth BC, Glenn TC (2014) Protocol: Preparation of an AMPure XP substitute (AKA Serapure). doi: 10.6079/J9MW2F26
  6. Fooden J (1996) Zoogeography of Vietnamese primates. Int J Primatol 17(5):845–899CrossRefGoogle Scholar
  7. Garrison E, Marth G (2012) Haplotype-based variant detection from short-read sequencing. arXiv:1207.3907 [q-bio.GN]
  8. Katoh M, Kuma M (2002) MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform. Nucleic Acids Res 30:3059–3066CrossRefPubMedPubMedCentralGoogle Scholar
  9. Kolleck J, Yang M, Zinner D, Roos C (2013) Genetic diversity in endangered Guizhou snub-nosed monkeys (Rhinopithecus brelichi): contrasting results from microsatellite and mitochondrial DNA data. PLoS One 8(8):e73647. doi: 10.1371/journal.pone.0073647 CrossRefPubMedPubMedCentralGoogle Scholar
  10. Kutty SN, Bernasconi MV, Šifner F, Meier R (2007) Sensitivity analysis, molecular systematics and natural history evolution of Scathophagidae (Diptera: Cyclorrhapha: Calyptratae). Cladistics 23:64–83CrossRefGoogle Scholar
  11. Lande R, Barrowclough G (1987) Effective population size, genetic variation, and their use in population management. In: Soulé ME (ed) Viable populations for conservation. Cambridge University Press, New York, pp 87–123CrossRefGoogle Scholar
  12. Langmead B, Salzberg SL (2012) Fast gapped-read alignment with Bowtie 2. Nat Methods 9:357–359CrossRefPubMedPubMedCentralGoogle Scholar
  13. Le KQ (2002) Distribution and conservation of Tonkin snub-nosed monkey (Rhinopithecus avunculus) in Du Gia Nature Reserve, Ha Giang Province, Northeast Vietnam. Fauna and Flora International-Indochina ProgrammeGoogle Scholar
  14. Le KQ, Dong TH, Nadler T (2015) Tonkin snub-nosed monkey Rhinopithecus avunculus (Dollman 1912). In: Schwitzer C, Mittermeier RA, Rylands AB, Chiozza F, Williamson EA, Wallis J, Cotton A (eds) Primates in peril: the world’s 25 most endangered primates 2014–2016. IUCN SSC PSG, IPS, CI, and Bristol Zoological Society, Arlington, pp 58–59Google Scholar
  15. Leese F, Held C (2011) Analysing intraspecific genetic variation: a practical guide using mitochondrial DNA and microsatellites. In: Held C, Koenemann S, Schubart C (eds) Phylogeography and population genetics in Crustacea. CRC, Boca Raton, pp 3–30CrossRefGoogle Scholar
  16. Li H, Durbin R (2009) Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25(14):1754–1760CrossRefPubMedPubMedCentralGoogle Scholar
  17. Lopez JV, Culver M, Stephens JC, Johnson WE, O’Brien SJ (1997) Rates of nuclear and cytoplasmic mitochondrial DNA sequence divergence in mammals. Mol Biol Evol 14:277–286CrossRefPubMedGoogle Scholar
  18. Ma C, Huang ZP, Zhao XF, Zhang LX, Sun WM, Scott MB, Wang XW et al (2014) Distribution and conservation status of Rhinopithecus strykeri in China. Primates 55:377–382CrossRefGoogle Scholar
  19. Meier R, Wong W, Srivathsan A, Foo M (2016) $1 DNA barcodes for reconstructing complex phenomes and finding rare species in specimen-rich samples. Cladistics 32:100–110CrossRefGoogle Scholar
  20. Miller W, Hayes VM, Ratan A, Petersen DC, Wittekindt NE, Miller J, Walenz B et al (2011) Genetic diversity and population structure of the endangered marsupial Sarcophilus harrisii (Tasmanian devil). PNAS 108(30):12348–12353CrossRefPubMedPubMedCentralGoogle Scholar
  21. Mittermeier RA, Cheney DL (1987) Conservation of primates and their habitats. In: Smuts BB, Cheney DL, Seyfarth RM, Wrangham RW, Strushsaker TT (eds) Primate societies. University of Chicago Press, Chicago, pp 477–490Google Scholar
  22. Nadler T (2014) Habitat disturbance and loss, and the primates of Vietnam. In: Nadler T, Brockman D (eds) Primates of Vietnam. Endangered Primate Rescue Center, Cuc Phuong National Park, Vietnam, pp 55–60Google Scholar
  23. Nsubuga AM, Robbins MM, Roeder AD, Morin PA, Boesch C, Vigilant L (2004) Factors affecting amount of genomic DNA extracted from ape faeces and the identification of an improved sample storage method. Mol Ecol 13:2089–2094CrossRefPubMedGoogle Scholar
  24. Osterholz M, Walter L, Roos C (2008) Phylogenetic position of the langur genera Semnopithecus and Trachypithecus among Asian colobines, and genus affiliations of their species groups. BMC Evol Biol 8:58–69CrossRefPubMedPubMedCentralGoogle Scholar
  25. Ratajszczak R, Can Dang Ngoc, Nhat Pham (1992) A survey for Tonkin snub-nosed monkey (Rhinopithecus avunculus) in the North Vietnam. FFI Preservation Society and WWF International, London and GlandGoogle Scholar
  26. Rohland N, Reich D (2012) Cost-effective, high-throughput DNA sequencing libraries for multiplexed target capture. Genome Res 22:939–946CrossRefPubMedPubMedCentralGoogle Scholar
  27. Roos C, Thanh Vu Ngoc, Walter L, Nadler T (2007) Molecular systematics of Indochinese primates. Vietnam J Primatol 1:41–53Google Scholar
  28. Roos C, Zinner D, Kubatko LS, Schwarz C, Yang M, Meyer D, Nash SD, Xing J, Batzer MA, Brameier M, Leendertz FH, Ziegler T, Perwitasari-Farajallah D, Nadler T, Walter L, Osterholz M (2011) Nuclear versus mitochondrial DNA: evidence for hybridization in colobine monkeys. BMC Evol Biol 11:77CrossRefPubMedPubMedCentralGoogle Scholar
  29. Spielman D, Brook BW, Briscoe DA, Frankham R (2004) Does inbreeding and loss of genetic diversity decrease disease resistance? Conserv Genet 5:439–448CrossRefGoogle Scholar
  30. Srivathsan A, Sha J, Vogler AP, Meier R (2015) Comparing the effectiveness of metagenomics and metabarcoding for diet analysis of a leaf-feeding monkey (Pygathrix nemaeus). Mol Ecol Res 15(2):250–261CrossRefGoogle Scholar
  31. Srivathsan A, Ang A, Vogler AP, Meier R (2016) Fecal metagenomics for the simultaneous assessment of diet, parasites, and population genetics of an understudied primate. Front Zool 13:17. doi: 10.1186/s12983-016-0150-4 CrossRefPubMedPubMedCentralGoogle Scholar
  32. Wisely SM, Buskirk SW, Fleming MA, McDonald DB, Ostrander EA (2002) Genetic diversity and fitness in black-footed ferrets before and during a bottleneck. J Hered 93:231–237CrossRefPubMedGoogle Scholar
  33. Yang M, Yang Y, Cui D, Fickenscher G, Zinner D, Roos C, Brameier M (2012) Population genetic structure of Guizhou snub-nosed monkeys (Rhinopithecus brelichi) as inferred from mitochondrial control region sequences, and comparison with R. roxellana and R. bieti. Am J Phys Anthropol 147:1–10CrossRefPubMedGoogle Scholar
  34. Yu L, Wang X, Ting N, Zhang Y (2011) Mitogenomic analysis of Chinese snub-nosed monkeys: evidence of positive selection in NADH dehydrogenase genes in high-altitude adaptation. Mitochondrion 11(3):497–503CrossRefPubMedGoogle Scholar
  35. Zhou X, Wang B, Pan Q, Zhang J, Kumar S, Sun X, Liu Z et al (2014) Whole-genome sequencing of the snub-nosed monkey provides insights into folivory and evolutionary history. Nat Genet 46:1303–1310CrossRefPubMedGoogle Scholar

Copyright information

© Japan Monkey Centre and Springer Japan 2016

Authors and Affiliations

  1. 1.Department of AnthropologyUniversity of Colorado BoulderBoulderUSA
  2. 2.Department of Biological SciencesNational University of SingaporeSingaporeSingapore
  3. 3.HanoiVietnam
  4. 4.HanoiVietnam
  5. 5.Southern Institute of EcologyVietnam Academy of Science and TechnologyHo Chi Minh CityVietnam

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