Conservation Genetics

, Volume 11, Issue 4, pp 1389–1404 | Cite as

Barcoding bushmeat: molecular identification of Central African and South American harvested vertebrates

  • Mitchell J. Eaton
  • Greta L. Meyers
  • Sergios-Orestis Kolokotronis
  • Matthew S. Leslie
  • Andrew P. Martin
  • George Amato
Research Article

Abstract

The creation and use of a globally available database of DNA sequences from a standardized gene region has been proposed as a tool for species identification, assessing genetic diversity and monitoring the legal and illegal trade in wildlife species. Here, we contribute to the Barcode of Life Data System and test whether a short region of the mitochondrial cytochrome c oxidase subunit 1 (COX1) gene would reliably distinguish among a suite of commonly hunted African and South American mammal and reptile species. We used universal primers to generate reference barcode sequences of 645 bp for 23 species from five vertebrate families (Crocodilidae, Alligatoridae, Bovidae, Suidae and Cercopithecidae). Primer cocktails yielded high quality barcode sequences for 179 out of 204 samples (87.7%) from all species included in the study. For most taxa, we sequenced multiple individuals to estimate intraspecific sequence variability and document fixed diagnostic characters for species identification. Polymorphism in the COX1 fragment was generally low (mean = 0.24%), while differences between congeneric species averaged 9.77%. Both fixed character differences and tree-based maximum likelihood distance methods unambiguously identified unknown and misidentified samples with a high degree of certainty. Barcode sequences also differentiated among newly identified lineages of African crocodiles and identified unusually high levels of genetic diversity in one species of African duiker. DNA barcoding offers promise as an effective tool for monitoring poaching and commercial trade in endangered species, especially when investigating semi-processed or morphologically indistinguishable wildlife products. We discuss additional benefits of barcoding to ecology and conservation.

Keywords

Barcode of life Caiman Crocodiles Cytochrome c oxidase subunit 1 (COX1Hunting Molecular forensics Primates Wildlife monitoring Maximum likelihood phylogeny Ungulates 

Notes

Acknowledgments

This work was supported by the Alfred P. Sloan Foundation and the Richard Lounsbery Foundation. The National Science Foundation and the American Museum of Natural History’s Research Experience for Undergraduates Program supported the laboratory work of GLM. The AMNH sponsored Aritra Datta and Arlene Amador to extract and sequence the USFWS crocodile skin products and we thank them for their efforts. Field support was provided to MJE by the Wildlife Conservation Society’s Congo and Gabon programs, the National Geographic Society, the Rufford Foundation, Lincoln Park Zoo’s Asia & Africa Fund and the Mac-Arthur Program of the University of Minnesota. MJE thanks Paul and Sarah Elkan (WCS-Congo), Debora Pires, and Congo field assistants Yamba Flavien, Bienvenu Kimbembe and Rufin Lekana. New World crocodilian samples were collected by Peter Brazaitis, Carlos Yamashita (IBAMA, Brazil) and George Rebelo (INPA, Brazil) and provided by G. J. Watkins-Colwell of the Peabody Museum. Ellen Bean and three anonymous reviewers greatly improved the clarity and scope of this manuscript.

Supplementary material

10592_2009_9967_MOESM1_ESM.htm (22 kb)
Supplementary material

References

  1. Albrechtsen L, David WMA, Paul JJ et al (2007) Faunal loss from bushmeat hunting: empirical evidence and policy implications in Bioko island. Environ Sci Policy 10:654–667CrossRefGoogle Scholar
  2. Avise JC (2000) Phylogeography: the history and formation of species. Harvard University Press, CambridgeGoogle Scholar
  3. Baker CS (2008) A truer measure of the market: the molecular ecology of fisheries and wildlife trade. Mol Ecol 17:3985–3998CrossRefPubMedGoogle Scholar
  4. Baker CS, Cipriano F, Palumbi SR (1996) Molecular genetic identification of whale and dolphin products from commercial markets in Korea and Japan. Mol Ecol 5:671–685CrossRefGoogle Scholar
  5. Baker CS, Dalebout ML, Lento GM et al (2002) Gray whale products sold in commercial markets along the pacific coast of Japan. Mar Mamm Sci 18:295–300CrossRefGoogle Scholar
  6. Bennett EL, Blencowe E, Brandon K et al (2007) Hunting for consensus: reconciling bushmeat harvest, conservation, and development policy in west and central Africa. Conserv Biol 21:884–887CrossRefPubMedGoogle Scholar
  7. Bickford D, Lohman DJ, Sodhi NS et al (2007) Cryptic species as a window on diversity and conservation. Trends Ecol Evol 22:148–155CrossRefPubMedGoogle Scholar
  8. Birstein VJ, Doukakis P, Sorkin B et al (1998) Population aggregation analysis of three caviar-producing species of sturgeons and implications for the species identification of black caviar. Conserv Biol 12:766–775CrossRefGoogle Scholar
  9. Brochu CA (2007) Morphology, relationships, and biogeographical significance of an extinct horned crocodile (Crocodylia, Crocodylidae) from the Quaternary of Madagascar. Zool J Linn Soc 150:835–863CrossRefGoogle Scholar
  10. Brower AVZ (1999) Delimitation of phylogenetic species with DNA sequences: a critique of Davis and Nixon’s population aggregation analysis. Syst Biol 48:199–213CrossRefPubMedGoogle Scholar
  11. Busack SD, Pandya S (2001) Geographic variation in Caiman crocodilus and Caiman yacare (Crocodylia: Alligatoridae): systematic and legal implications. Herpetologica 57:294–312Google Scholar
  12. Chomel BB, Belotto A, Meslin FX (2007) Wildlife, exotic pets, and emerging zoonoses. Emerg Infect Dis 13:6–11CrossRefPubMedGoogle Scholar
  13. Chung W, Steiper M (2008) Mitochondrial CO1I introgression into the nuclear genome of Gorilla gorilla. Int J Primatol 29:1341–1353CrossRefPubMedGoogle Scholar
  14. Davis JI, Nixon KC (1992) Populations, genetic variation, and the delimitation of phylogenetic species. Syst Biol 41:421–435Google Scholar
  15. Deagle BE, Eveson JP, Jarman SN (2006) Quantification of damage in DNA recovered from highly degraded samples–a case study on DNA in faeces. Front Zool 3:11CrossRefPubMedGoogle Scholar
  16. DeSalle R (2006) Species discovery versus species identification in DNA barcoding efforts: response to Rubinoff. Conserv Biol 20:1545–1547CrossRefPubMedGoogle Scholar
  17. Eaton MJ (2002) Subsistence wildlife hunting in a multi-use forest of the Republic of Congo: monitoring and management for sustainable harvest (Unpublished MS thesis), University of Minnesota, St. Paul, p 90Google Scholar
  18. Eaton MJ (2006) Ecology, conservation and management of the Central African dwarf crocodile (Osteolaemus tetraspis), a progress report pp 84–95. Crocodiles: Proceedings of the 18th working meeting of the IUCN-SSC Crocodile Specialist Group, IUCN, Gland, SwitzerlandGoogle Scholar
  19. Eaton MJ, Barr B (2005) Regional Africa report: Lac Tele, Rep. of Congo. Crocodile Specialist Group Bull 24:18–20Google Scholar
  20. Eaton MJ, Martin AP, Thorbjarnarson J et al (2009) Species-level diversification of African dwarf crocodiles (Genus Osteolaemus): a geographic and phylogenetic perspective. Mol Phylogenet Evol 50:496–506CrossRefPubMedGoogle Scholar
  21. Estes RD (1991) The behavior guide to African mammals: including hoofed mammals, carnivores, primates. The University of California Press, BerkeleyGoogle Scholar
  22. Fitzhugh K (2006) DNA barcoding: an instance of technology-driven science? Bioscience 56:462–463CrossRefGoogle Scholar
  23. Folmer O, Black M, Hoeh W et al (1994) DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Mol Mar Biol Biotechnol 3:294–299PubMedGoogle Scholar
  24. Funk DJ, Omland KE (2003) Species-level paraphyly and polyphyly: frequency, causes, and consequences, with insights from animal mitochondrial DNA. Annu Rev Ecol Evol Syst 34:397–423CrossRefGoogle Scholar
  25. Hajibabaei M, Smith MA, Janzen DH et al (2006) A minimalist barcode can identify a specimen whose DNA is degraded. Mol Ecol Notes 6:959–964CrossRefGoogle Scholar
  26. Hebert PDN, Cywinska A, Ball SL et al (2003a) Biological identifications through DNA barcodes. Proc R Soc Lond B Biol Sci 270:313–321CrossRefGoogle Scholar
  27. Hebert PDN, Ratnasingham S, deWaard JR (2003b) Barcoding animal life: cytochrome c oxidase subunit 1 divergences among closely related species. Proc R Soc Lond B Biol Sci 270:S96–S99CrossRefGoogle Scholar
  28. Hebert PDN, Penton EH, Burns JM et al (2004) Ten species in one: DNA barcoding reveals cryptic species in the neotropical skipper butterfly Astraptes fulgerator. Proc Natl Acad Sci USA 101:14812–14817CrossRefPubMedGoogle Scholar
  29. Hekkala E (2004) Conservation genetics at the species boundary: case studies from African and Caribbean crocodiles (genus Crocodylus) (Unpublished Ph.D. thesis), Columbia University, New York, p 142Google Scholar
  30. Ivanova NV, Zemlak TS, Hanner RH et al (2007) Universal primer cocktails for fish DNA barcoding. Mol Ecol Notes 7:544–548CrossRefGoogle Scholar
  31. Janzen DH, Hajibabaei M, Burns JM et al (2005) Wedding biodiversity inventory of a large and complex Lepidoptera fauna with DNA barcoding. Phil Trans R Soc Lond B 360:1835–1845CrossRefGoogle Scholar
  32. Johns GC, Avise JC (1998) A comparative summary of genetic distances in the vertebrates from the mitochondrial cytochrome b gene. Mol Biol Evol 15:1481–1490PubMedGoogle Scholar
  33. Kaila L, Stahls G (2006) DNA barcodes: Evaluating the potential of COX1 to diffentiate closely related species of Elachista (Lepidoptera: Gelechioidea: Elachistidae) from Australia. Zootaxa 1170:1–26Google Scholar
  34. Kerr KCR, Stoeckle MY, Dove CJ et al (2007) Comprehensive DNA barcode coverage of North American birds. Mol Ecol Notes 7:535–543CrossRefPubMedGoogle Scholar
  35. Kingdon J (1997) The Kingdon field guide to African mammals. Academic Press, LondonGoogle Scholar
  36. Kolokotronis SO, MacPhee RDE, Greenwood AD (2007) Detection of mitochondrial insertions in the nucleus (NuMts) of Pleistocene and modern muskoxen. BMC Evol Biol 7:67CrossRefPubMedGoogle Scholar
  37. Lanave C, Preparata G, Saccone C et al (1984) A new method for calculating evolutionary substitution rates. J Mol Evol 20:86–93CrossRefPubMedGoogle Scholar
  38. Lemos B, Canavez F, Moreira M (1999) Mitochondrial DNA-like sequences in the nuclear genome of the opossum genus Didelphis (Marsupialia : Didelphidae). J Hered 90:543–547CrossRefPubMedGoogle Scholar
  39. Marko PB, Lee SC, Rice AM et al (2004) Mislabeling of a depleted reef fish. Nature 430:309–310CrossRefPubMedGoogle Scholar
  40. Martin AP (1991) Application of mitochondrial DNA sequence analysis to the problem of species identification of sharks. NOAA NMFS 115:53–59Google Scholar
  41. McAliley LR, Willis RE, Ray DA et al (2006) Are crocodiles really monophyletic? Evidence for subdivisions from sequence and morphological data. Mol Phylogenet Evol 39:16–32CrossRefPubMedGoogle Scholar
  42. Milius S (2005) Bushmeat on the Menu: untangling the influences of hunger, wealth, and international commerce. Science News 167:138CrossRefGoogle Scholar
  43. Milner-Gulland EJ, Bennett EL, The SCB 2002 Annual Meeting Wild Meat Group (2003) Wild meat: the bigger picture. Trends Ecol Evol 18:351–357CrossRefGoogle Scholar
  44. Moura T, Silva MC, Figueiredo I et al (2008) Molecular barcoding of north-east Atlantic deep-water sharks: species identification and application to fisheries management and conservation. Mar Freshwater Res 59:214–223CrossRefGoogle Scholar
  45. Neigel J, Domingo A, Stake J (2007) DNA barcoding as a tool for coral reef conservation. Coral Reefs 26:487–499CrossRefGoogle Scholar
  46. Nowak RM (1999) Walker’s mammals of the world. John Hopkins University Press, BaltimoreGoogle Scholar
  47. Rach J, DeSalle R, Sarkar IN et al (2008) Character-based DNA barcoding allows discrimination of genera, species and populations in Odonata. Proc R Soc Lond B Biol Sci 275:237–247CrossRefGoogle Scholar
  48. Ratnasingham S, Hebert PDN (2007) BOLD: The barcode of life data system. Mol Ecol Notes 7:355–364CrossRefPubMedGoogle Scholar
  49. Redford KH (1992) The empty forest. Bioscience 42:412–422CrossRefGoogle Scholar
  50. Rodriguez F, Oliver JL, Marin A et al (1990) The general stochastic model of nucleotide substitution. J Theor Biol 142:485–501CrossRefPubMedGoogle Scholar
  51. Ross HA, Lento GM, Dalebout ML et al (2003) DNA surveillance: web-based molecular identification of whales, dolphins, and porpoises. J Hered 94:111–114CrossRefPubMedGoogle Scholar
  52. Rubinoff D (2006) Utility of mitochondrial DNA barcodes in species conservation. Conserv Biol 20:1026–1033CrossRefPubMedGoogle Scholar
  53. Rubinoff D, Cameron S, Will K (2006) A genomic perspective on the shortcomings of mitochondrial DNA for “barcoding” identification. J Hered 97:581–594CrossRefPubMedGoogle Scholar
  54. Schmitz A, Mansfeld P, Hekkala E et al (2003) Molecular evidence for species level divergence in African Nile Crocodiles Crocodylus niloticus (Laurenti, 1786). CR Palevol 2:703–712CrossRefGoogle Scholar
  55. Shapiro B, Drummond A, Rambaut A et al (2004) Rise and fall of the Beringian steppe bison. Science 306:1561–1565CrossRefPubMedGoogle Scholar
  56. Song H, Buhay JE, Whiting MF et al (2008) Many species in one: DNA barcoding overestimates the number of species when nuclear mitochondrial pseudogenes are coamplified. Proc Natl Acad Sci USA 105:13486–13491CrossRefPubMedGoogle Scholar
  57. Stamatakis A (2006) RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models. Bioinformatics 22:2688–2690CrossRefPubMedGoogle Scholar
  58. Stamatakis A, Hoover P, Rougemont J (2008) A rapid bootstrap algorithm for the RAxML web servers. Syst Biol 57:758–771CrossRefPubMedGoogle Scholar
  59. Tamura K, Nei M (1993) Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees. Mol Biol Evol 10:512–526PubMedGoogle Scholar
  60. Tamura K, Dudley J, Nei M et al (2007) MEGA4: Molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol 24:1596–1599CrossRefPubMedGoogle Scholar
  61. Thompson J, Higgins D, Gibson T (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680CrossRefPubMedGoogle Scholar
  62. Thorbjarnarson JB, Eaton MJ (2004) Preliminary examination of crocodile bushmeat issues in the Republic of Congo and Gabon, pp 236–247. Crocodiles: Proceedings of the 17th working meeting of the IUCN-SSC Crocodile Specialist Group, IUCN, Gland, SwitzerlandGoogle Scholar
  63. van Vliet N, Zundel S, Miquel C et al (2008) Distinguishing dung from blue, red and yellow-backed duikers through noninvasive genetic techniques. Afr J Ecol 46:411–417CrossRefGoogle Scholar
  64. Vasconcelos WR, Hrbek T, Da Silveira R et al (2006) Population genetic analysis of Caiman crocodilus (Linnaeus, 1758) from South America. Genet Mol Biol 29:220–230CrossRefGoogle Scholar
  65. Vuissoz A, Worobey M, Odegaard N et al (2007) The survival of PCR-amplifiable DNA in cow leather. J Archeol Sci 34:823–829CrossRefGoogle Scholar
  66. Witt JDS, Threloff DL, Hebert PDN (2006) DNA barcoding reveals extraordinary cryptic diversity in an amphipod genus: implications for desert spring conservation. Mol Ecol 15:3073–3082CrossRefPubMedGoogle Scholar
  67. Yan P, Wu X-B, Shi Y et al (2005) Identification of Chinese alligators (Alligator sinensis) meat by diagnostic PCR of the mitochondrial cytochrome b gene. Biol Conserv 121:45–51CrossRefGoogle Scholar
  68. Yang ZH (1994) Maximum likelihood phylogenetic estimation from DNA sequences with variable rates over sites: approximate methods. J Mol Evol 39:306–314CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2009

Authors and Affiliations

  • Mitchell J. Eaton
    • 1
    • 4
  • Greta L. Meyers
    • 2
  • Sergios-Orestis Kolokotronis
    • 3
  • Matthew S. Leslie
    • 3
  • Andrew P. Martin
    • 1
  • George Amato
    • 3
  1. 1.Department of Ecology and Evolutionary Biology, N122 RamaleyUniversity of ColoradoBoulderUSA
  2. 2.Department of Environmental ScienceBarnard CollegeNew YorkUSA
  3. 3.Sackler Institute for Comparative GenomicsAmerican Museum of Natural HistoryNew YorkUSA
  4. 4.Patuxent Wildlife Research CenterUS Geological SurveyLaurelUSA

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