DNA Barcodes pp 153-182 | Cite as

DNA Barcoding in Mammals

  • Natalia V. Ivanova
  • Elizabeth L. Clare
  • Alex V. BorisenkoEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 858)


DNA barcoding provides an operational framework for mammalian taxonomic identification and cryptic species discovery. Focused effort to build a reference library of genetic data has resulted in the assembly of over 35 K mammalian cytochrome c oxidase subunit I sequences and outlined the scope of mammal-related barcoding projects. Based on the above experience, this chapter recounts three typical methodological pathways involved in mammalian barcoding: routine methods aimed at assembling the reference sequence library from high quality samples, express approaches used to attain cheap and fast taxonomic identifications for applied purposes, and forensic techniques employed when dealing with degraded material. Most of the methods described are applicable to a range of vertebrate taxa outside Mammalia.

Key words

Mammalia Molecular diagnostics Molecular biodiversity Molecular methods DNA extraction PCR Primers Sequencing Cytochrome c oxidase subunit I 



We thank Judith Eger, Mark Engstrom, Burton Lim, Don Stewart, Charles Francis, Sergey Kruskop, Andrey Lissovsky, Vladimir Lebedev, Natalia Abramson, Ivan Kuzmin, Bernard Agwanda, Anna Bannikova, Ticul Alvarez, Fernando Cervantez, Curtis Strobeck, Jack Millar, and William Pruitt, Jr. for providing materials for analysis; Robert Hanner for advice on protocol development; Agata Pawlowski and Miranda Elliott for protocol testing; and Paul Hebert for administrative support. DNA analyses were performed at the Canadian Centre of DNA Barcoding, Biodiversity Institute of Ontario, and University of Guelph, and supported by grants to Paul Hebert from the Gordon and Betty Moore Foundation, Genome Canada through the Ontario Genomics Institute, (2008-OGI-ICI-03) the Canada Foundation for Innovation, the Ontario Innovation Trust, and the Natural Sciences and Engineering Research Council of Canada.


  1. 1.
    Reeder DM, Helgen KM, Wilson DE (2007) Global trends and biases in new mammal species discoveries. Occas Papers Mus Texas Technol Univ 269:1–35Google Scholar
  2. 2.
    Ceballos G, Ehrlich PR (2009) Discoveries of new mammal species and their implications for conservation and ecosystem services. Proc Natl Acad Sci 106:3841–3846PubMedCrossRefGoogle Scholar
  3. 3.
    Baker RJ, Bradley RD (2006) Speciation in mammals and the genetic species concept. J Mammal 87:643–662PubMedCrossRefGoogle Scholar
  4. 4.
    Bradley RJ, Baker RD (2001) A test of the genetic species concept: cytochrome-b sequences and mammals. J Mammal 82:960–973CrossRefGoogle Scholar
  5. 5.
    Hebert PDN, Cywinska A, Ball S, deWaard JR (2003) Biological identifications through DNA barcodes. Proc R Soc Lond B 270:313–321CrossRefGoogle Scholar
  6. 6.
    Hebert PDN, Ratnasingham S, deWaard JR (2003) Barcoding animal life: cytochrome c oxidase subunit 1 divergences among closely related species. Proc R Soc Lond B 270:S96–S99CrossRefGoogle Scholar
  7. 7.
    Broughton RE, Reneau PC (2006) Spatial covariation of mutation and nonsynonymous substitution rates in vertebrate mitochondrial genomes. Mol Biol Evol 23:1516–1524PubMedCrossRefGoogle Scholar
  8. 8.
    Folmer O, Black M, Hoeh W, Lutz R, Vrijenhoek R (1994) DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Mol Mar Biol Biotechnol 3:294–299PubMedGoogle Scholar
  9. 9.
    Simmons RB, Weller SJ (2001) Utility and evolution of cytochrome b in insects. Mol Phylogenet Evol 20:196–210PubMedCrossRefGoogle Scholar
  10. 10.
    Consortium for the Barcode of Life [Internet].
  11. 11.
    Meiklejohn C, Montooth K, Rand D (2007) Positive and negative selection on the mitochondrial genome. Trends Genet 23:259–263PubMedCrossRefGoogle Scholar
  12. 12.
    Pfunder M, Holzgang O, Frey J (2004) Development of microarray-based diagnostics of voles and shrews for use in biodiversity monitoring studies, and evaluation of mitochondrial cytochrome oxidase I vs. cytochrome b as genetic markers. Mol Ecol 13:1277–1286PubMedCrossRefGoogle Scholar
  13. 13.
    Lissovsky AA, Ivanova NV, Borisenko AV (2007) Molecular phylogenetics and taxonomy of the subgenus Pika (Ochotona, Lagomorpha). J Mammal 88:1195–1204CrossRefGoogle Scholar
  14. 14.
    Hajibabaei M, Singer GA, Clare EL, Hebert PDN (2007) Design and applicability of DNA arrays and DNA barcodes in biodiversity monitoring. BMC Biol 5:24PubMedCrossRefGoogle Scholar
  15. 15.
    Lorenz J, Jackson W, Beck J, Hanner R (2005) The problems and promise of DNA barcodes for species diagnosis of primate biomaterials. Philos Trans R Soc B 360:1869–1877CrossRefGoogle Scholar
  16. 16.
    Hajibabaei M, Singer G, Hickey D (2006) Benchmarking DNA barcodes: an assessment using available primate sequences. Genome 49:851–854PubMedCrossRefGoogle Scholar
  17. 17.
    Borisenko AV, Lim BK, Ivanova NV, Hanner RH, Hebert PDN (2008) DNA barcoding in surveys of small mammal communities: a field study in Suriname. Mol Ecol Resour 8:471–479PubMedCrossRefGoogle Scholar
  18. 18.
    Ivanova NV, Borisenko AV, Hebert PDN (2009) Express barcodes: racing from specimen to identification. Mol Ecol Resour 9:35–41PubMedCrossRefGoogle Scholar
  19. 19.
    Clare EL, Lim BK, Engstrom MD, Eger JL, Hebert PDN (2007) DNA barcoding of Neotropical bats: species identification and discovery within Guyana. Mol Ecol Notes 7:184–190CrossRefGoogle Scholar
  20. 20.
    Francis CM, Borisenko AV, Ivanova NV, Eger JL, Lim BK, Guillén-Servent A, Kruskop SV, Mackie I, Hebert PDN (2010) The role of DNA barcodes in understanding and conservation of mammal diversity in Southeast Asia. PLoS ONE 5:e12575PubMedCrossRefGoogle Scholar
  21. 21.
    Clare EL, Lim BK, Fenton MB, Hebert PDN (2011) Neotropical bats: estimating species diversity with DNA barcodes. PLoS ONE 6(7):e22648Google Scholar
  22. 22.
    Clare EL, Adams AM, Maya-Simoes AZ, Eger JL, Hebert PDN, Fenton MB. Cryptic species in Parnell’s Mustached Bat (Pteronotus parnellii): molecular, morphological and acoustic evidence. Mol Phylogenet Evol (in review)Google Scholar
  23. 23.
    Clare EL. Cryptic species? Patterns of maternal and paternal gene flow in 8 Neotropical bats. PLoS ONE 6(7):e21460Google Scholar
  24. 24.
    Maya-Simões AZ, Clare EL, Fenton MB (2010) Parnell’s mustached bat (Pteronotus parnellii): a morphologically cryptic species complex. Chiroptera Neotropical 16:130–132Google Scholar
  25. 25.
    Borisenko AV, Kruskop SV, Ivanova NV (2008) A new mouse-eared bat (Mammalia: Chiroptera: Vespertilionidae) from Vietnam. Russ J Theriol 7:57–69Google Scholar
  26. 26.
    Engstrom M, Murphy R, Haddrath O (1999) Sampling vertebrate collections for molecular research: practice and policies. In: Metsger D, Byers S (eds) Managing the modern herbarium: an interdisciplinary approach. Elton-Wolf, Vancouver, pp 315–330Google Scholar
  27. 27.
    DeSalle R, Amato G (2004) The expansion of conservation genetics. Nat Rev Genet 5:702–712PubMedCrossRefGoogle Scholar
  28. 28.
    Ruedas L, Salazar-Bravo J, Dragoo J, Yates T (2000) The importance of being earnest: what, if anything, constitutes a “specimen examined?”. Mol Phylogenet Evol 17:129–132PubMedCrossRefGoogle Scholar
  29. 29.
    Borisenko AV, Sones JE, Hebert PDN (2009) The front-end logistics of DNA barcoding: challenges and prospects. Mol Ecol Resour 9:27–34PubMedCrossRefGoogle Scholar
  30. 30.
    deWaard J, Ivanova N, Hajibabaei M, Hebert P (2008) Assembling DNA barcodes: analytical protocols. In: Martin C (ed) Environmental genomics, methods in molecular biology, vol 410. Humana Press, Totowa, 275–283Google Scholar
  31. 31.
    Ivanova N, deWaard J, Hebert P (2006) An inexpensive, automation-friendly protocol for recovering high quality DNA. Mol Ecol Notes 6:998–1002CrossRefGoogle Scholar
  32. 32.
    Borisenko AV (2008) Reconnaissance survey of the small mammal community in the Churchill Northern Studies Centre Area. Polar Barcode Life Newsl 1:4Google Scholar
  33. 33.
    Hajibabaei M, Smith MA, Janzen DH, Rodriguez J, Whitfield JB, Hebert PD (2006) A minimalist barcode can identify a specimen whose DNA is degraded. Mol Ecol Notes 6:959–964CrossRefGoogle Scholar
  34. 34.
    American Society of Mammalogists Animal Care and Use Committee (1998) Guidelines for the capture, handling, and care of mammals as approved by the American Society of Mammalogists. J Mammal 79:1416–1431CrossRefGoogle Scholar
  35. 35.
    Wilson DE, Cole FR, Nichols JD, Rasanayagam R, Foster MS (eds) (1996) Measuring and monitoring biological diversity. Standard methods for mammals. Smithsonian Institution Press, Washington; LondonGoogle Scholar
  36. 36.
    Lillestolen T, Foster N, Wise S (1993) Development of the National Marine Mammal Tissue Bank. Sci Total Environ 139:97–107PubMedCrossRefGoogle Scholar
  37. 37.
    Triant DA, DeWoody JA (2007) The occurrence, detection, and avoidance of mitochondrial DNA translocations in mammalian systematics and phylogeography. J Mammal 88:908–920CrossRefGoogle Scholar
  38. 38.
    Hanner R, Corthals A, Dessauer H (2005) Salvage of genetically valuable tissues following a freezer failure. Mol Phylogenet Evol 34:452–455PubMedCrossRefGoogle Scholar
  39. 39.
    Kilpatrick C (2002) Noncryogenic preservation of mammalian tissues for DNA extraction: an assessment of storage methods. Biochem Genet 40:53–62PubMedCrossRefGoogle Scholar
  40. 40.
    Nunley WC, Schuit KE, Dickie MW, Kinlaw JB (1972) Delayed, in vivo hepatic post-mortem autolysis. Virchows Arch Abt B Zellpath 11:289–302Google Scholar
  41. 41.
    Tomita Y, Nihira M, Ohno Y, Shigeru S (2004) Ultrastructural changes during in situ early postmortem autolysis in kidney, pancreas, liver, heart and skeletal muscle of rats. Leg Med 6:25–31CrossRefGoogle Scholar
  42. 42.
    Herdson P, Kaltenbach J, Jennings R (1969) Fine structural and biochemical changes in dog myocardium during autolysis. Am J Pathol 57:539–557PubMedGoogle Scholar
  43. 43.
    Ljungman M, Hanawalt PC (1992) Efficient protection against oxidative DNA damage in chromatin. Mol Carcinog 5:264–269PubMedCrossRefGoogle Scholar
  44. 44.
    Scheuerle A, Pavenstaedt I, Schlenk R, Melzner I, Rödel G, Haferkamp O (1993) In situ autolysis of mouse brain: ultrastructure of mitochondria and the function of oxidative phosphorylation and mitochondrial DNA. Virchows Arch B Cell Pathol 63:331–334CrossRefGoogle Scholar
  45. 45.
    Borisenko A, Dooh R (2007) An electronic lab book to facilitate high throughput DNA barcoding. CCDB Adv Meth Release 8:1Google Scholar
  46. 46.
    Ivanova N, Zemlak T, Hanner R, Hebert P (2007) Universal primer cocktails for fish DNA barcoding. Mol Ecol Notes 7:544–548CrossRefGoogle Scholar
  47. 47.
    Meyer CP (2003) Molecular systematics of cowries (Gastropoda: Cypraeidae) and diversification patterns in the tropics. Biol J Linn Soc 79:401–459CrossRefGoogle Scholar
  48. 48.
    Robins JH, Hingston M, Matisoo-Smith E, Ross HA (2007) Identifying Rattus species using mitochondrial DNA. Mol Ecol Notes 7:717–729CrossRefGoogle Scholar
  49. 49.
    Ward R, Zemlak T, Innes B, Last P, Hebert P (2005) DNA barcoding Australia’s fish species. Philos Trans R Soc B 360:1847–1857CrossRefGoogle Scholar
  50. 50.
    Messing J (1983) New M13 vectors for cloning. Methods Enzymol 101:20–79PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Natalia V. Ivanova
    • 1
  • Elizabeth L. Clare
    • 1
  • Alex V. Borisenko
    • 1
    Email author
  1. 1.Biodiversity Institute of Ontario & Integrative BiologyUniversity of GuelphGuelphCanada

Personalised recommendations