Marine Biology

, Volume 156, Issue 12, pp 2641–2647 | Cite as

DNA barcoding of Pacific Canada’s fishes

  • Dirk SteinkeEmail author
  • Tyler S. Zemlak
  • James A. Boutillier
  • Paul D. N. Hebert
Comment and Reply


DNA barcoding—sequencing a standard region of the mitochondrial cytochrome c oxidase 1 gene (COI)—promises a rapid, accurate means of identifying animals to a species level. This study establishes that sequence variability in the barcode region permits discrimination of 98% of 201 fish species from the Canadian Pacific. The average sequence variation within species was 0.25%, while the average distance separating species within genera was 3.75%. The latter value was considerably lower than values reported in other studies, reflecting the dominance of the Canadian fauna by members of the young and highly diverse genus Sebastes. Although most sebastids possessed distinctive COI sequences, four species did not. As a partial offset to these cases, the barcode records indicated the presence of a new, broadly distributed species of Paraliparis and the possibility that Paraliparis pectoralis is actually a species pair. The present study shows that most fish species in Pacific Canadian waters correspond to a single, tightly cohesive array of barcode sequences that are distinct from those of any other species, but also highlights some taxonomic issues that need further investigation.


Introgressive Hybridization Sequence Congruence Barcoding System Prime Cocktail Rapid Sample Processing 



This study was supported by the Canadian Barcode of Life Network through funding from NSERC and Genome Canada through the Ontario Genomics Institute. We thank the Canadian Department of Fisheries and Oceans and the Canadian Coast Guard for ship time and other support during the cruises where specimens were collected. We also thank Ken Fong, Graham Gillespie, Gavin Hanke, Katy Hind, John Klymko, and Dennis Rutherford for help with specimen collections and Mark Stoeckle for sharing his idea of the half-logarithmic do plots for genetic distances.

Supplementary material

227_2009_1284_MOESM1_ESM.pdf (855 kb)
(PDF 854 kb) A neighbour-joining tree of COI sequence divergences (K2P) in all 1,225 individuals of this study. Species names, BOLD process ID, Sample ID, sequence length, and numbers of ambiguous bases are given at branch tips
227_2009_1284_MOESM2_ESM.pdf (457 kb)
(PDF 456 kb)


  1. Andriashev AP, Pitruk DL (1993) Review of the ultra-abyssal (hadal) genus Pseudoliparis (Scorpaeniformes, Liparidae) with a description of a new species from the Japan trench. J Ichthyol 33:31–39Google Scholar
  2. Barrett RDH, Hebert PDN (2004) Identifying spiders through DNA barcodes. Can J Zool 83:481–491CrossRefGoogle Scholar
  3. Burns JM, Janzen DH, Hajibabaei M, Hallwachs W, Hebert PDN (2007) DNA barcodes of closely related (but morphologically and ecologically distinct) species of skipper butterflies (Hesperiidae) can differ by only one to three nucleotides. J Lepid Soc 61:138–153Google Scholar
  4. Froese R, Pauly D (2006) Fishbase. World Wide Web electronic publicationGoogle Scholar
  5. Garland ED, Zimmer CA (2002) Techniques for the identification of bivalve larvae. Mar Ecol Prog Ser 225:299–310CrossRefGoogle Scholar
  6. Gharrett AJ, Matala AP, Peterson EL, Gray AK, Li Z, Heifetz J (2005) Two genetically distinct forms of rougheye rockfish (Sebastes aleutianus) are different species. Trans Am Fish Soc 134:242–260CrossRefGoogle Scholar
  7. Hajibabaei M, Janzen DH, Burns JM, Hallwachs W, Hebert PDN (2006) DNA barcodes distinguish species of tropical Lepidoptera. Proc Natl Acad Sci USA 103:968–971CrossRefGoogle Scholar
  8. Hart JL (1973) Pacific fishes of Canada. Bull Fish Res Board Can 180:740Google Scholar
  9. Hebert PDN, Cywinska A, Ball SL, deWaard JR (2003a) Biological identifications through DNA barcodes. Proceedings of the Royal Society of London Series B-Biological Sciences 270:313–321CrossRefGoogle Scholar
  10. Hebert PDN, Ratnasingham S, deWaard JR (2003b) Barcoding animal life: cytochrome c oxidase subunit 1 divergences among closely related species. Proceedings of the Royal Society of London Series B-Biological Sciences 270:S96–S99Google Scholar
  11. Hebert PDN, Penton EH, Burns JM, Janzen DH, Hallwachs W (2004a) Ten species in one: DNA barcoding reveals cryptic species in the neotropical skipper butterfly Astraptes fulgerator. Proc Natl Acad Sci USA 101:14812–14817CrossRefGoogle Scholar
  12. Hebert PDN, Stoeckle MY, Zemlak TS, Francis CM (2004b) Identification of birds through DNA Barcodes. Public Libr Sci Biol 2:e312Google Scholar
  13. Hogg ID, Hebert PDN (2004) Biological identification of springtails (Hexapoda: Collembola) from the Canadian Arctic, using mitochondrial DNA barcodes. Can J Zool-Revue Canadienne De Zoologie 82:749–754CrossRefGoogle Scholar
  14. Hubert N, Hanner R, Holm E, Mandrak NE, Taylor E, Burridge M, Watkinson D, Dumont P, Curry A, Bentzen P, Zhang J, April J, Bernatchez L (2008) Identifying Canadian freshwater fishes through DNA barcodes. Public Libr Sci One 3:e2490Google Scholar
  15. Hyde JR, Vetter RD (2007) The origin, evolution, and diversification of rockfishes of the genus Sebastes (Cuvier). Mol Phylogen Evol 44:790–811CrossRefGoogle Scholar
  16. Ivanova NV, Dewaard JR, Hebert PDN (2006) An inexpensive, automation-friendly protocol for recovering high-quality DNA. Mol Ecol Notes 6:998–1002CrossRefGoogle Scholar
  17. Ivanova NV, Zemlak TS, Hanner R, Hebert PDN (2007) Universal primer cocktails for fish DNA barcoding. Mol Ecol Notes 7:544–548CrossRefGoogle Scholar
  18. Kai Y, Nakayama K, Nakabo T (2002a) Genetic differences among three colour morphotypes of the black rockfish, Sebastes inermis, inferred from mtDNA and AFLP analysis. Mol Ecol 11:2591–2598CrossRefGoogle Scholar
  19. Kai Y, Yagishita N, Ikeda H, Nakabo T (2002b) Genetic differences between two color morphotypes of redfish, Sebastes scythropus (Osteichthyes: Scorpaenidae). Species Divers 7:371–380CrossRefGoogle Scholar
  20. Kimura M (1980) A simple model for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 16:111–120CrossRefGoogle Scholar
  21. Kumar S, Tamura K, Nei M (2004) MEGA3: integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief Bioinform 5:150–163CrossRefGoogle Scholar
  22. Meyer CP, Paulay G (2005) DNA barcoding: error rates based on comprehensive sampling. Public Libr Sci Biol 3:2229–2238Google Scholar
  23. Moritz C (1994) Defining ‘evolutionarily significant units’ for conservation. Trends Ecol Evol 9:373–375CrossRefGoogle Scholar
  24. Narum SW, Buonaccorsi VP, Kimbrell CA, Vetter RD (2004) Genetic divergence between gopher rockfish (Sebastes carnatus) and black and yellow rockfish (Sebastes chrysomelas). Copeia 4:926–931CrossRefGoogle Scholar
  25. Nelson J (1994) Fishes of the world. Wiley, New YorkGoogle Scholar
  26. Pegg GG, Sinclair B, Briskey L, Aspden WJ (2006) MtDNA barcode identification of fish larvae in the southern great barrier reef, Australia. Sci Mar 70:7–12CrossRefGoogle Scholar
  27. Ratnasingham S, Hebert PDN (2007) The barcode of life database. Mol Ecol Notes 7:355–364CrossRefGoogle Scholar
  28. Roques S, Sevigny JM, Bernatchez L (2001) Evidence for broadscale introgressive hybridization between two redfish (genus Sebastes) in the North-west Atlantic: a rare marine example. Mol Ecol 10:149–165CrossRefGoogle Scholar
  29. Smith MA, Fisher BL, Hebert PDN (2005) DNA barcoding for effective biodiversity assessment of a hyperdiverse arthropod group: the ants of Madagascar. Phil Trans Royal Soc B-Biol Sci 360:1825–1834CrossRefGoogle Scholar
  30. Smith PJ, McVeagh MS, Steinke D (2008a) Application of DNA barcoding for the identification of smoked fish products. J Fish Biol 72:464–471CrossRefGoogle Scholar
  31. Smith PJ, Steinke D, McVeagh MS, Stewart AL, Struthers CD, Roberts CD (2008b) Molecular analysis of Southern ocean skates (Bathyraja) reveals a new species of Antarctic skate. J Fish Biol 73:1170–1182CrossRefGoogle Scholar
  32. Steinke D, Vences M, Salzburger W, Meyer A (2005) TaxI: a software tool for DNA barcoding using distance methods. Phil Trans Royal Soc B-Biol Sci 360:1975–1980CrossRefGoogle Scholar
  33. Ward RD, Holmes BH (2007) An analysis of nucleotide and amino acid variability in the barcode region of cytochrome c oxidase I (cox1) in fishes. Mol Ecol Notes 7:899–907CrossRefGoogle Scholar
  34. Ward RD, Zemlak TS, Innes BH, Last PR, Hebert PDN (2005) DNA barcoding Australia’s fish species. Phil Trans Royal Soc B-Biol Sci 360:1847–1857CrossRefGoogle Scholar
  35. Ward RD, Costa FO, Holmes BH, Steinke D (2008a) DNA barcoding shared fish species from the North Atlantic and Australasia: minimal divergence for most taxa but a likely two species for both Zeus faber and Lepidopus caudatus. Aquat Biol 3:71–78CrossRefGoogle Scholar
  36. Ward RD, Holmes BH, Yearsley GK (2008b) DNA barcoding reveals a likely second species of Asian sea bass (barramundi) (Lates calcarifer). J Fish Biol 72:458–463CrossRefGoogle Scholar
  37. Ward RD, Hanner R, Hebert PDN (2009) The campaign to DNA barcode all fishes, FISH-BOL. J Fish Biol 73:1–28Google Scholar
  38. Wong EHK, Hanner R (2008) DNA barcoding detects market substitution in North American seafood. Food Res Int 41:828–837CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2009

Authors and Affiliations

  • Dirk Steinke
    • 1
    Email author
  • Tyler S. Zemlak
    • 1
    • 2
  • James A. Boutillier
    • 3
  • Paul D. N. Hebert
    • 1
  1. 1.Canadian Centre for DNA Barcoding, Biodiversity Institute of OntarioUniversity of GuelphGuelphCanada
  2. 2.Department of BiologyDalhousie UniversityHalifaxCanada
  3. 3.Pacific Biological StationFisheries and Oceans CanadaNanaimoCanada

Personalised recommendations