, Volume 401, Issue 0, pp 239–254 | Cite as

Taxonomic and systematic assessment of planktonic copepods using mitochondrial COI sequence variation and competitive, species-specific PCR

  • A. Bucklin
  • M. Guarnieri
  • R.S. Hill
  • A.M. Bentley
  • S. Kaartvedt


Accurate taxonomic identification of species at all life stages is critical to understand and predict the processes that together determine marine community dynamics. However, zooplankton assemblages may include numerous sibling and congeneric species distinguished by subtle morphological characteristics. Molecular systematic databases, including DNA sequences of homologous gene regions for selected taxonomic groups, allow the design of rapid protocols to determine species' diversity and identify individuals. In this study, the DNA sequence of a 300 base-pair region of the mitochondrial cytochrome oxidase I (COI) gene was determined for eight species of three genera of calanoid copepods: Calanus finmarchicus, C. glacialis and C. helgolandicus; Neocalanus cristatus, N. flemingeri and N. plumchrus; and Pseudocalanus moultoni and P. newmani. The DNA sequences differed between congeneric species by 13 – 22% of the nucleotides; the protein sequences differed by zero to five amino acid substitutions. Both the DNA and amino acid sequences resolved the evolutionary relationships among congeneric species; relationships among the genera were not well-resolved by this region of mtCOI. Using the same conserved primers, the only amplification product for C. finmarchicus was an aberrant sequence (and putative pseudogene) which differed from the C. finmarchicus COI sequence by 36% of the nucleotides and 32 amino acid substitutions. Species-specific oligonucleotide primers were designed for Calanus spp. (which cannot be distinguished at larval stages) and Pseudocalanus spp. (which are difficult to distinguish even as adults). Individual copepods were identified using competitive, multiplexed species-specific polymerase chain reactions (PCR) in two studies of co-occurring sibling species. The first study confirmed the presence of three Calanus spp. in Oslofjord, Norway and found a predominance of C. helgolandicus. The second study determined patterns of distribution and abundance of Pseudocalanus spp. on Georges Bank in the NW Atlantic and showed that P. moultoni predominated in shallow and coastal waters, while P. newmani was more abundant in offshore regions flanking the Bank. Competitive, species-specific PCR is a useful tool for biological oceanographers. This simple, rapid, and inexpensive assay may be used to identify morphologically-similar individuals of any size and life stage, and to determine a species' presence or absence in pooled samples.

mitochondrial DNA cytochrome oxidase I competitive PCR copepoda taxonomy marine zooplankton 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Avise, J. C., 1994. Molecular Markers, Natural History and Evolution, Chapman and Hall, New York, NY. 511 pp.Google Scholar
  2. Banks, M., D. Hedgecock & C. Waters, 1993. Discrimination between closely related Pacific oyster species (Crassostrea) via mitochondrial DNA sequences coding for large sununit rRNA. Mol. Mar. Biol. Biotechnol. 2: 129–136.Google Scholar
  3. Bradford, J.M. & J. B. Jillett, 1974. A revision of generic definitions in the Calanidae (Copepoda, Calanoida). Crustaceana 27: 5–16.Google Scholar
  4. Bradford, J. M., 1988. Review of the taxonomy of the Calanidae (Copepoda) and the limits to the genusCalanus. Hydrobiologia 167/168: 73–81.Google Scholar
  5. Brown, J. M., O. Pellmyr, J. N. Thompson & R. G. Harrison, 1994. Phylogeny of Greya (Lepidoptera: Prodoxidae), based on nucleotide sequence variation in mitochondrial cytochrome oxidase I and II: congruence with morphological data. Mol. Biol. Evol. 11: 128–141.Google Scholar
  6. Bucklin, A., B. W. Frost & T. D. Kocher, 1992. DNA sequence variation of the mitochondrial 16S rRNA in Calanus (Copepoda; Calanoida): intra-and inter-specific patterns. Molec. Mar. Biol. Biotech. 1: 397–407.Google Scholar
  7. Bucklin, A., B. W. Frost & T.D. Kocher, 1995. Molecular systematics of seven species of Calanus and three species of Metridia (Calanoida; Copepoda). Mar. Biol. 121: 655–664.Google Scholar
  8. Bucklin, A., T. C. LaJeunesse, E. Curry, J. Wallinga & K. Garrison (1996a) Molecular genetic diversity of the copepod, Nannocalanus minor: genetic evidence of species and population structure in the N. Atlantic Ocean. J. mar. Res. 54: 285–310.Google Scholar
  9. Bucklin, A., R. Sundt & G. Dahle, 1996b. Population genetics of Calanus finmarchicus (Copepoda; Calanoida) in the North Atlantic. Proceedings of an ICES Workshop on a TransAtlantic Study of Calanus finmarchicus. Ophelia 44: 29–45.Google Scholar
  10. Bucklin, A., R. S. Hill, N. J. Mottola & A. M. Bentley, 1997a. Seasonal patterns of distribution and abundance of the copepods, Pseudocalanus moultoni and P. newmani, on Georges Bank: evidence for a dynamic balance between retention and loss. Internat. Cons. Expl. Seas Science Mtg., September, 1997. Background Paper T: 06.Google Scholar
  11. Bucklin, A., S. B. Smolenack, A. M. Bentley & P. H. Wiebe, 1997b. Gene flow patterns of the euphausiid, Meganyctiphanes norvegica, in the N. Atlantic based on DNA sequences for mitochondrial cytochrome oxidase I and cytochrome b. J. Plank. Res. 19: 1763–1781.Google Scholar
  12. Bucklin, A., A. M. Bentley & S. P. Franzen, 1998a. Distribution and relative abundance of the copepods, Pseudocalanus moultoni and P. newmani, on Georges Bank based on molecular identification of sibling species. Mar. Biol. (in press).Google Scholar
  13. Bucklin A., C. C. Caudill & M. Guarnieri, 1998b. Population genetics and phylogeny of marine planktonic copepods. Chapter 14. In: K. C. Cooksey (ed.). Molecular Approaches to the Study of the Ocean. London: Chapman & Hall: 303–318.Google Scholar
  14. Burton, R. S. & B.-N. Lee, 1994. Nuclear and mitochondrial gene genealogies and allozyme polymorphism across a major phylo253 genetic break in the copepod Tigriopus californicus. Proc. natn. Acad. Sci. 91: 5197–5201.Google Scholar
  15. Charlieu, J.-P., 1994. Distinction between almost-identical DNA sequences by polymerase chain reaction. Chapter 12. In H. G. Griffin & A. M. Griffin (eds), PCR Technology Current Innovations. CRC Press, Boca Raton, FL: 101–106.Google Scholar
  16. Clary, D. O. & D. R. Wolstenholme, 1985. The mitochondrial DNA molecule of Drosophila yakuba: nucleotide sequence, gene organization and genetic code. J. molec. Evol. 22: 252–271.Google Scholar
  17. Cunningham, C. W., N. W. Blackstone & L. W. Buss, 1992. Evolution of king crabs from hermit crab ancestors. Nature 355: 539–542.Google Scholar
  18. Davis, C. S, 1987. Zooplankton Life Cycles. In: Backus R. H. (ed.). Georges Bank, MIT Press, Cambridge, MA: 256–267.Google Scholar
  19. DeDecker, A. H. B., B. Z. Kaczmaruk & G. Marska, 1991. A new species ofCalanus (Copepoda, Calanoida) from South African waters. Ann. S. Afr. Mus. 101: 27–44.Google Scholar
  20. DeLong, E. F., G. S. Wickman & N. R. Pace, 1989. Phylogenetic strains: ribosomal RNA-based for the identification of single cells. Science 243: 1360–1363.Google Scholar
  21. Dixon, D. R., D. A. S. B. Jollivet, L. R. J. Dixon, J. A. Nott & P. W. H. Holland, 1995. The molecular identification of early lifehistory stages of hydrothermal vent organisms. In L. M. Parson, C. L. Walker & D. R. Dixon (eds.), Hydrothermal Vents and Processes, Geol. Soc. Spec. Publ. 87: 343–350.Google Scholar
  22. Engels, W., 1992. Amplify. Computer Freeware. Genetics Department, University of Wisconsin, Madison, WI 53706.Google Scholar
  23. Fell, J. W., 1995. rDNA targeted oligonucleotide primers for the identification of pathogenic yeasts in a polymerase chain reaction. J. Ind. Microbiol. 14: 475–477.Google Scholar
  24. Fleminger, A. & K. Hulsemann, 1977. Geographical range and taxomonic divergence in North Atlantic Calanus (C. helgolandicus, C. finmarchicus and C. glacialis). Mar. Biol. 40: 233–248.Google Scholar
  25. Folmer, O., M. Black, W. Hoen, R. Lutz & R. Vrijenhoek, 1994. DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metozoan invertebrates. Molec. Mar. Biol. Biotech. 3: 294–299.Google Scholar
  26. France, S. C. & T. D. Kocher, 1996. DNA sequencing of formalin-fixed crustaceans from archival research collections. Mol. Mar. Biol. Biotech. 5: 304–313.Google Scholar
  27. Frost, B. W., 1971. Taxonomic status of Calanus finmarchicus and C. glacialis (Copepoda), with special reference to adult males. J. Fish. res. Bd. Can. 28: 23–30.Google Scholar
  28. Frost, B. W., 1974. Calanus marshallae, a new species of calanoid copepod closely allied to the sibling species C. finmarchicus and C. glacialis. Mar. Biol. 26: 77–99.Google Scholar
  29. Frost, B. W., 1989. A taxonomy of the marine calanoid copepod genus Pseudocalanus. Can. J. Zool. 67: 525–551.Google Scholar
  30. Gibbs, R. A., P.-N. Nguyen & C. T. Caskey, 1989. Detection of single DNA base differences by competitive oligonucleotide priming. Nuc. Acids Res. 17: 2437–2448.Google Scholar
  31. Gocke, C. D., F. A. Benko & P. K. Rogan, 1998. Transmission of mitochondrial DNA heteroplasmy in normal pedigrees. Hum. Genet. 102: 182–186.Google Scholar
  32. Grainger, E. H., 1961. The copepods Calanus glacialis and Calanus finmarchicus (Gunnerus) in Canadian Arctic-Subarctic waters. J. Fish. Res. Bd. Can. 18: 663–678.Google Scholar
  33. Harasewych, M. G., S. L. Adamkewicz, J. A. Blake, D. M. Saudek, T. Spriggs & C. J. Bult, 1997. Phylogeny and relationships of pleurotomariid gastropods (Mollusca: Gastropoda): an assessment based on partial 18S rRNA and cytochrome c oxidase I sequences. Mol. Mar. Biol. Biotechnol. 6: 1–20.Google Scholar
  34. Hulsemann, K., 1991. Calanus euxinus, new name, a replacement name for Calanus ponticus Karavaev, 1894 (Copepoda: Calanoida). Proc. biol. Soc. Wash. 104: 620–621.Google Scholar
  35. Jacobs, H. T. & B. Grimes, 1986. Complete nucleotide sequences of the nuclear pseudogenes for cytochrome oxidase subunit I and the large mitochondrial ribosomal RNA in the sea urchin Strongylocentrotus purpuratus. J. mol. Biol. 187: 509–527.Google Scholar
  36. Jaschnov, W. A., 1955. Morphology, distribution and systematics of Calanus finmarchicus s.l. [Russ.] Zool. Zh. 34: 1210–1223.Google Scholar
  37. Juan, C., P. Oromi & G. M. Hewitt, 1995. Mitochondrial DNA phylogney and sequential colonization of Canary Islands by darking beetles of the genus Pimelia (Tenebrionidae). Proc. R. Soc. Lond. B Biol. Sci. 261: 173–180.Google Scholar
  38. Jukes, T. H. & C. R. Cantor, 1969. Evolution of protein molecules.In Munro, H. N. (ed.), Mammalian Protein Metabolism, Academic Press, New York: 21–31.Google Scholar
  39. Knowlton, N., 1993. Sibling species in the sea. Ann. Rev. Ecol. Syst. 24: 189–216.Google Scholar
  40. Kumar, S., K. Tamura & M. Nei, 1993. MEGA: Molecular Evolutionary Genetics Analysis, Version 1.0, Pennsylvania State University, University Park, PA 16802.Google Scholar
  41. Lunt, D. H., D. X. Zhang, J. M. Szymura & G. M. Hewitt, 1996. The insect cytochrome oxidase I gene: evolutionary patterns and conserved primers for phylogenetic studies. Insect Mol. Biol. 5: 153–165.Google Scholar
  42. McLaren, I. A., E. Laberge, C. J. Corkett & J.-M. Sevigny, 1989. Life cycles of four species of Pseudocalanus in Nova Scotia. Can. J. Zool. 67: 552–558.Google Scholar
  43. Mackas, D. L., H. Sefton, C. B. Miller & A. Raich, 1993. Vertical habitat partitioning by large calanoid copepods in the oceanic subarctic Pacific during spring. Progr. Oceanogr. 32: 259–294.Google Scholar
  44. Medeiros-Bergen, D. E., R. R. Olson, J. A. Conroy & T. D. Kocher, 1995. Distribution of holothurian larvae determined with species-specific genetic probes. Limnol. Oceanogr. 40: 1225–1235.Google Scholar
  45. Miller, C. B., 1988. Neocalanus flemingeri, a new species of Calanidae (Copepoda; Calanoida) from the subarctic Pacific Ocean, with a comparative redescription of Neocalanus plumchrus (Marukawa). Progr. Oceanogr. 20: 263–273.Google Scholar
  46. Olson, R. R., J. Runstadler & T. D. Kocher, 1991. Whose larvae? Nature 351: 357–358.Google Scholar
  47. Palumbi, S. R. & J. Benzie, 1991. Large mitochondrial DNA differences between morphologically similar Penaeid shrimp. Molec. Mar. Biol. Biotech. 1: 27–34.Google Scholar
  48. Parfait, B., P. Rustin, A. Munnich & A. Rotig, 1998. Coamplification of nuclear pseudogenes and assessment of heteroplasmy of mitochondrial DNA mutations. Biochem. Biophys. Res. Commun. 247: 57–59.Google Scholar
  49. Pedersen, B. V., 1996. A phylogenetic analysis of cuckoo bumblebees (Psithyrus, Lepeletier) and bumblebees (Bombus, Latreille) inferred from sequences of the mitochondrial gene cytochrome oxidase I. Mol. Phylogenet. Evol. 5: 289–297.Google Scholar
  50. Quesada, H., D. A. Skibinski & D. O. Skibinski, 1996. Sex-biased heteroplasmy and mitochondrial DNA inheritance in the mussel Mytilus galloprovincialis Lmk. Curr. Genet. 29: 423–426.Google Scholar
  51. Rychlik, W., 1992. OLIGO, Ver. 4.04. Computer software. National Biosciences, Inc, Plymouth, MN.Google Scholar
  52. Saitou, N. & M. Nei, 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4: 406–425.Google Scholar
  53. Sameoto, D. D., L. O. Jaroszynski & W. B. Fraser, 1980. BIONESS, a new design in multiple net zooplankton samplers. Can. J. Fish. aquat. Sci. 37: 722–724.Google Scholar
  54. Sevigny, J.-M., I. A. McLaren & B. W. Frost, 1989. Discrimination among and variation within species of Pseudocalanus based on the GPI locus. Mar. Biol. 102: 321–327.Google Scholar
  55. Skjoldal, H. R. & F. Rey, 1989. Pelagic production and variability of the Barents Sea ecosystem. In K. Sherman & L. M. Alexander (eds.), Biomass Yields and Geography of Large Marine Ecosystems AAAS Publ, 241–286.Google Scholar
  56. Stauffer, C., F. Lakatos & G. M. Hewitt, 1997. The phylogenetic relationships of seven European Ips (Scolytidae, Ipinae) species. Insect. Mol. Biol. 6: 233–240.Google Scholar
  57. Swofford, D. L., 1993. Phylogenetic analysis using parsimony (PAUP), Ver. 3.1, University of Illinois, Champaign.Google Scholar
  58. Tamura, K. & M. Nei, 1993. Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees. Mol. Biol. Evol. 10: 512–526.Google Scholar
  59. Wiebe, P. H., A. W. Morton, A. M. Bradley, R. H. Backus, J. E. Craddock, V. Barber, T. J. Cowles & G. R. Flierl, 1985.New developments in the MOCNESS, an apparatus for sampling zooplankton and micronekton. Mar. Biol. 87: 313–323.Google Scholar
  60. Wilson, A. C., R. L. Cann, S. M. Carr, M. George, U. B. Gyllensten, K. M. Helm-Bychowski, R. G. Higuchi, S. R. Palumbi, E. M. Proger, R. D. Sage & M. Stoneking, 1985. Mitochondrial DNA and two perspectives on evolutionary genetics. Biol. J. linn. Soc. 26: 375–400.Google Scholar

Copyright information

© Kluwer Academic Publishers 1999

Authors and Affiliations

  • A. Bucklin
    • 1
    • 2
    • 3
  • M. Guarnieri
    • 1
  • R.S. Hill
    • 1
  • A.M. Bentley
    • 1
  • S. Kaartvedt
    • 4
  1. 1.Ocean Process Analysis LaboratoryUSA
  2. 2.Department of ZoologyUSA
  3. 3.Graduate Program in GeneticsUniversity of New HampshireDurhamU.S.A
  4. 4.Department of Marine Zoology and Marine ChemistryUniversity of OsloBlindern, Oslo 3Norway

Personalised recommendations