The ZooGene Technical Approach

MtDNA sequence variation, and mtCOI in particular, has been shown to be useful to resolve evolutionary relationships among closely-related species groups for a wide range of taxa (Jacobs and Grimes, 1986; Brown et al., 1994; Lunt et al., 1996; Harasewych et al., 1997; see review in Avise, 1994). MtCOI sequences have also been used for population genetic studies for a wide variety of organisms. For most species, variation of mtCOI within a species is far less than variation between species, making the gene a diagnostic molecular systematic character. The selection of mtCOI as a target gene for the ZooGene effort results from previous molecular systematic and population genetic studies of copepods and euphausiids by Ann Bucklin. These studies have revealed mtCOI sequence variation within species to range from 1 - 2%, while variation between species ranged from 10 - 20% (Bucklin et al., 1998a, 1999, 2000b, unpublished data).

Preliminary studies have demonstrated the usefulness of mtCOI sequence data for molecular systematic and population genetic studies of calanoid copepods and euphausiids (Bucklin et al., 1997a, 1997b, 1998a, 1999, 2000a, 2000b). Among the calanoid copepods, we have focused initially on two families: the Calanidae (including 29 species of eight genera; Bradford, 1988) and the Clausocalanidae (including at least 28 species in six genera; Frost and Fleminger, 1968), which together include at least eight sibling species groups.

We have recently determined the DNA sequence of a ~ 650 base-pair (bp) region of mtCOI for 19 species of six calanoid genera (Bucklin et al., 1999, unpublished data) using modifications of techniques given in Folmer et al. (1994; and see Bucklin et al., 1999). Sequence differences between calanoid copepod species for this gene portion ranged from 8 - 21% (Bucklin, unpublished data). The mtCOI sequences resolved evolutionary relationships among congeneric species (shown in Fig. 1 [large version] as blue lines). MtCOI sequence variation also discriminated geographic populations of C. helgolandicus (from the Adriatic, North Sea, and Gulf Stream Extension) and C. pacificus (from the California Current and the North Pacific Central Gyre).

The selected MtCOI region was too variable to resolve relationships among genera (Bucklin et al., 1999), so a phylogenetic "backbone" - based on the more conserved sequence for nuclear small-subunit16S rRNA - was used to show the relationships among the genera (shown in Fig. 1 as red lines). The ~350 bp region of nuclear 16S rRNA differed by less than 1% between species. [Note: The phylogenetic tree shown in Fig. 1 was reconstructed by separate analyses of mtCOI and nuclear16S rRNA sequences using the Neighbor Joining algorithm (Kumar et al., 1993). Numbers at branchpoints are bootstrap values.]

Preliminary analysis of mtCOI variation among euphausiid species has focused on the genera Stylocheiron and Nematoscelis. A ~650 bp region of mtCOI clearly discriminated and resolved evolutionary relationships among eight species of these two genera (Bucklin et al., 2000b, unpublished data) and a ~350 bp region of nuclear 16S rRNA resolved relationships among the genera (Fig. 2 [large version] ). Among fourteen species from eight genera, DNA sequence differences ranged from 11 - 25% for mtCOI, while nuclear 16S rRNA differed by about 1% (Bucklin, unpublished data). [Note: Methods of tree reconstruction and explanations of the phylogenetic presentation are the same for Figs. 1 and 2].

These preliminary studies have confirmed that species of calanoid copepods and euphausiids - whether sibling species or species of different families - are readily distinguishable based on mtCOI sequence variation. Further, the levels and patterns of sequence variation are suitable for the design of rapid molecularly-based species' identification protocols, such as competitive species-specific PCR, which requires identification of unique and diagnostic sequence regions for each species (see Bucklin et al., 1997a, 1998a, 2000a). These studies led to our selection of the mtCOI gene as the basis for the comprehensive, world-wide ZooGene database for the molecular systematics and phylogeny of calanoid copepods and euphausiids.

Literature cited

Avise, J.C. (1994) Molecular Markers, Natural History and Evolution, Chapman and Hall, New York, NY. 511 pp.

Bradford, J.M. (1988) Review of the taxonomy of the Calanidae (Copepoda) and the limits to the genus Calanus. Hydrobiologia 167:73-81.

Brown, J.M., O. Pellmyr, J.N. Thompson, and 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.

Bucklin, A., R.S. Hill, M. Guarnieri (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. Background paper for Internat. Consortium Study of Sea, 1997 Scientific Meeting, ICES CM 1997/T:06.

Bucklin, A. , S.B. Smolenack, A.M. Bentley, and 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.

Bucklin, A., A.M. Bentley, and 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. 132:97-106.

Bucklin, A., C.C. Caudill, and M. Guarnieri (1998b) Population genetics and phylogeny of marine planktonic copepods. In: Molecular Approaches to the Study of the Ocean, Chapter 14. K.C. Cooksey (ed.). Chapman & Hall, London, UK. Pp. 303-317.

Bucklin, A., R.S. Hill, and M. Guarnieri. (1999) Taxonomic and systematic assessment of planktonic copepods using mitochondrial COI sequence variation and competitive, species-specific PCR. Special Issue, Molecular Ecology of Aquatic Communities (J.P. Zehr and M. Voytek, Eds.) Hydrobiol. 401:239-254.

Bucklin, A. (2000) Methods for Population Genetic Analysis of Zooplankton. Chapter 11 in: The Zooplankton Methodology Manual, International Council for the Exploration of the Sea. Academic Press, London. Pp. 533 - 570.

Bucklin, A., M. Guarnieri, D. McGillicuddy, and R.S. Hill (2000a) Spring-summer evolution of Pseudocalanus spp. abundance on Georges Bank based on molecular discrimination of P. moultoni and P. newmani. Deep-Sea Res. (In press)

Bucklin, A., S.B. Smolenack, J.J. Pierson, and P.H. Wiebe (2000b) Population genetic diversity and structure of the euphausiid, Stylocheiron elongatum, in the Gulf Stream, with a molecular phylogeny of six Stylocheiron.species (In review)

Folmer, O., M. Black, W. Hoen, R. Lutz, and 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.

Frost, B.W. and A. Fleminger (1968) A revision of the genus Clausocalanus (Copepoda: Calanoida) with remarks on distributional patterns in diagnostic characters. Bull. Scripps Inst. Oceanogr. 12:1-235.

Harasewych, M.G., S.L. Adamkewicz, J.A. Blake, D.M. Saudek, T. Spriggs, and 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.

Jacobs, H.T. and 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.

Kumar, S., Tamura, K., and Nei, M. (1993) MEGA: Molecular Evolutionary Genetics Analysis, Version 1.0. Pennsylvania State University, University Park, PA.

Lunt, D.H., D.X. Zhang, J.M. Szymura, and G.M. Hewitt (1996) The insect cytochrome oxidase I gene: evolutionary patterns and conserved primers for phylogenetic studies. Insect Mol. Biol. 5:153-165.