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

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.