Multiplexed species-specific PCR protocol to discriminate four N. Atlantic Calanus species, with an mtCOI gene tree for ten Calanus species

Abstract.

Accurate species identification is the cornerstone of any ecological study – yet this fundamental step is not always possible for marine zooplankton. Routine species' identification, especially of juvenile andlarval stages, is difficult for Calanus species (Copepoda; Calanoida) in the N. Atlantic Ocean, where two or three species may co-occur. A rapid, simple, and inexpensive molecularly based protocol to identify individual copepods of any life stage has beendeveloped. This protocol will routinely identify four Calanus species in the N. Atlantic and will help to accurately understand the role of each species in coastal and open ocean ecosystems. The DNA sequence of a 633 bp region of the mitochondrial cytochrome oxidase I (mtCOI) was determined for ten Calanus species: C. australis (Brodsky, 1959), C. chilensis (Brodsky, 1959), C. finmarchicus (Gunnerus, 1770), C. glacialis (Jaschnov, 1955), C. helgolandicus (Claus, 1863), C. hyperboreus (Krøyer, 1838), C. marshallae (Frost, 1974), C. pacificus (Brodsky, 1948), C. simillimus (Giesbrecht, 1902), and C. sinicus (Brodsky, 1965). MtCOI sequences were used to design species-specific oligonucleotide primers for C. finmarchicus, C. glacialis, C. helgolandicus, and C. hyperboreus and to optimize a competitive, multiplexed, species-specific PCR (SS-PCR) protocol to discriminate the four species. This corrects and improves a previously published protocol for three Calanus species (Bucklin et al. 1999: Hydrobiologia 401:239–254), unambiguously identifying individual copepods and copepodites from diverse geographic regions of the four species' ranges. In order to further examine the pattern of mtCOI evolution within Calanus (an important consideration for molecular systematic characters), consensus mtCOI sequences were used to reconstruct phylogenetic relationships among the ten species; the mtCOI gene tree agreed with morphological and molecular (based on mt16S rRNA) phylogenies, except that the affiliation of C. sinicus could not be resolved.