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

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, i s difficult for Calanus species (Copepoda; Calanoida) in the N. Atlantic Oc ean, where two or three species may co-occur. A rapid, simple, and inexpens ive 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 deter mined for ten Calanus species: C. australis (Brodsky, 1959), C. chilensis ( Brodsky, 1959), C. finmarchicus (Gunnerus, 1770), C. glacialis (Jaschnov, 1 955), C. helgolandicus (Claus. 1863), C. hyperboreus (Kroyer, 1838), C. mar shallae (Frost, 1974), C. pacificus (Brodsky, 1948), C. simillimus (Giesbre cht. 1902), and C. sinicus (Brodsky, 1965). MtCOI sequences were used to de sign species-specific oligonucleotide primers for C. finmarchicus, C. glaci alis, C. helgolandicus, and C. hyperboreus and to optimize a competitive, m ultiplexed, species-specific PCR (SS-PCR) protocol to discriminate the four species. This corrects and improves a previously published protocol for th ree Calanus species (Bucklin et al. 1999: Hydrobiologia 401:239-254). unamb iguously identifying individual copepods and copepodites from diverse geogr aphic regions of the four species' ranges. In order to further examine the pattern of mtCOI evolution within Calanus (an important consideration for m olecular systematic characters), consensus mtCOI sequences were used to rec onstruct phylogenetic relationships among the ten species; the mtCOI gene t ree agreed with morphological and molecular (based on mt 16S rRNA) phylogen ies, except that the affiliation of C. sinicus could not be resolved.