Using DNA barcoding and phylogenetics to identify Antarctic invertebrate larvae: Lessons from a large scale study.

Ecological studies of the diversity and distribution of marine planktonic larvae are increasingly depending on molecular methods for accurate taxonomic identification. The greater coverage of reference marine species on genetic databases such as GenBank and BoLD (Barcoding of Life Data Systems; www.boldystems.org); together with the decreasing costs for DNA sequencing have made large scale larval identification studies using molecular methods more feasible. Here, we present the development and implementation of a practical molecular approach to identify over 2000 individual marine invertebrate larvae that were collected in the Ross Sea, Antarctica, during the austral summer over five years (2002-2007) as part of the LGP (Latitudinal Gradient Project). Larvae for molecular ID were morphologically identified to belong to the Phyla Mollusca, Echinodermata, Nemertea and Annelida (Class Polychaeta), but also included unidentified early developmental stages which could not be assigned a specific taxon (e.g., eggs, blastulae). The use of a 100μm mesh plankton net makes this one of the first larval identification studies to simultaneously consider both embryos and larvae. Molecular identification methods included amplification of up to three molecular loci for each specimen, a pre-identification step using BLAST with GenBank, phylogenetic reconstructions and cross-validation of assigned Molecular Operational Taxonomic Units (MOTUs). This combined approach of morphological and molecular methods assigned about 700 individuals to 53 MOTUs, which were identified to the lowest possible taxonomic level. During the course of this long-term study we identified several procedural difficulties, including issues with the collection of larvae, locus amplification, contamination, assignment and validation of MOTUs. The practical guidelines that we describe here should greatly assist other researchers to conduct reliable molecular identification studies of larvae in the future.