DNA barcoding of annelids from tidal flats of the Japanese Archipelago

We conducted DNA barcoding of various Japanese coastal annelids, targeting the mitochondrial 16S rRNA (16S) and nuclear 28S rRNA (28S) genes. Specimens were collected from 10 tidal-flat sites, which have been selected as monitoring sites for the Monitoring Sites 1000 Project by the Ministry of the Environment, Japan, ranging from Hokkaido to Ishigaki Island, Okinawa. Partial 16S and/or 28S gene sequences of 360 specimens were obtained. Morphology-based identification revealed 131 species of 39 families, whereas 149 genetically distinct lineages were recognized based on 16S. Among these, four species, Marphysa hongkongensa (Eunicidae), Spio picta, Pygospio elegans (Spionidae), and Loimia tuberculata (Terebellidae), were recorded from Japan for the first time. Discrepancies between morphology-based and DNA-based identifications suggest that some lineages may represent previously overlooked cryptic species distinct from currently valid species. Large genetic distances (3–15%) were detected between the 16S sequences of 12 morphologically identified species in the present study and those deposited in GenBank under the same species name. Generally, genetic distances were smaller in 28S than in 16S, consistent with previous studies on animals. The 28S sequences failed to discriminate lineages in Cirriformia and Nephtys, suggesting that 28S should be applied with caution as a marker for DNA barcoding of annelids.