An elongated COI fragment to discriminate botryllid species and as an improved ascidian DNA barcode Other

Botryllids are colonial ascidians widely studied for their potential invasiveness and as model organisms, however the morphological description and discrimination of these species is very problematic, leading to frequent taxonomic misidentifications of specimens.To facilitate species discrimination, we developed new barcoding primers for the amplification of a COI fragment of about 860 bp (860-COI), which is an extension of the common Folmer's barcode region. Our 860-COI was successfully amplified in 168 worldwide-sampled botryllid colonies and allowed not only discriminating known and cryptic species, but also identifying undescribed species and resurrecting old species currently in synonymy. Our data also suggest that clade D of the model organism Botryllus schlosseri could be assigned to Botryllus renierii. Remarkably, the 860-COI showed a higher resolving power than the Folmer's fragment within clade A of B. schlosseri. Indeed, within this clade the 860-COI significantly supported sub-clades unidentified by the Folmer's fragment and recognized at least two of them as candidate species. Thus, we conclude that, as a DNA barcode, 860-COI works better than the Folmer's fragment at the shortest evolutionary distances. Finally, we showed that our new primers allow an easy amplification of 860-COI even in non-botryllid ascidians, suggesting their wider usefulness in ascidians.