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

Abstract

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 specimen misidentifications. To facilitate species discrimination and detection of cryptic/new species, 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 177 worldwide-sampled botryllid colonies. Combined with morphological analyses, 860-COI allowed not only discriminating known species, but also identifying undescribed and cryptic species, resurrecting old species currently in synonymy, and proposing the assignment of clade D of the model organism Botryllus schlosseri to Botryllus renierii. Importantly, within clade A of B. schlosseri, 860-COI recognized at least two candidate species against only one recognized by the Folmer's fragment, underlining the need of further genetic investigations on this clade. This result also suggests that the 860-COI could have a greater ability to diagnose cryptic/new species than the Folmer's fragment at very short evolutionary distances, such as those observed within clade A. Finally, our new primers simplify the amplification of 860-COI even in non-botryllid ascidians, suggesting their wider usefulness in ascidians.

Identification and characterization ofBotrylloides(Styelidae) species from Aotearoa New Zealand coasts

“…The presence of two larger lateral tentacles given for B. anceps from Israel also confirms the genetic affinity regarding our assignment [27]. Besides a recent study, there is no recently reported record of B. jacksonianum [28]. The authors stated that B. jacksonianum which was synonymized with B. leachii by Kott [45, 46] is not accurate and essentially a distinct species.…”

“…This means barcoding of these animals is often not easy and can result in contamination. A new primer pair called 860-COI was designed to control for these issues [28, 47]. It provides sufficient resolution for the barcoding of botryllid ascidians even if they are cryptic or a new species.…”

First Record of Colonial Ascidian, Botrylloides diegensis Ritter and Forsyth, 1917 (Ascidiacea, Stolidobranchia, Styelidae), in South Korea

“…In this study, we selected the mitochondrial cytochrome c oxidase subunit 1 (COI) for the detection of one-and two-toned color B. diegensis in South Korea. The COI was identified as the marker of choice for species discrimination [28] and has been effectively used for detecting NIS [8,29] and botryllids [27,30]. As a result, we recognized the presence of B. diegensis in South Korea based on DNA barcoding analysis.…”

Phylogeographic and morphological analysis ofBotrylloides nigerHerdman, 1886 from the northeastern Mediterranean Sea

“…as a Botrylloides sp .) due to the high similarities between the sister species of this taxon (Brunetti, 2009; Reem et al, 2018; Nydam et al, 2021; Salonna et al, 2021). Although the type of larval incubation and the structure of pyloric caecum have been reported to be support species differentiation (Brunetti, 2009), these features are very challenging to assess precisely during punctual field sampling.…”