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

Marika Salonna,Francesco Mastrototaro,Marissa Mcnamara,Merrick Ekins,Dorothée Huchon,Michal Haddas-Sasson,Federica Montesanto,Carmela Gissi,Fabio Gasparini

doi:10.1038/s41598-021-83127-x

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.

Highlights

The subfamily Botryllinae consists of small colonial ascidians of the genera Botryllus and Botrylloides
The 18S rDNA, the mitochondrial cytochrome b gene, and the entire mitochondrial genome have been used to try to elucidate the relationships within the subfamily Botryllinae[13,29,30] and within the family Styelidae[19], or to detect botryllid cryptic s pecies[31]. Most of these studies have left the overall Botryllinae phylogeny poorly resolved, because of the low support of several nodes in the reconstructed t rees[19,29], but they have been decisive for discriminating species and detecting cryptic speciation events. Both nuclear and mitochondrial markers have demonstrated the weaknesses of the Botryllus schlosseri morphological taxonomy, revealing that B. schlosseri is a “species complex” consisting of five genetically highly divergent clades, each corresponding to a cryptic species and with a peculiar geographic d istribution[9,18,19,20,32,33]
Apart from few additional strongly supported nodes within clade A (Table 4), the remaining parts of the 860-cytochrome oxidase subunit I (COI) tree remain unresolved or are reliably supported only by Bayesian inference. These results demonstrate that the 860-COI fragment efficiently discriminates between Botryllinae species and is able to resolve relationships between very closely related specimens, i.e., within the B. schlosseri clade A

Summary

Introduction

The subfamily Botryllinae (family Styelidae) consists of small colonial ascidians of the genera Botryllus and Botrylloides. The 18S rDNA, the mitochondrial cytochrome b (cob) gene, and the entire mitochondrial genome (mtDNA) have been used to try to elucidate the relationships within the subfamily Botryllinae[13,29,30] and within the family Styelidae[19], or to detect botryllid cryptic s pecies[31] Most of these studies have left the overall Botryllinae phylogeny poorly resolved, because of the low support of several nodes in the reconstructed t rees[19,29], but they have been decisive for discriminating species and detecting cryptic speciation events. It is crucial to fully resolve its taxonomic status

Methods

Results

Discussion

Conclusion