The genus Ciona is an interesting ‘taxonomic case’ because its evolutionary history and taxonomy have not yet been resolved completely. In this study, we present new findings, describing specimens of an unidentified Ciona species collected along the north-eastern coasts of Sardinia (Tyrrhenian Sea, Mediterranean Sea). Applying an integrative taxonomic approach, based on the joint examination of morphological and molecular traits, we identify these specimens as a new species, Ciona intermedia sp. nov. Morphological comparisons and peculiarities of the habitat first revealed that these Ciona specimens have intermediate characters compared with other Ciona species. Molecular characterization (based on three mitochondrial regions: two already used for discriminating Ciona cryptic species and a newly developed one) confirmed that our specimens could not be assigned to any previously molecularly-characterized species. Both molecular phylogenetic reconstructions and morphological data clearly indicate C. intermedia as sister clade of Ciona edwardsi. Our findings add further complexity to the taxonomy of Ciona, underlying the importance of an integrative taxonomic approach for the study of the evolutionary history of this enigmatic genus.

In order to accelerate the isolation and characterisation of structurally new or novel secondary metabolites, it is crucial to develop efficient strategies that prioritise samples with greatest promise early in the workflow so that resources can be utilised in a more efficient and costeffective manner. We have developed a metrics-based prioritisation approach using exact LC-HRMS which uses data for 24,618 marine natural products held in the PharmaSea database. Each sample was evaluated and allocated a metric score by a software algorithm based on the ratio of new masses over the total (sample novelty), ratio of known masses over the total (chemical novelty), number of peaks above a defined peak area threshold (sample complexity), and peak area (sample diversity). Samples were then ranked and prioritized based on these metric scores. To validate the approach, 8 marine sponges and 6 tunicate samples collected from the Fiji Islands were analysed, metric scores calculated and samples targeted for isolation and characterisation of new compounds. Structures of new compounds were elucidated by spectroscopic techniques, including 1D and 2D NMR, MS and MS/MS. Structures were confirmed by Computer Assisted Structure Elucidation methods (CASE) using the ACD/Structure Elucidator Suite.

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.

We report here one of the first records of the non-indigenous colonial ascidian Symplegma brakenhielmi (Michaelsen, 1904) in the western Mediterranean Sea. Colonies of this invasive species have been collected in 2014 and 2018 along North-eastern Sardinia coasts (Olbia, Italy). Further colonies have been observed in 2016 in the Mar Piccolo basin (Gulf of Taranto, Italy). Both areas are strongly influenced by anthropogenic activities such as commercial shipping and aquaculture, suggesting these human-mediated pathways the most likely vectors of introduction. In both areas, the colonies present two different color phenotypes, the yellow and the red type, with the yellow coloration never found before in the Mediterranean Sea. Morphological and DNA barcode analyses of the collected specimens show that both these color types belong to the same species. Phylogenetic and species delimitation analyses based on the DNA barcode confirm our identification as S. brakenhielmi, but also indicate a surprisingly high similarity with published sequences of other two species, including the co-generic species Symplegma rubra Monniot, 1972. Morphological and molecular examination of a large number of samples of these species would be need in the near future to clarify this issue.