DNA Barcoding and Molecular Taxonomy Confirm the Occurrence of Polyclinum constellatum (Tunicata, Ascidiacea) from Malta

Background/Objectives: The Mediterranean Sea is highly susceptible to marine bioinvasions, with non-indigenous ascidians frequently introduced and dispersed via maritime traffic. Here, we report the first records of the non-indigenous ascidian Polyclinum constellatum Savigny, 1816 in Maltese waters, identified within Marsaxlokk Harbour, which hosts a major international transshipment hub in the Central Mediterranean. Situated at a crossroads of global shipping lanes, Malta serves as a critical geographic sentinel for monitoring the occurrence and spread of non-indigenous species within the basin. Methods: Two colonies of P. constellatum were collected in 2022 and 2023 respectively. Given the condition of the sampled colonies, the high morphological plasticity and cryptic diversity within the genus Polyclinum Savigny, 1816, species identification was established using molecular taxonomy through DNA barcoding against reference sequences. Results: The obtained sequences from the two colonies were identical and confirmed the species’ identity through a 100% match in GenBank and BOLD. Comparative analysis revealed that the 678 bp sequences obtained here differed by 1 bp from previously documented Mediterranean records while matching others from more distant locations. The structure of the haplotype network based on the homologous sequences, together with the occurrence of multiple mitochondrial haplotypes across the Mediterranean Sea that are shared with geographically distant regions rather than neighbouring Mediterranean localities, supports a scenario involving multiple introductions of P. constellatum into the region, likely associated with maritime transport. Conclusions: These findings highlight the importance of integrating genetic markers into biosecurity monitoring frameworks to resolve taxonomic identities of cryptic taxa, improve understanding of invasion pathways and assess genetic diversity associated with the establishment and spread of non-indigenous species in novel environments.