Evaluating and ITS2 dual barcoding for molecular delimitation and taxonomic insights in Wilson, 1932 (Harpacticoida: Ectinosomatidae) along Turkish Coasts.

BACKGROUND

Accurate species delimitation is essential in morphologically conservative taxa such as harpacticoid copepods, in which cryptic diversity may go unnoticed without molecular data. The genus , common along the Turkish coastline, comprises two species: and , with overlapping ranges and subtle morphological differences. This study aimed to assess species boundaries and uncover potential hidden diversity within using the dual-marker DNA barcoding approach.

METHODS

Specimens of were collected from the Mediterranean, Aegean, and Black Sea coasts of Türkiye. Nuclear DNA from a total of 46 individuals were amplified and sequenced for both mitochondrial cytochrome oxidase I () and nuclear internal transcribed spacer 2 (ITS2) markers. sequences were analysed for haplotype diversity, phylogenetic relationship, and species delimitations. ITS2 sequences were subjected to evaluation with regard to nucleotide diversity, secondary structure, and compensatory base changes (CBCs), using both sequence- and structure-based approaches. The concatenated dataset and species tree reconstruction (StarBEAST2) were employed to test gene tree-species tree congruence.

RESULTS

The analyses revealed a high level of haplotype diversity (21 haplotypes) and the presence of three molecular operational taxonomic units (MOTUs) within and one MOTU within , consistent with the geographic distribution patterns. ITS2 sequences exhibited relatively more conservation with nine haplotypes. These sequences revealed informative structural variation, including CBCs among candidate species. The species delimitation approaches reliably supported the identification of four to seven MOTUs, which corresponded to geographic populations. The analyses of the concatenated dataset supported four well-supported candidate species, and yielded congruent species trees, with high posterior probabilities. Morphological comparisons among MOTUs revealed subtle differences in female P5 structure and anal somite ornamentation among lineages, while MOTUs were morphologically indistinguishable.

CONCLUSION

This study provides the first integrative application of and ITS2 barcoding in and within Harpacticoida overall, combining DNA sequences and structure, and morphological data for species delimitation. The results demonstrate that is effective for detecting geographic differentiation and haplotype diversity, whereas ITS2 contributes structural resolution and potential markers of reproductive isolation through CBCs. These findings suggest the presence of a species complex within and confirm the distinct status of . Dual-marker barcoding, particularly incorporating ITS2 secondary structure, represents a valuable tool for taxonomic studies in morphologically conservative copepod groups.