DNA barcoding of Messor ants of Bulgaria with insights into their taxonomic diversity

In this study, we analyse ants of the genus <i>Messor</i> using DNA barcode sequences, with a particular focus on the Bulgarian fauna. A total of 85 COI sequences were examined, including 84 from <i>Messor</i> specimens and one from <i>Aphaenogaster</i>, which was used as an outgroup. Of these, 81 sequences were newly generated, while four were retrieved from GenBank. The majority of specimens were collected in Bulgaria (61), with additional samples from Greece (13), Türkiye (4), Albania (1) and North Macedonia (2), providing broader genetic and geographic representation.Althogether, 11 <i>Messor</i> morphospecies were identified, based on specimens used for molecular analysis. To assess the degree of congruence between morphological and molecular data, six species delimitation analyses were conducted: RESL, GMYC, ASAP, ABGD, bPTP and mPTP. In addition, haplotype network analysis of all sequences identified 35 distinct and coherently clustered haplotypes, providing insights into genetic diversity.The COI barcode region successfully distinguished <i>Messor wasmanni</i> Krausse, 1910, <i>M. oertzeni</i> Forel, 1910 and <i>M. ibericus</i> Santschi, 1931. In contrast, species pairs, such as <i>M. atanassovii</i> Atanassov, 1982 and <i>M. creticus</i> Salata & Borowiec, 2019, as well as <i>M. ponticus</i> Steiner et al., 2018 and <i>M. hellenius</i> Agosti & Collingwood, 1987, could not be reliably differentiated using COI data. Furthermore, <i>Messor structor</i> (Latreille, 1798) showed high intraspecific genetic diversity. Finally, the <i>structor</i> and <i>instabilis</i> species groups were recovered with moderate to high support in both Maximum Likelihood and Bayesian Inference analyses, confirming that <i>M. oertzeni</i> and <i>M. hellenius</i> belong to the <i>structor</i> group.Our results provide a reference for future research and underscore the value of integrative taxonomic approaches in ant biodiversity studies.