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

Background: Despite ongoing efforts to catalogue European ant species, studies focusing on the genetic diversity of Balkan ants remain limited. An integrative approach combining morphology, genetics, ecology and biogeography is preferable for accurately identifying species and resolving taxonomic uncertainties, particularly amongst challenging insect taxa, such as the ants in the genus Messor (Hymenoptera, Formicidae). New information: In this study, we analyse ants of the genus Messor using DNA barcode sequences, with a particular focus on the Bulgarian fauna. A total of 85 COI sequences were examined, including 84 from Messor specimens and one from Aphaenogaster, 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 Messor 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 Messor wasmanni Krausse, 1910, M. oertzeni Forel, 1910 and M. ibericus Santschi, 1931. In contrast, species pairs, such as M. atanassovii Atanassov, 1982 and M. creticus Salata & Borowiec, 2019, as well as M. ponticus Steiner et al., 2018 and M. hellenius Agosti & Collingwood, 1987, could not be reliably differentiated using COI data. Furthermore, Messor structor (Latreille, 1798) showed high intraspecific genetic diversity. Finally, the structor and instabilis species groups were recovered with moderate to high support in both Maximum Likelihood and Bayesian Inference analyses, confirming that M. oertzeni and M. hellenius belong to the structor group.Our results provide a reference for future research and underscore the value of integrative taxonomic approaches in ant biodiversity studies.