LAUSR

Simple Summary The application of molecular methods in the studies of biological diversity revealed that there are many more species than we can recognise by their appearance. Species pairs or groups, which are difficult to recognise and are known as cryptic species, may differ in their ecological traits, place in trophic networks, and their functional role in ecosystems. It is important to separate cryptic species, particularly if we apply species composition or other diversity metrics for an assessment or monitoring of the ecosystem state or human pressures and impacts. In our study, we revealed a new cryptic species of aphid-hunting wasp that is virtually indistinguishable from a well-known one by morphology. These two species can be easily segregated using molecular characters, and presumably they differ also by nesting habitat. Abstract Upon exploring the mitotype diversity of the aphid-hunting wasp, Diodontus tristis, we revealed specimens with highly divergent mitotypes from two localities in Lithuania and nesting in clayey substrate, while the specimens with typical mitotypes were found nesting in sandy sites. The comparison of inter- and intra-specific distances and application of delimitation algorithms supported the species status of the clay-nesting populations. Using a set of DNA markers that included complete or partial sequences of six mitochondrial genes, three markers of ribosomal operon, two homeobox genes, and four other nuclear genes, we clarified the phylogenetic relationships of the new cryptic species. The endosymbiotic bacteria infestation was checked, considering the option that the divergent populations may represent clades isolated by Wolbachia infection; however, it did not demonstrate any specificity. We found only subtle morphological differences in the new clay-nesting species, D. argillicola sp. nov.; the discriminant analysis of morphometric measurements did not reliably segregate it as well. Thus, we provide the molecular characters of the cryptic species, which allow confident identification, its phylogenetic position within the genus, and an updated identification key for the D. tristis species group.