Unveiling the Identity of Unio crassus (Bivalvia: Unionidae) in Central and Eastern Europe

Background/Objectives: Freshwater mussels (Bivalvia: Unionida) are among the most endangered faunal groups. This study examines the genetic diversity of Unio crassus, a species critically endangered in Europe. Methods: Using novel (nadh3-tRNA-nadh2) and existing (cox1) mitochondrial markers, we identified four genetic lineages distributed across Central and Southern Europe. The genetic analysis aligned with a recent integrative approach that recognized U. crassus as a complex of 12 geographically structured species. Results: Genetic clusters identified in the present study are grouped within U. crassus s. str., U. nanus, and U. bruguierianus clades. Moreover, our results support stronger internal differentiation within U. nanus and U. bruguierianus than within U. crassus s. str. Further analyses confirmed the widespread and overlapping distribution ranges of both U. crassus s. str. and U. nanus. Moreover, mitochondrial data are consistent with an expanded distribution of the mitochondrial lineage corresponding to U. bruguierianus and support its expanded range covering also the Crimean Peninsula and western part of Ukraine. Conclusions: Results of time-calibrated phylogenetic analyses conducted for identified clusters were in line with those derived from advanced analyses based on mitogenome sequencing. We showed that selected mitochondrial markers are sufficient to make computations of divergence times within the U. crassus complex. The present study suggests that historical climatic events and glacial refugia played a significant role in the divergence within selected freshwater mussels. The findings highlight the importance of genetic analyses in revealing cryptic diversity within threatened species and managing their conservation. Data are deposited in GenBank (accession numbers KT598034–KT598185).