Hidden Lineage Diversity in Hydrochara (Coleoptera: Hydrophilidae): An Integrative Study from Floodplain Ecosystems of South-Eastern Europe

The genus Hydrochara (Coleoptera: Hydrophilidae) comprises large-bodied water beetles associated with shallow, well-vegetated freshwater habitats and is characterised by considerable taxonomic complexity. While Hydrochara caraboides is relatively well studied in western and central Europe, lineage diversity and species boundaries within the genus remain poorly resolved in eastern and south-eastern Europe. This study uses an integrative approach combining mitochondrial DNA data, morphometric analyses, and male genital morphology to investigate Hydrochara populations in continental Croatia. Specimens were collected from floodplain and lowland aquatic habitats across major river basins, morphologically identified and verified using cytochrome oxidase subunit I (16S) sequences through comparison with reference data from public databases (GenBank and BOLD). Molecular analyses confirmed the presence of H. caraboides and Hydrochara flavipes in continental Croatia. A single specimen from the upper Drava River basin (CROH030-26) formed a distinct mitochondrial lineage positioned between H. caraboides and Hydrochara dichroma in the COI phylogeny. Morphometric analyses showed extensive overlap between this specimen and H. caraboides, indicating no clear differentiation in external body size. In contrast, examination of male genitalia revealed an intermediate aedeagus morphology with transitional characters between H. caraboides and H. dichroma. Haplotype network analysis revealed a star-like structure with a dominant central haplotype shared by most H. caraboides specimens and several low-frequency variants, while the divergent specimen occupies a peripheral position, separated from the main cluster by multiple mutational steps. These results indicate that H. caraboides is a genetically heterogeneous taxon comprising multiple divergent mitochondrial lineages, suggesting that lineage diversity within this species may be underestimated. By integrating molecular and morphological evidence, this study provides new insights into the lineage diversity of Hydrochara in floodplain ecosystems of south-eastern Europe and highlights the importance of integrative approaches for resolving species boundaries and informing freshwater biodiversity conservation.