Deep molecular divergence and shallow morphological differentiation: three new pseudocryptic Gammarus (Crustacea: Amphipoda) species from the Carpathian region

Abstract Freshwater amphipods often include highly divergent molecular lineages with limited morphological variation, resulting in many reported cryptic species. However, most of these cases rely solely on mitochondrial markers, lacking nuclear and morphological characters. This study integrates multilocus phylogenetics and morphological examination to describe three new species in the Gammarus balcanicus complex from Romania. Gammarus balcanicus consists of narrowly endemic lineages spanning south-eastern Europe to Central Asia, many of which remain undescribed due to morphological stasis. The newly described species, Gammarus carausui sp. nov., G. dobrogicus sp. nov., and G. pinkfloydorum sp. nov., show significant divergence at mitochondrial and nuclear levels, along with consistent, yet subtle morphological differences compared to each other and to topotypical G. balcanicus from Montenegro. These species diverged in the Late Miocene, pre-dating Quaternary glaciations. Despite extensive sampling, two are known only from their type localities in the Carpathians, while G. dobrogicus is endemic to the Dobrogea plateau. Considering global threats to freshwater habitats, these species may already be endangered. This work highlights the role of molecular tools in resolving cryptic species’ taxonomy and highlights the Carpathians as a crucial refugial region for relict pre-Quaternary fauna.