Cryptic diversity in groundwater amphipods of Iran: insights from morphological and molecular approaches

Groundwater ecosystems host a diverse yet poorly understood fauna, largely due to their inaccessibility and the prevalence of cryptic species. These organisms often exhibit convergent adaptations (troglomorphy), such as loss of pigmentation and eyes, which can mask true phylogenetic relationships and impede species delimitation based solely on morphology. This study investigates the diversity of three populations of the stygobiont amphipod genus Niphargus from springs in Iran using an integrative taxonomic approach, combining molecular data (mitochondrial COI and nuclear 28S rRNA genes) with traditional morphological analysis. Phylogenetic reconstructions placed all three populations within the N. alisadri subclade. Despite minimal differentiation in the 28S marker and only subtle morphological divergence, species delimitation analyses (ASAP and bPTP) of COI sequences and genetic distance calculations identified the populations as comprising two putative new species. We describe these as N. hamedanensis sp. nov. and N. arakensis sp. nov. The small but significant genetic and inferred functional differences between these species and N. alisadri suggest a recent evolutionary divergence, potentially driven by allopatric isolation in higher altitude refugia. This discovery of cryptic diversity highlights the necessity of molecular methods for uncovering groundwater biodiversity and presents a model system for studying the initial stages of speciation in subterranean environments.