DNA Barcoding Reveals Hidden Genetic Diversity and Environmental Correlates of Lineage Differentiation in Neozavrelia Goetghebuer & Thienemann, 1941 (Diptera: Chironomidae)

Species delimitation and biodiversity assessment in Chironomidae are frequently hindered by morphological conservatism and widespread cryptic diversity. Neozavrelia (Diptera: Chironomidae), a genus inhabiting specialized freshwater microhabitats such as springs and hygropetric seepages, remains poorly understood with respect to its genetic diversity and the environmental processes underlying lineage differentiation. Clarifying lineage diversity is a prerequisite for evaluating the ecological factors associated with genetic differentiation. In this study, we compiled a global COI DNA barcode dataset comprising 137 sequences from newly generated specimens and publicly available records in the Barcode of Life Data Systems (BOLD). Genetic diversity was evaluated using neighbor-joining (NJ) analysis and Automatic Barcode Gap Discovery (ABGD), whereas environmental and geographic drivers of genetic differentiation were assessed using Mantel tests, distance-based redundancy analysis (db-RDA), variation partitioning, and hierarchical partitioning. A total of 26 molecular operational taxonomic units (MOTUs) were identified, with most lineages forming well-supported clades in the NJ tree. Genetic differentiation was significantly associated with both environmental and geographic distances, although environmental variables explained a substantially greater proportion of the observed variation. Hierarchical partitioning identified slope, annual cloud cover, mean precipitation amount of the coldest quarter, and frost frequency as the principal environmental predictors of lineage differentiation. Our findings reveal substantial COI genetic diversity within Neozavrelia and suggest that environmental heterogeneity, particularly variation in water availability and thermal conditions, is significantly associated with lineage differentiation. This study expands the global COI DNA barcode reference library for Neozavrelia and provides new insights into ecological and evolutionary factors associated with diversification in freshwater chironomids.