Phylogenetic analysis of Chinese Enidae: Inference from genome-wide analyses and morphological studies on three selected genera

Land snails of the family Enidae (Gastropoda, Stylommatophora, Pupilloidea) exhibit specific regional distributions, primarily in southern Gansu and northwestern Sichuan, China. Although numerous morphological studies have been conducted, molecular and integrative evidence remains insufficient for resolving the classification of species with uncertain taxonomic status. More rigorous phylogenetic investigations are necessary to clarify the evolutionary biology of Chinese Enidae in this biodiversity hotspot. In the present study, we examined three Enidae genera—Subzebrinus, Pupinidius, and Petraeomastus—collected from 19 localities around Wenxian and Wenchuan in the adjoining regions of Gansu and Sichuan, China. We employed genome-wide double-digest restriction site-associated DNA sequencing (ddRAD-seq) and machine learning-based image clustering for integrative phylogenetic and morphological analyses of these genera. Phylogenetic trees constructed from genome-wide single nucleotide polymorphism (SNP) data revealed species-level monophyly but genus-level polyphyly, highlighting the need for taxonomic revision. Image clustering identified distinct shell morphology clusters that closely aligned with phylogenetic relationships, suggesting highly conserved phylogeny and morphology within species, as supported by geographical differentiation and localization within species. However, molecular and morphological divergences were also detected in certain Pupinidius and Petraeomastus species. This integrative analysis of genome-wide phylogenetic data and shell morphology clustering provides a solid foundation for future evolutionary studies of Chinese Enidae.