Molecular phylogeny of the operculated land snail family Pupinidae (Caenogastropoda, Cyclophoroidea) in mainland Southeast Asia
The operculated land snail family Pupinidae from mainland Southeast Asia has been systematically revised based on shell morphology. Despite previous morphological studies, the evolutionary relationships within this family remained unclear. This study represents the first comprehensive molecular phylogeny of this snail group, utilizing two mitochondrial (COI and 16S rRNA) and two nuclear (5.8S rRNA + ITS2 and 28S rRNA) genetic markers. Additionally, we conducted phylogenetic analyses of Pupina species from 1,106 loci generated through double-digest restriction site-associated DNA sequencing (ddRADseq). It turned out that Southeast Asian Pollicaria emerged as a sister clade to Central American Aperostoma of the Megalomastomatidae, leading to the resurrection of the Pollicariidae. Among the remaining pupinid genera, Tortulosa was nested within the Coptocheilus clade, while Pupina and Pupinella were not monophyletic. The previously recognized Pupina arula species group was found to be monophyletic and was reclassified into Tylotoechus (formerly a Pupina subgenus), based on distinctive conchological characters such as an extending parietal tooth from a parietal callus and a wide, outward-curving posterior canal. However, some Pupina and Tylotoechus species were not retrieved as monophyletic, suggesting the presence of multiple cryptic species. Divergence time estimation indicated that the Pupinidae split could date back to the Late Triassic to Early Cretaceous, with the first diversification of pupinid genera occurring during the Middle Jurassic and Early Cretaceous. This successful reconstruction of a robust phylogeny using ddRADseq loci demonstrates the significant potential of RADseq techniques in elucidating the evolutionary relationships of deeply divergent taxa.