A confocal microscopy study of the gross morphology and musculature of the copulatory apparatus in Baikalian acroloxids (Mollusca: Hygrophila)

Acroloxidae is a family of freshwater limpets with about 50 species in five genera, four of which are endemic or nearly endemic to Lake Baikal. Although Acroloxidae are affiliated with other freshwater pulmonates in the superfamily Lymnaeoidea, recent evidence suggests that they may be phylogenetically distinct. This study examined eight species of Baikalian acroloxids from three genera (Baicalancylus, Gerstfeldtiancylus and Pseudancylastrum) using phalloidin staining and confocal microscopy to gain a better understanding of the morphology and musculature of the acroloxid copulatory apparatus and evaluate its significance for the taxonomy and phylogenetic position of the family. The acroloxid copulatory apparatus includes a sac-like penis sheath housing the penis, a glandular appendage (flagellum) and a preputium, with two internal structures (sarcobellum and velum) at the junction between the penis sheath and preputium. The musculature of the penis sheath and preputium in all Baikalian acroloxids and the penial musculature in some of the species were shown to have a three-layered arrangement with outer and inner circular layers and a middle longitudinal layer, which is likely plesiomorphic for the Lymnaeoidea. In Gerstfeldtiancylus, the penial muscle arrangement is reversed (outer and inner longitudinal and middle circular layers), which may represent a synapomorphy of this genus. In Gerstfeldtiancylus benedictiae and three species of Pseudancylastrum, the penial musculature is further specialized, becoming four-layered, with the middle layer subdivided into two. The ejaculatory canal opens laterally on the penis in all the species studied, which contrasts with previous descriptions of Gerstfeldtiancylus and Baicalancylus kobeltii and suggests that their diagnoses need revision. Three morphological types of the velum were identified in the studied species, indicating its greater variability than previously thought. The results of the study demonstrate the significant potential of confocal microscopy to provide more detailed information on acroloxid morphology compared to conventional light microscopy.