Our study contains the first molecular phylogeny of Chinese enids based on the mitochondrial markers cytochrome oxidase c (COI) and 16S rRNA (16S). We have sequenced 19 species belonging to 10 out of the 12 currently accepted genera. A new species, Petraeomastus limenghuai Chen, Dai, Wu & Ouyang, sp. nov., is described from the Lancangjiang River Valley in southwest China based on comparative morphology and molecular phylogeny. The currently accepted classification of Chinese enids relies largely on shell morphology and is deemed to require systematic revision.

Biodiversity, dry-hot river valley, land snails, phylogeny, taxonomy

Enidae Woodward, 1913, encompasses small to large species from Eurasia, northern Africa, and northern Australia (Wu 2018). China represents a diversity hotspot of the family, harboring about 185 described species and subspecies (Heude 1882, 1885; Ancey 1883, 1884; Hilber 1883; Möllendorff 1901; Annandale 1923; Yen 1938, 1939; Chen and Zhang 2000, 2001; Wu and Wu 2009; Wu and Zheng 2009; Wu and Gao 2010; Zhang et al. 2010; Wang and Wu 2012; Wu and Fang 2012; Wu and Xu 2012; Fang and Wu 2013; Wang and Wu 2013; Wu and Xu 2013; Wu 2018; Chen 2020; Chen et al. 2024a, 2024b). To date, all taxonomic studies have been based exclusively on morphology, with no molecular genetic data being available (Wu 2018; Chen 2020; Chen et al. 2024a, 2024b). Several Chinese genera as currently delineated exhibit overlapping morphological characteristics, hampering an unambiguous classification. The distribution of Enidae in China is highly uneven, with most species restricted to dry-hot sections of several river valleys in the southwest (Wu 2018), including the Bailongjiang River Valley, the Fujiang River Valley, the Minjiang River Valley, the Daduhe River Valley, the Jinshajiang River Valley, and the Lancangjiang River Valley. Among these valleys, the Lancangjiang River Valley is one of the most remote and poorly studied. There has been almost no subsequent study after several species were described from here in the late 19^th to early 20^th centuries (Heude 1882; Ancey 1883; Hilber 1883; Ancey 1884; Heude 1885; Annandale 1923). The species composition of a large section of the Lancangjiang River Valley is still unclear.

During land snail surveys in 2023, we discovered a group of enid specimens with ribbed shells from the Lancangjiang River Valley that did not resemble any known species and were challenging to place in any genus. Based on a combination of comparative morphology and molecular phylogenetic analysis, we describe these snails as a new species of the genus Petraeomastus Möllendorff, 1901. The discovery contributes to our understanding of the morphological variations within Petraeomastus in China, suggesting that further exploration of the species diversity of Enidae in the Lancangjiang River Valley may yield additional insights. Furthermore, the significant morphological variation within the same genus and the similar morphology between different genera indicate that the current taxonomic classification of Chinese Enidae may be problematic, requiring an integrative, systematic revision.

Specimens were collected from southern China in 2023. Living specimens were initially frozen at -20 °C for 12 hours and subsequently thawed at room temperature for 12 hours to facilitate the extraction of soft parts. The soft parts were then fixed with 70% ethanol. Empty shells were cleaned, dried, and preserved at 4 °C. Soft parts were transferred from 70% alcohol to 10% alcohol and softened at 4 °C for 5 hours before dissection. Photographs were taken by camera and edited in Adobe Photoshop CC 2015 (Adobe, San Jose, US). Maps were made in ArcGIS Pro (Esri, Redlands, US).

Genomic DNA was extracted from foot tissues preserved in 70% ethanol using a TIANamp Marine Animals DNA Kit (Tiangen Biotech, China). The quality and concentration of the DNA were checked using 1% agarose gel electrophoresis and NanoDrop 2000 (Thermo Scientific, USA). Partial cytochrome c oxidase subunit 1 (COI) and 16S ribosomal RNA (16S) were amplified and sequenced for molecular phylogenetic analyses. Polymerase chain reaction (PCR) systems, conditions, and primer pairs are listed in Table 1. Sequences were aligned using MEGA v. 6.0 (Tamura et al. 2013) and checked manually. The accession numbers of other species and newly obtained sequences are given in Table 2.

Phylogenies reconstructed by the dataset combined two genes using maximum likelihood (ML) and Bayesian inference (BI). Camaena detianensis Zhou & Lin, 2016, Pupilla muscorum (Linnaeus, 1758), and Achatinella sowerbyana Pfeiffer, 1855, were used as the outgroups for rooting the trees. ML analyses were performed in IQ-TREE v. 1.6.12 (Minh et al. 2013) using the Ultrafast bootstrap approach (Minh et al. 2013) with 10,000 iterations. The most appropriate model of sequence evolution (GTR+I+G) was selected under PartitionFinder2 v. 1.1 (Lanfear et al. 2017). Bayesian inference (BI) analysis was conducted in MrBayes v. 3.2.6 (Ronquist et al. 2012). The most appropriate model of sequence evolution (GTR+I+G) was selected under ModelFinder (Kalyaanamoorthy et al. 2017). Four simultaneous runs with four independent Markov Chain Monte Carlo (MCMC) algorithms were implemented for 10 million generations, and trees were sampled every 10,000 generations with a burn-in of 25%. The convergence was checked with the average standard deviation of split frequencies <0.01 and the potential scale reduction factor (PSRF) ~ 1. Trees were visualized in FigTree v.1.4.3 (http://tree.bio.ed.ac.uk/software/figtree/).

Only four enid species in China have a ribbed shell: Clausiliopsis clathratus (Möllendorff, 1901), Clausiliopsis senckenbergianus Yen, 1939, Holcauchen multicostatus Chen, Xie, Wang & Wu, 2024, and Serina xirong Chen, Dai, Wu & Ouyang, 2024 (Möllendorff 1901; Yen 1939; Wu 2018; Chen et al. 2024a, 2024b). Petraeomastus limenghuai sp. nov. can be distinguished from C. clathratus and C. senckenbergianus by absence of a tooth (vs. the presence of a parietal tooth). It resembles H. multicostatus and S. xirong, which also lack a tooth, but differs from them by having stronger ribs and a larger shell (height 19.8–21.2 mm vs. 8.3–10.5 mm in H. multicostatus and 9.7–11.0 mm in S. xirong). It further differs from H. multicostatus by having a more pointed shell and from S. xirong by having sparser ribs. Within the genus, the new species differs from congeners by the ribbed shell (vs. smooth in all congeners), the long flagellum (vs. short in P. breviculus, P. heudeanus, and P. platychilus), and the unexpanded diverticle (vs. diverticle absent in P. breviculus; expanded in P. heudeanus, P. moellendorffi, P. mucronatus, P. platychilus, and P. semifartus).

The shell morphology of Petraeomastus limenghuai sp. nov. is so unique that it is difficult to place it in any genus based on shell morphology alone. It also lacks diagnostic characteristics in its reproductive anatomy. Wu (2018) proposed that the penial pilaster is a relatively stable character in different genera, but we failed to dissect it because it was too thin (< 0.2 mm). However, the molecular phylogeny strongly supported placement in Petraeomastus (bootstrap supports = 99, posterior probabilities = 1). All known species of Petraeomastus have a broad, cylindrical to conical, and smooth shell (Fig. 2B–D), which is significantly different from that of Petraeomastus limenghuai sp. nov. The shell morphology of land snails is frequently influenced by environmental selection, and the characters play a pivotal role in regulating the water and heat budget, thereby preventing desiccation (Cowie and Jones 1985; Chiba 2004; Pfenninger et al. 2005; Giokas et al. 2014). Considering that all Chinese Enidae with ribbed shells are distributed in the dry-hot river valleys (Möllendorff 1901; Yen 1939; Wu 2018; Chen et al. 2024a, 2024b), the special character that evolved independently in four genera may have been developed to adapt to the extremely dry and hot environment. The ribs on the shell could increase its surface area and may promote heat dissipation. Giokas et al. (2014) found that the ribbed shells in Albinaria Vest, 1867, retain more water on their shell surface, and the smooth shells exhibit lower water permeability. A similar phenomenon may also occur in Chinese Enidae, although further statistical study is required to confirm it. Currently, the proportion of known Chinese enid species with a ribbed shell is very low. Further comprehensive surveys of several dry-hot river valleys in southwest China might reveal additional species of Enidae with ribbed shells.

Although 10 out of 12 genera of Enidae in China were included in this study, only 19 out of 185 species and subspecies were included. More comprehensive sampling is needed in future research. Currently, the taxonomy of enid genera in China is primarily based on shell morphology (Wu 2018). However, the shell morphology of land snails is frequently influenced by environmental factors (Cowie and Jones 1985; Chiba 2004; Pfenninger et al. 2005; Giokas et al. 2014) and is not stable. Some genital diagnostic characters of Chinese Enidae have been summarized (Wu 2018), but there is overlap between different genera in several characteristics. Currently, the diagnostic characters of Chinese Enidae are insufficiently defined at the genus level, as there is considerable overlap in characters among different genera. This presents a significant challenge in reliably distinguishing some genera from each other. The three clades of Pupinidius, which render the genus nonmonophyletic in the molecular phylogenies, exhibit a high similarity in shell morphology. Upon examination of a substantial number of specimens of the three clades, it is challenging to identify any reliable diagnostic characters that distinguish them, both in terms of shell and genitalia. As a rock-dwelling group, the similar morphology between the three clades of Pupinidius may be attributed to convergent evolution due to similar environments. The inconsistency between the morphological characters and the molecular phylogenies also occurred in the new species described in this study and is possible to be prevalent in Chinese Enidae. The currently accepted classification of Chinese enids, which relies largely on shell morphology, may be problematic and is deemed to be in need of systematic revision.

We thank Menghua Li (Sichuan Agricultural University), Jinsheng Mou (China Agricultural University, Sichuan Agricultural University), Kaichen Ouyang (Kunming), and Shiyang Feng (Sichuan Agricultural University) for assistance in collecting specimens; Frank Köhler, Ruud Bank, and Aydin Örstan for their valuable comments on the manuscript. This study was supported by the National Natural Science Foundation of China under Grant No. 32360132, No. 31772412, the research project of the Zhejiang Natural History Museum under Grant No. 2024001, and the Biodiversity Monitoring Project of Xixi National Wetland Park of Hangzhou.