A molecular phylogenetic study was conducted on genus Grandinenia, based on COI and 16S sequences. A total of eight out of 26 species in the genus, as well as three unidentified species were sequenced. Phylogenetic results supported the monophyly of Grandinenia and the validity of all sampled species and subspecies. A new species, Grandinenia jiangjilini Chen, Lin, Wu & Ouyang, sp. nov., from Guangxi, southern China is identified and described, based on morphological comparison and molecular phylogeny. The discovery indicates that the inflated-fusiform shell is not isolated in genus Grandinenia and the species diversity of the genus still remains to be explored.

Door snails, karst landscape, phylogeny, taxonomy

The Guangxi Zhuang Autonomous Region is situated in southern China and is renowned for its distinctive landscape and rich biodiversity. The karst landscape in this region provide a suitable habitat for land snails. The well-developed and exposed limestone have nurtured diverse rock-dwelling gastropod groups, with genus Grandinenia Minato & Chen, 1984 of subfamily Garnieriinae Boettger, 1926 being the most diverse and widespread in the region (Nordsieck 2012a, 2012b, 2012c, 2016; Lin and Lin 2022).

The subfamily Garnieriinae Boettger, 1926 is a group of medium to large-sized door snails distributed from Myanmar to southern China. It is characterised by a furrowed neck, projected and unattached, so-called apostrophic peristome and a lunella-type lunellar region (Nordsieck 2007). There are few molecular phylogenetic studies on the Garnieriinae and the limited studies suggest its close relationship with Synprosphyminae Nordsieck, 2007 and Phaedusinae Wagner, 1922 (Uit de Weerd and Gittenberger 2013; Mamos et al. 2021; Uit de Weerd et al. 2023). Currently, Garnieriinae consist of seven genera, three of which are recorded in China: Garnieria Bourguignat, 1877, Grandinenia Minato & Chen, 1984 and Tropidauchenia Lindholm, 1924 (Nordsieck 2012a, 2012b, 2012c). The genus Grandinenia is defined by the furrowed neck, the distinctly expanded and unattached peristome, the lunella-type lunellar and the inferior lamella separated from the superior lamella (Nordsieck 2007, 2012a). It is distributed in Laos, Vietnam and southern China and consists of 26 known species (Nordsieck 2012a, 2012c, 2016; Grego et al. 2014; Lin and Lin 2022). Guangxi is the centre of diversity of the genus, with a total of 17 species recorded (Nordsieck 2012a, 2012c, 2016; Lin and Lin 2022). Most of these species were described in recent years, indicating that the species diversity has been poorly known in the past. Currently, the taxonomy of genus Grandinenia mainly rely on shell morphology, only two out of 26 species have been sequenced, but without detailed molecular phylogenetic studies.

In this study, we conducted the molecular phylogenetic analysis of genus Grandinenia, based on partial COI and 16S sequences and described a new species with a peculiar morphology from Guangxi, southern China. The discovery of this new taxon further increases the species diversity of land snails in Chinese karst landforms.

Samples were collected from Guangxi and Guangdong, China from 2022 to 2023. Living specimens were initially frozen at -20 °C for 12 hours and subsequently thawed at room temperature for 12 hours to extract the soft parts. The soft parts were then fixed in 70% ethanol. Empty shells were cleaned, dried and preserved at 4 °C. All specimens were deposited in the School of Life Sciences, Nanchang University (Nanchang, Jiangxi, China). Photographs were taken by a Sony^® Alpha a6500 Digital 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 on 1% agarose gel electrophoresis and NanoDrop 2000 (Thermo Scientific, USA). Partial cytochrome c oxidase subunit 1 (COI) and partial 16S ribosomal RNA (16S) gene segments 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 newly-obtained sequences and other species are given in Table 2.

Phylogenies were reconstructed by the dataset combined COI and 16S genes using Maximum Likelihood (ML) and Bayesian Inference (BI). Five clausiliid species were used as outgroups for rooting the tree. ML analyses were performed in IQ-TREE v. 1.6.12 (Minh et al. 2013) using the Ultrafast fast bootstrap approach (Minh et al. 2013) with 10000 reiterations. The most appropriate model of sequence evolution (GTR+I+G for COI, GTR+G for 16S) was selected under PartitonFinder2 v. 1.1 (Robert et al. 2017). Bayesian Inference (BI) 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 (Subha et al. 2017). Four simultaneous runs with four independent Markov Chain Monte Carlo (MCMC) were implemented for 10 million generations and trees were sampled every 1000 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 visualised in FigTree v.1.4.3.

The placement of the new species within Grandinenia is supported by both morphology (inferior lamella separated from superior lamella) and molecular phylogeny. The absence of a comprehensive description of the genitalia, as well as the dearth of illustrations of lamellae and genitalia in the most original descriptions of Grandinenia species, precludes the possibility of detailed comparison of the new species with most other congeners for these two characters. The comparison of the shell morphology of the new species with that of eight congeners collected in this study revealed that the lamellae of them are highly similar. In contrast to Tropidauchenia, no variation in the fusion or separation of lamellae was identified between Grandinenia species. The new species is preliminarily distinguished from G. fuchsi, G. pseudofuchsi, G. takagii and G. mirifica by the presence of penial caecum. However, the shell appearance of the new species is sufficiently distinctive that it can be readily distinguished from all other congeners through a simple comparison. Grandinenia jiangjilini sp. nov. can be easily distinguished from all other congeners by the teleoconch with broad, blunt and sparse wrinkles (ribs) (vs. without or with thin and dense ribs). Furthermore, except for G. mirifica, the remaining 25 species of Grandinenia exhibit a relatively slender shell (Fig. 2C). Grandinenia jiangjilini sp. nov. can be easily distinguished from them by the different shell shape (inflated-fusiform vs. slender-fusiform). Grandinenia jiangjilini sp. nov. is most similar to G. mirifica by similar inflated-fusiform shell (Fig. 2A, B), but differs by the broad and sparse wrinkles (ribs) on teleoconch (vs. thin and dense), peristome not reflected (vs. reflected), protoconch preserved (vs. decollated), shell semi-translucent and fragile (vs. opaque and solid) and the different shell colour (yellowish-brown vs. yellowish-white). Grandinenia jiangjilini sp. nov. is also somewhat similar to G. dautzenbergi (Morlet, 1892) and G. yulinensis Nordsieck, 2012, but differs by the more inflated shell, the stronger ribs on neck, the deeper suture, the thinner-walled and more fragile shell and the broad, blunt and sparse wrinkles (ribs) on teleoconch (vs. with very weak ribs to even smooth).

The validity of Grandinenia jiangjilini sp. nov. was also supported by the molecular phylogeny. It forms a distinct lineage and has a distant relationship with G. mirifica. The molecular phylogenetic relationships of genus Grandinenia do not correspond to the morphological similarities. The important characters of shell including shell shape, integrity, ribs, thickness and spiral ribbon, have homoplasiously evolved more than once. The species like G. rex, G. fuchsi and G. ignea with clearly similar smooth, thin, fragile and semi-translucent shells with spiral ribbons, do not form a monophyletic group. The same phenomenon also occurs in species with similar thick and ribbed shells. The result shows that the shell appearance of Grandinenia is an effective means of distinguishing species, but does not reflect the interspecies affinities. The phylogenetic relationship also did not demonstrate a clear geographical correlation overall. Grandinenia magnilabris from the middle northern Guangxi (the westernmost distribution of the sampled species) is the earliest diverging lineage. Grandinenia ignea from north-eastern Guangxi sistered with G. sp. 2 from the central region. In addition, G. fuchsi from north-eastern Guangxi was sistered with the clade, which consists of four species from the central region and one from the south-eastern region. The distribution pattern may be attributed to multiple independent diffusions from west to east in history. Only two lineages exhibited a certain geographical correlation, G. sp. 1 and G. ookuboi pulchricosta from Shanglin and G. mirifica, G. gastrum gastrum and G. gastrum laticosta from Guigang, which formed monophyletic lineages, respectively. Due to the limited species included in this study, extensive and continuous sampling in future studies may help further analysis of the phylogeny and elucidation of the reasons for its distribution pattern formation.

The variation of shell morphology is frequently selected by environmental factors (Chiba 2004; Rolán-Alvarez 2007; Giokas et al. 2014). The shell characters of land snails play a pivotal role in regulating the water and heat budget, thereby preventing desiccation (Cowie and Jones 1985; Pfenninger et al. 2005; Giokas et al. 2014). The discovery of the new species shows that the special inflated-fusiform shells are not isolated in genus Grandinenia. Phylogenetic result indicates that the new species and G. mirifica do not form a monophyletic group, suggesting that the similar shell shape between them may be the result of convergent evolution. Nordsieck (2012a) proposed that the inflated-fusiform shell of G. mirifica is an adaptation to a special habitat, but did not specify what this habitat is. For Grandinenia, it is probable that the inflated-fusiform shells have increased resistance to desiccation and ultraviolet radiation compared to the slender-fusiform shells, although the precise mechanism of action remains unclear. Through field observation, it was found that the two shell shapes of Grandinenia correspond to two life strategies. The majority of Grandinenia species with slender-fusiform shells burrow into crevices to hibernate during the dry season (Fig. 4C). It is challenging to find them during the dry season, but a considerable number of individuals can be observed during the rainy season in the same area. In contrast, the two species with inflated-fusiform shells primarily hibernate on rock surfaces which can be found throughout the year, regardless of precipitation patterns (Fig. 4A, B, D).

The discovery of new taxon or just new species indicates that the species diversity of Grandinenia in Guangxi still remains to be explored. Nine species of Grandinenia have been recoded within a few dozen kilometres of the new species’ type locality (Nordsieck 2012a, 2012c, 2016; Lin and Lin 2022) and several specimens which may represent other undescribed species were found during the field survey. Extensive exploration of land snails in Guangxi should be strengthened in the future which may lead to the discovery of yet-to-be-described species. In addition, the protection of Grandinenia should be a priority. As a rock-dwelling land snail, the survival of Grandinenia depends on the exposed rock environment under the forest. Large-scale mining of limestone and agricultural reclamation in Guangxi pose a threat to it. It was even found that some peaks that used to be rich in Grandinenia have been completely blasted and disappeared. The field survey conducted in 2024 revealed that the native shrubs in the type locality of Grandinenia jiangjilini sp. nov. had been completely cut down (Fig. 7). This has resulted in the environment of the rock walls becoming more exposed and dry and has led to a significant reduction in the population size of Grandinenia jiangjilini sp. nov. It is imperative that further protection measures are implemented without delay, otherwise a significant number of unique species may be lost.

We thank Ji-Lin Jiang (Zhaoqing), Meng-Hua Li (Sichuan Agriculture University), Chen-Yu Fei (Guangzhou) and Shi-Yang Feng (Sichuan Agriculture University) for assistance in collecting specimens; Frank Köhler, Zhe-Yu Chen, Barna Páll-Gergely, Miklós Szekeres and Anna Sulikowska-Drozd for their valuable comments for the manuscript. This study was supported by the National Natural Science Foundation of China under Grant No.32360132, No.31772412, the research project of Zhejiang Natural History Museum under Grant No.2024001 and the Biodiversity Monitoring Project of Xixi National Wetland Park of Hangzhou.