The camaenid land snail genus Camaena is widely distributed throughout Southeast Asia. Thirteen species are found in China alone. Among these, C. cicatricosa (Müller, 1774) is the most widely distributed species, including four subspecies, C. c. ducalis (Ancey, 1885), C. c. inflata (Möllendorff, 1885), C. c. obtecta (Fischer, 1898) and C. c. connectens (Dautzenberg & Fischer, 1906). The systematics of these taxa is revised herein based on comparative shell morphology and anatomy as well as analyses of DNA sequences of two mitochondrial genes (COI, 16S rRNA) and one nuclear marker, ITS2. We found that all subspecies form well-supported clades in a molecular phylogeny and are well-differentiated from each other by genetic distances that are consistent with amounts of interspecific differentiation. In addition, they clearly differ from each other in reproductive features. Based on these observations, we elevate all four subspecies to the rank of full species. Moreover, based on morphological and mitochdondrial differentiation, we describe a new species, Camaena poyuensis sp. n. from Guangxi, China. The new species conspicuously differs from its sibling species C. cicatricosa in having a larger and more depressed shell, a completely covered umbilicus, more or less purplish peristome, an obtuse angle at the junction of the basal and columellar lip, longer pedunculus of the bursa copulatrix, thicker epiphallus and penis, and short conic verge. Previous named species are also redescribed on their shell and anatomical characters, because the original descriptions are uninformative.

Land snail, Gastropoda , camaenid, taxonomy, anatomy, molecular phylogeny

The genus Camaena Albers, 1850, with the type species Helix cicatricosa Müller, 1774, is distributed throughout Southeast Asia where it occurs in southern China, Indo-China, Eastern India, the Philippines and Sulawesi (Zilch 1959–1960). In China, this genus is found mainly in the provinces of Hainan, Guangdong, Guangxi, Yunnan, Guizhou, Hunan, Fujian and Sichuan, but has not been recorded from north of the Yangtze River. Thirteen species of Camaena have been recorded in China (Pilsbry 1894, Yen 1939, Richardson 1985, Chen and Gao 1987, Chen 1990, Chen and Zhang 1999, Schileyko 2003, Wang et al. 2014).

The shells of all but two Chinese species are dextral. Among the two sinistral species, C. seraphinica Heude, 1890 is known only from the type locality, Dingan Town, Tianlin County (formerly “Si-lin”), Guangxi Province. The type locality of the second sinistral species, Camaena cicatricosa cicatricosa (Müller, 1774) is unknown, but specimens exhibiting typical features, such as a sinistral, umbilicated, subcarinated, depressed-globular, yellowish shell with numerous chestnut coloured bands, are widely distributed throughout southern China (Möllendorff 1885, Pilsbry 1891). They can be found in Guangdong, Guangxi, Yunnan, Guizhou, Hunan and Hong Kong (Yen 1939, Chen and Gao 1987, Zhang 2008, Wang et al. 2014).

So far, anatomical characters of sinistral camaenids have not been studied except for C. cicatricosa (Schileyko 2003). The shell morphology of C. cicatricosa is variable. Four infraspecific names have been proposed, these being C. c. ducalis (Ancey, 1885) and C. c. inflata (Möllendorff, 1885) from Guizhou, China, C. c. obtecta (Fischer, 1898), and C. c. connectens (Dautzenberg & Fischer, 1906) from northern Vietnam. These subspecies differ from the nominate form in shell features, such as shell dimensions and shape, openness of umbilicus, sharpness of peripheral angle, convexity of whorls and the presence of a hump beside umbilicus. Camaena c. ducalis is distinguished from other subspecies by its much larger and stronger malleated shell, a more dilated columellar margin and an almost completely covered umbilicus (Ancey 1885). Camaena c. inflata differs from the nominate form in having a much more globular shell, with obsolete peripheral angle and more inflated and gibbous last whorl and nearly closed umbilicus (Möllendorff 1885). Camaena c. obtecta is characterized by having a more globular shell, with weak peripheral angle, a completely closed umbilicus and a nearby umbilical hump (Fischer 1898). Camaena c. connectens differs from C. c. cicatricosa by having fine and tight granules on shell (Dautzenberg and Fischer, 1906). These taxa have previously been treated either as synonyms, varieties or subspecies of C. cicatricosa by different authors based on comparative shell morphology (Pilsbry 1891, 1894, Fischer and Dautzenberg 1904, Dautzenberg and Fischer 1906, Yen 1939, Zilch 1964, Chen and Gao 1987, Schileyko 2011) without reaching a consensus.

Owing to the incongruent delimitation of species in the past, which reflect exclusive reliance on shell characters and the incomplete description of these species, an up-dated revision using modern techniques and species delimitation is required. The mtDNA COI (cytochrome c oxidase subunit I) gene is a commonly used marker for the DNA barcode identification system and is potentially useful for species discovery and identification (Hebert et al. 2003). Additional genetic markers on mitochondrial and nuclear genome have been used alongside COI fragment (e.g., Wu et al. 2008, Criscione and Köhler 2014). The present study aims to resolve the phylogenetic relationships of C. cicatricosa, to correctly delimit its closely related allies and to describe a putatively new species based on comparative analyses of morphological and molecular characters.

This study is based on material collected by the authors at several sites in China (Fig. 1). Live adults were drowned in water for 12–24 hours, then boiled briefly in hot water to ensure their death. Soft body was preserved in 95% ethanol and stored at -40 °C. Empty shells were cleaned and preserved at room temperature. Samples have been deposited in the State Key Laboratory of Molluscan Quarantine and Identification, FJIQBC. Shells were measured to 0.01 mm using electronic calipers. Standard shell parameters were measured on 10–74 specimens per species following Dillon (1984). Genitalia of adult snails were dissected under a dissecting microscope (ZEISS Stemi 2000). The terminology used for the reproductive system follows Gómez (2001). More than three specimens of each species have been dissected.

Figure 1. 

Map of locations of Camaena species. C. cicatricosa: A Nanning, Guangxi, China B Guiping, Guangxi, China C Yangchun, Guangdong, China D Gaoming, Canton, Guangdong, China E Yingde, Guangdong, China F Shantou, Guangdong, China. C. obtecta: G Buhaitun, Jinxi, Guangxi, China H Longbang, Jingxi, Guangxi, China I Cao Bang, Vietnam (type locality). C. inflata: J Qianlin park, Guiyang, Guizhou, China K Ziyun, Guiyang, Guizhou, China. C. connectens: L Tianbao, Malipo, Yunnan, China M Ha Giang, Vietnam (type locality). C. poyuensis sp. n.: N Poyue, Bama, Hechi, Guangxi, China (type locality). C. seraphinica: O Dingan, Tianlin, Guangxi, China (type locality).

A piece of foot muscle tissue of about 0.05 g was used for DNA extraction. The muscle tissue was bathed in sterile water for 3–6 hours to remove residual alcohol. Genomic DNA was isolated using a DNeasy Blood and Tissue Kit (Qiagen, Beijing), examined by agarose gel electrophoresis, and stored at -20 °C for further use. Three specimens from each sampling locality were used for DNA extraction. Fragments of the partial mitochondrial cytochrome c oxidase subunit 1 (COI) and 16S rRNA (16S), and the internal transcribed spacer 2 (ITS2) region of nuclear ribosomal DNA were amplified by PCR using the primer pairs and amplification conditions listed in Table 1.

Both strands of PCR products were purified and sequenced by use of the PCR primers. After sequencing, raw sequence files were proof-read based on chromatograms and assembled in BioEdit 7.2 (Hall 1999). ITS2 sequences were annotated by using HMMer (Eddy 1998) and ITS2 Database (Koetschan et al. 2010). Sequence alignments were generated using ClustalW implemented in MEGA6 (Tamura et al. 2013). In the 16S alignment, sequences of ambiguous alignment were removed using Gblocks v. 0.91b (Castresana 2000), with the minimum number of sequences for a conserved position set to 22, the minimum number of sequences for a flanking position set to 36, the maximum number of contiguous non-conserved positions set to 8, the minimum length of a block set to 4, and no gap positions are allowed. Sequences were checked for saturation using the test implemented in DAMBE 5.3 (Xia et al. 2003, Xia and Lemey 2009, Xia 2013). Pairwise p-distances between taxa were calculated using MEGA6 under the option pairwise deletion of gaps. Prior to the model-based phylogenetic analyses, the best-fitted model of nucleotide substitution was determined for each gene separately using the Akaike Information Criterion calculated with jModelTest v2.1.7 (Darriba et al. 2012, Guindon and Gascuel 2003). Sequences of the three genes were then concatenated into one partitioned data set. Unique sequences were identified using DAMBE 5.3. Maximum likelihood (ML) analyses were conducted using RaxML v8.2.4 (Stamatakis 2014) by applying the GTRGAMMAI model, with parameters estimated from the data, to separate partitions for each gene. The branch support of the ML tree was estimated by using the bootstrapping criteria autoMRE (Majority Rule Criterion) implemented in RAxML. Bradybaena sequiniana (Heude, 1885) (Bradybaenidae) and Cornu aspersum (Müller, 1774) (Helicidae) were used as outgroup. Two additional dextral Camaena species were also analyzed for comparisons.

The systematics of four of the five subspecies of C. cicatricosa has been revised in this study. The genetic distances of the COI barcoding region between C. cicatricosa, C. inflata, C. obtecta, C. connectens and the new species agree with the interspecific genetic distances of other camaenid groups, such as, for example, the Australian camaenid Kimberleytrachia (0.055–0.161, Criscione and Köhler, 2014), the Japanese camaenid Luchuhadra (0.003–0.205, Kameda et al. 2007) and the Taiwanese camaenid Satsuma (0.006–0.150, Wu et al. 2008). In addition to genetic distance, phylogenetic topography and morphological differences also support that they are distinct species. Nomenclatural acts were applied to these taxa, which include raises of taxonomic rank of three species and description of a new species.

Live specimens of two Chinese sinistral Camaena were not collected in the present study: C. c. ducalis and C. seraphinica. Their systematic position are not revised and remained as their current taxonomic ranks. Camaena c. ducalis was named based on a single specimen collected from Kouy-Yang-Fou (nowadays Guiyang), Guizhou. No further specimens were confirmedly recorded since its publication. The present authors and Prof. Tai-Chang Luo (a malacologist based on Guizhou Normal University, Guiyang, personal communication) have spent decades in biodiversity survey in Guizhou, but no shell fullfil the original descriptions were collected (Luo et al. 2003). This taxon is characterized in its large shell width of 74 mm (Ancey 1885). The shell width of known species of sinistral Camaena (Camaena) are hardly larger than 62 mm. Owing to its rarity and unusual size, this subspecies is possibly merely a gigantic variation of one of the Camaena species or extinct. Camaena seraphinica (Fig. 3E) differs from the C. cicatricosa species complex by having a totally open umbilicus, a strongly descending aperture, a non-malleated surface, and a white shell background with few wide bands.

The molecular data also confirms the widely distributed C. cicatricosa a well-delineated species by including samples from subtropical lowland and hill region of Guangdong and eastern Guangxi Province. An extreme example is that individuals from Shantou and Nanning, populations that are about 1000 km apart (Fig. 1), shared the same sequences of the three genes. The large distributional range of this species and short genetic distances among populations are probably due to its adaptation to mankind-disturbed environments, such as farmland and forest ecotone. They have the higher probability to be passively transported large distances and to establish a new population, through human activities (Aubry et al. 2006, Guiller and Madec 2010). Besides, species of Camaena demonstrate different life history strategies in preliminary field and laboratory observations. Camaena cicatricosa laid more but smaller eggs (10–25 eggs each clutch, 0.2–0.25 g each egg) than C. inflata (5–9 eggs each clutch, 0.38 g each egg) (Zhang 2008, Xiao 1989). The former also has shorter gestation period (5–36 days) between the last copulation and the first egg-laying than the latter (2 months). Organisms having higher fecundity and, hence, higher abundance tend to be more competitive species (Stearns 1992). This may partly explain the dominance of C. cicatricosa in these areas. A thorough sampling for a phylogeographic analysis and comparative studies of life history of C. cicatricosa and its allies can provide a considerable insight into the evolutionary processes of C. cicatricosa.

Most of Camaena (Camaena) are distributed in the area of southwestern China and northern Vietnam, where is located at northern part of the Indo-Burma Hotspot and at transition to the Mountains of Southwest China Hotspot (Myers et al. 2000). Three of the species, C. obtecta, C. connectens and C. poyuensis sp. n., studied in the present research are distributed in the Indo-Burma Hotspot. The complex topography, varied physical conditions and a wide diversity of ecosystems in these mountainous areas have likely resulted in allopatric and sympatric speciation (Harl et al 2014, Criscione and Köhler 2016) and hence, a high biodiversity of land snails is expected (Yen 1939, Schileyko 2011).

The present molecular data set using three genetic markers supports the previous separations of C. cicatricosa based exclusively on shell morphology. However, more work is needed to sort out some systematic issues: (1) the taxonomy and phylogenetic relationship of all of the Camaena species, especially those inhabit around the border between Vietnam and China (2) the mechanism of speciation of Camaena in this area (3) the phylogeography of C. cicatricosa, the most widely distributed species in China.

We sincerely thank associate professor Wei-Hong Zhang of the University of Xinjiang, professor Tai-Chang Luo of Guizhou Normal University, and professor De-Niu Chen of Institute of Zoology, Chinese Academy of Science for helpful comments on the manuscript. We are grateful to Dr. R. Janssen of SMF for taking photographs of type specimens; to Muséum national d’Histoire naturelle, Paris, France for open access to the digitized photograph of type specimens. We thank anonymous reviewers for their constructive comments on an earlier manuscript. This research is supported by National Natural Science Foundation of China (31372162; 31040072), National Science Council, Taiwan (NSC 98-2621-B-390-001) and Public Science and Technology Research Funds Projects of General Administration of Quality Supervision, Inspection and Quarantine of the People’s Republic of China (No. 201410076; 2015IK042).