Descriptions of four new dextral land snails of the genus Camaena (Gastropoda, Eupulmonata, Camaenidae) from south China

Abstract In this study, four new dextral camaenid from China are reported, based on shell morphology, reproductive system anatomy, and molecular phylogenetic analyses: Camaenafuningensis Zhou, Wang & Lin, sp. nov., Camaenagaolongensis Zhou, Wang & Lin, sp. nov., Camaenamaguanensis Zhou, Wang & Hu, sp. nov., and Camaenayulinensis Zhou, Wang & Hu, sp. nov. Detailed descriptions of the morphological characteristics including shells and genitalia, DNA sequences, and living environments of the four new species are provided, with further comparisons with congeners.

There are 24 species of the genus distributed in southern China belonging to two subgenera, Camaena and Camaenella.Twenty-three species belong to Camaena (Yen 1939;Chen and Zhang 1999;Schileyko 2003;Ai et al. 2016;Ding et al. 2016), and only one species is in Camaenella (Pilsbry 1894;Yen 1939;Chen and Zhang 1999).The subgenus Camaenella was treated as a synonym of Camaena or as a genus in its own right by some scholars (Chen and Gao 1987;Chen and Zhang 1999).In this article, Camaenella will be considered as a valid subgenus.
Camaena species are divided into a sinistral group and a dextral one.They are usually characterized by a moderately solid shell with scar-like protrusions or malleations, 4.5-5.5 slightly convex whorls, a brown or yellow surface with red or puce spiral bands, and reflexed aperture margins (Schileyko 2003;Ai et al. 2016).The classification of Camaena has mainly relied on the shell features.Anatomical and molecular studies of Camaena are rare, except for the sinistral and the newly described species (Chen and Zhang 1999;Ai et al. 2016;Ding et al. 2016;Páll-Gergely et al. 2016;Wu et al. 2019).Historically, the classification of this genus is rather confused.For the sinistral group, the taxonomic status has always been controversial, and scientific names have been revised repeatedly.Ding et al. (2016) revised C. cicatricosa as four species, C. cicatricosa, C. inflata (Möllendorff, 1885), C. obtecta (Fischer, 1898), and C. connectens (Dautzenberg & Fischer, 1906), and described one new species C. poyuensis Zhou, Wang & Ding, 2016 using morphological and molecular studies.In the same year, Ai et al. (2016) described two new species C. lingyunensis Zhou &Lin, 2016 andC. detianensis Zhou &Lin, 2016 according to shell morphology, reproductive system and molecular biology.Thus, the sinistral Camaena group contains 12 species or subspecies to date (Schileyko 2003;Ai et al. 2016;Ding et al. 2016).The dextral group can be divided into three informal subgeneric groups according to the morphological characteristics of the shell, especially the shape and location of the carina.
In this study, the authors have examined many specimens collected in Guangxi and Yunnan in southern China between 2013 and 2015, and discovered four new dextral species on the basis of morphological, anatomical, and molecular evidence, and living environments.

Materials and methods
Specimens were collected by the authors from several sites in China (Fig. 1).The longitude and latitude were recorded using a GPS.The map was established by MapInfo Professional 15.0.The live adults were drowned in water for 12-24 hours, and then killed in hot water.Soft bodies were preserved in 95% ethanol and stored at -20 °C.Empty shells were cleaned and preserved at room temperature in the Key Laboratory of Molluscan Quarantine and Identification of Fuzhou Customs District, Fujian, China (GACC).
Shells were measured to 0.1 mm using electronic calipers.Standard shell parameters were taken following Dillon (1984).All adult specimens of each species were measured.Live sexually mature specimens were dissected for the examination of reproductive system under a dissecting microscope (ZEISS Stemi 2000).Terminology for reproductive system follows Gómez (2001).The basal direction starts from the reproductive opening while that of verge starts from the epiphallus following Hwang et al. (2018).
Approximately 30 mg of the foot muscle was used for DNA extraction.The foot muscle was bathed in sterile water for 3-6 hours to remove residual alcohol.Genomic DNA was isolated using Qiagen DNeasy Blood & Tissue kit (Qiagen, Beijing), examined by agarose gel electrophoresis and ultra-micro spectrophotometer (Implen NP80, Germany), then stored at -20 °C for further use.The partial mitochondrial cytochrome c oxidase subunit 1 (COI) was amplified by PCR using apt primer pairs, reaction system, and amplification condition listed in Table 1.The PCR products were analyzed using 1.2% agarose gel electrophoresis.
After sequencing, raw sequences were proof-read on chromatograms and aligned into contigs using BioEdit 7.2 (Hall 1999).Sequence alignments were generated using ClustalW implemented in MEGA6 (Tamura et al. 2013).A total of 35 sequences were used in this study, 23 sequences of which were newly generated and deposited in GenBank (Table 2), and the remainder referenced in Wu et al. (2008), Ding et al. (2016), Ai et al. (2016), andHu et al. (2019).Pairwise p-distances between taxa were calculated using MEGA6 (Tamura et al. 2013) and were compared with the currently known intra and inter-specific differentiation values (p-distances) of Camaenidae (Criscione and Köhler 2014;Ai et al. 2016;Ding et al. 2016).Neighbor-Joining and Minimum-Evolution analyses based on COI sequences were performed using MEGA6 (Tamura et al. 2013).Amphidromus atricallosus (Gould, 1843) (Camaenidae) was used

Molecular analysis
In  Inter and intra-specific P-distances from COI gene of seven species were calculated and are listed in Table 3.According to the results of the target gene COI, the p-distances between C. funingensis sp.nov., C. gaolongensis sp.nov., C. maguanensis sp.nov., and C. yulinensis sp.nov.and other dextral Camaena were 0.068-0.200,0.075-0.203,0.068-0.198and 0.092-0.202respectively.
Etymology.The name of the new species refers to the type locality.
Diagnosis.Shell.Shell dextral, large, thin, fragile and lucent, low, and flat conical.4.5 whorls, the front whorls increasing slowly.Spire relatively low.Body whorl rapidly  expanded.Shell light yellowish brown with clear growth lines and spiral bands on the surface.Apex quite blunt.Suture shallow.The protoconch surface smooth, and some short clear growth lines near the inner side of suture under 32 × stereomicroscope.Body whorl with carinate periphery, and a thin reddish brown band on the carina and several sparse bands below the carina.Aperture lunate, slightly descending.Peristome reflected, white, thin, sharp.Columellar lip reflected.Umbilicus reddish brown, large, only 1/5 covered.Inner lip attached to the body whorl, forming translucent callus.Soft body.Yellowish brown with irregular black lines and spots.Tentacles dark.
Reproductive system.Bursa copulatrix oval and large with long and tapering pedunculus, expanded at the base.Flagellum long, tapering distally.Vas deferens short and thin.Epiphallus long, slightly thick.Penis retractor muscle medium length and slender, becoming wider at the end.Penis swollen and long, with longitudinal, slightly corrugated, strong and widely spaced pilasters internally.Verge ovate, opened terminally, and one clear crack on the verge surface extending from the terminal to the base.
Habitat.The species was found on limestone.Distribution.Only known from the type locality.
Remarks.Camaena funingensis sp.nov. is characterized by a more oblate shape, lower spire, thin and fragile shell, and yellowish brown coloration, which are clearly different from the other dextral camaenids except C. longsonensis (Morlet, 1891), C jinpingensis Chen, Zhang &Li, 1990, andC. vorvonga (Bavay &Dautzenberg, 1900) (Chen et al. 1990;Schileyko 2011).The shells of the above four species are distinct from C. funingensis in the following ways: (1) The umbilicus of C. funingensis sp.nov. is only 1/5 covered, while that of C. longsonensis is almost covered by reflected columellar lip leaving only a narrow slit, and that of C. jinpingensis is fully covered.(2) C. funingensis sp.nov.has several reddish brown bands at the bottom of the body whorl in addition to those on the carina, while only one thin reddish brown band is present on the carinate periphery of C. vorvonga.(3) For C. funingensis sp.nov., the verge is ovate and has one clear crack on the surface extending to the base, which makes it stand out other dextral camaenids.
Camaena gaolongensis sp.nov. is distinguishable from C. funingensis sp.nov. in having no spiral band.For C. maguanensis sp.nov., there is no band on the carinate periphery of the body whorl except for several below the carina.Moreover, the verge of C. maguanensis sp.nov. is small and circular.Camaena yulinensis sp.nov.differs to C. funingensis sp.nov. in having a conical verge and flesh-colored peristome.
P-distances of the COI gene between C. funingensis sp.nov.and the other camaenids are 0.068-0.200(Table 3), and those between C. funingensis sp.nov.and C. gaolongensis sp.nov., C. maguanensis sp.nov.and C. yulinensis sp.nov.are 0.075, 0.068 and 0.094 respectively.All of these P-distances exceed the maximum intra-specific value 0.059 in the family Camaenidae.On the phylogenetic tree, these four new species are adjacent, hence it is reasonable to designate this as a new species.
Etymology.The name of the new species refers to the type locality.
Diagnosis.Shell.Shell dextral, large, thick, strong, low, and flat conical.4.5 whorls, the front whorls increasing slowly.Spire relatively low.Body whorl rapidly expanded.Shell dark brown with clear and dense growth lines on the surface.Apex quite blunt.Suture shallow.The protoconch surface smooth with scale marks, and some short growth lines clear near the outer side of suture under 32 × stereomicroscope.Body whorl with acute and carinate periphery, but no spiral band.Aperture Ushaped.Peristome reflected, white and thick.Columellar lip reflected.Umbilicus reddish brown, open, large, and only 2/5 covered.Inner lip attached to the body whorl, forming translucent callus.
Soft body.Brown with irregularly black lines and spots.Tentacles dark.
Reproductive system.Bursa copulatrix oval and medium sized with long pedunculus, expanded at the base, becoming thinner at the distal end.Flagellum long and smooth, tapering distally.Vas deferens long and thin.Epiphallus medium length and thick.Penis retractor muscle short, slender basally but wide and flat distally.Penis thick and medium length.Inner penial wall supporting longitudinal, stronger, and more widely spaced pilasters, smooth basally, curved distally.Verge irregularly conical, opened basally, extending from the base to the end, with several slanted wrinkles on the surface.
Habitat.It is common in primary forest and loess areas, but it has not been found on the reclaimed lands outside the primary forest.
Distribution.Only known from the type locality.
Remarks.Camaena gaolongensis sp.nov. is clearly different from other dextral camaenids by its quite thick, low, flat, and dark brown conical shell resembling a flying saucer (Chen et al. 1990, Schileyko 2011).Additionally, the longitudinal pilasters on the inner penial wall are stronger and more widely spaced, as well as smooth at the base but curved at the end, which are also distinct from the other dissected Camaena snails (Ding et al. 2016, Ai et al.2016 3-5 Type material.Holotype.[FJIQBC 19405] Shell height 19.2 mm, shell width 39.0 mm, height of aperture 12.0 mm, width of aperture 16.5 mm, 16 April 2015, collected from the type locality.
Apex quite blunt.Suture shallow.The protoconch surface smooth, some short growth lines visible near the two sides of suture under 32 × stereomicroscope.Last whorl with quite acute carina at periphery and a shallow groove-like depression above and below the carina.No band on the carina, but several reddish brown and sparse spire bands below the carina.Aperture crescent-shaped.Peristome reflected, white and thick.Columellar lip reflected.Umbilicus reddish brown, open, large and only 2/5 covered.Inner lip attached to the body whorl, forming translucent callus.
Soft body.Light yellowish brown with black lines.Tentacles dark.
Reproductive system.Bursa copulatrix oval, small, with quite long and tapering pedunculus.Flagellum long, tapering distally.Vas deferens long and thin.Epiphallus medium thickness and length.Penis retractor muscle very short and slender.Penis long with a short protrusion at the middle.Inner penial wall with longitudinal, slightly straight and smooth pilasters.Verge circular, somewhat small, opened basally, extending from the base to the end.
Habitat.The species was found on limestone in Maguan county of Yunnan province, China.
Distribution.Only known from the type locality.
Remarks.Camaena maguanensis sp.nov. is clearly different from other dextral camaenids with a lower conical shell.In particular, C. maguanensis sp.nov.has a large and open umbilicus, which distinguishes it from C. longsonensis and C. jinpingensis.Although the umbilicus of C. maguanensis sp.nov. is similar to that of C. vorvonga, some differences are obvious.For example, C. maguanensis sp.nov.has no spiral band on the carinate periphery of the body whorl but some spaced bands at the base.The shell of C. maguanensis sp.nov. is yellowish, but that of C. gaolongensis sp.nov. is dark brown.On the other hand, C. maguanensis sp.nov.has a circular and slightly smaller verge.
P-distances of the COI gene between this new species and the other dextral species are 0.068-0.198(Table 3), and that between C. maguanensis sp.nov.and C. yulinensis sp.nov. is 0.108, also exceeding 0.059 (currently the maximum differentiation value (p-distance) of Camaenidae) (Criscione and Köhler 2014), and the topology of the phylogenetic tree also supports the new species.
Paratype.[FJIQBC 19461-19466] 6 specimens: 3 live adults, 3 empty adult shells, 4 November 2013; [FJIQBC 19468-19472]      Etymology.The name of the new species refers to the type locality.Diagnosis.Shell.Shell dextral, large, thin, fragile, and slightly lucent, low and flat conical.4.5 whorls, the front whorls increasing slowly.Spire relatively low.Body whorl rapidly expanded.Shell light yellowish with clear and dense growth lines and spiral bands on the surface.Apex quite blunt.Suture shallow.The protoconch surface smooth for most individuals, but a few are rough.Growth lines clear near the outer side of suture under 32 × stereomicroscope.Last whorl with carinate periphery, a thin reddish brown spiral band on the carina, and many reddish brown spiral bands of different thickness on the upper and lower parts.Aperture lunate.Peristome reflected, fleshcolored, thin, sharp.Columellar lip reflected.Umbilicus reddish brown, open, large, and only 1/3 covered.Inner lip attached to body whorl, forming translucent callus.
Soft body.Pale yellow with irregular black lines.Tentacles dark brown.
Reproductive system.Bursa copulatrix oval, large, with long and tapering pedunculus.Flagellum long and slightly thick, tapering distally.Vas deferens short and thin.Epiphallus medium length and slightly thick.Penis retractor muscle short and wide.Penis short and swollen at distal 1/3, with longitudinal, thin, smooth pilasters internally.Verge conical, large, opened terminally, with some irregular wrinkles on the surface.
Habitat.The species was found on limestone in Yulin city, Guangxi province.Distribution.Only known from the type locality.
Remarks.Camaena yulinensis sp.nov.differs from C. longsonensis and C. jinpingensis in the key characteristic of large open umbilicus.This new species not only has spiral bands with different thickness on the body whorl but also has a flesh-colored peristome compared to C. vorvonga.The differences between this species and the other three new Camaena species herein have already been described above.
P-distances of the COI gene between C. yulinensis sp.nov.and the other dextral congeners ranges from 0.092 to 0.202 (Table 3) and the phylogenetic topology tree supports the establishment of this new species.

Discussion
We describe four new species of dextral Camaena snails, namely C. funingensis sp.nov., C. gaolongensis sp.nov., C. maguanensis sp.nov.and C. yulinensis sp.nov., which Verge opening terminally, one clear crack on the surface extending from the end to the base basally, one crack on the side surface extending from the base to the end basally, one crack on the surface extending from the base to the end terminally are distinguished from their congeners by their shell morphologies, especially the low and flat shell shape, the large open umbilicus, the acute and carinate periphery of the body whorl, as well as features in their reproductive systems and molecular characteristics.Among the first three new species, the differences of shells and genitals are obvious.Although C. funingensis sp.nov.and C. yulinensis sp.nov.are similar in shell morphology except size, color and umbilicus, the former has an ovate and terminally opened verge and one clear crack on the surface extending from the end to the base, as well as strong and widely spaced penis pilasters, that distinguish it from C. yulinensis sp.nov.with a conical verge, thin penial inner pilasters and without crack on the surface (Figs 3,(5)(6)(7)(8)(9).Nonetheless, the two similar-shaped species are relatively distantly related genetically (Fig. 2).Some scholars have considered genetic distance as one of the more important pieces of evidence used for identifying new species and revising species; for example, in the Asian camaenids Luchuhadra (Kameda et al. 2007) and Satsuma (Wu et al. 2008), the Australian camaenid Kimberleytrachia (Criscione and Köhler 2014), and Camaena (Ai et al. 2016;Ding et al. 2016).In the present study, the p-distances between C. funingensis sp.nov., C. gaolongensis sp.nov., C. maguanensis sp.nov., C. yulinensis sp.nov., and the other dextral Camaena was substantial: 0.068-0.200,0.075-0.203,0.068-0.198,and 0.092-0.202respectively for the mitochondrial COI barcoding region (Table 3).These numbers exceed the intra-specific differentiation values (p-distances) of Camaenidae (for Camaena, minimum 0.00, maximum 0.018 in Ding et al. (2016), minimum 0.00, maximum 0.019 in Ai et al. (2016), for Kimberleytrachia, minimum 0.00, maximum 0.059, mean 0.026 in Criscione and Köhler (2014).Based on these considerations, inter-specific differentiation supports the recognition of the four new species.
In the phylogenetic analyses, C. vorvonga and C. longsonensis, which were placed in informal subgeneric group I, have a close relationship, while they are distant from C. jinpingensis that originally also belonged to group I.In the future, more species and sequences will be needed for a more robust analysis of camaenid phylogeny.
During our long-term field investigations, we observed that most Camaena species have a narrow distribution and a low population density, and only inhabit primary forests.An exception to this is C. cicatricosa, which is widespread and has high population densities (Ai et al. 2016;Ding et al. 2016).In recent years, with the development of the Chinese economy, areas of primary forest have been decreasing and the habitats of Camaena species are becoming increasingly restricted and threatened.Therefore, it is necessary to maximize forest protection, prevent deforestation, and prevent excessive tourist development to preserve the biodiversity of these terrestrial mollusks and other animals and plants.

Figure 2 .
Figure 2. Neighbor-Joining and Minimum-Evolution trees based on analysis of the COI sequences.Numbers beside nodes indicate bootstrapping support (%) for the main clades, based on 1000 replicates.

Figure 8 .
Figure 8. Reproductive system of the snail Camaena maguanensis sp.nov.(FJIQBC 19405, Huazhige, Maguan, Yunnan, China) A reproductive organ B penis C verge D inner penial wall.The arrow indicates opening position of the verge.

Figure 9 .
Figure 9. Reproductive system of the snail Camaena yulinensis sp.nov.(FJIQBC 19460, Longquan cave, Yulin, Guangxi, China) A reproductive organ B penis C verge D inner penial wall.The arrow indicates opening position of the verge.

Table 1 .
Primer pairs and PCR conditions used in the analyses of the COI gene of Camaena.

Camaenidae Pilsbry, 1895 Camaena Albers, 1850 Type species. Helix
The bootstrap support of each species exceeds 50%.The sinistral camaenids were clearly clustered together.The four dextral new species have the closest phylogenetic relationship to each other and are sister species with C. vorvonga.From the tree structure, branch length and comparison of the known species, the phylogenetic trees supported C. funingensis sp.nov., C. gaolongensis sp.nov., C. maguanensis sp.nov., and C. yulinensis sp.nov.as new species.Moreover, the four new species all had a closer genetic relationship with each other than with any other Camaena species studied here.cicatricosa Müller, 1774, subsequent designation by Martens 1860.

Table 3 .
Inter and intra-specific P-distances of the COI sequences on dextral Camaena species.
Camaena species are 0.075-0.203(Table3), and those between C. gaolongensis sp.nov., C. maguanensis sp.nov., and C. yulinensis sp.nov.are 0.085 and 0.104 respectively.Combining the topological structure of the phylogenetic tree, the new species C. gaolongensis sp.nov. is distinct from other dextral Camaena species.
). P-distances of the COI gene between C. gaolongensis sp.nov.and other dextral

Table 5 .
Diagnostic comparisons of morphological characters of the four new species.