Systematic revision of Stegodera Martens, 1876 (Gastropoda, Stylommatophora, Camaenidae), with description of a new genus

Abstract The monotypic genus Stegodera Martens, 1876 is systematically revised based on anatomical and morphological examination of freshly collected specimens. A new species from southern Hunan, which resembles Stegoderaangusticollis, is confirmed to represent a new genus evidenced by comparative shell morphology and anatomy as well as by molecular phylogenetic analyses. The new genus might be more closely related to Stegodera and Nesiohelix Kuroda & Emura, but differs anatomically from the latter two genera by the absence of a dart apparatus.


Introduction
Stegodera Martens, 1876 is a monotypic camaenid genus endemic to southern Hubei and northern Jiangxi, China (Zilch 1960;Richardson 1983). Since the original description (Martens 1875a), only a few additional specimens of this species have been reported (Heude 1882; Chen and Gao 1987;Qian et al. 2008;Qian and Zhou 2014). The typical shell character states of Stegodera are "shell sinistral, disk-shaped, with low spire and open, deep umbilicus; solid, opaque, brown. Inner whorls slowly increasing, regular; latter half of the last whorl distorted, straightened, covering the preceding whorl above. Aperture very oblique, crescentic, toothless; peristome reflexed; throat very much contracted" (Pilsbry 1895), while the anatomy of this genus kept unknown. Recently, we received several fresh specimens of Stegodera angusticollis (Martens, 1875) from southeastern Hubei. In another recent field investigation in southern Hunan, Dr Lu Qiu found two land snail specimens belonging to a species, which is conchologically similar to Stegodera angusticollis, but has been found to differ from it in anatomical characteristics.

Materials and methods
A piece of foot tissue was cut from the living animal and preserved in 99.7% ethanol for molecular analysis. Then the animal was relaxed by drowning in water before being transferred to 70% ethanol for fixation, which was replaced with ethanol of the same concentration after three days. Photographs of the shell and reproductive system were taken using a Canon camera with Macro lens, and then modified in Adobe Photoshop CC 2018. The shells were measured with digital vernier calipers to the nearest 0.1 mm. Measurement abbreviations: S Dmaj shell major diameter; S Dmin shell minor diameter; S H Shell height.
Whorls were counted as described by Kerney and Cameron (1979). Directions used in descriptions: proximal, towards the genital atrium; distal, away from the genital atrium.

Molecular phylogenetics
Muscle tissue was obtained from eleven species in this study (Table 1), including Stegodera angusticollis and the single paratype of Pseudostegodera qiului gen. et sp. nov. Genomic DNA was extracted by using Tiangen DNA Extraction Kit (for SYS samples. Abbreviations see below) and TIANamp Marine Animals DNA Kit (for HBUMM samples). Two mitochondrial genes, partial 16S ribosomal RNA gene (16S) and partial cytochrome C oxidase 1 gene (CO1), were amplified. Primers used for 16S were 16SA/16SB (Palumbi et al. 1991), and for CO1 were LCO1490/HCO2198 (Folmer et al. 1994). PCR amplifications were performed in a 20 μl (for SYS samples) / 50 μl (for HBUMM samples) reaction volume with the cycling conditions of an initial denaturing step at 94° C for 2 min, 35 cycles of denaturing at 94° C for 30 s, annealing at 58° C (for 16S)/ 50° C (for CO1) for 30 s and extending at 72° C for 30 s, and final extending step of 72° C for 10 min. PCR amplicons were inspected on a 1% agarose gel for quality and fragment size, then were purified, and sequenced on an automated sequencer.
For phylogenetic analysis, sequences from seven camaenid species and one out-group species were obtained from GenBank and incorporated into our dataset (Table 1). Cornu aspersum is used as the out-group of the studied Camaenidae, following the previous studies (Wang, Xiao et al. 2014;Wang, Yang et al. 2014;Huang et al. 2015) considering that the branch including Helicidae is a close group of Bradybaenidae + Camaenidae (Asian and Australasian) + Polygyridae (Wade et al. 2007;Razkin et al. 2015). DNA sequences of the two genes were aligned respectively by the Clustal W algorithm with default parameters (Thompson et al. 1997) in MEGA 7.0.26 (Kumar et al. 2015). The substitution saturation assessment for CO1 genes was done using DAMBE 7.0 (Xia 2018) employing the methods introduced by Xia et al. (2003) and Xia and Lemey (2009). The poorly aligned positions and divergent regions of the alignment of 16S were eliminated using Gblocks 0.91b (Castresana 2000), a concatenated matrix of 20 (including outgroup, Table 1)×703 bp was used for the subsequent analyses. Model selection was performed by "Models" in MEGA 7.0.26 (Kumar et al. 2015). The data set was analyzed using Bayesian Inference (BI) in MrBayes 3.2.4 (Ronquist et al. 2012) and the Maximum Likelihood analysis in raxmlGUI 2.0 beta (Edler et al. 2019) (Fig. 1). In Bayesian Inference analysis, three independent runs were conducted, each of which was performed for 10,000,000 generations and sampled every 1000 generations with the first 25% samples discarded as burn-in. Convergence of the Markov Chain Monte Carlo simulations was assessed using Tracer v1.7 (Rambaut et al. 2018), verifying that all ESS values exceeded 200. In addition, we repeated these analyses for a dataset containing a larger number of species (Table 1) (Fig. 1B).

Phylogeny
The substitution saturation assessment for CO1 sequences suggested that the first and second codon positions were relatively conserved but that the third codon po-  Table 1) based on partial mitochondrial 16S and CO1 sequences. Numbers near nodes indicate Bayesian posterior probabilities. The squares on the right of each taxon name indicate its character states. Abbreviations: PS: penial sheath; EpP: epiphallic papilla; PC: penial caecum; Fl: flagellum; DS: dart sac apparatus A phylogram without Australian camaenids B the results of ancestral states (tagged on branch in red, blue, or black) reconstruction mapping on the phylogram with some Australian camaenid genera added. Orange part: the topologies supported by the results respectively by using both Bayesian Inference and Maximum Likelihood methods. Character states are from Schileyko (2003) and Wu (2019). Scale bars for substitutions per site.
sitions revealed sequence saturation and are therefore not suitable for phylogenetic inference (for both symmetrical and asymmetrical trees, Iss > Iss.c, ps < 0.001). Our final molecular dataset contained nineteen sequences of partial 16S and CO1 genes. After eliminating poorly aligned positions and divergent regions of the alignment of 16S using Gblocks 0.91b (Castresana 2000), a concatenated matrix of 20 (including outgroup, Table 1)×703 bp was used for the subsequent analyses. The models "T92 + G" and "GTR + G + I" were chosen as the best nucleotide substitution models for 16S (lnL = -1877.8, BIC = 4100.7) and CO1 (lnL = -1399.1, BIC = 3216.9), respectively. The model of the combined dataset is "TN93 + G + I" (lnL=-3387.6, BIC = 7192.6). The phylograms produced by both Maximum Likelihood Inference and Bayesian Inference based on partial 16S + partial CO1 sequences are topologically identical in major branches (Fig. 1). All trees reveal a primary division (posterior probability PP = 1) between penial caecum-bearing Satsuma A. Adams, 1868 (Camaeninae) and the remaining studied groups, a mixture of so-called camaenine and bradybaenine genera that usually have no penial caecum. The monophyly of Bradybaeninae is not supported because neither Nesiohelix Kuroda & Emura, 1943nor Dolicheulota Pilsbry, 1901, and Euhadra Pilsbry, 1890, all well-known bradybaenine genera, are not included in the clade where most bradybaenine genera stay. The monophyly of Camaeninae, which is morphologically characterized by the absence of dart sac apparatus is also not supported when considering that Satsuma is situated most basally on the phylograms and meanwhile Camaena Albers, 1850 and its sister group Amphidromus Albers, 1850 are deeply nested in the clade that comprises most bradybaenine genera. Stegodera E. Martens, 1876 is found to be the sister group of Nesiohelix with strong nodal support (PP = 1). In one phylogram (Fig. 1A), Pseudostegodera gen. nov. is a sister group of clade Steogera + Nesiohelix and in the other, it is in an unresolved trichotomy with these two taxa.
As suggested by the analyses of character evolution, the ancestral character states among most studied camaenids, except Satsuma, Pseudostegodera gen. nov., Stegodera, and Nesiohelix, are: penial sheath, epiphallic papilla, flagellum and dart sac apparatus present, penial caecum absent (Fig. 1B). The penial sheath has been lost in all the studied taxa at least for five times. The epiphallic papilla has been lost at least for four times but regained once in Tatemelon. Amongst the ingroup, the dart sac was acquired for three times but lost in Camaena, Amphidromus and the Australian camaenids. The flagellum has been lost twice, once in Exiligada Iredale, 1939 Pilsbry (1905) formally established its independent status. Stegodera and Traumatophora Ancey, 1887 were considered to be closely related, and Traumatophora was once treated as a subgenus of Stegodera (Pilsbry 1905;Gude 1920). Pilsbry (1905) believed that the two shallow grooves in the throat area of Stegodera are likely to be homologous to the dentition in Traumatophora.
In light of the genital system, Stegodera shares with Nesiohelix the most important character states like the presences of flagellum, numerous tubes of mucous glands and the papilla distally leading to mucous glands on the inner wall of the accessory sac, and the absences of the epiphallic papilla, poly-layered structure in dart sac apparatus and membranous sac surrounding terminal genitalia (Wu 2019). The close relationship between them in genitalia is supported by the phylogenetic result of this work (Fig. 1A, B). Figures 1, 2  Diagnosis. Body whorl completely covering partial penultimate whorl. Penial sheath weakly present. Epiphallic papilla absent. Dart sac apparatus and flagellum present. Mucous glands with numerous tubes.
General anatomy (Fig. 4A-D). Head wart absent. On internal body wall of head region between ommatophore insertions with tiny pits rather than glands (Fig. 4C). Each side of mantle edge with a leaf-shaped appendage (Fig. 4A, B). Body reddish brown, central dorsa with light longitudinal stripes. Sole dirty white. Jaw arcuate; with 12 more or less projecting ribs (Fig. 4D).
Genitalia (Figs 5A-C, 6A, B). Penis sheath present but thin and very short. Penis medially slightly thickened, moderately long, ~ 8.8 mm, externally simple. Inside penis with several thin longitudinal pilasters. Epiphallus slightly longer than penis. A very thin membranous sac wrapping distal half of penis with distal end connecting basal penial retractor muscle (Fig. 6B). Flagellum cylindrical, tapering. Vas deferens thin throughout. Dart sac apparatus distally inserting on vagina. Dart sac small. Dart not observed. Membranous sac surrounding terminal genitalia absent. Accessory sac relatively large. Tubes of mucous glands more than twenty, neatly inserting in a single row on distal and same side of dart sac on accessory sac (Figs 5C, 6B). From proximal to distal accessory sac, mucous gland tubes increased gradually in length (Figs 5C, 6B). Papilla distally leading to mucous glands on inner wall of accessory sac integrated into one long thick spongious pilaster on the side of mucous gland tube insertion (Fig. 6A). Bursa copulatrix ball-shaped.
Ecology. This species was observed under litter layer, on nearby rocks and crevices. Distribution. Central China: Hubei, Jiangxi (Fig. 2).
Remarks. The peristome of Stegodera angusticollis is in chestnut in fresh specimens, but in most museum specimens this color gets faint to white. In Stegodera angusticollis, the compressed part of body whorl usually completely cover the penultimate whorl, while in Pseudostegodera qiului gen. et sp. nov. the corresponding part is not so compressed and the compressed part of body whorl can hardly cover the penultimate whorl. Some authors (Chen and Gao 1987;Qian et al. 2008;Qian and Zhou 2014) recorded this species in Jiangsu, Zhejiang and Anhui, but voucher specimens from these provinces were neither recorded therein nor being found in the mollusk collection deposited in IZCAS where some of them once worked. Therefore, the accuracy of these records needs to be further confirmed. Diagnosis. Shell sinistral. Apical whorls with dense fine ribs gradually becoming granules. The last 1 / 8 to 1 / 4 body whorl compressed, only partly covering penultimate whorl. Umbilicus broad. Head wart absent. Each side of mantle edge with a leafshaped appendage. Penis sheath present. Penis externally simple. Epiphallic papilla absent. Flagellum present. Dart sac apparatus absent. Remarks. Conchologically the new genus can be distinguished from Stegodera by the detached body whorl and penultimate whorl. The new genus is supported to be the closest relative of a clade containing Stegodera + Nesiohelix as based on the Maximum Likelihood/Baysian Inference phylogeny shown in Fig. 1A. However, when the Australian camaenids were included in the analysis, the relationships between the new genus, Stegodera + Nesiohelix, and all remaining taxa except Satsuma remained unresolved (Fig. 1B). The new genus is conchologically similar to Stegodera, however, it differs in certain reproductive characters (see above). We consider that the genital anatomy is more informative than shell features in helicoid systematics (e.g., Criscione and Köhler 2013). In addition, the loss of the dart sac apparatus in Bradybaeninae and/or Camaeninae is not a frequent event during the evolution of Camaenidae (Fig. 1B). Based on these two considerations, we consider it is reasonable to distinguish this new camaenid taxon as a monotypic genus represented by the new species described below. Diagnosis. Body whorl incompletely covering partial penultimate whorl. Dart sac apparatus absent. Flagellum present.
General anatomy (Fig. 4E-H). Eversible head wart absent. On internal body wall of head region between ommatophorous insertions with tiny pits rather than glands (Fig. 4F). Each side of mantle edge with a leaf-shaped appendage (Fig. 4G, H). Body reddish brown, central dorsa with light longitudinal stripes. Sole dirty white. Jaw arcuate, with ~ 14 more or less projecting ribs (Fig. 4E). Genitalia (Fig. 7A-D). Penis sheath short but well developed. Penis somewhat swollen on proximal half, externally simple. In the middle of penis, internally with a single strong longitudinal pilaster almost as thick as epiphallus, ~ 2 mm long (Fig. 7D). Epiphallic papilla absent (Fig. 7D). Flagellum cylindrical, tapering. Vas deferens thin throughout, approximately as long as penis. Dart sac apparatus absent. Bursa copulatrix duct thickened basally. Bursa copulatrix duct longer than 50 mm. Bursa copulatrix rod-shaped.
Etymology. This new species is named after Dr Lu Qiu [邱鹭], who provided the specimens and field data.
Ecology. This species was found under rotten wood. Distribution. Only known from the type locality (Fig. 2).
Remarks. See under the genus.

Discussion
The comparison of the phylograms (Fig. 1) suggests the phylogeny obtained in this paper is more or less robust when the Australian camaenids, which form a monophyly together with the Asian camaenids (as suggested by Scott 1996), are considered. The phy- logenetic position of Satsuma agrees with that suggested by many authors (Chiba 1999: fig. 3;Wade et al. 2007: fig. 2). As indicated by some authors, Satsuma is the sister group of the common ancestor of Bradybaeninae and Camaeninae ( Fig. 1; Wade et al. 2007: fig. 2), rather than a typical camaenine (sensu Azuma 1995) or bradybaenine (sensu Schileyko 2004). The monophyly made by Stegodera, Nesiohelix and the new genus is the sister group of all the remaining bradybaenines and camaenines involved in this study. It suggests that neither the Camaeninae including Satsuma (sensu Azuma 1995) nor the Bradybaeninae including Nesiohelix (sensu nearly all the authors) are monophyletic. The current work also corfirms that so-called important characters of genital system, including penial sheath, epiphallic papilla, penial caecum, flagellum, and dart sac apparatus, have homoplasiously evolved more than once in the studied terminal taxa and their ancestral nodes (Fig. 1B). In other words, widespread homoplasious changes in morphology (Hirano et al. 2014; Fig. 1) including dart sac apparatus based on which Bradybaeninae and Camaeninae are distinguished, rendering the basis of the establishment of Bradybaeninae particularly feeble, explaining that no agreement on the subdivision within Camaenidae was reached to date (Nordsieck 2002;Schileyko 2003Schileyko , 2004Wade et al. 2006Wade et al. , 2007Gittenberger et al. 2012;Bouchet et al. 2017).