Asian Herpetological Research 2014, 5(3): 137–149 DOI: 10.3724/SP.J.1245.2014.00137

Reclassification of ningshaanensis Yuan, 1983 (Ophidia: ) into a New , Stichophanes gen. nov. with Description on Its Malacophagous Behavior

Xiaohe WANG1, Kevin MESSENGER2, Ermi ZHAO1* and Chaodong ZHU3*

1 Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, Sichuan, 2 Department of Biological and Environmental Sciences, Alabama A and M University, Normal 35801, Alabama, USA 3 Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China

Abstract The complete mitochondrial cytb gene and the partial nuclear c-mos gene of Oligodon ningshaanensis Yuan, 1983 were sequenced and used for reconstructing the phylogenetic relationship of this taxon amongst alethinophidian . Its strong affinity to the New World subfamily and the Old World Thermophis baileyi was inferred. Hemipenial morphology found by authors conflicts with the original description and its similarity with those of the dipsadid snakes is in accordance with our molecular results. Feeding tests show that O. ningshaanensis is a malacophagous predator, which is another matchless character for this species. This behavior is described and compared with other known slug- and snail-feeding snakes. The discovery of the particular position of our subject indicates that erecting a new genus is necessary accommodate this unique species.

Keywords Stichophanes gen. nov. ningshaanensis, Dipsadinae, molecular phylogeny, hemipenial morphology, mollusks-predator

1. Introduction have already been included, it is possible to employ the known molecular inferences to elucidate the phylogenetic There has been much recent progress on higher-level position for some of the lesser-known species when the phylogenies based on different molecular datasets subject presents many unique characters. (Slowinski and Lawson, 2002; Lawson et al., 2005; Vidal Hemipenial morphology has functioned to obtain et al., 2007, Vidal and Hedges, 2009; Wiens et al., 2012; traditional morphological character, with its importance Pyron et al., 2011, 2013; Pyron and Burbrink, 2012). in the systematics of snakes already acknowledged Molecular work has rapidly produced new phylogenies by the end of 19th century (Cope, 1894, 1895). It of Colubridae, the largest and most speciose group fits the widely accepted biological species concept among snakes. Approximately half of the known snake and does not correlate with ecological factors or species have already been sequenced and incorporated behavior. Dowling (1967) considered the hemipenial in phylogenetic reconstructions. While most subgroups features to be more reliable than other morphological characteristics. Before modern methods, trying to * Corresponding author: Prof. Ermi ZHAO, from the Department of diagnose monophyletic subgroups within Colubridae was , Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu, Sichuan, China, with his research focusing on based primarily on the morphology of the hemipenes. systematics herpetology; Prof. Chaodong ZHU, from the Division of Along with fast developing molecular research, Zaher Functional Insect Groups Evolution, Institute of Zoology, Chinese et al. (2009) subsequently reworked the classification Academy of Sciences, Beijing, China, with his research focusing on systematic entomology, evolutionary biology, computational of caenophidian snakes as a higher-level taxonomic phylogenetics, pollination biology and biological control. category. Hemipenial features continue to be used in E-mail: [email protected] (Ermi ZHAO); [email protected] (Chaodong ZHU) describing, defining and diagnosing new species and Received: 4 April 2014 Accepted: 11 July 2014 genera (Zaher, 1999; Myers, 2003; Sawaya and Sazima, 138 Asian Herpetological Research Vol. 5

2003; Passos et al., 2005; Myers and McDowell, 2014). 2. Material and Methods Oligodon ningshaanensis Yuan, 1983 was described from one adult and two subadult specimens collected 2.1 The material used in this study NAFU071227: in Huoditang, Ningshan County, Shaanxi Province, adult male, collected from type locality, Huoditang, People’s Republic of China. It was assigned to the genus Ningshan, Shaanxi Province without known date, fixed Oligodon based on some diagnostic characters, and was and preserved in Northwest Agriculture and Forestry distinguished from “the closely related O. catenata” by: University, Yangling, Shaanxi Province. 1) presence of a pair of internasals; 2)prefrontals not in CIB100191: adult female, collected from type locality contact with second supralabial; 3) less ventrals; and in 2008, fixed and preserved in Chengdu Institute of 4) more subcaudals (Yuan, 1983). The genus Oligodon Biology, Chinese Academy of Sciences, Chengdu, Fitzinger 1826, the Kukri snakes, also known as the Small- Sichuan Province. headed snakes in Chinese, currently contains 74 small- CIB100192–100200: newborn specimens in 2009, from to-medium sized species, most of which are nocturnal. two clutches laid by females from Shennongjia, Hubei Oligodon’s feeding preference for eggs is recorded in Province, deposited in Chengdu Institute of Biology, detail (Minton, 1963; Hu and Zhao, 1987; Coleman et al., Chinese Academy of Sciences. 1993; Zhao et al., 1998). Their diet relies on the strongly 2.2 DNA extraction, amplification, and sequencing We recurved hind teeth, which resemble kukri knives. Given extracted DNA from alcohol fixed muscle tissue from their wide distribution throughout Southeast Asia, most CIB100191 and CIB100192 using the QIAGEN DNeasy studies only involve a few species of the genus. Even Blood & Tissue Kit according to the manufacturer’s though there are an increasing number of investigations instructions. The entire mitochondrial cytochrome b gene that target the phylogeny among snake species, (cytb) was amplified by using the primers L14910 (de affinities of the neglected O. ningshaanensis (Yuan, Queiroz et al., 2002) and H16064 (Burbrink et al., 2000). 1983) remain mysterious amongst all previous work The partial oocyte maturation factor gene (c-mos) was looking at congeners (Green et al., 2010; Lawson et al., amplified and sequenced by using the primers S77 and 2005; Pryon et al., 2011, 2013). S78 (Lawson et al., 2005). PCR was performed with the Little work has been done on this species since its 50 μL system (2 μL template, 1 μL of each primers, 25 μL initial description, with the exception of examining the dNTPs mix and 21 μL ddH O) and the following reaction microstructure of the skin, which revealed a canaliculated- 2 parameters: predenaturation at 94°C for 4 min, followed type microstructure (Li and Liang, 2007), a trait that is by 40 cycles of 30 s denaturation (94°C), 30 s annealing shared with many different subfamilies of Colubridae (55°C–60°C), and a 1 min extension (72°C) with a final (Prince, 1982). Since 2006, a series of field surveys in extension at 72°C for 7 min. Then the PCR products Shennongjia, Hubei province, discovered large number were sent to a sequencing company for purification and of O. ningshaanensis. This material represented the first sequencing (ABI PRISM 3730xl Genetic Analyzer). The documentation of large population of the species since sequences generated in this study have been deposited in its discovery, as well as a new record for this province GenBank (Accession Numbers: KJ638715–KJ638718). (Yang et al., 2009). During these surveys, we found a lot of characters conflicting with the designation of this 2.3 Phylogenetic analyses For our analyses, we mainly species within Oligodon (e.g., slim body shape, lack of follow the dataset used by Lawson et al. (2005). For most an upturned snout, lack of the “kukri” teeth, etc.). Its prey Colubroid families, subfamilies, and some important taxa, made of terrestrial snails and slugs is different from most the cytb and c-mos sequences of subpunctatus colubrid snakes, and especially Oligodon. Compared (Genebank Accession Numbers: KC347471, KC347411) to the number of species that prey on vertebrates, and Thermophis baileyi (EU496923, EU496922) malacophagous behavior is seldom seen among snakes were added. The translated protein sequences were (Laporta-Ferreira and Salomão, 2004). All of these aligned by using MUSCLE (Edgar, 2004) in MEGA6 observations brought an ineluctable investigation on the (Tamura et al., 2013) with default settings. Phylogenetic basic information of this neglected and previously “rare” reconstruction was conducted on the combined cytb species. Here we use the mitochondrial and nuclear DNA and c-mos genes by using Maximum Likelihood (ML) to reconstruct its phylogenetic relationship. We are also method on RAxMLBlackbox (http://embnet.vital-it.ch/ using morphological data to supplement its distinction raxml-bb/) (Stamatakis et al., 2008) and RAxML7.2.5 from Oligodon. (Stamatakis, 2006). Cylindrophis ruffus was employed as No. 3 Xiaohe WANG et al. Stichophanes gen. nov. and Malacophagous Behavior 139 the outgroup. The alignment was partitioned by the codon autopotamic), earthworms, and planaria. position of different genes and the GTRCAT strategy was used by all six partitions on the web server for 3. Results obtaining a best-scoring tree. This initial tree was passed to the RAxML 7.2.5 for an additional search with 1000 3.1 Phylogenetic position of O. ningshaanensis As standard bootstrap replicates. The bipartition information we chose one of the henophidian species to be outgroup, was overlaid on the best-scoring tree based on multiple the tree estimated from the aligned cytb and c-mos bootstrap trees. genes presents a higher-level caenophidian phylogeny (Figure 1). Though there was limited taxon sampling, 2.4 Hemipenis preparation The hemipenis from the the members of the well-known main snake groups topotypic specimen NAFU071227 was prepared. We were clustered together, respectively. However, the followed the method presented by Pesantes (1994), topological structure among the main clades of our best- but modified by Zaher (1999) and Jiang (2010) to scoring ML tree is not well supported by the overlaid prepare the hemipenis from preserved specimens. bootstrap values (BS < 70% on some nodes). The tree is Removal of the right hemipenis was carefully done, approximately congruent with many previous molecular and the organ was placed into 3% KOH solution studies on different datasets (Lawson et al., 2005; Pyron for six hours in a 25°C water bath. The capsule and and Burbrink, 2012; Pyron et al., 2011, 2013; Vidal et al., muscles were gradually taken away during this period 2007; Vidal and Hedges, 2009; Wiens et al., 2012; Zaher as the tissue gets softer. When the hemipenis became et al., 2009) except the position of small subfamilies of as flexible as a fresh organ, it was incised to enlarge the Colubridae (Calamariinae, and opening at the base of asulcate side. Then round- Sibynophiinae). tip-forceps were used to evert the whole structure. Among Colubridae, which contains the species Instead of gelatin or agar, the organ was filled with and O. ningshaanensis, three absorbent cotton. The terminology follows Zaher (1999). clades (, Dipsadinae and ), each 2.5 Clearing and Staining The adult female specimen with a large number of species separate, are clearly (CIB100191) was used in this section. After dehydrating separated from each other. The clade ((Dipsadinae + the snake without skin, gastrointestinal tract or gonads Pseudoxenodontinae) + Sibynophiinae) and Natricinae in 100% alcohol for two days, the specimen was placed form one main clade of Colubridae. Calamariinae clusters into a stain solution made from 40 mL acetic acid, 60 with (Colubrinae + Grayiinae) to form another. Oligodon mL 100% ethanol and 20 mg alcian blue for 40 hours. cinereus nested within Colubrinae. However, two Then a series of alcohol solution (25%, 50%, 75% and specimens from different localities of O. ningshaanensis 100%) were used to wash the stain gradually away for are placed within the subfamily Dipsadinae as the sister one day each, respectively, until the muscle became group of all the New World dipsadid species. Thermophis white. Then the specimen was carefully moved into baileyi stays as a basal branch of Dipsadinae; several a mixture of 100 mL of 0.3 mol/L KOH solution and other molecular works confirm this position (He et al., 5 mg alizarin red for 20 hours for coloring the bones. 2009; Pyron et al., 2011, 2013). The support value on the Later, the colored specimen was placed into a gradient node between Dipsadinae and Pseudoxenodontinae is low glycerol solution (25%, 50%, 75% and 100%) for one day (BS 56%), but on the basis of the bootstrap value (BS each, respectively, until the bones were transparent. The 100%) of the 50% majority-rule consensus tree, this node preserved specimen was then placed in a 0.1% phenol is firmly supported. solution in glycerol (Chen and Wang, 2002). 3.2 Hemipenial morphology When retracted, the 2.6 Feeding Behavior Observation CIB100191–CIB hemipenes of O. ningshaanensis reach the 12th subcaudal 100200 were involved in this simple behavioral test. and are forked at level of the the 9th. The lobes twist The specimens were kept in separate boxes (30 × slightly to the central line of tail. After complete eversion, 18 × 10 cm3 for adults and 10 × 10 × 10 cm3 round for the total length of the right hemipenis reaches 25 mm. The newborn specimens) with water dishes and shelters. hemipenis is bilobed, semicapitate and semicalyculate. Different vertebrate and invertebrate preys were offered, The lobes are not equally long on both hemipenes. The including mice (various sizes), fish, frogs (small species sulcus spermaticus is bifurcated at around the proximal and tadpoles), lizards ( and ), insects one-third of the hemipenial body and centrolineal along (crickets, moth and moth larva), mollusks (terrestrial and both lobes. Outside sulcus spermaticus both lobes are 140 Asian Herpetological Research Vol. 5

67/100 Spilotes pullatus Phyllorhynchus decurtatus Masticophis flagellum Salvadora mexicana 70/100 Tantilla relicta Sonora semiannulata Drymarchon corais 59/100 Oxybelis aeneus Opheodrys aestivus Oligodon cinereus 83/100 Thelotornis capensis Philothamnus heterodermus Gonyosoma oxycephalum 96/100 Dinodon rufozonatum Lycodon zawi 55/100 100/100 Crotaphopeltis tornieri 68/100 Dipsadoboa unicolor Colubrinae 72/100 Telescopus fallax 54/100 atra 86/100 Boiga dendrophila Lytorhynchus diadema Rhynchophis boulengeri 90/100 Macroprotodon cucullatus 71/100 97/100 Hierophis viridiflavus 89/100 Eirenis modestus 77/100 Spalerosophis diadema 83/100 Hemorrhois hippocrepis 95/100 Platyceps karelini 100/100 Cemophora coccinea 99/100 obsoletus Colubridae 84/100 100/100 Coronella girondica Gastropyxis smaragdina Ahaetulla fronticincta Grayia smythii Grayiinae 98/100 pavimentata Pseudorabdion oxycephalum Calamariinae 80/100 tigrinus 80/100 Amphiesma stolatum 98/100 punctulatus 75/100 100/100 olivacea 100/100 Afronatrix anoscopus Natricinae 99/100 natrix 100/100 dekayi 100/100 rigida Thamnophis godmani Sibynophiinae karlschmidti Pseudoxenodontinae 56/100 Thermophis baileyi 100/100 St n NSh 56/100 St n ShNJ Oligodon ningshaanensis 94/100 Farancia abacura 60/100 Carphophis amoenus 97/100 Diadophis punctatus Heterodon simus Contia tenuis Dipsadinae 100/100 Hypsiglena torquata Rhadinaea flavilata 100/100 Magliophis exiguum 100/100 Borikenophis portoricensis 100/100 Helicops angulatus Hydrops triangularis Cerberus rynchops Naja kaouthia Homalopsidae Dendroaspis polylepis Ophiophagus hannah Bungarus fasciatus 56/100 86/100 Notechis ater 98/100 Laticauda colubrina 94/100 Sinomicrurus japonicus Micrurus fulvius 94/100 Oxyrhabdium leporinum 82/100 Bothrophthalmus lineatus 96/100 97/100 Lamprophis fuliginosus 79/100 Mehelya unicolor Lycophidion ornatum Prosymna visseri 80/100 werneri 75/100 Homoroselaps lacteus Atractaspis microlepidota 71/100 Alluaudina bellyi Duberria lutrix 99/100 Ditypophis vivax Leioheterodon modestus Ithycyphus miniatus 100/100 100/100 Mimophis mahfalensis 100/100 Psammophis condanarus Malpolon monspessulanus 59/100 Psammodynastes pulverulentus 95/100 Pseudaspis cana Pythonodipsas carinata 91/100 100/100 viridis 100/100 Agkistrodon piscivorus 97/100 Cerastes cerastes 66/100 Daboia russelii 99/100 67/100 Atheris nitschei Bitis nasicornis Pareas macularius Pareatidae granulatus 90/100 Boa constrictor Acrochordidae Casarea dussumieri Uropeltis phillipsi Cylindrophis ruffus

0.2

Figure 1 The best-scoring maximum-likelihood tree derived from cytb and c-mos genes. The support values (≥0.50 on the overlaid best tree / ≥0.70 on the 50% majority-rule consensus tree) are left above nodes. The Oligodon cinereus is labeled with a diamond and two O. ningshaanensis specimens were labled with triangles. The subfamilies of Colubridae and the families of are labeled beside trees by vertical bars. almost covered with identical reticular-arranged lobular calyces on the lobes. The first pair of lateral large papillae calyces, the areas of which are gradually enlarged appear with a noncapitulate basal region of both lobes. towards the proximal region and disappeared at half of Nearly symmetrical vertically directed conspicuous large the hemipenial body. Laterally, the outer reticular calyces spines cover the main body of asulcate hemipenis and increase in height for merging into the body calyces on spread to the proximal sulcate surface to form two slender the asulcate side and large spines at the merging region. thorny triangles pointing to the apex (Figure 2). The intrasulcar calyces are almost latitudinally shaped 3.3 Feeding behavior of O. ningshaanensis Though with a small longitudinal area near the fork. The asulcate many prey types were offered, only terrestrial slugs and side is nude medially and has irregularly arranged body snails were accepted. When earthworms and planaria No. 3 Xiaohe WANG et al. Stichophanes gen. nov. and Malacophagous Behavior 141 were offered, the snake showed some interest with the snake moves closer and targets the slug’s head with tongue flicking and actively staring at the prey item. the neck in an arch and constant tongue flicking. Once it However, none were attacked, and after several seconds grasps the slug, the snake lifts the slug off the ground and the snake stopped showing interest and moved away. starts to ingest immediately. After ingestion, the snake No ontogenetic change was found on their preference rubs the mucosa from the sides of its mouth on nearby or hunting behavior. The mollusk species accepted were substrate. as follows, slugs: Deroceras laeve and Phiolomycus The process of preying on a snail is initially very bilineatus; snails: Bradybaena fruticola, B. fruticum, B. similar with that of a slug – the snake targets the , ravida, B. similaris, Euphaedusa sp., and Laeocathaica bites into the anterior portion and lifts it off the ground. prionotropis Typically the snake will then try to find a crack, or anchor For hunting a slug (Figure 3), when the prey is noticed, point, in which to attempt to pull the snail out of its shell.

Figure 2 The right hemipenis of Oligodon ningshaanensis. A and C: Lateral views; B: Asulcate view; D: Sulcate view with .

Figure 3 Sequence of slug-preying behavior by a hatchling Oligodon ningshaanensis specimen. A: Gazing at the slug with tongue flickering; B: Targeting at the anterior end of the slug; C: Biting the head of slug. 142 Asian Herpetological Research Vol. 5

If it cannot find anything in the surrounding habitat, the lower jaw. The paired chin shields are not completely snake may hold the snail in the air for quite some time bilaterally symmetrical either in the shallow-groove- (more than 20 min). There was no mandible insertion present specimens (Figure 6). This partial loss of mental observed, as seen in other snail specialists (Figure 4). The groove in O. ningshaanensis is very similar with Sibon entire snail or most of the soft body was ripped out and dimidiatus (Günther, 1872) and S. nebulatus (Linnaeus, the shell left behind. 1758) from South and Middle America (Peters, 1960). It is worth noting that the different species of snails in this test were diversified in shape. Euphaedusa sp., with 4. Discussion a quite slender helical shell, got attacked immediately by the adult specimen CIB100191, but the snail was too 4.1 Indication from the molecular results The tree slender for the snake to find any snag for pulling it out obtained in this work figures out the phylogenetic of its shell. Eventually, the snake gave up and the snail position of O. ningshaanensis clearly, though the was released alive. Laeocathaica prionotropis, whose position of Sibynophiinae and its relationship with shells are left-handed, rotating chirally from Bradybaena Pseudoxenodontinae and Dipsadinae conflict with the sp., were taken successfully by the snakes. It is difficult previous molecular and morphological research (Zhang to find a sufficient sample size of chiral snail species for et al., 1984; Zaher, 1999; Pyron and Burbrink, 2012; a feeding experiment with statistical significance, but Chen et al., 2013; Pyron et al., 2013). Interestingly, O. anecdotally, no difference was noticed on the level of ningshaanensis is not a sister species of Thermophis success during hunting B. ravida and L. prionotropis. baileyi, but that of the American Dipsadinae, with Thermophis being their most common ancestor in the 3.4 Dentition The specimen examined (CIB100191) Old World. The close relationship between Thermophis has 5 teeth on the maxillary subequal in size. On the and Dipsadinae (including the former Xenodontinae, pterygoid 16 teeth decrease in size posteriorly, and 12 on following Vidal et al., 2010 and the works later on) is well the dentary do likewise. The 10 teeth on palatine increase known based on several molecular works (He et al., 2009; slightly in size posteriorly. All teeth point backward. No Huang et al., 2009; Pyron et al., 2011) and morphological significant asymmetry was observed in either size or investigation(Malnate, 1953; Zaher, 1999; Zhang density of the teeth (Figure 5). et al., 1984). Eventually this genus was moved into the 3.5 Mental groove Either a shallow or no mental groove subfamily Dipsadinae by Pyron et al. (2013). Oligodon were met in the examined specimens of O. ningshaanensis cinereus, a typical member of the genus Oligodon (see between two pairs of chin-shield. In NAFU071227, the Green et al., 2010; Pryon et al., 2013), is clustering with chin-shields touch each other tightly and their inward the Colubrinae species. The far relationship between angles do not contact each other in the central line of the O. ningshaanensis and Oligodon requires a necessary

Figure 4 The position of holding different snails by the adult specimen. The contained snails are A: Bradybaena ravida; B: Laeocathaica prionotropis. No. 3 Xiaohe WANG et al. Stichophanes gen. nov. and Malacophagous Behavior 143

Figure 5 Dentition of Oligodon ningshaanensis. reclassification of the former species. in North Asia from the New World. Whether these two Vidal et al. (2000) suggested an Asian-North mysterious relict groups of snakes present the ancestor or American origin of xenodontines (meaning differently descendant of the American dipsadines remains unknown from dipsadines) in the early Tertiary. This result is from the limited data. also mentioned by He et al. (2010) that the divergence 4.2 Conflicts with the original hemipenial description time of Thermophis from other xenodontine snakes is The hemipenis we examined is very different from the ~10–28 mya. The topologies of our results may agree with an Asian origin of the American Dipsadinae. original description (Yuan, 1983) and those quoted While the phylogenetically controversial position of later on (Zhao, 2006; Zhao et al., 1998). The text of the Pseudoxenodon, as a group in Dipsadinae according original description is translated as: “proximal region thin, to Zhang and Huang (2013), entered or returned to the smooth, cylindrical, taken 1/4 of the total length; middle Old World from the New World across the Bering Land region enlarged, sulcus apparent; distal region bifurcate, Bridge during the early Tertiary and the warm mid- sulcus spermaticus reaches the node; on the asulcate side, Miocene. Different from the dispersal of Natricinae at two rows of little spines located symmetrically from the similar time (Guo et al., 2008), the distributions of the middle body of hemipenis to the top of both lobes.” O. ningshaanensis and Thermophis, the central montane According to Yuan (per. comm.), the type specimens were region and the northwest plateau of China respectively, in the collection of Northwest Agriculture and Forestry are separated by large geographically diversed territory University. More than 20 years absence of herpetological 144 Asian Herpetological Research Vol. 5

these populations seems to be necessary. Nonetheless, no intraspecific variation on sulcus spermaticus division or orientation was observed for any documented species. Therefore, we trend to treat the original description as a possible mistake. 4.3 On the hemipenial morphology Oligodon, as one of the largest snake genera, presents lots of variation in the morphology of the hemipenes. Some Oligodon members, which cluster and firmly belong to Colubrinae from the molecular results (Green, 2010; Pryon et al., 2011, 2013), present deeply forked hemipenes, which are relatively rare for this subfamily. Excluding O. ningshaanensis, 16 species possess the forked hemipenes (Green, 2010; Zhang et al., 1984; Zhao, 1998), and most are deeply forked. The hemipenes of O. dorsalis and O. taeniolatus are 1/3 and 2/5 bifurcate, respectively, but these two species were not included in the molecular sampling to elucidate their position between the single and forked groups. According to the presence of single sulcus spermaticus (with some exception), of well-developed calyces present on the distal hemipenis (characters for Colubrinae, Zaher, 1999; Zaher et al., 2009), and of the nearly symmetric ornamentation on the sulcus and asulcus sides (Character for Oligodon, Jiang, unpublished data), the hemipenial morphology of O. ningshaanensis does not agree with the morphology that is associated with Figure 6 Comparision of Mental groove between Oligodon Oligodon, and other Colubrinae species. ningshaanensis and Orthriophis moellendorffi. Dorsal view of head in NAFU071227 (A), CIB100192 (B), Orthriophis moellendorffi Compared with other subfamilies of Colubridae, adult specimen (C) and new born specimen (D) from private the hemipenis of O. ningshaanensis can be easily collection. distinguished from Calamariinae (nude hemipenial body), Natricinae (hemipenial calyces absent), reseach in this university brought huge losses for the Pseudoxenodontinae (spinulate lateral and sulcate preserved specimens. We visited their museums in surfaces) and Sibynophiinae (a sharply curved U-shaped 2013 but found no specimens left except NAFU071227 without identification. Staff of the university provided convolution of the sulcus spermaticus). Characters information about broken bottles labeled “Oligodon”. The like “bifurcate sulcus spermaticus, armed with well- type specimens appear to be lost, thus, we were unable developed spines, which are developed from the marginal to get the original material for re-examination. Although papollae of calyculi” described by Cope (1894) for some cases for intraspecific variation in hemipenes have Xenodontinae, “a row of enlarged lateral spines on each been recorded, the differences found were not particularly side of the hemipenis, and hemipenial lobes with distinct different in these cases, at least among specimens that differentially ornamented regions (a sulcate capitulum share same/close locality (e.g. shape [T-shaped or and an asulcate nude or weakly calyculate region)” by Y-shaped, Keogh, 1999, Zaher and Prudente, 1999; lobes Zaher (1999) are obvious in O. ningshaanensis. wider or thinner, Zaher et al., 2005], length [Cole and From large number of descriptions on hemipenial Hardy, 1981; Keiser, 1974] and ornamentation [Cole morphology by Zaher (1999) for the Xenodontinae and and Hardy, 1981; Keogh, 1999; Prudente and Passos, Dipsadinae, that of O. ningshaanensis is seemingly 2010]). The only exception we are aware of is Calamaria similar with that of Arrhyton vittatum and A. taeniatum lumbricoidea, which has been found with single or forked from Cuba. Independent evidence demonstrates the close hemipenis from different geographic distributions (Inger relationship between the American subfamily Dipsadinae and Marx, 1962). But a reconsideration of the status of and this Old World species. No. 3 Xiaohe WANG et al. Stichophanes gen. nov. and Malacophagous Behavior 145

4.4 Erection of a new genus, Stichophanes, gen. nov. the dorsal head, loreal absent, preocular 1, postocular 2, The relationships depicted by our analyses on both temporal 1+2 (Figure 7 B), rostral broad, supralabial 6, molecular and morphological data support the position of 3rd and 4th entering the eye, infralabial 6, 4th the largest, Oligodon ningshaanensis in the subfamily Dipsadinae (at sublingual 2 pairs, mental groove inconspicuous. Body least incertae sedis in the Dipsadinae as advised by Zaher scales smooth, 13 dorsal rows throughout the trunk, et al. [2009]). A new genus is required for this unique ventrals 159–170, anal divided, subcaudals 64–73 pairs. species, which we name here as Stichophanes gen. nov. The species exhibits sexual dimorphism, both in size Etymology: From the Greek στίχος (stichos, a row or a and color. Males are shorter as adults with a longer tail line of a book) + the suffix -φανής ( -phanes, appearing, (24% to 27% of the total body length), and dorsal color conspicuous), alluding both to the slenderness of the body olive to brown in life. Females are longer with a shorter and its straight dorsal lines throughout the trunk and tail. tail (21% of the total body length), and dorsal color is Gender masculine. yellowish-brown in life (Figure 7 A). Preserved specimens Common English Name: line-shaped snake. are dark bluish brown in formalin. Head darker, from Common Chinese Name: 线形蛇属 . which originate two dark lines extending on 4th rows Type Species: Stichophanes ningshaanensis [Ningshan of dorsals (D4) throughout the body and tail, other two line-shaped snake ( 宁陕线形蛇 )]. thin dark lateral lines from neck to vent located between Content: Monotypic. D1 and D2. At the posterior head a large elongated dark Stichophanes ningshaanensis (Yuan, 1983) new comb. spot fades along the central dorsal row (Figure 7 A and Definition and Diagnosis: No similarity with any other D). Belly pale yellow with two lines of black little dots Old World Colubridae species. Small terrestrial colubrid. forming ventral dashed stripes (Figure 7 C). Head blunt, pupil of eye round, neck distinguishable Hemipenis bilobed, semicapitate and semicalyculate, from head, body slender. All 9 large shields present on with deeply forked sulcus spermaticus and large lateral

Figure 7 Stichophanes ningshaanensis in life. A: Dorsal view of anterior body demonstrating sexually dimorphic coloration; B: Lateral view of head; C: Ventral dots; D: full-body photo displaying the dorsal stripes; E: a female specimen sitting among leaf litter. 146 Asian Herpetological Research Vol. 5 spines. Maxillary teeth 5 or 6. This species is oviparous, elegans terrestris (Britt et al., 2009). The snail-eating laying between 8–9 eggs in a clutch. pareatid species have more teeth on the right mandible Distribution: Shaanxi Province: Huoditang, Ningshan than the left for the dextral (clockwise) snail houses County; Hubei Province: Muyu, Pingqian and Guogong- which are predominant in nature (Hoso et al., 2007). It is ping, Shennongjia Nature Reserve. difficult to provide any suitable comparison with a near Habit and Habitat: Stichophanes ningshaanensis is a sister group as Britt et al. (2009) used for Thamnophis to diurnal leaf litter dweller feeding on terrestrial gastropods. prove if the dentition of S. ningshaanensis is more slender Living specimens were found among dead leaves near than that of the non-malacophagous predator. However, roads and trails, and usually in proximity to water (Figure the symmetry is noticeable, as in most other snail or 7 E). The species lives in moderately high elevations slug eaters in Dipsadinae, and reflects that the feeding (1550 to 2022 m) and are most active in late June and behavior developed lately (not earlier than the other early July. dipsadid malacophagous species). Note: Type specimens are lost. Mental groove absence is a very important character in the Pareatidae (Guo et al., 2011) and the genus Dipsas 4.5 Comparison of hunting skills Approximately (Peters, 1960). According to Peters (1960) loss of the 180 known snake species consume mollusks as their mental groove adds rigidity for the slug-eating snakes. diet (Laporta-Ferreira and Salomão, 2004). Among And the rigidity perhaps compensates in part for the (revised after Pyron et al., 2011, 2013), increased mobility of all the toothed bones. Though except the well-known family Pareatidae and many Dipsas species do not consume mollusks exclusively species from Dipsadinae (Peters, 1960), species in (Ray et al., 2012), their common diet with Pareatidae Lamprophiidae (Duberria, Gans, 1975) and Natricinae displays their convergence to some extent. Given the (Storeria, Judd, 1954, Rossman and Myer, 1990; distance between S. ningshaanensis and the Dipsadini Thamnophis, Britt et al., 2006, 2009;) were documented species (Figure 1), phylogeny reconstructed by many as malacophagous predators. Although all genera previous works (Pinou et al., 2004; Sheehy, 2012; Vidal mentioned above include species able to hunt slugs, et al., 2010; Zaher et al., 2009; Pyron et al., 2013), S. preying on snails is not handled by all of them. The ningshaanensis might have obtained this diet and the exclusive snail-eater Pareas developed high specialization related characters independently in the Dipsadinae. on extracting the snail from the gastropod helical shell (Gotz, 2002; Hoso and Hori, 2008; Hoso et al., 2007). In Acknowledgements We are grateful to Huoditang the Dipsadinae, inserting mandibles are also employed Forestry Station and Shennongjia Nature Reserve for by Dipsas (Gans, 1975; Peters, 1960; Sazima, 1989) allowing us to collect specimens. We would like to thank and Sibynomorphus (Agudo-Padrón, 2013; Laporta- Xianhua TIAN, Lei PAN, Junhai XU, Yang YU and Ferreira and Salomão, 2004), but the morphological and Shanzhong GONG, Dong XIE, and Linsen YANG for behavioral asymmetry in Pareatidae for efficient handling their great help in the field work; Cong WEI and Hong dextral shell has not been documented. Beside this, “snag HE for their effort in searching the preserved specimens in and drag” strategy is another method used by Sibon and NAFU; Guanfu WU, Ke JIANG, Fang YU, Chen WANG, Tropidodipsas (Sheehy, 2012), and now it is known from Wenqing ZHOU, Xiuwei LIU and Tianjuan SU for the S. ningshaanensis. Similarly, the “snag” is also used by help with experimental skill and laboratorial technique; (Rossman and Myer, 1990) — “substrate” Jiatang LI and Robert MURPHY for their advice on the and “torsion.” The long time holding and searching of S. structure of this article; Sergey RYABOV for collecting ningshaanensis was very similar as for Storeria when no and identifying different mollusks. We thank Yujia snag was offered: “the snakes pushed the snails around ZHANG and Robert SCHLEICHER for their kindly the terrarium for more than 30 min before abandoning the revision on the language of this manuscript. We also thank feeding attempt”. the anonymous reviwers for their precious comments and suggestion to improve the quality of the paper. 4.6 The characters associated with the feeding behavior Slender teeth are required for grasping the References mollusks with efficiency in spite of their slimy secreta. This character is very common among the malacophagous Agudo-Padrón A. I. 2013. 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