Revision of three camaenid and one bradybaenid species (Gastropoda, Stylommatophora) from China based on morphological and molecular data, with description of a new bradybaenid subspecies from Inner Mongolia, China

Abstract We have revised the taxonomy of three camaenid and one bradybaenid species from China and described one new subspecies of the genus Bradybaena (Family Bradybaenidae) from Inner Mongolia, China. The genitalia of three Satsuma (Family Camaenidae) species S. mellea stenozona (Moellendorff, 1884), S. meridionalis (Moellendorff, 1884), comb. n. and S. uncopila (Heude, 1882), comb. n. assigned to the genus Bradybaena previously,lack a dart sac and mucous glands. Moreover, the molecular phylogeny has revealed close relationships between the three species and the genus Satsuma. Two species, S. stenozona (Moellendorff, 1884) from Fuzhou and Ganesella citrina Zilch, 1940 from Wuyi Mountain, are considered as synonymous and should be a subspecies of S. mellea mellea (Pfeiffer, 1866) because of the morphological and molecular similarities. Meanwhile, the other two are placed in the genus Satsuma: S. meridionalis (Moellendorff, 1884), comb. n. and S. uncopila (Heude, 1882), comb. n. G. virgo Pilsbry, 1927 differs from species of the genera Ganesella and Satsuma not only in its shell, but also in anatomical characters, such as having a dart sac and mucous gland, and lacking a flagellum. Additionally, phylogenetic analyses highly support the sister relationship with other Bradybaena species. Thus, placement of G. virgo Pilsbry, 1927 in the genus Bradybaena issuggested.


Introduction
The land snail families Camaenidae and Bradybaenidae are extremely specious, and both families are difficult groups to deal with in terms of taxonomy. The camaenids occur across a wide geographical area from the northern to southern hemisphere, such as China, Japan, Taiwan, Philippines, Indonesia, New Guinea, Australasia, America (Scott 1996;Cuezzo 2003). There is no special synapomorphy which is characteristic of this group (Scott 1996). Usually, they are defined by the absence of dart sac and dart-related organs on the female genitalia (Pilsbry 1939). Morphological studies and molecular phylogeny are contradictory about the monophyly or paraphyly of the group (Scott 1996;Cuezzo 2003;Wade et al. 2007). The bradybaenids have maximal diversity in Southeast and East Asia, Northwest America and Europe, including China, Russian Far East and Siberia, Japan, Korean, Taiwan, Philippines, Indonesia (Pilsbry 1900;Zilch 1959Zilch -1960Minato 1985;Chang and Hwang 2000;Hsieh et al. 2006). Generally, the bradybaenids are identified based on the presence of dart sac and dartrelated organs, however, some studies have suggested that the absence of dart-related organs occurred in a number of lineages (Davison et al. 2005;Wade et al. 2007;Hirano et al. 2014). Moreover, certain incongruence among the morphology, taxonomy, and molecular phylogeny of the bradybaenid land snails has been found (Hirano et al. 2014). These studies indicate that traditional morphology-based systematics may largely stray from molecular phylogeny, hence, the combination of morphology, anatomy and molecular studies is quite essential in biological classification.
The traditional classification of Ganesella relies predominantly on shell features. Purportedly characteristic features are, for instance, a thin, high, lustrous and conical shell, white to pale brown shell color, and a slightly descending body whorl (Yen 1939;Zilch 1966;Chen and Gao 1987). Most Chinese Ganesella species are conchologically more similar to Satsuma in having a conical to depressed conical shell of corneous color. Chinese species are often confused with the genera Plectotropis Martens, 1860, Aegista Albers, 1850 and Bradybaena Beck, 1837 of Bradybaenidae owing to the morphological similarity of shells. Clearly, our knowledge of the Chinese species remains comparatively poor (Zhou et al. 2011). In order to contribute to a better understanding of their taxonomy, three species from China currently placed in Bradybaena and one species currently placed in Ganesella are revised on the basis of morphological, anatomical and molecular evidence. One new subspecies of the genus Bradybaena from Inner Mongolia, China is described for the first time.

Material and methods
Material. This study is based on material collected by the authors from several sites in China (Fig. 1). Live adults were drowned in water for 12-24 hours, then killed in hot water, preserved in 75% or 95% ethanol, and stored at -20°C. Samples have been deposited in the State Key Laboratory of Molluscan Quarantine and Identification, Fujian Entry-Exit Inspection & Quarantine Bureau, Fuzhou, China (FJIQBC).  (Pilsbry, 1927) 6 Bradybaena virgo mongolia subsp. n. Methods. Shells were measured to 0.1 mm using electronic calipers. Standard shell parameters were taken following Dillon (1984). More than 15 specimens of each species were measured.
Genitalia of adult snails were dissected under a dissecting microscope (ZEISS Discovery V20). All drawings were traced with the aid of a Canon 550D digital camera. Terminology for reproductive system follows Gómez (2001). More than three specimens of each species were dissected.

Results and discussion
Reproductive system. Penis slender, with a short penial caecum near the penis retractor. Epiphallus as wide as penis, half as long as penis. Flagellum short, about 1/5 of length of epiphallus. Penis retractor muscle thin and long. Vas deferens short. Free oviduct moderately long, slightly inflated. Vagina short. Pedunculus of bursa copulatrix inflated at base, fusiform. Bursa copulatrix oval.
Ecology. One of the collected sites, Yuhua Hole, Jiangle, Fujian belongs to a Karst land form (limestone), all others are on Danxia land forms (acidic soil). Snails generally live under rotten branches and fallen leaves in forests, and actively crawl on trees during rainy seasons. Population density is generally not high in these locations. In Fuzhou, snails become active in early April, brisk in May and June, lie dormant in the soil by the end of October; juveniles and eggs aestivate during winter. Newly hatched snails will grow into adult in 7-8 months, then mate and spawn, about 100-200 eggs at once. Eggs are large, 1.5-2.0 mm in diameter.
Remark. This species has been placed in Bradybaena for a long time. Based on a study of the types, Zilch (1966) transferred it to the genus Ganesella, assuming a close relationship with G. mellea mellea (Pfeiffer, 1866) (=Satsuma mellea) from Taiwan and G. citrina Zilch, 1940 from Wuyi Mountain. However, his classification was not refuted subsequently (Wu 1999;Chen and Zhang 2004). Eventually, this species was classified as a member of the genus Satsuma by Zhou et al. (2011) for a lack of accessory sac as well as mucous gland, but the authors didn't provide any molecular evidence.
In the present study, the phylogenetic analyses based on COI showed close phylogenetic relationships and short genetic distances between specimens identified as S. stenozona, G. citrina and S. mellea (Fig. 5). The shell features of S. stenozona from Fuzhou and G. citrina from Wuyi Mountain do not reveal obvious differences. The differences mentioned by Zilch (1940), such as the shell dimensions and color bands, are mere variations between individuals and populations. The molecular phylogeny also indicated that S. stenozona and G. citrina were sister taxa. Therefore, we consider G. citrina a synonym of S. stenozona. S. mellea and S. stenozona may be considered as geographical races of the same species for the rather low amounts of morphological and molecular difference (Chang 1981;Zhou et al. 2011). Hence, we classified S. stenozona as a subspecies of S. mellea. (Moellendorff, 1884) Shell. Sinistral, medium sized; about 11.0 mm in height, 15.2 mm in width, thin but solid, yellowish-brown in color, depressed conic; 5 1 / 2 whorls. Surface with dense growth lines and weak spiral lines. Spire slightly low conical, slowly increasing, slightly convex. Body whorl fast expanding, quite convex. With slight, slender and dull red band on periphery of body whorl for most specimens. Periphery bluntly angulated. Aperture descending and elliptical. Peristome thin, sharp, slightly reflected. Inner lip with thin callus. Columellar lip short, reflected, slightly covering umbilicus. Umbilicus deep, round, and about 1/5 of width of shell.

Satsuma meridionalis
Reproductive system. Penis thick and short, with an expanded base. Penial caecum short. Epiphallus slender, about 2/3 of length of penis. Flagellum short and small, about 1/10 of length of epiphallus. Penis retractor muscle thick and wide. Vas deferens long and slender. Oviduct thin. Vagina longer than penis, expanding at posterior end. Pedunculus of bursa copulatrix expanding at base. Bursa copulatrix oval.
Ecology. The species usually lives in the wet bushes and grass near farmland, especially on limestone cliffs and in cracks with more humus, or under rotten branches and fallen leaves; occasionally within human settlements. This snail is sensitive to low temperature, aestivates from November to March. Animals often feed on all kinds of crops, especially tender shoot and leaf.

Satsuma uncopila
Shell. Sinistral, medium sized, about 11.5 mm in height, 16.8 mm in width, thin, fawn colored, conical. Whorls 5. Surface with short and diagonal growth lines, and weak spiral lines. Spire higher. Body whorl fast increasing, expanding but not descending at the front. Periphery smooth, not convex. Apex obtuse. Suture deep. Aperture elliptical. Peristome slightly thickened, reflected, white, occasionally reddish-brown. Columellar lip reflected, slightly covering umbilicus. Umbilicus narrow and small.
Ecology. The snail ordinarily lives in the wet bushes and grass on hills, especially in places that are rich in humus, under rotten branches and fallen leaves; also frequently found on limestone cliffs and in cracks.
Ecology. The snail often lives on damp pastures, especially near ditch, or in grass.
Remark. This species is the first intermediate host of Eurytrema pancreaticum, a parasite of humans and livestock (Tang et al. 1979;Tang et al. 1980;Gu et al. 1990).
Recently, several studies on bionomics and control measures of the snail have been published. However, the taxonomic status has been unclear (Zhu et al. 1989;. Originally assigned to the Camaenidae, G. virgo has subsequently been transferred to the Bradybaenidae based on anatomical and shell features by Kuroda (1941Kuroda ( , 1949 and Habe (1956). This treatment, however, has been widely neglected by Chinese workers. In the present study, we dissected several specimens collected in Haerbin and Jidong, Heilongjiang, and found that anatomical characters were in concordance with the description of Kuroda (1941Kuroda ( , 1949 and Habe (1956). In addition, the molecular phylogeny confirmed close relationships with other species in Bradybaena. Thus, G. virgo is correctly placed in Bradybaena. Etymology. For the type locality, adjective.
Description. Dextral, small sized, thin but solid, semi-translucent, lustrous, globular. Whorls 6 on average, with conical spire. Shell light yellow or white in color, with some dense and well-developed growth lines. Spiral lines on body whorl weak. Apex sharp. Suture deep. Last whorl constricted, expanded towards the base, convex, comprising about 3/4 of shell high. Aperture elliptical. Peristome reflected, with white, thickened callus inside. Inner lip and columellar lip reflected, partly covering umbilicus. Umbilicus narrow, deep.
Reproductive system. Penis long. Flagellum absent. Penis retractor muscle slender, moderately long. Vas deferens moderately long. Oviduct short and thick. Vagina short. Dart sac inflated, thick. Accessory sac small. Two mucus glands. Pedunculus of bursa copulatrix slender, but not long. Bursa copulatrix oval.
Ecology. The snail usually lives on damp pastures, especially in tall and dense grass, i.e., Achnatherum splendens. However, it is difficult to collect this animal because of serious grassland degradation in Inner Mongolia.
Remark. The new subspecies resembles B. virgo virgo in morphology, but the two subspecies can be differentiated by the following characteristics: (1) The subspecies mongolia has a smaller shell (shell height 5.5-7.0 mm, width 6.4-7.5 mm) than B. virgo virgo (shell height 12.0 mm, width 13.5 mm), (2) it has two mucus glands instead of one in the nominate form, and (3) its umbilicus is wider (about 1/9 of the shell width) than in the nominate form (about 1/12 of the shell width).

Molecular analysis
Twenty-three partial sequences of COI were analyzed. The aligned sequences contained no indels and were deposited in GenBank (Table 1). The molecular phylogeny was based on the analysis of 615 unambiguously aligned nucleotide sites, of which 253 were variable and 233 were parsimony informative. According to the Akaike information criterion, the general time reversible model with a proportion of invariable sites and a gamma shaped distribution of rates across sites (GTR + I + G) was the best-fitting model of sequence evolution. All other settings for ML analysis were kept as default.
The ML tree (Fig. 5) presented two major clades corresponding to the families Camaenidae and Bradybaenidae, respectively. B. virgo virgo originally classified in Ganesella belonged to a clade of taxa in Bradybaena, and this agreed with the anatomical result. Thus the placement of this species in Bradybaena is suggested.
The clade of taxa in the family Camaenidae contained three subclades, Satsuma species from Taiwan in group A, S. largillierti from Japan and species from southeast China and north Taiwan in group B. In addition, species in group B were divided into two subgroups, including subgroup B1 with sinistral shell and subgroup B2 with dextral shell, and this is consistent with the study on the reproductive system above. Therefore, the two species B. meridionalis from Luofushan, Guangdong and B. uncopila from Hangzhou, Zhejiang in subgroup B1, which were originally classified in Bradybaenidae, should be assigned to the family Camaenidae. On the other hand, S. stenozona from Fuzhou and G. citrine from Wuyi Mountain in subgroup B2 appeared monophyletic. There were low amounts of morphological difference between species from Fujian and S. mellea from Taiwan with geographic isolation. In view of the above, the two taxa from Fujian are revised as a subspecies of S. mellea (Fig. 5).
In the present study, three camaenids and one bradybaenid from China were revised on the base of morphological and molecular characters, but the systematics of the remaining Chinese species in the superfamily Camaenoidea are still problematic. Camaenids and bradybaenids may be more complex than we have previously suspected. In the future, more samplings will be required to resolve this problem.