Conchological differentiation and genital anatomy of Nepalese Glessulinae (Gastropoda, Stylommatophora, Subulinidae), with descriptions of six new species

Abstract Eleven species of Glessulinae belonging to the genera Glessula Martens, 1860 (three species) and Rishetia Godwin-Austen, 1920 (eight species) are reported from Nepal, six of which are new to science and are described here, viz., G. tamakoshi Budha & Backeljau, sp. n., R. kathmandica Budha & Backeljau, sp. n., R. nagarjunensis Budha & Naggs, sp. n., R. rishikeshi Budha & Naggs, sp. n., R. subulata Budha & Naggs and R. tribhuvana Budha, sp. n. and two are new records for Nepal viz. G. cf. hebetata and R. cf. mastersi. The relation between the shell height-width ratio and the structure of the proximal part of the male reproductive organs in Glessulinae is explored. Illustrations and a key for the identification of the Nepalese Glessulinae are provided, including the first record of a spermatophore in Rishetia.

Snails were drowned in water and preserved in 90% ethanol, which was changed at least once within a week after collection. All new material is deposited in the Central Department Zoology Museum of Tribhuvan University (CDZMTU), Nepal. The numbers of shells in the samples are indicated after the registration numbers. The letter 'P' indicates that the sample consists of live-collected specimens preserved in ethanol. Illustrations were made using a camera lucida mounted on a stereomicroscope. Features of the interior of the penis were drawn from photographs by Rajman Maharjan (Natural History Museum, Kathmandu). Whorls were counted and shells were measured (in mm) with electronic digital callipers as described by Kerney and Cameron (1979). All descriptions in this paper are based on material collected in Nepal.
We avoided the shell terms 'protoconch' and 'teleoconch' because the demarcation between these shell parts is unclear in glessulines. So we used the first whorl and second whorl for 'protoconch'and the other whorls for 'teleoconch'. The term 'apical whorls' is used for the first three or more whorls together. The terminology of the reproductive organs was modified from Tompa (1984). We arbitrarily differentiate the proximal parts of the male reproductive organs as 'epiphallic caecum', being the structure positioned close to where the vas deferens joins the epiphallus, and as 'flagellum', being the other sac-like structure connected to the epiphallus. The internal surface of the penis is referred to as smooth or folded to avoid ill-defined functional terms such as 'stimulator' and 'pilaster'. In this paper we refer proximal part toward the free end of the male genitalia and the distal part closer toward genital orifice.
Type and other reference material was examined in the collections at the Natural History Museum, London (NHM), the University Museum of Zoology, Cambridge (UMZC), the Royal Belgian Institute of Natural Sciences, Brussels (RBINS) and the Zoological Museum of Moscow State University (ZMMU), Moscow, Russia.
Genitalia (n = 1) (Fig. 4). Vas deferens widens towards the spermoviduct and opens into the terminal part of the small, pear shaped epiphallus. Flagellum comb-like with numerous notches in the comb, the terminal notch comparatively wider. The basal end of the penis cylindrical, widening from the middle to the proximal end (Fig. 4). Vagina very short, nearly 1/5 th of the length of the penis. The penial retractor muscle close to the flagellum. The gametolytic sac is elongated, connected to the gametolytic duct by a narrow neck. The convoluted mass of the hermaphroditic duct is thick and compact and the albumen gland in the dissected samples is short.
Remarks. The sculpture, thick peristome and size of the Nepalese specimen match with the original description of G. hebetata Godwin-Austen, 1920 (p. 49, pl. 162, fig. 26). The genitalia suggest that G. cf. hebetata is closely related to G. ochracea Godwin-Austen, 1918, andG. orophila (Reeve, 1849), all sharing a similar comb-shaped flagellum. Yet, the shell of G. ochracea is larger (SH 21.25,SW 9.25) and shows a sharper striation (Godwin-Austen 1920). In contrast, with respect to G. orophila, Godwin-Austen (1920, p. 4) questioned the identity of the genitalia figured under this name by Semper (1873, pl. 12, fig.14-16). Hence, there is no other comparative data available with respect the genital anatomy of G. cf. hebetata, including of G. hebetata itself. However, the shell of the single specimen of putative G. hebetata from Nepal looks similar to the figure of Godwin-Austen (1920, p.49, pl. CLXII, fig. 26) which is why this specimen is referred to as G. cf. hebetata. Distribution. Only known from the type locality. Etymology. The species name refers to the type locality Tamakoshi River valley. Shell. Measurements (n = 4): SH 17.7-19.5 mm, SW 9.2-9.6 mm, HA 8.6-8.8 mm, WA 4.9-5.0 mm, Wh 7.0-8.0; holotype: SH 19.5 mm, SW 9.6 mm, HA 8.8 mm, WA 4.9 mm, Wh 7.8. The largest shell measured 19.5 mm, approx. 2× higher than wide, solid, ovate-conic, light yellowish. Surface glossy, with widely spaced incised radial striations, stronger towards the suture and faint at the middle to lower part of the whorls. The first whorl smooth, blunt with fine and dense striations near the suture. Sides convex, suture fairly impressed. Aperture nearly ovate, 1.7× higher than wide, peristome simple and thick, columellar margin abruptly truncate, columella slightly curved. Radula (Fig.13A). Teeth pointed, lateral cusps not distinct. Central tooth smaller and with a narrower base than the lateral teeth.

Glessula tamakoshi
Genitalia (n = 3) (Fig. 5). Vas deferens with a constant diameter. Flagellum wide comb-like with numerous notches ending in a short hook at the free end. Vagina very short nearly 1/6 th length of the penis. Gametolytic sac is oval, connected to the gametolytic duct by a short neck. The penial retractor muscle close to the flagellum. The mass of the hermaphroditic duct highly developed in all dissected samples. Interior of penis with two bulky masses of 'brain-like folds' (Fig. 5B).
Remarks. Conchologically, G. tamakoshi sp. n. is similar to South Indian G. indica Gude, 1914 (genitalia unknown) and Sri Lankan G. serena (Benson, 1860). Yet, G. indica has a much wider shell and relatively more whorls (>9 whorls), while the penis of G. serena contains one longitudinal fold and two folds proximal to this longitudinal fold. Schileyko (1999, p. 542, fig. 711) referred the longitudinal fold to as pilaster and the two proximal folds to as the spiral stimulators. In G. tamakoshi sp. n. the interior of the penis contains a bulky masses of brain-like folds (Fig. 5B).
Genitalia (n = 5) (Fig. 6). Flagellum very short tubular sac; there is a very short knob-like epiphallic caecum. Penis cylindrical, basal end comparatively narrow, swollen at the middle and proximal portions cylindrical. Epiphallus basally swollen and the proximal portion tapering. The penial retractor muscle far apart from the flagellum. Gametolytic sac balloon-like, separated from the gametolytic duct by a narrow neck. A mature dissected specimen contained 4-5 gelatinous eggs in the spermoviduct. Another mature specimen contained a spermatophore in the gametolytic sac. This is the first observation of a glessuline spermatophore (Fig. 6B). Vagina short, nearly half the length of the penis. Albumen gland elongate and yellowish; hermaphroditic duct thinner than the albumen gland in the observed specimens.

Rishetia kathmandica
Shell. Measurements (n = 17): SH 24.8-41.1 mm, SW 7.8-9.3 mm, HA 7.1-8.5 mm, WA 4.1-5.4 mm, Wh 11.0-13.0; holotype: SH 28.1 mm, SW 8.1 mm, HA 7.1 mm, WA 4.1 mm, Wh 12; approx. 4× higher than wide, elongately turreted, colour ruddy, regularly sculptured. The first whorl pointed ( Fig. 2E1) with distinct and regular radial riblets, more prominent on the first few whorls, ribs much stronger towards the suture; middle whorls of the shell show incised radial striation. Suture shallow, sides moderately flat. Aperture small, oval, 1.6× higher than wide, peristome thin, columellar margin slightly convex, thinly calloused, white. Radula (Fig. 13B). Central tooth very small, lacks cusps, lateral teeth tricuspid. Animal (Fig. 14E). Grey black, with dark tentacles. Body minutely papillate. The sole is lighter than the body. Genitalia (n = 5) (Fig. 7A-B). The flagellum is a long cylindrical sac; the epiphallic caecum is a simple sac, shorter than the flagellum. Penis cylindrical and longer than the epiphallus. The penial retractor muscle far apart from the flagellum. Interior wall of the penis with distinct rectangular zigzag folds (Fig. 7A). Three specimens contained 12-14 juvenile shells in the spermoviduct. Vagina about as long as the penis. Gametolytic sac pear shaped with a neck that is not distinct from the Gametolytic duct. Hermaphroditic duct is connected closer towards the free end of the albumen gland.
Genitalia (n = 2) (Figs 8A). Vas deferens long with a constant diameter. The flagellum is a simple sac. Epiphallic caecum is longer than the flagellum. Epiphallus much shorter than the penis. The penial retractor muscle far apart from the flagellum. Gametolytic duct slender, ending into a balloon-like gametolytic sac, which is slightly longer than the gametolytic duct. Vagina as long as the penis. Interior wall of the penis with a strong fold.
Remarks. A single specimen of Rishetia sp. was collected from the Shorea robusta forest at Bosikharka, Dhanding (CDZMTU078P). Its shell is similar to R. mastersi Godwin-Austen, 1920, but it is slightly smaller and has relatively stronger radial ribs. The shape of its flagellum and epiphallus is similar to that of R. mastersi too (Figs 8A, 8C), but the interior of its penis shows three longitudinal folds (Fig. 8D), whereas in R. mastersi there is only one longitudinal fold (Fig. 8B). Distribution. Nagarjun-Shivapuri National Park and Phulchowki Hill, Central Nepal. Etymology. The name is derived from the type locality Nagarjun Forest. Shell. Measurements (n = 4): SH 33.2-38.4 mm, SW 10.6-11.9 mm, HA 10.2-10.8 mm, WA 5.5-6.2 mm, Wh 10-11.0; holotype: SH 38.0 mm, SW 12.0 mm, HA 10.5 mm, WA 6.3 mm, Wh 11; approx. 3.3× higher than wide, elongately turreted, thick, reddish-brown or dark chestnut colour. Surface striated with obliquely raised riblets on the first 2-3 whorls, later whorls with comparatively thin and dense sculpture. The first whorl smooth, rounded. Sides rather flat, suture shallow. Aperture ovate, 1.8× longer than wide, peristome thin, inner side of the aperture thickened and whitish, columella slightly truncate at the base. Radula (Fig. 13E). Central tooth very small, lacks cusps, lateral teeth shouldered. Genitalia (n = 2) (Fig. 9). All specimens have a long vas deferens. Flagellum and epiphallic caecum are reduced to two very short knobs (Fig. 9A). Penis cylindrical, with a nearly constant diameter in its proximal half, distally it rapidly expands into a bulbous section before the diameter contracts to somewhat less than the proximal penis, narrowing slightly distally. The penial retractor muscle far apart from the flagellum. The inner surface contains weakly convoluted folds (Fig. 9B). Epiphallus length about 1/3 of penis length. Vagina nearly as long as the penis. Gametolytic duct cylindrical, ends into a simple gametolytic sac. Hermaphroditic duct is loosely convoluted and connected at the middle of the albumen gland.

Rishetia nagarjunensis
Remarks. The shell of R. nagarjunensis sp. n. is similar in size and shape to that of sympatric R. kathmandica sp. n. However, the shell of R. kathmandica sp. n. is more slender and has more whorls than R. nagarjunensis sp. n. Conversely, R. nagarjunensis sp. n. has a wider body whorl, a comparatively more robust shell, and stronger radial ribs than R. kathmandica sp. n. The genitalia of both species are consistently different due to the reduced knob-like flagellum and epiphalic caecum in R. nagarjunensis sp. n., as well as by the inner surface of the penis, which in R. nagarjunensis sp. n. shows three loosely convoluted folds, whereas in R. kathmandica sp. n. it shows distinct rectangular zigzag folds (Fig. 7A1).  Radula (Fig. 13C). Central tooth very small, lateral teeth tricuspid with the central cusp bifurcated.
Animal (Fig. 14E). Dark grey with a heavily papillated body. The foot is light, showing weak transverse lines on the sole.
Genitalia (n = 2) (Figs 10A-B). Vas deferens with a constant diameter over its entire length. Epiphallic caecum nearly as long as the flagellum. Epiphallus long, but shorter than the penis. The penial retractor muscle far apart from the flagellum. Vagina as long as the penis. Gametolytic sac is nearly round, separated by a neck from the gametolytic duct. Albumen gland very small in the dissected specimens. The internal surface of the penis smooth with several longitudinal convoluted folds (Fig. 10B).
Genitalia (n = 1) (Fig. 11). Vas deferens very long, enters into the epiphallus at some distance from the base of the flagellum. The flagellum is long and C-shaped. Epiphallic caecum very short. Epiphallus approx. as long as the penis. The basal part of the penis is comparatively wider than the proximal part. The penial retractor muscle far apart from the flagellum. Gametolytic sac elongated. The vagina is nearly as long as the penis. Albumen gland elongated and hermaphrodite duct is connected at the middle of the gland.
Remarks. R. subulata sp. n. is similar to R. hastula (Benson, 1860) and R. tribhuvana sp. n., but R. subulata sp. n. has a wider body whorl and more blunt first whorl than R. hastula, while its 2 nd and 3 rd whorls have equal diameters (unequal diameters in R. hastula) (Fig. 2 D1 and J1). The body whorl in R. subulata sp. n. is much wider than in R. tribhuvana sp. n. (Fig. 2 J and K). Moreover, the flagellum of R. subulata sp. n. much larger than the epiphallic caecum, whereas in R. tribhuvana sp. n. the flagellum and epiphallic caecum are of similar sizes. In R. hastula the flagellum and the epiphallic caecum are very small. Etymology. The name refers to the type locality, Tribhuvan University garden Shell. Measurements (n = 12): SH 7.5-11.0 mm, SW 2.7-3.0 mm, HA 2.5-3.0 mm, WA 1.4-2.0 mm, Wh 8.5-9.5; holotype: SH 10.1 mm, SW 3.0 mm, HA 2.5 mm, WA 2.0 mm, Wh 9; approx. 3.3× higher than wide, slender, thin, dull brown. Surface striated with radial fine ribs, much denser on the 3 rd and 4 th whorls; ribs widely separated on the penultimate and body whorls. The first whorl blunt and smooth. Spire rounded; suture deep. Aperture ovate elliptical; approx. 2× higher than wide, peristome thin; columellar margin calloused whitish, obliquely truncate at the base.

Rishetia tribhuvana
Genitalia (n = 4) (Fig. 12). Vas deferens long. Flagellum slightly shorter than the epiphallic caecum. The length of the flagellum nearly two-thirds of that of the epiphallus. The basal part of the penis narrower than the proximal part. The vagina nearly as long as the penis. The penial retractor muscle far apart from the flagellum. The gametolytic sac is rounded and distinct from the gametolytic duct. The hermaphrodite duct is connected at the base of the albumen gland.
Remarks. R. tribhuvana sp. n. is the smallest Rishetia species in Nepal. Conchologically, it is "intermediate" between R. hastula (Benson, 1860) and R. roberti Godwin-Austen, 1920. R. tribhuvana sp. n. differs from both these species by its weaker radial sculpture compared to R. hastula and its narrower body whorl compared to R. roberti. Moreover, the illustrations of Godwin-Austen (1920, pl. CLXIII, fig. 10) suggest that R. roberti has a rounded first whorl, whereas it is nearly flat in R. tribhuvana sp. n. The flagellum and epiphallic caecum are well-developed and nearly equal in size in R. tribhuvana sp. n., whereas in R. hastula the flagellum is very small and the epiphallic caecum is only a minute-knob. R. tribhuvana sp. n. and R. hastula are known from geographically different locations. The former is a hill species reported above 1300 m, while the latter is known only from the plain below 300 m elevation. R. roberti is also a hill species from Richila peak, Sikkim. The presence of R. tribhuvana sp. n. in the garden of Tribhuvan University is due to a historical connection between this garden and the Champadevi hill Forest, about 4 km south 300-400 m higher from the university premises. This connection has been lost because of human settlement.   First two whorls with spiral lirae (Fig. 2A1), flagellum hand-shaped (Fig. 3)  Adult shell height < 15 mm, flagellum comb-shaped, vas deferens becomes wider towards spermoviduct (Fig. 4) (Fig. 6) ... R. hastula 7(5) Epiphallic caecum longer than flagellum (Fig. 8)  Epiphallic caecum nearly as long as flagellum (Fig. 10) ... R. rishikeshi sp. n.

Discussion
Godwin-Austen (1920) differentiated Rishetia from Glessula sensu stricto on the basis of the male reproductive organs of four Glessula species G. ochracea, G. orobia var. major, G. orophila and G. inornata, and two species of Rishetia, R. longispira (type species) and R. garoense Godwin-Austen, 1920. He reported that Glessula has a comb-like flagellum and Rishetia has a simple sac-like flagellum. Probably based on the elongated shell of R. longispira he assigned several other slender and elongated Himalayan species to Rishetia. Therefore we explored the relation between shell form and genitalia in 10 species of Glessula and 10 species of Rishetia (Table 1). This suggests that the male reproductive organ of Glessula and Rishetia differs by: (1) the shape of the flagellum, being hand-or comb-shaped without an epiphallic caecum in Glessula (Figs 3-5) vs. a simple knob or tubular sac with an epiphallic caecum in Rishetia (Figs 6-12), (2) the penial retractor inserting close to the flagellum and epiphallus in Glessula vs. penial retractor inserting far from the flagellum near the penis/epiphallus junction in Rishetia, and (3) the very short epiphallus in Glessula vs. the comparatively longer epiphallus in Rishetia. Recent data on the genitalia of G. ceylanica (the type species of Glessula) shows that this species has a very distinct hand-like flagellum with a small thumb and five fingers (D. Raheem, pers communication). Taken altogether, it appears as if these genital characters are correlated with the shell height/width ratio, such that the shell height/width ratio in Glessula is < 2.5, while in Rishetia it is always > 2.5, at least in the species listed in Table 1. Also the shell sculpture tends to differ between Glessula and Rishetia, with Glessula usually having a somewhat weaker sculpture than Rishetia. The extent to which this putative differentiation between Glessula and Rishetia can be maintained when data on more species from a wider geographic range become available remains to be established. Finally, Schileyko (1999) recognised the Glessulidae Godwin-Austen, 1920 as a distinct family and introduced the Rishetiinae (with Rishetia as type genus) as a new subfamily within the Subulinidae. He also included the genera Eutomopeas Pilsbry, 1946, Tortaxis Pilsbry, 1906and Bacillum Theobald, 1870 within Rishetiinae based on the presence of some form of truncation of the columella. It remains to be decided whether this classification (and separation) of Glessula and Rishetia will stand a phylogenetic analysis. This study illustrated the spermatophore of R. hastula, the first observation of a spermatophore in Glessulinae. This observation adds to the accumulating evidence that in contrast to Tompa's (1984) suggestion that Achatinidae and their relatives do not produce spermatophores, these structures may be not uncommon in the Achatinoid clade of the Stylommatophora. Indeed, spermatophores have been reported in Achatinidae (Plummer 1975), Subulinidae (Baker 1945, Marcus 1968, Naggs 1994, Medeiros et al. 2013) and Streptaxidae (de Winter et al. 1999, Gerlach and van Bruggen 1999, de Winter and Vastenhout 2013, Rowson and Tattersfield 2013. Table 1. Qualitative correlation between shell height/width ratio and proximal part of the male genital parts in Glessula and Rishetia (n refers to the number of shells measured).