Systematic review of the land snail genus Neocepolis Pilsbry, 1891 (Pulmonata: Camaenidae) from north Vietnam

By examination of the shell, radula and genital morphology and comparison with type material of the two known species, we reassess the status of Neocepolis Pilsbry, 1891, a poorly known genus of Vietnamese camaenids. Previously recognised solely on the basis of shell characters, we identify features of genital morphology that confirm the specific status of Neocepolis meracha (Mabille, 1888) and N. morleti (Dautzenberg and d'Hamonville, 1887) and the validity of Neocepolis as a distinct genus within a clade that includes Camaena and Camaena (Camaenella).


Introduction
The Camaenidae exemplify the land snail fauna of Indochina in being diverse and rich in endemic species (Vermeulen & Maassen 2003). The family is also representative of the regional fauna in being little studied and poorly known. This particularly applies to the endemic camaenid genus Neocepolis Pilsbry, 1891, which consists of just two described species for which relationships with other camaenid genera are unknown. Early studies on Neocepolis were conchological investigations largely carried out by nineteenth century French naturalists such as P. Dautzenberg, H. Fischer, L. d'Hamonville and J. Mabille, and the group has not been revised for about 100 years. Material from recent surveys in North Vietnam allowed us to review the status of Neocepolis and examine features of the internal anatomy for the first time. We consider that it is important to set our findings on record as a step towards gaining an understanding of this difficult group.

Material and methods
Snails were sampled from several localities in northern Vietnam. Adult shells were measured for height (H), diameter (D) and whorl count (Kerney & Cameron 1979). Shell height/shell width ratios h/d ratio) were calculated as a measure of shape (Kerney & Cameron 1979). Radulae were extracted and examined under a scanning electron microscope (JEOL, JSM-5410 LV), recording shape and teeth formulae, following Sutcharit and Panha (2006). Features of the genitalia were examined in at least three examples of each of the two species.

Diagnosis
Shell brown to corneous, globose to depressed, dextral, umbilicate or rimate; surface with striate growth lines. Aperture slightly rounded and descending; lip thickened and expanded; parietal callus thin and transparent. Columella straight, slightly dilated and with obtuse tooth; typically with strong fold inside outer lip, represented externally by a deep longitudinal groove. Genitalia possess large or small penial verge, long penis and vagina, and short epiphallus and flagellum. Radula possesses a unicuspid and triangular central tooth, bicuspid lateral teeth, tricuspid marginal teeth; ribbed jaw. Pallial anatomy sigmurethrous.
Neocepolis merarcha differs from N. morleti in exhibiting a slightly more dome-shaped, brownish shell, slightly narrower umbilicus and with a groove marked on the outside wall. The genitalia are very similar but, on the basis of material examined by us, N. merarcha possesses a slightly shorter atrium, smaller penial verge and penial wall without the transverse pilaster present in N. morleti.

Measurements
See Table I.

Shell
Shell globose, opaque and rimate; light brown; periostracum thin; surface with regular ribs, rather smooth below periphery. Embryonic shell small, surface smooth ( Figure 2A). Spire dome shaped; apex obtuse; sutures shallow but slightly more impressed on last whorl. Whorls 6K, slowly widening and increasing regularly. Last whorl evenly rounded or with very slight peripheral keel. Close to aperture, a transverse groove below the periphery corresponds with lamella inside outer lip. Aperture round, internally brownish. Peristome subcircular, subvertical, descending; parietal callus transparent. Lip whitish, expanded, slightly thickened with short reflection. Columella curved, brownish, expanded across umbilicus, and an obtuse columella tooth, marked externally by a shallow pit ( Figure 1A, B).

Pallial system
Typically sigmurethran and without mantle gland ( Figure 4A). Heart (auricle (au) and ventricle (ve)) located left of kidney (on right in figure). Pulmonary cavity approximately five times longer than broad. Pulmonary vein (puv) and venation on lung (l) roof distinct and well developed. Kidney (k) elongated and slender, and extending from posterior of  cavity for approximately one-third length of pulmonary cavity. Ureter (ur) sigmoid, closed tube arising from apex of kidney, extending along right side of kidney, recurving adjacent to rectum (r). Anus (a) adjacent to mantle collar (mc) ( Figure 4B).

Genitalia
Atrium (at) short (n54). Penis (p) cylindrical, long and reaching similar length to vagina; proximally enlarged; distally tapering to narrow tube. Epiphallus (e) narrow and short; flagellum (fl) slightly longer than epiphallus. Vas deferens (vd) a long and small tube, extending from free oviduct to distal epiphallus. Penial retractor muscle (pr) long and slightly thickened ( Figure 5B). Internally, penial verge (pv) small, conical and long; surface with thin longitudinal ridges and wrinkles. Penial wall ribbed, forming a series of swollen longitudinal pilasters (pp); proximally, wall relatively smooth. Smooth pilasters occupy the middle of the penial chamber and encircle tip of penial verge. ( Figure 5C).
Vagina (v) a cylindrical tube held in position by series of muscle bands rising from foot floor. Gametolytic duct (gd) long; proximally broader and cylindrical; distally tapering to small, long tube and terminating with swollen gametolytic sac (gs). Free oviduct (fo) relatively long; oviduct (ov) enlarged with curled lobules. Albumen gland (ag) curved ligulate ( Figure 5B). Hermaphroditic duct (hd) convoluted and connected to head of talon (ta) ( Figure 5D).

Other features
Preserved specimens of N. merarcha exhibit a long and narrow foot, blackish integument and mantle cavity, dorsally a pale stripe runs along the entire body length. The lung roof exhibits a uniform distribution of black pigmentation. All examined specimens exhibited a longitudinally divided head wart, located between the superior tentacles.

Distribution
Neocepolis merarcha has a restricted distribution, early records were from Ha Long, Lang Son and That Khé, northern Vietnam (Mabille 1888; Pilsbry 1891; Dautzenberg & Fischer 1908). Our material was collected from Cuc Phuong National Park, Ninh Binh Province in a limestone karst area covered with evergreen forest, located about 160 km south of Hanoi.

Remarks
Neocepolis merarcha as understood by us, exhibits a wide range of shell form, associated with several named ''varieties'' of uncertain status based on degree of shell elevation and nature of apertural barrier (Dautzenberg & Fischer 1908). The material collected by us closely corresponds with the nominotypical form, distinguished from the other five ''varieties'' by possession of an elevated or dome-shaped shell and obtuse columella tooth (Mabille 1888;Dautzenberg & Fischer 1908). Additional material is needed before the status of these ''varieties'' can be assessed meaningfully. One specimen exhibited 22 mite-cysts on the lung roof and distributed from around the kidney base, being most abundant proximal to the pneumostome.

Measurements
See Table I.
Jaw with strong and prominent vertical ribs, variable in number, very thin transverse growth lines present ( Figure 5E).

Pallial system
Typically sigmurethrous; the structure of kidney and lung similar to that of N. merarcha.
Atrium relatively long (n53). Penis long cylindrical; proximally enlarged and curved at penial verge; distally tapering to narrow tube. Epiphallus (e) narrow and short; flagellum (fl) relatively long, almost the same size as epiphallus. Vas deferens (vd) a small tube extending from free oviduct to distal epiphallus. Penial retractor muscle relatively long ( Figure 5F).
Internal wall of penis corrugated and exhibiting a series of thickened and swollen transverse and longitudinal pilasters, which form a fringe around penial verge tip. Penial verge very large, long conic, curved and with nearly smooth surface. Longitudinal pilasters proximal to genital orifice possesses thin to nearly smooth ridges ( Figure 5G).
Vagina (v) narrow, cylindrical and longer than penis, held in position by series of muscles originating from foot floor and inserted on external wall of vagina. Gametolytic duct (gd) long, extending from vagina as a proximally enlarged cylindrical tube; abruptly tapering distally to narrow tube connected to gametolytic sac (gs). Free oviduct (fo) relatively long; oviduct (ov) compact and enlarged to form lobular alveoli. Albumen gland (ag) curved ligulate ( Figure 5F). Hermaphroditic duct (hd) contracted, convoluted and connected proximally to talon head (ta) ( Figure 5H).

Distribution
N. morleti was previously known only from along the road between Bach Ninh and Lang Son Provinces (Dautzenberg & d'Hamonville 1887;Mabille 1887;Pilsbry 1891). Our specimens were collected from Huu Lien Nature Reserve, Lang Son Province, a limestone karst area about 90 km north of Hanoi, and from Cuc Phuong National Park, Ninh Binh Province, about 160 km south of Hanoi.

Remarks
Pilsbry (1891) considered that N. morleti was closely related to Camaena platyodon (Pfeiffer, 1846); the two species sharing the similar shell characters of a depressed shell, toothed columella and descending aperture. Our observations of the genitalia, especially the long penis, large penial verge, triangular central tooth, and ribbed jaw are consistent with there being a close relationship between these two species. However, N. morleti differs from C. platyodon in having a quite distinct shell form which is monochrome with a ribbed surface, a longer vagina and a transverse penial pilaster (Pilsbry 1891;this study).
The body and head wart in the preserved specimens of N. morleti are similar to the former species in almost all characters. Only the slightly light brownish skin and lung roof differ from that of the N. merarcha.

Taxonomic affinity of Neocepolis
Conchologically, Neocepolis and Camaena share a descending aperture, reflected lip and elevated spire. Pilsbry (1894) placed Neocepolis as a subgenus of Camaena and suggested it might be closely related to Camaena (Camaenella) or Obba. A comparison of morphological characters of Camaena, Camaena (Camaenella), Neocepolis and Obba is given in Table II. Absence of penial verge, epiphallus longer than penis, smooth jaw, nearly flat shell, horizontal and deep descending aperture, continuous lip, and feather-like glandular appendix (sensu Pilsbry 1894, p. 108) are important characters in distinguishing Obba from Camaena, Camaena (Camaenella) and Neocepolis. The presence of a penial verge, epiphallus shorter than penis, ribbed jaw, depressed to conic shape, discontinuous aperture, and an Indochinese distribution suggest a close relationship between Camaena, Camaena (Camaenella) and Neocepolis. However, an aperture bearing a small columella tooth provides only an apparent affinity between Neocepolis and Camaena (Camaenella) and the resemblance is probably due to homoplasy. In Neocepolis the columella tooth corresponds to an external depression and the parietal callus is thin. In Camaena (Camaenella) the columellar tooth is squarish, a corresponding external depression is absent and the parietal callus forms a thick cord.
Although camaenids are considered to be an ancient group with a fossil record extending to the Lower Cretaceous (Smith et al. 2004), camaenids have a generally conservative range of morphological forms. Although species often exhibit considerable variation, subtle differences have been interpreted as having more significance in camaenid systematics than they might have in other groups and have been widely used to form the basis for recognising camaenid genera (Zilch 1960). We accept that this is a valid approach and on the basis of current evidence consider that Zilch (1960) Richardson (1985) and Vaught (1989) should be followed in recognising Neocepolis as a distinct genus. However, the internal anatomy and radular morphology of most species attributed to these groups are poorly known. More species need to be examined and molecular methods applied before the status of these camaenid genera and phylogenetic relationships between them can be fully evaluated.