Taxonomic revision of the Elephant Pupinid snail genus Pollicaria Gould, 1856 (Prosobranchia, Pupinidae)

Abstract The status of species currently assigned to the Southeast Asian Elephant Pupinid snail genus Pollicaria Gould, 1856 is reassessed. Shell, radular and reproductive morphology are investigated and analysed with reference to karyotype patterns previously reported and to distribution patterns among the species. Six previously described species are recognised: Pollicaria gravida (Benson, 1856), Pollicaria myersii (Haines, 1855), Pollicaria mouhoti (Pfeiffer, 1862), Pollicaria elephas (Morgan, 1885), Pollicaria crossei (Dautzenberg & d’Hamonville, 1887) and Pollicaria rochebruni (Mabille, 1887). A new subspecies, Pollicaria mouhoti monochroma ssp. n.,is proposed and a dichotomous key to species is provided.


Introduction
Land operculate snails of the family Pupinidae generally possess a pupoid shell shape and exhibit a wide range of shell height from 5-50 mm. Apart from size, their often distinctive shells can also be distinguished from other members of the Cyclophoroidea by unique features of the genitalia, notably the long bursa copulatrix (Wenz 1938, Tielecke 1940. About 20 extant genera range from South Asia, East Asia to Southeast Asia, Melanesia, Micronesia and part of Australia (Solem 1959, Vaught 1989, Stanisic 1998, Stanisic et al. 2010. Fossil representatives are known from the European Cretaceous (Naggs and Raheem 2005) and British Eocene (Sandberger 1873). They generally occur in tropical forest and most commonly and abundantly in limestone areas. Fourteen pupinid genera have been recorded from Indochina (Kobelt 1902), including the very distinctive Elephant Pupinid genus Pollicaria Gould, 1856 which is endemic to the region.
Hitherto, nine nominal species of the Pollicaria have been described (Crosse 1885, Kobelt 1902, Gude 1921, Pain 1974. Pollicaria, as "Hybocystis", was first revised by Crosse (1885) and by Fischer (1885) who detailed the anatomy. Crosse (1885) recognized four species of Pollicaria and separated those species into two species groups, which are now unrecognized. Subsequently, two additional species were described from Vietnam (Dautzenberg and ďHammonville 1887, Mabille 1887a). These six nominal species were revised by Kobelt (1902) and more recently Pain (1974). Relying solely on shell morphology, Kobelt (1902) placed P. crossei into synonymy with P. rochebruni. Pain (1974), partly followed Kobelt's classification but recognized only three species: P. gravida (Benson, 1856), P. myersii (Haines, 1855) and P. elephas (Morgan, 1885), placing P. mouhoti into synonymy with P. myersii. However, Pain's study was of limited value because it was based on an examination of few specimens and populations and did not examine the type specimens. Hence the true status of species still remains unresolved. Apart from the studies of Crosse (1885) and Fischer (1885) none of the subsequent studies on Pollicaria have used anatomical data or studied type material.
The large shell size (up to 50 mm in height) and distinctive yellow to orange body colour render Pollicaria very distinctive and easily recognizable, although some confusion might arise from the helicoid shape exhibited by juveniles. The fact that populations are often widely scattered and highly localized may account for their having been little studied and consequently poorly known (Crosse 1885, Kobelt 1902. Recently, karyotypic studies and preliminary allozyme analysis (Kongim et al. 2009 have indicated that the species placed in synonymy by Kobelt (1902) and Pain (1974) should be recognized as distinct species.
Herein, we provide the first critical and comprehensive revision of Pollicaria based on a detailed morphological study of newly collected specimens and their comparison with type material.

Materials and methods
Snails were collected and distributions recorded, mostly from limestone areas throughout Thailand, Laos, Vietnam and Peninsular Malaysia. Species identifications were made by comparison with type material, primarily at The Natural History Museum (London), Muséum National ďHistoire Naturelle (Paris), and University Museum of Zoology Cambridge (Cambridge). Living snails were photographed before examining the external and internal morphological characters. Adult shells were measured for height, diameter and whorl number. Features of the genitalia were examined for between 5 to 10 individuals of each species. Radulae were extracted and examined using a Scanning Electron Microscope (JEOL, JSM-5410 LV), and radular teeth shape and formulae were described.
External features: As recorded in the literature, Pollicaria was found to possess a yellow-orange to pale orange body, usually with dark orange cephalic tentacles (Fig.  2). Body colour variation within species appeared to be largely confined to patches of dark-brown or blackish spots spread across areas of the head and dorsal foot. Such variation may be present between different populations or can occur on different growth stages within populations.
The foot (ft) is broad and short; cephalic tentacles (ct) long with dark eye spots (e) located at outer base; snout broad. Animal dioecious; genital groove present at right side running downwards from pallial gonoduct. Male with conical external penis (p) on the right side (penis usually broad and enlarged in breeding season) located below cephalic tentacles, and with seminal groove (sg) on penis (Fig. 3A); female with only genital groove on the right side disappear external penis. Operculum (op) attached to opercular lobe or disk posterior-dorsally on foot (Fig. 3B).
No external anatomical features were found to exhibit useful taxonomic characters. Internal anatomy: The internal anatomical description of P. mouhoti mouhoti collected form Tam Wungdang, Nern Maprang, Phitsanulok, Thailand serves as being representative of the genus. Kidney (k) a brownish lobule, constricted-triangular in shape. Heart located on the left side of kidney; pericardium (pcd) thin, atrium (at) slightly larger than ventricle (ven). Lung cavity (lc) with reticulated vessels. Stomach (st) embedded in dark brown lobulated digestive gland (dg). Rectum (rt) large, attached to genital apparatus (prostate gland or uterus), and tapering anteriorly to anus (an), which opens close to mantle collar edge. Mantle edge (me) smooth and slightly thickened. Columellar muscle (cm) large, broad, thickened and whitish ( Fig. 3A-D).
Testis (ts) with branched tubules, bright orange, occupying around 2-3 whorls from apex. Vas deferens thin and slender-straight tube attached to prostate gland at around two-third of its length proximal to external penis. Prostate gland (pg) large, long and slender, pale yellowish; proximally with genital opening. Seminal groove (sg) small, distinct and connected from genital opening on the right side of snail to external penis. External penis (pen) digitiform, short, located posteriorly, below cephalic tentacles (Fig. 3A, C).
Ovary bright orange multi-lobulated gland embedded in digestive gland. Pale yellow oviduct (ov) extends from ovary to uterus (ut) near the base of seminal receptacle. Bursa copulatrix (bc) cream to whitish long pouch that receives and digests the spermatophore case. Uterus (ut) large, curved pea-pod shape, posterior end rounded and anterior end tapering with genital opening (Fig. 3D).
Spermatophore tadpole shaped, about 20 mm long. Anterior portion or head of spermatophore (h) is a swollen pouch with thickened wall that is packed with sperm. Posterior portion or tail (t) tapering to slender tube is about half of the total length (Fig. 3E).
Both male and female genital organs of all species except P. gravida were examined and no distinguishing species-level taxonomic characters were found.   dum Benson, 1856. Although Benson (1860) noted that Hybocystis was a junior subjective synonym of Pollicaria Gould, 1856, the name Pollicaria was widely overlooked prior to Kobelt's (1902) review of cyclophoroideans and both Wenz (1938) and Pain (1974) continued to mistakenly cite Megalomastoma gravidum Benson, 1856 as the type species. With only the doubtful inclusion of Cyclostoma myersii Haines, 1855 and Cyclostoma chrysalis Pfeiffer, 1852 in the original description of Pollicaria, the type species of Pollicaria was unequivocally fixed in the original publication by monotypy.
Diagnosis. Shell pupoid, small to large (shell height 35-50 mm), thickened and solid. Shell smooth or malleated sculpture from almost white to pale yellow, reddish brown and nearly black; periostracum generally thick. Whorls 5-7, last whorl expanded, body whorl distorted when adult; sutures weakly impressed. Aperture rounded, shallow to absent posterior angled groove; peristome continuous and thickened; lip duplicated and reflexed; umbilicus narrow. Operculum multi-lamellar calcareous plate. Radula taenioglossate with seven teeth in each transverse row.
Distribution. Accepted records are confined to Burma: Moulmein, Damontha, Tavoy and Tenasserim (Benson 1856, 1859, Stoliczka 1871, Crosse 1885, Kobelt 1902, Gude 1921, Pain 1974. Records from Northern Vietnam of P. crossei and P. rochebruni are considered to be distinct species. Remarks. Otopoma blennus Benson, 1856 and Cyclostoma pollex Gould, 1856 have long been considered as junior synonyms of P. gravida and this classification has been followed by a number of authors (Hanley and Theobald 1870, Sowerby 1878, Crosse 1885, Kobelt 1902, Pain 1974). Subsequently, P. crossei and P. rochebruni from Vietnam were also placed into synonymy with this species (see Pain 1974). However, examination of the type specimens of these three species (Figs 4A, B; 5D, F) dem-onstrated that P. gravida could be distinguished from P. crossei and P. rochebruni by having a whitish to yellowish shell colour with swollen whorls, impressed sutures and with the last whorl flattened ventrally (Table 1). Furthermore, P. gravida is mainly  Gude (1921), fig. 29 restricted to the western edge of the Pollicaria distribution in Tavoy and Tenasserim of Burma, and does not overlap with the two Vietnamese species in the east (Pain 1974). Unfortunately, none of the live specimens of P. gravida were examined cytogenetically for additional discrimination of these three species.  -Crosse, 1885: 191-193, pl. 11, fig. 4. Pollicaria myersii -Habe, 1964: 114, pl. 2, fig. 13. Pain 1974 Description. Shell: Shell large, reddish brown to light orange. Periostracum thin, corneous; shell surface usually with fine malleations on upper half of last whorl. Aperture almost circular with a shallow posterior angled groove. Peristome yellow, parietal declining shoulder absent. Lip thickened, broadly expanded, reflexed, with concentric margin.
Radula: Radular teeth arranged in v-shaped rows, each transverse row with 7 teeth (2-1-1-1-2). Central tooth with well developed central cusp and one smaller lateral cusp on each side; central cusp large, elongate with pointed tip. Lateral teeth with 2 cusps, outer cusp largest and elongate shape with pointed tip, and with relatively small pointed tip of inner lateral cusps. Inner and outer marginal teeth with 2 cusps; central cusp large, flanked by small inner lateral cusps.
Distribution: The type locality of this species was given as the broad location of "Siam" (see Haines 1855). Subsequently, P. mouhoti was synonymised with P. myersii (von Martens 1867, Pain 1974) thus expanding the distribution of P. myersii beyond its historical range. However, in this study the distribution of the species is restricted to limestone areas of Vientiane to Luang Prabang, Laos, and probably the northern part of Thailand.
Remarks: The syntype AMNH 43629 could not be traced (Siddal and Watson, personal communication). Due to the proximity of the geographic distributions and similarity in shell morphology of the two species, P. mouhoti have long been considered a junior synonym of P. myersii (see Pain 1974). However, P. myersii can be distinguished from P. mouhoti by an elongated purple to pale orange shell with thin periostracum, rounded aperture and very fine wrinkles on the dorsal part of the last whorl (Table 1, Fig. 4F, K). P. myersii differs from P. gravida, P. rochebruni and P. crossei by having a larger shell, no apertural groove and noticeable wrinkles on last whorl (Tables 1, 2).
Distribution. The type locality of P. mouhoti was given as Laos Mountain, Cambodia. However, subsequent records of this species were from Thailand, Laos and Cambodia (Pfeiffer 1862, Crosse 1885, Kobelt 1902, Solem 1966. Remarks. von Martens (1867) and Pain (1974) synonymised this species with P. myersii and stated that all Pollicaria specimens collected from Thailand should be regarded as this species. However, examination of the type specimens of P. mouhoti (Fig.  4H) showed that it was clearly distinct from P. myersii in shell shape, sculpture and colour pattern. The major distinguishing shell characters of P. mouhoti are the smaller shell size, purplish shell colour, bright orange spire, expanded bright orange to red apertural lip and bold wrinkles on the dorsal side of last whorl (Tables 1, 2). In addition, the chromosome analysis shows a clear difference in karyotype patterns between these two species (Kongim et al. 2009(Kongim et al. , 2010. Hence, P. mouhoti is removed from the synonymy of P. myersii and reinstated as a distinct species. ( Nongbuadang, Chaiyaphum: CUMZ 1528, 1529, 1551, 1571, 1576, 1582, 1585ZMMSU 0003, 0020-4, 0027, 0029. Phu Phachit, Chaiyaphum: ZMMSU 0013. Tam Tao, Nernmaprang, Phitsanulok: CUMZ 1558. Tam Wungdang, Nernmaprang, Phitsanulok: CUMZ 1533 (Fig. 4I) Description. Shell: This nominotypical subspecies is characterized by the large shell size (Table 2). Shell with last whorl and penultimate whorl purple to black; first to third whorls distinct yellow to bright orange. Lip expanded, red to orange.

Pollicaria mouhoti mouhoti
Radula: Taenioglossate radula, teeth arrangement with central, lateral and marginal teeth shape similar to P. myersii. Differences include a central tooth with well developed central cusp and lateral cusp on each side; lateral teeth triangular in shape with a pointed tip; inner marginal teeth composed of 3 cusps; central cusp flanked with small inner and outer lateral cusps.
Distribution. This subspecies occupies the southern limit of the species' range in Cambodia and several localities in Loei, Phitsanulok, Chaiyaphum, Khon Kaen, Phetchabun Nakhon Ratchasima and Saraburi Provinces in Thailand.

Pollicaria mouhoti monochroma
Other material examined. Etymology. From the Greek monos = one or single, and chroma = color of the skin; referring to the characteristic uniform dark brown to blackish spire color of the shell.
Description. Shell: Shell relatively small, pupoid, monochrome purple to black. Periostracum thin and transparent. Whorls 5-6; sutures moderately impressed; apex obtuse; spire short. Last whorl large about two-thirds of shell height, flattened in front. Shell surface rough with malleations on upper half of last whorl. Aperture almost circular, shallow posterior angled groove present; peristome continuous, yellow to pale orange. Lip thickened, broadly expanded; umbilicus narrow. Operculum thick, calcareous, concentric, exterior little concave.
Radula: Taenioglossate radula, teeth arrangement with central, lateral and marginal teeth shape similar to the nominotypical subspecies.
Distribution. Pollicaria mouhoti monochroma ssp. n. is restricted to the northern limit of the species' distribution in Loei, Phetchabun, Chaiyaphum and Nongbua Lumphoo Provinces.
Remark. Pollicaria mouhoti monochroma ssp. n. can be distinguished from the nominotypical subspecies by having a much smaller, entirely black to purple shell (Tables 1, 2) and a distinct karyotype pattern (see Kongim et al. 2009Kongim et al. , 2010. The shell size and shape of this subspecies are similar to that of P. gravida and P. crossei, but the purple shell is a distinguishing characteristic. Shell character variations can be observed in the Phu Pha Lom, Loei Province population. These individuals exhibit a relatively larger shell than the typical populations (Table 2), however, the monochrome black shell and similar karyotype pattern indicate that they belong to this subspecies (Kongim et al. 2009 -Kobelt, 1902: 289. Laidlaw 1928: 33. van Benthem Jutting 1960: 12. Pain 1974: 176, pl. 6, fig. 1, 3. Abbott 1989: 46, 1 figure. Chan 1997 fig. 1-2.  Single syntype specimen is designated as the lectotype of Hybocystis jousseaumei Morgan, 1885 MNHN 21308 (Fig. 5B) Description. Shell: Shell large, elongate pupoid uniform yellow to orange. Periostracum thin, corneous; shell surface with fine growth lines and last whorl with distinctly strong pitting dorsally. Whorls 6-7 whorls; sutures impressed; apex obtuse. Last whorl large about two-third of shell height, flattened in front. Aperture rounded, with shallow to deep posterior angle groove. Peristome continuous, little elevated, yellow to orange, internal parietal declining shoulder absent. Lip thickened, duplicated, and with distinct growth ridges; umbilicus narrow. Operculum thick, calcareous, concentric.
Radula: Taenioglossate radula, teeth arrangement with central, lateral and marginal teeth shape similar to P. myersii. Minor differences are the well-developed central cusp with one to three small lateral cusps of the central tooth, and the slightly elongate and slender central cusp of the inner marginal teeth.
Distribution. This species has a restricted distribution and is known only from limestone outcrops in Perak, Peninsular Malaysia (Morgan 1885a, b). Material collected for this study was from Kinta valley, Perak, and the southern part of the species' historical range in Bukit Chintamani, Selangor, Peninsular Malaysia is considered to be this locally endemic species.
Remarks. The locally endemic Pollicaria elephas is confined to a few limestone outcrops in Peninsular Malaysia and shows several unique shell characters that separate it from its congeners. The major distinguishing characters of this species are the very large, monochrome yellowish to pale orange shell with the last whorl distorted ventrally and sculptured with scattered, deep pits dorsally; and rounded and thickened aperture. (Table 2, Fig. 5A-C). Morgan (1885a, b) proposed two nominal species of Pollicaria from Perak, which differed mainly by the shell size (larger shell Hybocystis elephas and smaller shell Hybocystis jousseaumei). In the first revision of this genus, Crosse (1885) assumed that they were the same species and recognized only P. elephas. Thereafter P. jousseaumei was recognized as a synonym of P. elephas (Kobelt 1902, Pain 1974. Examination of the type specimens (Fig. 5A, B) confirmed P. jousseaumei as junior synonym of P. elephas. Moreover, the recent land snail survey in Perak, Peninsular Malaysia recorded both large and small shell forms of the species from the same localities.  5D) and other specimens as paralecto- Description. Shell: Shell medium-sized, pupoid, red-brown. Periostracum thick, corneous; shell surface smooth. Whorls 5-6; sutures moderately impressed; apex obtuse; spire short. Last whorl large about two-thirds of shell height, distorted and flattened in front, ventrally rounded. Aperture rounded, shallow to absent posterior angled groove present. Peristome continuous, with thin parietal declining shoulder internally. Lip thickened, little expanded, margin moderately duplicated with thin growth ridges; umbilicus narrow. Operculum concentric, thick, calcareous, multi-spiral plate.
Radula: Taenioglossate radula, teeth arrangement with marginal teeth shape similar to P. myersii. Major differences are in the central teeth which have multiple cusps: the central cusp relatively short and small, flanked by 1-3 tapered lateral cusps; and inner marginal teeth with 3 cusps: the central cusp large with a convex tip, flanked by small and pointed inner cusps, the outer lateral cusp very small to nearly wanting.
Distribution. The previous records of this species were from Tonkin (Mabille 1887a, b); Babe National Park, Bac Kan, Vietnam (Yamazaki et al. 2007) Remarks. Based on the similarity in shell morphology, Pain (1974) placed P. rochebruni into the synonymy of P. gravida. However, examination of the type specimens of P. rochebruni indicate that it is a distinct species (see also P. gravida). P. rochebruni can be distinguished from the latter species by having a larger red-brown to purple-black shell with flattened whorls and shallow sutures, while P. gravida usually has smaller pale orange shell with convex whorls and impressed sutures (Tables 1, 2). P. rochebruni differs from the sympatric P. crossei in both shell size and colour (Tables 1, 2, Fig. 5F) as well as having a distinct karyotype pattern (see Kongim et al. 2010).
Radula: Taenioglossate radula, teeth arrangement with central, lateral and marginal teeth similar in shape to P. myersii.
Remarks. Pollicaria crossei has long been recognized as a subjective synonym of either P. rochebruni or P. gravida (Kobelt 1902, Pain 1974. However, the relatively smaller bright orange shell with thick, brown periostracum of P. crossei are a combination of characters that distinguish it from P. rochebruni. The bright orange shell with flattened whorls and shallow sutures distinguish it from P. gravida (Table 1, Fig. 3A). Moreover, the karyotypic study of the smaller shell form of P. gravida sensu lato indicated a distinct species recognized as P. crossei (see Kongim et al. 2010).