Taxonomic note on Trichelix horrida (Pfeiffer, 1863) from Laos, with a type catalogue of Moellendorffia, Trichelix, and Moellendorffiella (Heterobranchia, Camaenidae)

Abstract Land snail surveys conducted in northern Laos between 2013 and 2014 have led to the discovery of a living population of Trichelix horrida (Pfeiffer, 1863). This species has never been recorded from specimens other than the types, and its distribution and anatomy have remained essentially unknown. The genitalia and radula morphology are documented here for the first time and employed to re-assess the systematic position of this species: the unique morphological characters of T. horrida are a penis similar in length to the vagina, a small and triangular penial verge, gametolytic organs extending as far as the albumen gland, head wart present, and unicuspid triangular radula teeth. The type locality of this species was believed to be from “Lao Mountains, Camboja,” and is restricted herein to be Luang Phrabang Province, northern Laos. The assignment of species to either of three genera, Trichelix Ancey, 1887, Moellendorffia Ancey, 1887, and Moellendorffiella Pilsbry, 1905, based solely on information provided in their original descriptions is difficult. The type specimens of all nominal species presently placed in either of these three genera are examined and illustrated herein. Comparison with the primary type specimens will assist future revisions aiming to resolve the systematics of these taxa. In addition, we transfer Moellendorffia faberiana (Möllendorff, 1888) to the genus Moellendorffiella.

mens of all recognized taxa belonging to the genera Moellendorffia, Trichelix, and Moellendorffiella Pilsbry, 1905 are included for comparisons and because the species identification could not have been possible without comparison with the type specimens.

Materials and methods
Shells and living specimens were collected in a limestone forest in Luang Phrabang Province, northern Laos. The live specimens were photographed, euthanized (AVMA 2013), and then transferred to 70% (v/v) ethanol for fixation and preservation. The genitalia of three specimens were dissected and examined under a stereomicroscope. Drawings were made with a camera lucida. Radulae were extracted, soaked in 10% (w/v) NaOH, and then examined under scanning electron microscopy (SEM; JEOL, JSM-6610 LV). The formulae and morphology of radula were observed, recorded, and described. Adult shells were used to measure the shell height and shell width, and to count the number of whorls. The voucher specimens are now deposited in the Chulalongkorn University Museum of Zoology (CUMZ) and in the collection at the National University of Laos.

ANSP
Academy  Description. Shell small to medium-sized, flattened to concave, rather thin, umbilicate, and corneous to brownish. Spire shrunken; embryonic shell nearly smooth; following whorls granulated and with short to long periostracal hairs arranged in oblique rows along the lines of growth. Last whorl rounded and descending anteriorly. Aperture ventral or subvertical; trigonal or subcircular; with strong or weak barriers inside the aperture at upper periphery and below periphery, and externally marked with strong to weak longitudinal furrows. Peristome expanded and continuous or discontinuous; parietal callus thin or thickened and little elevated.
Genitalia typical of camaenids, without either dart apparatus or accessory glands. Penis and epiphallus long, penial verge present, and flagellum short. Internal wall of penis and vagina with longitudinal pilasters.
Radular teeth arranged in V-shaped rows; central and lateral teeth triangular. Remarks. The genus is currently comprised of six nominal species (Schileyko 2003;Minato 2011). Two species occur in northern Laos and southern China (Fig. 3), viz. T. horrida and T. biscalpta (Heude, 1885), and one has been recorded from Taiwan, viz. T. hiraseana (Pilsbry, 1905). Three species occur on the Amami Islands, Central Ryukyu Islands, Japan, viz. T. eucharista (Pilsbry, 1901), T. diminuta (Pilsbry & Hirase, 1905) and T. tokunoensis (Pilsbry & Hirase, 1905 Type material. Three specimens originating from H. Cuming's collection with the original label stating the taxon name and collection location in Pfeiffer's handwriting are present in the malacological collection of the NHMUK. Of these specimens, the one most closely matching the measurements given in the original description is here designated as the lectotype NHMUK 20200202/1 (Fig. 7B) to stabilize the name. The other two shells from the same lot become paralectotypes NHMUK 20200202/2 to 20200202/3. Trichelix horrida was originally described based on specimens collected by H. Mouhot, with "Lao Mountain, Camboja" as the published type locality. Our survey following Mouhot's itinerary in the south-western part of Cambodia yielded no specimens that could be identified in this genus. This record type locality seems to be imprecise. On the other hand, our survey in the northern part of Laos, where Mouhot had visited Luang Phrabang in 1861, recorded populations of this species in Muang Ngoi about 90 km north of Luang Phrabang City. Therefore, we restricted the known distribution and propose Luang Phrabang Province, Laos as the correct type locality for this species.
Shell. Shell medium-sized, dextral, slightly thin, translucent, depressed globose, biconcave shaped (dorsoventrally concave), and deeply umbilicate. Whorls 5-6, slightly convex, and increasing regularly; suture depressed, spire concave, looking like umbilicus. Embryonic shell large with very fine growth lines. Following whorl with corneous to brownish periostracum; upper surface with long hairs arranged in oblique rows; lower surface with slightly shorter hairs and few hairs around umbilicus. In worn specimens, shell surface possesses rough rows of tubercles running obliquely and descending, relatively smooth around umbilicus. Last whorl well rounded and little convex below periphery. Last whorl descending about ¼ whorl from aperture, and constriction occurs close to apertural lip. Aperture ear-shaped and opened subventrally; lip margin pale corneous, little thickened, and continuously expanded. External furrow aligns with internal apertural lamella or fold. Upper periphery marked with two furrows arranged spirally and correspond with palatal lamella and fold; below periphery with one furrow close to lip aligned with basal lamella. Parietal callus thickened, elevated, emarginated, and obtusely projecting inward. Umbilicus wide, but narrower than apex side which is cascade-shouldered.
Genitalia. Atrium (at) short; penis (p) long; proximally with penial verge and enlarged fold at penial verge base; distally similar in length as proximally and somewhat slender tube. Epiphallus (e) slightly enlarged and almost the same length as penis. Flagellum (fl) very short and small. Vas deferens (vd) a small tube, follows vagina and penis, and connects distally on epiphallus and free oviduct. Penial retractor muscle (pr) slightly thickened and long ( Fig. 2A).
Internal wall of penis ribbed by a series of swollen longitudinal penial pilasters (pp). Smooth pilasters line introverts penial chamber and encircles penial verge tip. Penial verge (pv) small, short conic with smooth surface (Fig. 2B). Vagina (v) of similar length to proximal penis and held in position by series of muscles attached to foot floor. Gametolytic organ (duct and sac) long, cylindrical, and extending as far as albumen gland. Gametolytic duct (gd) as wide as gametolytic sac (gs) for most of its length but narrows before reaching gametolytic sac. Free oviduct (fo) short, about half of vagina length; oviduct (ov) small. Prostate gland (pg) and oviduct (ov) developed; hermaphroditic duct long and convoluted tube; albumen gland solid and tongue shape ( Fig. 2A).
Internal wall of vagina possesses several longitudinal vaginal pilasters (vp). Pilasters with smooth surface and line entire vaginal chamber (Fig. 2B).
Animal. Live animal covered with blackish-brown reticulated skin and dorsally with whitish stripe in middle of the body. A small curved head wart (hw) is located between the posterior tentacles ( Fig. 2C). Foot narrow and long; mantle edge greyish; tentacles brownish, and lower tentacles pale brown. Mantle cavity possesses blackish pigmentation. Live snails possess short to long periostracal hairs, which mostly disappear in worn shells or old snails.
Distribution. Trichelix horrida was previously known only from the type locality ("Lao Mountain, Cambojia" [Cambodia or Laos]). The specimens examined herein were collected from limestone karst in Muang Ngoi Town, about 90 km north of Luang Phrabang City.
Our sampling locality was characterized by monsoonal karst landforms with high humidity. The snails occurred in tropical moist deciduous forest. There was heavy rain before our visit in August 2014. The snails were active, crawling or sitting on moist rotten logs among the limestone outcrops.
Remarks. Trichelix horrida is distinctly different in shell morphology from all other Moellendorffia species by having a concave spire, rounded last whorl, and two furrows arranged spirally on the upper periphery (Table 1). In contrast, Moellendorffia species tend to have flattened to elevated spires, rounded to shouldered last whorls, and two furrows arranged vertically on the periphery. Trichelix horrida differs from the other congeners in having two short furrows on the last whorl and an elevated parietal callus (Fig. 7B), while T. biscalpta and T. hiraseana tend to have a long furrow on the last whorl and unelevated parietal callus (Figs 6C, 7A). In addition, T. hiraseana has a relatively long, drumstick-shaped flagellum, while the type species has a very short protrusion (see Minato 2011 for a comparison). In addition, Chloritis bifoveata (Benson, 1850) from Myanmar and Thailand, C. diplochone Möllendorff, 1898 from Laos and Thailand, and C. vinhensis Thach & Huber, 2018 from Vietnam differ from T. horrida by having a thin parietal callus, with a shell constriction occurring about half a whorl from the aperture (absent in C. vinhensis), and without apertural dentition Páll-Gergely and Neubert 2019;Páll-Gergely et al. 2020).

Discussion
The newly collected material from Laos presents valuable additional information for the taxonomic position of Trichelix and its congeners. The relationship of Trichelix with Moellendorffia and Moellendorffiella has been suggested based on shell and genital anatomy characters ). The shrunken spire and one or two furrows located on the upper periphery are the unique characteristics of Trichelix. At present, Trichelix s.l. has a wide distribution across Indochina to Taiwan, southern China, and the Central Ryukyu Islands of Japan (Fig. 3).
The genus Trichelix s.l. appears to be a heteromorphic assemblage, as noted by Schileyko (2003), based on both shell and genital anatomy characters. The genus comprises the continental group and the Central Ryukyu group. The continental group includes three nominal species from northern Laos (the type species), Taiwan (T. hiraseana), and southern China (T. biscalpta). They all have prominent palatal lamellae arranged spirally on upper periphery, strong columellar lamella, and vagina almost the same length as penis. The Central Ryukyu group contains three nominal species: T. eucharista, T. tokunoensis, and T. diminuta; they lack the parietal lamella and have a very weak or absent columellar lamella, and the vagina is relatively longer than the penis (Habe 1957;Minato 1971Minato , 1980Minato , 2011Schileyko 2003). The unique genital characters of T. eucharista are: penis about half of vagina length and vagina with constrictions; T. tokunoensis possesses two penial retractor muscles, a very small epiphallus and penis about one-third of the vagina length; T. diminuta has the penis about half of the vagina length and the gametolytic duct bears constrictions (Habe 1957;Minato 1971Minato , 1980Minato , 2011Schileyko 2003). These unique and distinct genital characters are likely to be apomorphic traits and would be the main reproductive barrier among these species. It is very likely that the three species inhabiting the Central Ryukyu Islands of Japan do not belong to the same genus as the continental and Taiwanese species. However, with so few synapomorphic traits among these Central Ryukyu Islands species, the confidence in defining distinct lineages remains low. Therefore, we refrain from describing a genus without additional evidence from molecular analyses.

Catalogue of type specimens of Moellendorffia, Trichelix, and Moellendorffiella
In the following catalogue list, the primary type specimens (i.e., holotype, lectotype, and syntype/s) along with secondary type specimens (paratype/s and paralectotype/s) of Moellendorffia, Trichelix, and Moellendorffiella species are provided. The speciesgroup names are arranged by alphabetical order. The references for the usage of each taxon name have been comprehensively provided by Richardson (1985), Zilch (1966), and Minato (2011). The name in the original combination is given with the bibliographic information or the original description. The type locality is given, and if possible, the modern name and/or regional names of the type locality are provided in square brackets. The current taxonomic status includes the generic placement, whether a valid name or synonym. If necessary, remarks are given on the status of type specimens, authorships, availability of name, notes on the type locality, and other useful comments. Diagnosis. Shell flattened to globose-conic, and umbilicate. Periostracum thick and covered with short to long hairs. Last whorl rounded to shoulder and descending anteriorly. Aperture trigonal or squarish, entirely free from preceding whorl; with barriers inside, and externally marked with furrows. Parietal wall elevated to form prominent nodule; one or two palatal lamellae (two lamellae arranged vertically); one columellar lamella.
Remarks. The genus Moellendorffia can be distinguished from Trichelix s.l. in having low conical to elevated spire, one or two furrows (arranged vertically) on periphery and elevated parietal callus, while Trichelix s.l. has a concave spire. In addition, the continental-Trichelix have one or two furrows (arranged spirally) on the upper periphery and little elevated parietal callus, and the Central Ryukyu-Trichelix performs very weak or absent furrows, and a thin parietal callus.
Remarks. The lectotype was designated in Zilch (1974: 194) and illustrated for the first time in this study.

II. Genus Trichelix Ancey, 1887
Type species. Helix horrida Pfeiffer, 1863; by original designation. Diagnosis. Shell flattened to concave, spire shrunken and umbilicate. Periostracum covered with short hairs. Last whorl well rounded and descending anteriorly. Aperture subcircular, without barrier or with barriers inside, and externally marked with furrows. Parietal callus thin, with cord at margin or a little elevated to form nodule; two palatal lamellae arranged spirally; one columellar lamella.
Remarks. The genus Trichelix s.l. can be distinguished from Moellendorffiella by having concave spire, and short to long periostracal hairs, while Moellendorffiella have flat spire and without periostracal hair. The Central Ryukyu-Trichelix have a thin parietal callus with cord and very weak furrows below the periphery, and the continental-Trichelix have an elevated parietal callus with a nodule, and there are one or two furrows (arranged spirally) on the upper periphery. In comparison, Moellendorffiella has a thin parietal callus and one furrow on periphery. Type specimens. Syntype MCZ 167125 (two shells, Fig. 6C).

Helix biscalpta
Remarks. The original description does not clearly state how many specimens were available to the author, and a unique name-bearing type was not explicitly designated. Heude's (1885) original description included a single illustration and one set of shell measurements. Johnson (1973: 17) used the term "paratypes" for a lot of two shells from the MCZ collection, but this does not constitute a valid holotype designation (ICZN 1999: Articles 73.1.1 and 73.2 and Recommendation 73F). The MCZ museum registration book states "Cotype"; these are also considered to be syntypes.
Type specimens. Lectotype ANSP 81221 (one shell, Fig. 6E) from Oshima, Osumi. Remark. The lectotype was designated in Baker (1963: 245). In the original publication, the type locality was recorded as "Oshima" (=Island) which cannot be precisely located. The original label accompanying the lectotype states "Oshima, Osumi" (= historical name of Kagoshima). Habe (1957) examined the radula and genital anatomy based on a specimen from Amami Oshima, Kagoshima. Therefore, the type locality of this species is probably in the area of the Amami Islands, Kagoshima Prefecture.
Type specimens. Syntypes NHMUK 19991540 (two shells, Fig. 7C). Remarks. Ancey (1906: 128) stated that specimens sent by Mr Rouyer were often with doubtful or inaccurate locality records, where C. malangensis Bullen, 1905 was described based on Mr Rouyer's collection. The type locality was mentioned as "Malang Java," which is erroneous and should be ignored (Ancey 1906;Gude 1907). Ancey (1906: 128) also noticed this species was similar to Moellendorffia eucharista (Pilsbry, 1901) and does not occur in Java. Gude (1907: 228) compared the type specimen of C. malangensis with the Moellendorffia eucharista (Pilsbry, 1901) from Japan and found no differences in any of the shell characters.
Remarks. The original description did not clearly state how many specimens were available to Pilsbry, although  Diagnosis. Shell flattened and umbilicate. Periostracum thin, corneous. Last whorl shouldered and descending anteriorly. Aperture subcircular with barriers inside and externally marked with furrows. Parietal callus thin; one palatal lamella; one columellar lamella.
Remarks. The genus Moellendorffiella differs from Moellendorffia in having one furrow on periphery, parietal callus thin, and without periostracal hair. While, Moellendorffia has one or two furrows on periphery, parietal callus elevated with nodule and short to long periostracal hairs.
Remarks. The distinguishing characters are depressed conic spire, aperture with elevated parietal callus, furrows on periphery and below periphery. Therefore, we move this species to the genus Moellendorffiella.

Species inquirenda
mariae (Nobre, 1909)  Remarks. This nominal species was described by Nobre (1909) based on material collected from Angola on the west coast of Africa. Based on the shell morphology, Nobre (1909) attributed this taxon to the Southeast Asian endemic genus Stegodera (Moellendorffia). Later, Richardson (1985) placed this species under the genus Moellendorffia. However, the record of Moellendorffia on the east coast of Africa (Ethiopian Realm) are far outside of the known range of the genus. Thus, further study and anatomical examination are needed to relocate this nominal species into the suitable nominal genus, very probably a Sculptaria Pfeiffer, 1855 (Sculptariidae).
Remarks. The species was described based on one specimen. The holotype has a relatively small shell (shell width 8 mm) compared to other recent congeners. This species possesses a smooth shell surface and a narrow umbilicus. The outer surface of the last whorl probably has one spiral furrow on the periphery and two spiral furrows below the periphery. These characters suggest the possibility that it is closely related to the genus Traumatophora Ancey, 1887 (see Wu 2019 for a comparison).