﻿Rediscovering the dancing semislug genus Cryptosemelus Collinge, 1902 (Eupulmonata, Ariophantidae) from Thailand with description of two new species

﻿Abstract Knowledge of Thai semislugs remains scarce, especially the dancing semislug genus Cryptosemelus. Prior to the present study, only a single species has been recognized with little available information. To address this knowledge gap, we surveyed for semislugs in western and southern Thailand, which yielded three species belonging to the genus Cryptosemelus. The little-known type species C.gracilis is redescribed herein, including a comparison with the type specimens. Two additional species, C.betarmonsp. nov. and C.tigrinussp. nov., are described as new to science. All three species are characterized by differences in their genital anatomy, especially with respect to anatomical details of the penis, epiphallus, and spermatophore. In addition, C.tigrinussp. nov. differs from C.gracilis and C.betarmonsp. nov. in the mantle color pattern.


Introduction
Becoming a slug through the reduction of the shell has occurred multiple times among the stylommatophoran land snails; this has occurred particularly frequently within the limacoid snail families Ariophantidae Godwin-Austen, 1883, Helicarionidae Bourguignat, 1877, and Urocyclidae Simroth, 1889(Solem 1966Hausdorf 1998;Hyman et al. 2007;Hyman and Köhler 2020). This process, so-called 'limacization', has also produced intermediate forms between slugs and snails, known as 'semislugs', which are characterized by the presence of an external shell with a reduced number of whorls, and a loss of the ability to completely withdraw its body under the protective shell (Collinge 1902;Blanford and Godwin-Austen 1908;Solem 1966;Barker 2001;Schileyko 2003;Schilthuizen and Liew 2008;Hyman and Köhler 2020). In addition, semislugs tend to show extensive development of the mantle lobes that can completely cover its shell, providing an increased surface area for gaseous exchange (Tillier 1983;Barker 2001). Located in the center of the Indo-Burma biodiversity hotspot (Myers et al. 2000), Thailand harbors a large number of snails, slugs, and semislugs belonging to the Ariophantidae and Helicarionidae (Solem 1966;Panha 1996;Hemmen and Hemmen 2001), of which several semislug genera have never been systematically revised since the seminal work of Solem (1966).
In the past decade, knowledge of the species diversity of Thailand's ariophantid snails has dramatically improved, and many genera/species have been described and their systematics have been revised (i.e., Pholyotha et al. 2020Pholyotha et al. , 2021. For the poorly known genus Cryptosemelus Collinge, 1902 such a systematic revision had been pending so far as there had been no additional records on this taxon since its first description. Collinge (1902) only gave a very brief description without providing any details on the genitalia, which bear important characters in semislug taxonomy (Blanford and Godwin-Austen 1908;Solem 1966;Hyman and Ponder 2010;Hyman and Köhler 2020). This monotypic genus was described based on specimens collected during the 'The University of Cambridge Expedition to the North-Eastern Malay States and Upper Perak', known as the 'Skeat Expedition, 1899-1900' (Gibson-Hill et al. 1953, which visited a region now situated in southern Thailand and the northern part of the Peninsular Malaysia. In addition, Cryptosemelus has been referred to as the 'dancing semislug' because of the fidgety or dance-like movement that it makes when it is disturbed or attacked (Collinge 1902).
The purely shell-based taxonomy of the semislug groups provides insufficient evidence for their systematic classification because the highly reduced and rather featureless shells provide a dearth of informative characters. Convergence in shell characters has been demonstrated in Australian helicarionids (Hyman and Köhler 2020). The lack of reproductive information has created a profound inaccuracy in their recognition and delimitation (e.g., Blanford and Godwin-Austen 1908;Solem 1966;Schilthuizen and Liew 2008;Hyman and Ponder 2010;Hyman and Köhler 2020). At present, anatomy-based approaches or those integrating molecular analyses are likely to more successfully resolve these taxonomic problems. Because the systematic revision of Cryptosemelus has never been undertaken for over a century, to fill this gap, we present here valuable data on the genus Cryptosemelus, especially the genitalia, spermatophore, mantle extensions, and radula. This paper includes a re-description of its type species, C. gracilis Collinge, 1902, and the descriptions of two additional new species.

Materials and methods
This study is based on voucher specimens deposited in the Chulalongkorn University Museum of Zoology, Bangkok, Thailand and new materials collected during the rainy season from western and southern Thailand (Fig. 1). Prior to preservation, collected semislug specimens were photographed in life. Each semislug was then euthanized following the standard two-step method protocols (American Veterinary Medical Association 2020), and then fixed in 95% (v/v) ethanol for morphological work. Species identification was made based on the literature, i.e., Collinge (1902), Blanford and Godwin-Austen (1908), and Solem (1966), and then compared with available type specimens and the reference collection of the University Museum of Zoology, Cambridge. For the descriptive work, adult shells were measured for size using a vernier caliper and counting the number of whorls. Three to ten specimens of each species were dissected and examined under an Olympus SZX2-TR30 stereoscopic light microscope. Shell and genitalia were imaged using a Nikon camera (DSLR D850) with a Nikon 105 Macro lens (AF-S VR Micro-Nikkor 105 mm f/2.8G IF-ED) and the inner sculpture of the genitalia was imaged using a stereo microscope with Cell'D Imaging Software. Radulae were extracted, soaked in 10% (w/v) sodium hydroxide, cleaned with distilled water, and then imaged by scanning electron microscopy (SEM; JEOL, JSM-6610 LV).
Descriptions of all new species herein are attributed to the first author. Type material and other voucher specimens are deposited in the Chulalongkorn University Museum of Zoology (CUMZ), Bangkok, Thailand and additional paratype specimens are deposited at the Natural History Museum, London, United Kingdom (NHM or NHMUK-when citing specimen lots deposited in the NHM) and the Zoological Reference Collection (ZRC) of the Lee Kong Chian Natural History Museum, National University of Singapore, Singapore.
In the descriptions of the genitalia, the term 'proximal' refers to the region closest to the genital opening, while 'distal' refers to the region farthest away from the genital opening.
Animal with reticulated skin, pale grayish, brownish, blue-gray, and blackish body marked by conspicuous oblique lines running downwards and backwards. Mantle extensions well-developed and divided into two shell lobes and two dorsal lobes. Shell lobes entirely covering shell or retracted when disturbed; left and right shell lobes usually with same color as body and with or without irregular stripes; right shell lobe (rsl) broad and triangular; left shell lobe (lsl) narrow triangular and relatively small-sized. Right dorsal lobe (rdl) ovate to crescent-shaped and left dorsal lobe (ldl) undivided, larger, and crescent-shaped. Sole tripartite and lateral foot margin present. Caudal horn absent.
Genitalia with slightly short to moderately long penis, thin penial sheath, long to very long epiphallus, penial retractor muscle attached to epiphallus, and short to slightly long gametolytic duct. Epiphallic caecum, flagellum, and dart apparatus absent. Spermatophore with complex branching spines.
Radular teeth arranged in a wide U-shape with symmetrical tricuspid central tooth, asymmetrical tricuspid lateral teeth with square to oblong base-plate, and bicuspid marginal teeth with oblong plate.
Remarks. Originally, Collinge (1902) referred this genus to the family Girasiidae, but later it was suggested to be placed under the subfamily Parmarioninae of the family Zonitidae (Blanford and Godwin-Austen 1908). Thiele (1931) then reclassified this genus, placing it under the subfamily Helicarioninae of the family Ariophantidae. This familial classification was then widely accepted and followed by subsequent authors except with the distinct subfamilial classification in which Zilch (1959) and Vaught (1989) placed Cryptosemelus as a member of the subfamily Macrochlamydinae, while Schileyko (2003) arranged it under the subfamily Parmarioninae. Regardless of the phylogenetic study, the higher classification of Cryptosemelus is still equivocal. Therefore, in this study, we follow the most recent gastropod classification that placed Cryptosemelus under the Ostracolethinae of the Ariophantidae (Bouchet et al. 2017). Collinge (1902) additionally described another two monotypic semislug genera, Apoparmarion and Paraparmarion, from Peninsular Malaysia based on specimens from the Skeat Expedition. These two genera differ from the genus Cryptosemelus mainly based on the number of shell whorls and mantle extensions, shape of the caudal horn, and genital structure. The genus Apoparmarion has a very reduced shell with about two whorls, with mantle extensions rising upon the shell on all sides with the right shell lobe posteriorly large, wing-like, and covering the apex of the shell, a prominent caudal horn, and genitalia with both a flagellum and dart apparatus ( Fig. 2A , B;Collinge 1902). In contrast, Cryptosemelus has a reduced shell of about 3 to 4 whorls, with well-developed mantle extensions with the right shell lobe covering the apex and larger than the left shell lobe, a tail with no caudal horn, and genitalia without flagellum and dart apparatus. For further comparison, Paraparmarion and Cryptosemelus share a similar reduction in the number of shell whorls and the disappearance of the caudal horn, but Paraparmarion has only a right shell lobe (Fig. 2E, F;Collinge 1902), whereas Cryptosemelus has both right and left shell lobes (Fig. 2C, D;Collinge 1902). Unfortunately, the genitalia of the genus Paraparmarion have never been examined for comparison. A future search for additional specimens of the genus Paraparmarion is necessary for elucidating its relationship with the genus Cryptosemelus. Diagnosis. Shell globose and pale golden amber. Animal with blue-gray body. Genitalia with large vagina and elongated epiphallus with two small diverticula. Inner sculpture of penis with a small papilla near atrium. Spermatophore with a head filament of several spines and long tail filament with a cluster of small spines at the tip.
Vagina (v) large, cylindrical, and approximately half of penis length. Gametolytic sac (gs) bulbous ( Fig. 4B with spermatophore); gametolytic duct (gd) rather  short, cylindrical, and somewhat broader at its base. Free oviduct (fo) cylindrical, approximately one and a half times penis length, and encircled with thick tissue in middle (Fig. 4B).
Spermatophore long (Fig. 5). Sperm sac (ss) enlarged and elongate ovalate. Head filament (hf ) large and divided into two major branches located opposite: first branch bearing one small bifid spine, and second branch containing several bifid spines Radula (Fig. 10A). Teeth arranged in a wide U-shape with half row formula: 1-(19-20)-38 teeth. Central tooth square base-plate with symmetrical tricuspid; mesocone large and triangular shape; ectocones small and pointed cusps. Lateral teeth asymmetrical tricuspid, inner teeth square base-plate and then gradually become elongate-shaped at outer teeth. Inner lateral teeth with mesocone large, triangular, and with pointed cusp; ectocone larger than endocone and located near tooth base. Outer lateral teeth: mesocone and ectocone large and pointed tip; endocone very small to nearly absent. Marginal teeth starting at approximately teeth numbers 19 to 20 with obliquely elongate bicuspid; endocone large and pointed tip; ectocone small lanceolate shape with pointed cusp. Outermost marginal teeth shorter and smaller than inner teeth.
External appearance (Figs 1, 2C, D, 4A). Living animal with reticulated skin, blue-gray to blackish body marked by conspicuous oblique grooves running downwards. Four mantle extensions well-developed and same color as body. Shell lobes enlarged to cover entire shell; left shell lobe (lsl) smaller than right shell lobe (rsl); left dorsal lobe (ldl) larger than right dorsal lobe (rdl). Sole divided into three parts longitudinally. Caudal horn absent.
Distribution, habitat, and behavior observations. Cryptosemelus gracilis can be found in Satun, Yala, Songkhla, and Pattani Provinces in southern Thailand (Fig. 1). We searched after rain and found the semislug populations normally hiding under the slope of rocks or the tree trunks, and sometimes climbing on the rocks or low branches of plants. When the semislug are disturbed, they escape by quickly flipping and wagging their tail, and then falling on the floor. Information on its natural predators and parasites remains scarce, but the carnivorous slug genus Atopos and streptaxid snails were found sympatrically with this semislug.
Remarks. A specimen of C. gracilis was first discovered from 'Bukit Besar', the Malay Language, which means 'Big Mountains' in Thai Language (Skeat 1901;Annandale and Robinson 1906). However, this type locality is now referred to as the Namtok Sai Khao National Park area that is situated on the boundary of Pattani, Yala, and Songkhla Provinces in southern Thailand.
In this study, we examined specimens from Satun Province, which are identical to the syntype in having a blue-gray body with prominent oblique lines running downwards on the posterior body, large right shell lobes that covered the apex of the shell, and no caudal horn. Benthem Jutting (1949) provisionally attributed three semislug specimens from Telom Valley, Gunong Siku, Pahang State (1,000 m altitude) as C. gracilis s.l., but this was without any description or illustration. Based on our observation, all recognized Cryptosemelus species generally have a restricted distribution, and tend to occur at low altitudes near the mean sea level. Therefore, we consider that those semislug specimens from Pahang State probably belong to a distinct taxon from C. gracilis s.s. However, this semislug population needs to be re-examined to confirm their taxonomic status.  Diagnosis. Shell depressedly subglobose and pale yellowish. Animal with grayish body. Genitalia with penial caecum, small vagina, and elongated epiphallus. Inner sculpture of penis with papilla and penial caecum. Spermatophore with a row of branching spines. (Fig. 3C, D). Shell depressedly subglobose, small size (width up to 7.4 mm, height up to 4.1 mm), thin, smooth, polished, pale yellowish with olive tinge. Whorls 3½-4, rapidly increasing; body whorl large and well-rounded at periphery. Spire slightly elevated; suture little impressed. Aperture oblique, diagonal, roundly ovate; peristome thin, simple. Columellar margin simple. Umbilicus imperforate.
Vagina (v) cylindrical, and slightly shorter than a half of penis length. Gametolytic sac (gs) bulbous ( Fig. 6B with spermatophore); gametolytic duct (gd) cylindrical, enlarged at base, and then gradually reduced in diameter to gametolytic sac. Free oviduct (fo) long, cylindrical, approximately two times total penis length, and encircled with thick tissue in middle (Fig. 6B).
Spermatophore incomplete (sperm sac and tail filament missing). Head filament (hf) with nine branching spines arranged in a single row along the head filament section (Fig. 7). Radula (Fig. 10B). Teeth arrangement and shape similar to those of C. gracilis. Teeth with half row formula: 1-(27-28)-37 teeth. Central tooth square base-plate with symmetrical tricuspid. Inner lateral teeth square base-plate with asymmetrical tricuspid; outer lateral teeth oblong to elongate tricuspid. Marginal teeth elongate bicuspid. Marginal teeth starting at about teeth numbers 27-28; outermost teeth shorter and smaller than inner teeth.
External appearance (Figs 1, 6A). Living animal with reticulated skin, pale to dark grayish body marked by prominent, oblique, pale brownish lines running downwards. Four mantle extensions well-developed and same color as body. Shell lobes enlarged to cover almost entire shell; right shell lobe (rsl) larger than left shell lobe (lsl); right dorsal lobe (rdl) smaller than left dorsal lobe (ldl). Foot sole divided into median and lateral planes. Caudal horn absent.
Etymology. The specific name "betarmon" is from the Greek word meaning a dancer and refers to the fidgety movements or dance-like movements of living semislugs found in the field after being disturbed.
Distribution, habitat, and behavior observations. Cryptosemelus betarmon sp. nov. is restricted to the limestone outcrops in Prachuap Khiri Khan Province, Thailand (Fig. 1). During the rainy season, but with low precipitation, the semislugs were found inactive under the decaying leaf litter or sometimes inside the hole of decaying wood. This semislug species also moved quickly as well as quickly flipping and wagging its tail to escape after being disturbed. The data on its natural enemies are unknown, but the carnivorous snail, Haploptychius sp. (Streptaxidae), was found at a high abundance in the type locality.
Remarks. This new species is a small-sized Cryptosemelus species which has a subglobose and pale yellowish shell with an olive tinge, and genitalia with a penial caecum and without an epiphallic diverticulum. Compared to the type species, this species has a globose and pale golden amber shell, genitalia with two small diverticula on the epiphallus, and no penial caecum. Diagnosis. Shell globose, pale yellowish. Animal with brownish body, shell lobes pale yellowish-orange and flanked with irregular black bands. Genitalia with long penis and vagina and epiphallus with granulated surface near vas deferens; penial caecum and penial verge present. Inner sculpture of penis: proximal part with one thickened longitudinal fold; distal part with irregular folds. Spermatophore with smooth head filament and long tail filament with several delicate, branching spines.
Spermatophore long and twisted cylindrical tube (Fig. 9). Head filament (hf ) elongate tube with smooth surface (Fig. 9B). Sperm sac (ss) enlarged, elongate ovate with unclear boundary between sperm sac and tail filament. Tail filament (tf ) very long and enlarged tube with series of long and delicate branching spines arranged in a row, and then near the tip having multiple rows of short branching spines (Fig. 9C, D).  (Fig. 10C). Teeth arrangement and shape similar to those of C. gracilis. Teeth with half row formula: 1-(38-39)-44 teeth. Central tooth square base-plate with symmetrical tricuspid. Inner lateral teeth square base-plate with asymmetrical tricuspid; outer lateral teeth with oblong to elongate teeth with tricuspid. Marginal teeth elongate bicuspid. Marginal teeth starting at about teeth numbers 38-39; outermost teeth shorter and smaller than inner teeth. External appearance (Figs 1, 8A). Living animal with reticulated skin, pale to dark brownish body marked with prominent, oblique, dark brownish lines running downwards. Mantle extensions well-developed. Shell lobes pale yellowish-orange, painted with irregular black stripes, and enlarged to cover entire shell; right shell lobe (rsl) large (square shape in preserved specimen); left shell lobe (lsl) small (triangular shape in preserved specimen); left dorsal lobe (ldl) larger than right dorsal lobe (rdl). Foot sole divided into median and lateral planes. Caudal horn absent.

Radula
Etymology. The specific name is a Latin word "tigrinus", a noun in apposition referring to the dark stripes on shell lobes, which is similar to the color pattern of the tiger.
Distribution, habitat, and behavior observations. Cryptosemelus tigrinus sp. nov. can be found on the limestone hills in Phang-Nga Province (Fig. 1). This new semislug species has a high activity level, and is abundant in moist weather conditions after rain. They were seen hanging, crawling, or slowly climbing on the wet surface of the limestone rocks, tree trunks, and limestone shrubs. This new species also has an escape behavior similar to the other congeners. Its predators are unknown, but the carnivorous slug Atopos sp. (Rathuisiidae) and Discartemon sp. (Streptaxidae) were sympatric with the new species.
Remarks. Cryptosemelus tigrinus sp. nov. differs from C. gracilis and C. betarmon sp. nov. in having pale yellow-orange banded shell lobes and a well-developed penial verge, whereas C. gracilis and C. betarmon sp. nov. have monochrome shell lobes and do not have a penial verge.

Discussion
The three character states of (i) reduced shell, (ii) presence of the stimulator with a calcareous dart, and (iii) attachment of the penial retractor muscle directly to the epiphallus rather than to the epiphallic caecum are characteristic for members of the Ostracolethinae, family Ariophantidae (Hausdorf 1998). This study contains the first anatomical investigation of Cryptosemelus and we found that its genital anatomy is consistent with a membership in this subfamily. This finding is corroborated by evidence from shell and reproductive characters as outlined above (dart possibly secondarily reduced). With regard to the absence of a dart apparatus, this character together with the epiphallic caecum and flagellum appears to have been lost and gained repeatedly and convergently among the Ariophantidae and the limacoid snails in general (Hausdorf 1998;Schileyko and Semenyuk 2018).
The shell morphology of the genus Cryptosemelus is similar to that of several semislug genera on mainland Southeast Asia that consist of Apoparmarion, Cryptaustenia Cockerell, 1891, Durgella Blanford, 1863, and Paraparmarion. However, the absence of a caudal horn, which is a unique character shared between Cryptosemelus and Paraparmarion, distinguishes these two semislug genera from the others (Collinge 1902;Blanford and Godwin-Austen 1908;Solem 1966;Schileyko 2002Schileyko , 2003.
Cryptosemelus was stated to differ from Paraparmarion in that the left shell lobe is well-developed, whereas it is missing in the latter (Collinge 1902). However, the presence and absence of the left shell lobe can simultaneously occur within congeneric species in the ariophantid snail genus Sarika Godwin-Austen, 1907 . Hence, the relationship between Cryptosemelus and Paraparmarion remains uncertain since they were consecutively described in the same publication without genital information (Collinge 1902). However, the genitalia of Cryptosemelus have been examined herein and its generic status is confirmed. The genital morphology of all species of Cryptosemelus examined herein all show no epiphallic caecum, flagellum, and dart apparatus. In comparison, a flagellum occurs only in Apoparmarion, while an epiphallic caecum occurs only in Durgella (Collinge 1902;Blanford and Godwin-Austen 1908;Solem 1966;Schileyko 2002Schileyko , 2003.
Regarding the dart apparatus, the main role of which is for stimulation during courtship behaviour, this character has been used as a distinguishing character among the limacoid genera, i.e., Hemiplecta Albers, 1850 (with dart apparatus) vs. Falsiplecta Schileyko & Semenyuk, 2018 (without dart apparatus), or Macrochlamys Gray, 1847 (with dart apparatus) vs. Syama Godwin-Austen, 1908 (without dart apparatus). These sibling genera have a similar external appearance except for the dart apparatus (Blanford and Godwin-Austen 1908;Schileyko 2002Schileyko , 2003Schileyko and Semenyuk 2018). However, it is widely accepted that the dart apparatus could be present or absent within the same genus, i.e., Cryptaustenia and Durgella (Blanford and Godwin-Austen 1908;Solem 1966;Schileyko 2002Schileyko , 2003. Moreover, no molecular phylogeny has been implemented to test the monophyly of the genera Cryptaustenia and Durgella. In this study, the shell lobes and genitalia (penis and epiphallus) are considered as taxonomically informative and these can be used to distinguish all Cryptosemelus species. In addition, these characters might reflect the relationships among three species of Cryptosemelus. Our results indicated that C. gracilis is closely related to C. betarmon sp. nov. even though the distribution of C. gracilis is closer to C. tigrinus sp. nov. (Fig.  1). In support, the shell lobes of C. gracilis and C. betarmon sp. nov. have a monotone color and the internal wall of the penis has a papilla located close to atrium, whereas C. tigrinus sp. nov. has shell lobes with dark stripes and a large, conical penial verge. In addition, the undulated surface on the epiphallus of the two species is located close to the penial retractor muscle, while in C. tigrinus sp. nov. this character is located near the vas deferens. Furthermore, the color pattern of the shell lobes and the reproductive tracts of C. tigrinus sp. nov. are very distinct and unique, which could possibly be recognized as different subgenera or genera. However, we refrain from nominating this because at present it is without support from a molecular framework. Thus, future studies need more materials from other members of Cryptosemelus and will also require combining with molecular phylogenetic analyses to investigate this hypothesis.
Predator-prey interactions are recognized as major processes promoting morphological and behavioral diversity (Vermeij 1987;Chiba 2007;Morii et al. 2016;Sugiura 2016;Le Ferrand and Morii 2020). Land snails are preyed upon by a wide range of predators (e.g., rodents, birds, snakes, insects, and molluscs). Their own anti-predator adaptations, a passive defense by pulling their body into their shell and an active behavior by swinging their shell around, are well documented and are potentially associated with differences in shell traits (Vermeij 1987(Vermeij , 1993Chiba 2007;Baalbergen 2014;Liew and Schilthuizen 2014;Morii et al. 2016;Morii and Wakabayashi 2017;Le Ferrand and Morii 2020). In contrast, the evolution of slug and semislug forms have sacrificed the protection offered by possession of a shell for the mobility, fast body movements, and ability to occupy very small spaces afforded by the reduction or elimination of the shell (Solem 1974;Barker 2001;Wiktor 2002;Dedov et al. 2019). In our study, this dancing semislug genus showed an anti-predator behavior by quick dance-like movements or the sudden flipping and wagging tail movement and the fast movement away from the location of threat. This behavior has also been recorded in several helicarionid semislugs: Cryptaustenia species in Papua-New Guinea, Laocaia species in Vietnam, and Muangnua species in Thailand (Wiktor 2002;Dedov et al. 2019;Tumpeesuwan and Tumpeesuwan 2019). approved by the Chulalongkorn University Animal Care and Use Committee (CU-ACUC) under the approval number 1723018. Additionally, special thanks go to anonymous reviewers for providing valuable comments and suggestions on the manuscript.