Integrative taxonomy of insular land snails of the genus Sicradiscus Páll-Gergely, 2013 (Gastropoda, Plectopylidae) with description of a new species

2.1 Sample collection

In total, 64 Sicradiscus snails were newly collected. Twenty-five specimens of S. pallgergelyi sp. nov., 17 of S. hirasei, and 21 of S. ishizakii were examined morphologically; among these specimens, six individuals of S. pallgergelyi sp. nov., six of S. hirasei, and three of S. ishizakii were used for the molecular phylogenetic analysis (Table 1). In addition to the newly collected specimens, the morphological examination was performed for a lectotype of S. hirasei and a paratype of S. ishizakii. The field survey was conducted by the first and second authors from four localities in Japan and Taiwan, in 1997–2020 (Table 1; Figure 1). Sicradiscus hirasei was collected from crevices in the limestone on the ground in the inland natural forest of Miyako Island. On Iriomote Island, the new species was found in the inland natural forest at an elevation of around 200 m; however, S. pallgergelyi sp. nov. was not found from three limestone areas at low elevations whose environments are similar to the habitat of S. hirasei in Miyako Island. All of the snails collected by the first and second authors were found on the forest ground by eye. Because S. hirasei is protected by a Miyakojima City ordinance on Miyako Island, the snails were collected with permission by the city. In addition to the three insular species, a specimen of the continental S. schistoptychia (the type species of Sicradiscus) was included in the phylogenetic analysis.

Specimens from Iriomote and Miyako Islands were separated into shells and soft parts after being boiled in hot water at 80°C for 12 s. The extracted soft bodies were frozen until dissection after cutting off the foot tip. Separated tissues were preserved in 99% ethanol for molecular phylogenetic analysis. Snails from Taiwan were preserved in 95% ethanol after boiling. The newly collected specimens in this study were deposited in the Zoological Collection of Kyoto University, Kyoto, Japan (KUZ) and Hungarian Natural History Museum, Budapest, Hungary (HNHM).

2.2 Morphological examination

Morphometric characters of the newly collected specimens and the type specimens of S. hirasei and S. ishizakii were examined. Fully matured adults were observed and measured following the methods in previous studies (Páll-Gergely, Hunyadi, et al., 2015). The nomenclature of plicae and radulae follows Páll-Gergely and Hunyadi (2013) and Páll-Gergely (2018b), respectively. Newly collected specimens were first photographed using a Nikon D7100 camera with a Tamron SP 90 mm f/2.8 1:1 macro lens for Nikon. All specimen measurements were obtained from digital images of dorsal, ventral, and lateral sides of shells using ImageJ 1.51k (Schneider et al., 2012). The apertural callus length was obtained from photos taken of the callus in the frontal position and was measured at the longest part of the callus. The spire angle was measured as the angle between the lines connecting shoulders of the apical and body whorls (Figure 2a). The umbilicus width was measured by extending the line connecting the base of the thickened aperture with the center of the spiral (Figure 2b). The whorl number of adult shells was counted in intervals of 0.25 following the method described by Sawada and Nakano (2021). The numbers of specimens used for each character are shown in Table 2.

After obtaining measurements, microstructures of the protoconch and ventral side were observed in 10 adult shells of each species with a Hitachi TM1000 scanning electron microscope (SEM; Hitachi). The plical and lamellar morphology was studied in eight specimens of each species by carefully cracking open the shells. These inner structures of holotype of S. pallgergelyi sp. nov. were observed from the outside of the shell using transmitted light. Additionally, soft parts of three S. ishizakii specimens were extracted by breaking shells. Six S. hirasei, three S. ishizakii, and eight Iriomote Island specimens were dissected under a Leica M125C stereoscopic microscope. The reproductive organ structure of the Japanese snails was photographed after fixation with 70% ethanol. Following the dissection of reproductive organs, the radulae were extracted by soaking oral tissues in 1 M sodium hydroxide solution for a day. Extracted radulae were coated with gold using a JFC-1200 Fine Coater (JEOL) and photographed with an SEM.

A canonical variate analysis (CVA) was conducted to clarify the relationships of shell measurements among the three Sicradiscus species. The CVA was performed on the datasets using five measurements, apertural callus length, shell diameter, shell height, and whorl number, and spire angle. Missing values of apertural callus length were substituted with the measurement average. The CVA data were also used to assign specimens with a leave-one-out cross-validation to estimate the expected actual error rates in classifying the insular Sicradiscus species. The CVA was conducted with PAST 4.04 (Hammer et al., 2001).

Abbreviations. Museum collections: ANSP, Academy of Natural Sciences, Philadelphia, USA; NCP, paratype collection of the Nishinomiya Shell Museum, Hyogo, Japan. Morphometrics: BWL, body whorl length; CL, apertural callus length; D, shell diameter; EN, the number of embryos; H, shell height; HD, the proportion of H to D; SA, spire angle; UD; the proportion of umbilicus width to D; UW, umbilicus width; WN, whorl number. Radulae: EcL, ectocone of a lateral tooth; EcM, ectocone of a marginal tooth; EnL, endocone of a lateral tooth; EnM, endocone of a marginal tooth.

2.3 PCR and DNA sequencing

The phylogenetic relationships of the insular Sicradiscus species and the continental S. schistoptychia were estimated using a mitochondrial marker, a fragment of the cytochrome c oxidase subunit I (COI) gene. Genomic DNA was extracted from muscle tissue of feet following the method described in Okamoto et al. (2006). A 706 bp section of the mitochondrial COI gene which contains the DNA barcoding region was amplified in 16 Sicradiscus snails by polymerase chain reaction (PCR) using a Takara Ex Taq Kit (Takara Bio) and the primers shown in Table 3 with a GeneAmp PCR System 9700 (Thermo Fisher Scientific). The thermocycling regime was an initial denaturation step at 96°C for 1 min, followed by 35 cycles of 1 min at 96°C, 30 s at 42°C, and 1 min at 72°C, and a final extension at 72°C for 7 min. The PCR products were purified as described by Okamoto and Hikida (2009).

Nucleotide sequencing was conducted for PCR products using a BigDye Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific). The sequencing mixtures were heated to 96°C for 1 min, followed by 25 cycles at 96°C (10 s each), 45°C (30 s each), and 60°C (4 min each). Sequencing was then performed using an Applied Biosystems 3130xl Genetic Analyzer (Thermo Fisher Scientific). The final alignment for the phylogenetic tree reconstructions included 16 sequences which had a length of 655 sites (see Alignment S1). The sequences were deposited in the International Nucleotide Sequence Database Collaboration through the DNA Databank of Japan (LC638856–LC638871).

2.4 Molecular phylogenetic analyses

Phylogenetic trees were reconstructed using maximum likelihood (ML) with IQ-TREE v. 1.6.12 (Nguyen et al., 2015). The following best-fit models for each partition of the COI sequence were identified based on the corrected Akaike information criterion and the greedy algorithm: HKY+F for the 1^st position, K2P for the 2^nd position, and F81+F for the 3^rd position. The robustness of the ML phylogenetic tree was inferred by 1000 non-parametric bootstrap replicates. Mean genetic distances among the three insular Sicradiscus species were estimated with the Kimura two-parameter (K2P) in MEGA v. 10.2.4 (Stecher et al., 2020).

3.1 Morphological analyses

The morphometric character measurements are described in Table 2. The EN, H, UD, and WN measurements largely overlapped among the three Sicradiscus species. Although S. ishizakii shared similar BWL, D, HD, and UW measurements with S. pallgergelyi sp. nov., these characters distinguished S. hirasei from the two former species. The characters of CL and SA separated the three species with a slight overlap.

The first and second canonical components explained 90.5% and 100% of the total variance, respectively (Table 4). The variance of CV1 was mainly explained by SA and D. The two canonical components clearly distinguished S. hirasei from the other two congeners. The CVA results also showed that the shell morphology of S. pallgergelyi sp. nov. partly overlapped with that of S. ishizakii (Figure 3). The results of the leave-one-out cross-validation correctly classified 100%, 71.4%, and 88.0% of S. hirasei, S. ishizakii, and S. pallgergelyi sp. nov., respectively.

The upper whorl surface of the protoconch was smooth or very finely ribbed in S. hirasei and the new species, whereas S. ishizakii usually possessed remarkable ribs across its entire protoconch surface (Figure 4a,e,i). Sicradiscus ishizakii was also clearly distinguishable from its two congeners by the microstructure of the shell's ventral side (Figure 4b,f,j). Sicradiscus ishizakii had a glossed ventral side with rudimental radial striations, whereas the two other species had strong spiral striations reticulated with radial sculptures.

The lamellae shapes were approximately similar in the two Japanese species, although their morphology showed large variation (Figures 4c,g,k and 5a,c,e,g,i,k,m,o,q,s,u,w,y,aa,ac,ae,ag,ai,ak,am,ao,aq,as,au). The anterior lamellae of S. hirasei and S. pallgergelyi sp. nov. were narrow and elongated, and T-shaped (Figure 4c,k). Alternatively, S. ishizakii had thick C-shaped anterior lamellae (Figure 4g). Among the three species, no significant differences were detected in the plicae (Figures 4d,h,l and 5b,d,f,h,j,l,n,p,r,t,v,x,z,ab,ad,af,ah,aj,al,an,ap,ar,at,av), radulae (Figure 6), general reproductive organ morphology, and inner penial wall (Figure 7a–c,e,g). Prominent folds were observed in the inner vaginal wall of S. ishizakii, whereas fine ventral structures were observed in the two Japanese species (Figure 7d,f,h).

3.2 Molecular phylogenetic analyses

The 655-bp COI section was successfully sequenced for six S. hirasei (LC638865–LC638870), six S. pallgergelyi sp. nov. (LC638859–LC638864), three S. ishizakii (LC638856–LC638858), and one S. schistoptychia (LC638871). All S. hirasei snails had the same haplotype, whereas S. ishizakii and the new species had two different haplotypes each. In the obtained ML tree (Figure 8), each of the Japanese species appears as a strongly supported clade (BS = 100%). A well-supported clade was also formed by the Japanese species (BS = 98%). The two Japanese species had the closest relationship (1.6% of mean K2P distance), and the mean K2P distances among S. ishizakii and the two other species were 6.7% (vs. S. pallgergelyi sp. nov.) and 7.0% (vs. S. hirasei).

3.3 Systematics

It was suggested that a genetic distance of more than 3% is an appropriate boundary between intra- and interspecific differentiation of the COI gene (Hebert et al., 2003). This COI distance is generally accepted in Stylommatophora (e.g., Criscione et al., 2012; Kameda et al., 2007). However, it is also known that the interspecific distances can be as low as 1% in stylommatophoric snails (Davison et al., 2009). In contrast, 18.4% of the genetic distances have been revealed within Orcula dolium (Harl et al., 2014). Although the genetic divergence between S. hirasei and the new species was 1.6% and is thus lower than 3%, they were clearly discriminated by their shell morphology. Their unique shell morphology and the substantial difference in their habitats indicate that they adapted to their habitats upon allopatric divergence and are on independent evolutionary trajectories (see section 4). Therefore, we consider S. pallgergelyi sp. nov. an independent species, although further genetic study is needed.

Plectopylidae Möllendorff, 1898

Sinicolinae Páll-Gergely, 2018

Sicradiscus Páll-Gergely, 2013

Type species: Plectopylis schistoptychia Möllendorff, 1886 by original designation

Sicradiscus hirasei (Pilsbry, 1904)

Tables 2 and 5; Figures 4a–d, 5a–p, 6a–c, 7c,d and 9a–c

• Plectopylis (Sinicola) hirasei Pilsbry, 1904: 58–59; Minato, 1980: 88–89, figures 7–10; Minato, 1988: 135; Azuma, 1982: 210, figure 624; Azuma & Azuma, 1984: 89–90, unnumbered figure (genital anatomy); Higo & Goto, 1993: 481; Nature Conservation Division, Department of Environmental Affairs, Okinawa Prefectural Government, 2017: 457.
• Plectopylis hirasei—Pilsbry & Hirase, 1904: 616; Iwakawa, 1919: 207; Brooks & Brooks, 1931: 212; Hirase & Taki, 1951: pl. 124, figure 6; Baker, 1963: 215; Hsieh, 2003: 143, unnumbered figure (adult shell).
• Sicradiscus hirasei—Páll-Gergely & Hunyadi, 2013: 2, 50, 57; Páll-Gergely & Asami, 2014: 558–559; Páll-Gergely, Hunyadi, et al., 2015: 4, 107; Páll-Gergely, 2018a: 86–89, figure 1b,c,f–h; Páll-Gergely, 2018b: 105.

TABLE 5. Comparison of morphological characters between the three Sicradiscus species examined in this study

Characters	Sicradiscus hirasei	Sicradiscus ishizakii	Sicradiscus pallgergelyi sp. nov.
Body whorl length (BWL, mm)	Small (2.4 ± 0.1)	Large (2.6 ± 0.2)	Large (2.6 ± 0.1)
Apertural callus length (CL, mm)	Middle (0.36 ± 0.06)	Short (0.32 ± 0.08)	Long (0.44 ± 0.04)
Shell diameter (D, mm)	Small (5.8 ± 0.2)	Large (6.5 ± 0.4)	Large (6.5 ± 0.2)
The proportion of shell height to shell diameter (HD)	Large (0.52 ± 0.01)	Small (0.48 ± 0.01)	Small (0.49 ± 0.01)
Spire angle (SA, degrees)	Small (138.9 ± 3.0)	Large (149.1 ± 3.1)	Middle (144.9 ± 2.5)
Umbilicus width (UW, mm)	Small (2.2 ± 0.1)	Large (2.5 ± 0.2)	Large (2.5 ± 0.2)
Surface of the protoconch	Smooth or very finely ribbed	Remarkably ribbed	Smooth or very finely ribbed
Gloss of the shell ventral side	Absent	Present	Absent
Microstructure of the shell ventral side	Both spiral and radial striations	Only radial striations	Both spiral and radial striations
Anterior lamellae	Narrow, elongated, T-shaped	Thick, C-shaped	Narrow, elongated, T-shaped

Note

• Measurements show mean ± SD.

Material examined. Lectotype, ANSP 87632 (photos examined). Newly collected materials. 17 adult shells collected from crevices in the limestones on the ground in the inland natural forest in Miyako Island, Miyakojima City, Okinawa Prefecture, Japan on October 18, 2020, KUZ Z3942.

Amended diagnosis. Shell very small, lenticular, thin, dextral (D 5.8 ± 0.2 mm [mean ± SD]; H 3.0 ± 0.1 mm) with slightly keeled body whorl; apex moderately protruding (HD: 0.52 ± 0.01; SA: 138.9 ± 3.0 degrees); periostracal folds short, on dorsal side of shell (HL 0.40 mm); teleoconch surface matte, not glossy, with strong ribs and spiral striations on ventral side; parietal callus mildly elevated (CL: 0.36 ± 0.06 mm); apertural fold very weak or absent; parietal wall with slightly curved and elongated anterior lamella and weaker vestigial posterior one; palatal plicae in one row, slender, rounded, and oblique; reproductive system with penial caecum; inner penial wall with longitudinal folds that form pocket-like pouches.

Measurements of lectotype. Adult shell: Measurements: BWL 2.3 mm; D 5.7 mm; H 3.0 mm; HD 0.53; SA 129.3 degrees; UD 0.39; UW 2.2 mm; WN 5.25.

Description of newly collected materials. Adult shell: Measurements are shown in Table 2. Shell lenticular, with bluntly shouldered body whorl and with conical and moderately protruding apex; light brown, translucent; 5.25–5.50 whorls separated by shallow suture; first 0.25 whorl smooth or very finely ribbed; remaining 1.75–2.00 whorl finely ribbed (Figure 4a); radial and spiral lines of comparable strength on dorsal side; same structure on the upper half of the body whorl; ventral side with stronger radial sculpture and spiral striations (Figure 4b); periostracal fold visible in fresh specimen on keel (HL 0.40 mm); umbilicus deep and wide; apertural margin thickened and strongly reflected; parietal callus mildly to strongly elevated; aperture without or with very weak fold connected to the parietal callus.

Lamellae and plicae (Figures 4c,d and 5a–p): Parietal wall with two vertical lamellae; moderately curved and elongated anterior lamella variable from near straight line to C- to T-shaped line; dorsal end of anterior one elongated anteriorly and posteriorly; weaker vestigial posterior one usually connected to anterior one; palatal plicae in one row with six horizontal or oblique plicae; first and sixth plicae on dorsal side small and horizontal; 6^th plicae usually split in 2 horizontal parts; second longest and most slender; thick third one slender to rounded; thick, rounded, and oblique 4^th and 5^th rarely connected with weak prominence.

Radulae (Figure 6a–c): pointed central teeth same length as or slightly longer than EcL; lateral tooth in six rows with pointed EcL, EnL, EnL 2–2.5 times longer than EcL; 8–10 row marginal tooth bear pointed EcM, EnM. EnMs with incisions 1/4 to 1/6 the length of the tooth 2.5–3 times longer than EcM.

Genitalia: Atrium short; penis consisting of thicker proximal and slimmer distal portions, of comparable length; internally with elevated longitudinal folds that form pocket-like pouches (Figure 7c); calcareous crystals observed in penis lumen in three specimens of six examined ones; penial caecum well-developed, approximately half as long as proximal part of penis; slender elongated retractor muscle inserts on apical part of penial caecum; epiphallus joining penis laterally at base of caecum; vas deferens slender, long, distal end gradually thickened to the base of spermoviduct; vagina with slightly thickened vaginal bulb, slightly slender near atrium; several weak fibers attaching vaginal bulb to body wall; inner vaginal wall with fine folds (Figure 7d); calcareous crystals found in vagina lumen in two specimens; narrow stalk of the bursa copulatrix branch off from the base of spermoviduct connect to elongated oval bursa copulatrix; narrow diverticulum start from slight distal portion of stalk of the bursa copulatrix extends almost the same length as the end of bursa copulatrix; spermoviduct long, approximately the same thickness as the proximal portion of penis; uterus contained 1–5, well-developed embryo consisting of 2.5 whorls; talon relatively small; albumen gland short.

Remarks. The reproductive anatomy of S. hirasei was first shown in Azuma and Azuma (1984). Although the genital characteristics examined in that study excluded the penial caecum, vaginal bulb, and diverticulum, those characters were recently described (Páll-Gergely, 2018a). Reproductive organs of the newly collected snails in this study almost corresponded to those in Páll-Gergely (2018a) because a penial caecum, vaginal bulb, and diverticulum were present. However, the vaginal bulb was not well developed and was almost as thick as the vagina.

Sicradiscus hirasei can be discriminated from S. pallgergelyi sp. nov. by its smaller shell with more protruding apex (larger HD, smaller SA) and less elevated parietal callus (middle CL; Table 5). The keeled body whorl and absence of strong folds in the aperture distinguishes S. hirasei from the western group species of the genus and S. transitus. Sicradiscus hirasei is also distinct from S. ishizakii and the other continental congeners by the matte ventral side, a slightly curved and elongated anterior lamella, and palatal plicae in one row consists of rounded and oblique plicae.

Sicradiscus pallgergelyi Sawada & Nakano, sp. nov.

Tables 2 and 5; Figures 4i–l, 5ag–av, 6g–i, 7b,g,h,j, 9g–l and 10.

(New Japanese name: Yaeyama-itokakemaimai)

http://zoobank.org/urn:lsid:zoobank.org:act:E3250AC4-6494-438C-B591-1F5C99F9AA0E

Material examined. Holotype. KUZ Z3947, adult shell collected from gaps in fallen leaves on the ground in the inland natural forest at an elevation of around 200 m in Iriomote Island, Taketomi Town, Yaeyama County, Okinawa Prefecture, Japan, on October 15, 2020. Paratypes. Four specimens collected with the holotype, KUZ Z3948–Z3951. Additional materials. Twenty adult specimens collected with the holotype, KUZ Z3952.

Diagnosis. Shell small, lenticular, rather thick, dextral (D 6.5 ± 0.2 mm; H 3.2 ± 0.1 mm) with bluntly keeled body whorl; apex moderately protruding (HD 0.49 ± 0.01; SA 144.9 ± 2.5 degrees); periostracal folds on dorsal side of shell (HL 0.38 ± 0.08 mm); teleoconch surface matte, not glossy, with strong ribs and spiral striations on ventral side; callus very strongly elevated (CL 0.44 ± 0.04 mm); apertural fold absent; parietal wall with slightly curved and elongated anterior lamella and weaker vestigial posterior one; palatal plicae in one row with rounded and oblique plicae; reproductive system with penial caecum; inner penial wall with longitudinal folds that form pocket-like pouches.

Description of holotype. Adult shell (Figure 9g–i): Measurements: BWL 2.6 mm; D 6.3 mm; EN 6; CL 0.47 mm; H 3.1 mm; HD 0.49; SA 147.7 degrees; UD 0.39; UW 2.5 mm; WN 5.50. Shell lenticular; with slightly keeled body whorl and with conical and moderately protruding apex; pale yellow, translucent; whorls separated by rather deep suture; first 0.25 whorl smooth; remaining 1.75 whorl very finely ribbed; radial and spiral lines of comparable strength on dorsal side; same structure on the upper half of the body whorl; ventral side with stronger radial sculpture and spiral striations; periostracal folds visible in fresh specimens on keel (HL 0.42 mm); umbilicus deep and wide; apertural margin thickened and strongly reflected; parietal callus very strongly elevated; aperture without fold connected to the parietal callus.

Parietal lamellae: Not examined.

Palatal plicae: one row with 6 horizontal or rounded plicae; first and sixth plicae on dorsal side small and horizontal; second longest, most slender, and horizontal; remaining ones thick, rounded, and oblique.

Radula: pointed central teeth same length as or slightly longer than EcL; lateral tooth in seven rows with pointed EcL, EnL, EnL three times longer than EcL; nine row marginal tooth bear pointed EcM, EnM. EnMs with incisions 1/5 the length of the tooth three times longer than EcM.

Genitalia: Atrium short; penis consisting of thicker proximal and slimmer distal portions, of comparable length; internally with elevated longitudinal folds that form pocket-like pouches; penis lumen without calcareous crystals; penial caecum well-developed, approximately half as long as proximal part of penis; slender elongated retractor muscle inserts on apical part of penial caecum; epiphallus joining penis laterally at base of caecum; vas deferens slender, long, distal end gradually thickened to the base of spermoviduct; vagina with slightly thickened vaginal bulb, slightly slender near atrium; several weak fibers attaching vaginal bulb to body wall; inner vaginal wall with fine folds; vagina lumen without calcareous crystals; narrow stalk of the bursa copulatrix branch off from the base of spermoviduct connect to elongated oval bursa copulatrix; narrow diverticulum start from slight distal portion of stalk of the bursa copulatrix extends almost the same length as the end of bursa copulatrix; spermoviduct long, approximately the same thickness as the proximal portion of penis; uterus contained 6, well-developed embryo consisting of 2.5 whorls; talon relatively small; albumen gland short.

Variation. Adult shell (Figure 9j–l): Measurements are shown in Table 2. Shell pale yellow or light to dark brown; first 0.25–0.50 whorl smooth; remaining 1.50–1.75 whorl very finely ribbed; parietal callus moderately to very strongly elevated; aperture without or with very weak fold connected to the parietal callus.

Lamellae and plicae (Figures 4k,l and 5ag–av): Parietal wall with two vertical lamellae; slightly to moderately curved and elongated anterior lamella variable from near straight line to C- to T-shaped line; dorsal end of weaker vestigial posterior one elongated anteriorly and posteriorly and usually connected to anterior one; palatal plicae in one row with 6 horizontal or rounded plicae; first and sixth plicae on dorsal side small and horizontal; second longest and most slender; remaining ones thick, rounded, and oblique.

Radulae (Figure 6g–i): lateral tooth in 6–7 rows with pointed EcL, EnL, EnL 2–2.5 times longer than EcL; nine rows marginal tooth bear pointed EcM, EnM. EnMs with incisions 1/4 to 1/6 the length of the tooth 2.5–3 times longer than EcM.

Genitalia (Figure 7b,g,h,j): calcareous crystals found in penis lumen in 3 specimens of 8 examined ones; calcareous crystals found in vagina lumen in 1 specimen; uterus contained 3–6, well-developed embryo consisting of 2.5 whorls.

Distribution and ecology. The new species was found from a narrow area in the interior of Iriomote Island, Japan. The fossil recorded from Ishigaki Island and identified as S. hirasei may belong to this species (Nature Conservation Division, Department of Environmental Affairs, Okinawa Prefectural Government, 2017).

Etymology. The specific name is dedicated to Dr. Barna Páll-Gergely, who greatly contributed to the systematics of the family Plectopylidae.

Remarks. The new species can be discriminated from S. hirasei by the larger shell size (larger BWL, D, H, UW) of the former species (Table 5). The new species also possesses a flatter shell (smaller HD, larger SA) and a more developed parietal callus (larger CL). Although the shell morphology of S. pallgergelyi sp. nov. is similar to that of S. ishizakii because it has a larger and flatter shell (similar values in BWL, D, H, HD, UW), the new species exhibits a more protruding apex (larger SA) and a more developed parietal callus (larger CL) than the Taiwanese species. The new species has a shell whose ventral side is not glossy, and has strong ribs and spiral striations. In contrast, the ventral side of S. ishizakii has a glossy appearance because it lacks spiral striae.

The matte ventral side of the new species distinguishes this species from its congeners. The new species is distinguishable from S. feheri, S. mansuyi, S. invius, S. secures, which belong to the western group of Sicradiscus and S. transitus by the presence of keeled body whorls and absence of strong folds from the aperture. The new species is also distinct from seven Chinese and Vietnamese congeners, S. feheri, S. invius, S. cutisculptus, S. schistoptychia, S. transitus, S. diptychia, S. securus, and S. mansuyi by the following internal structures: elongated and slender anterior lamella; vestigial and slender posterior lamella; and palatal plicae in one row consists of rounded and oblique plicae that are not usually connected.

4.1 Genital morphology of insular Sicradiscus species

The genus Sicradiscus has been subdivided into two species groups based on shell characters (Páll-Gergely & Hunyadi, 2013). The new species is clearly a member of the eastern group of the genus based on its slightly keeled body whorl and no fold in the aperture. Observations of reproductive organs in previous studies (Azuma & Azuma, 1984; Páll-Gergely, 2018a; Páll-Gergely & Asami, 2014; Páll-Gergely & Hunyadi, 2013; Páll-Gergely, Hunyadi, et al., 2015) revealed that each group has different genital characteristics (Páll-Gergely, 2018a). The western group species have a very weakly developed penial caecum and pockets in their inner penial wall, whereas the eastern group species have a well-developed penial caecum and parallel folds in their inner penial wall.

In the present study, the general morphology of S. hirasei genitalia was mostly concordant with that of Páll-Gergely (2018a) (see Remarks of S. hirasei). However, the previously overlooked penial caecum and diverticulum were discovered in the reproductive organs of S. ishizakii. The new species also possessed a clearly distinguishable penial caecum. Accordingly, it is even more likely that the members of the eastern group share the characteristics of a well-developed penial caecum, although the reproductive anatomy of S. cutisculptus and S. diptychia is still not known.

This study revealed the presence of slit-like pockets in the inner penial wall of the three examined species. In contrast to the penial caecum results, these pockets were not previously detected in the eastern group. As indicated in Páll-Gergely, Hunyadi, et al. (2015), the inconsistencies between the present and previous observations may be due to seasonal changes because the samples were collected in different seasons [Páll-Gergely (2018a), December; this study, October]. Therefore, the inner penial wall structure is unlikely to significantly differ between the groups, and further investigation is necessary to clarify the seasonal change in these reproductive structures.

Calcareous granules were observed in the inner penial and/or vaginal wall of the three Sicradiscus species. It was suggested that the penial granules function as a mating apparatus for disposable males and are lost when snails bear offspring (Páll-Gergely, Hunyadi, et al., 2015). In this study, however, all nine individuals with granules on the inner penial wall possessed embryos, and no significant difference was detected in the number or size of embryos between the snails with or without the granules. The vaginal granules observed in the three Sicradiscus species have only been previously described from two other species in the family, Halongella schlumbergeri (Morlet, 1886) and Halongella fruhstorferi (Möllendorff, 1901). These granules have also been proposed to be seasonal and associated with mating season (Páll-Gergely, Hunyadi, et al., 2015). In this analysis, some snails had penial and vaginal granules and some did not, even within the same population collected on the same day; this indicates that the reproductive cycle of each snail may also affect granule appearance.

4.2 Morphological evolution of insular Sicradiscus species

The present morphological examination discriminated the three Sicradiscus species with the measurements of the apertural callus length and the spire angle. The four other quantitative characters separated S. hirasei from the other species. CVA based on the measurements of five characters also differentiated S. hirasei from S. pallgergelyi sp. nov. and S. ishizakii, which indicates that S. hirasei has a unique shell morphology. Moreover, S. ishizakii was distinguished from S. hirasei and the new species by the ventral sculpture of its shell and characteristics of its protoconch, lamellae, and plicae. The molecular phylogenetic analyses revealed that shell measurements of the insular Sicradiscus species seem to have higher plasticity than shell structure. In addition, the ventral sculpture characteristics can be estimated to be a common trait of Japanese lineages, and the Taiwanese species share characteristics with the continental congeners.

The CL measurements were largest in the new species and smallest in S. ishizakii. Although the function of the apertural callus is still unclear, it may help prevent natural enemies from entering the aperture if the apertural folds serve a similar function in land snails as they do in Vertiginidae and Diapheridae (Gittenberger, 1995; Solem, 1972). In the genus Satsuma A. Adams, 1868, it was suggested that strong apertural folds evolved by predation pressure from snail-eating snakes of family Pareatidae in the Ryukyu Islands and Taiwan (Hoso & Hori, 2008). Even though snakes are not predators of Sicradiscus species due to the snails' small size, morphological evolution in the apertural callus may be promoted by predation pressures from snail-eating fireflies, which occur on each of the Ryukyu Islands (Ohba, 2004). The relationship between aperture size and water loss revealed that the apertural callus may also prevent water loss from the aperture by reducing the amount of air that passes through the aperture (Machin, 1967).

The proportion of shell height to shell diameter of S. hirasei found from gaps in limestone was larger than that of the new species, which was found in gaps in fallen leaves on the ground. Land snails with a low-spired shell tend to prefer low-angle or horizontal surfaces, whereas the species with intermediate shell shapes are active on a wide variety of angles (Cain & Cowie, 1978; Cameron, 1978). It was also theorized that a low-spired shell is well-balanced in both horizontal and vertical substrates (Okajima & Chiba, 2009). In addition, it is known that a flat shell morphology is more advantageous in narrow spaces such as rock crevices (Goodfriend, 1986). Because Sicradiscus species are small, it is unclear whether shell balance significantly contributes to shell morphology. However, the present results of the larger HD of the rock dweller may be explained by their habitat use: S. hirasei is active on rocks with various angular conditions, whereas the other species are restricted to the vicinity of the horizontal ground. It was also shown that land snails in limestone areas tend to thicken their shells in response to calcium availability (Owen, 1965), although the S. hirasei shells were the thinnest among the three species. Because shell thickness is also related to predation pressure (Moreno-Rueda, 2009), different predation pressures among islands may potentially affect the shell thickness of Sicradiscus species.

The two Japanese species are phylogenetically closely related, which indicates that major morphological shifts occurred in the two species, even though Sicradiscus species are estimated to experience only slight conchological changes (Páll-Gergely & Hunyadi, 2013). The aforementioned morphological differences between S. hirasei and the new species strongly indicate that the common ancestor of the Japanese lineage expanded to different islands and underwent different selection pressures, which resulted in speciation.

4.3 Radular morphology

The radular morphology of Sicradiscus was examined in S. ishizakii and four other species by Chang and Ookubo (1999) and Páll-Gergely, Hunyadi, et al. (2015), and the latter study also reported that the shape of the radulae was highly preserved among related genera. In this examination, significant differences were not observed in radular morphology among the three Sicradiscus species, and the S. ishizakii radulae corresponded to those described by Chang and Ookubo (1999). The radulae of the three species consisted of a relatively large central tooth, a lateral tooth with ectocones as large as cusps of a central tooth, and tricuspid marginal teeth with pointed cusps and deep incisions between the cusps; these characteristics are similar to those of the radulae of other congeners and members of Sinicola and Gudeodiscus (Páll-Gergely, Hunyadi, et al., 2015).

4.4 Taxonomic position of other insular populations of Sicradiscus

The new species was discovered in a well-preserved natural forest at an elevation of approximately 200 m on Iriomote Island. In addition to the record on the island, fossil specimens previously identified as S. hirasei were found on Ishigaki Island between Miyako Island and Taiwan (Nature Conservation Division, Department of Environmental Affairs, Okinawa Prefectural Government, 2017). Although only fossil specimens have been collected from Ishigaki Island, extant Sicradiscus snails may be distributed on the island because the natural forests are preserved on the island and are similar to the habitat of the new species. Taking into account the similarity of the land snail faunas between the two islands (Habe & Chinen, 1974), the fossil records on Ishigaki Island likely belong to S. pallgergelyi sp. nov.

The length of the apertural callus distinguishes the three insular species. Although the apertural structure may facilitate self-protection, a population without callus was also found in central Taiwan (Lee & Chen, 2003). This population also has a smaller number of ribs on the ventral side of the shell compared with S. ishizakii of northern Taiwan and the Japanese species. Because there are other species in northern and central Taiwan (Hsieh, 2003) and the length of the callus may be related to different selection pressures, this central population may be an independent species with a unique evolutionary history. The systematic position of the populations in Ishigaki Island and central Taiwan should be examined in future studies.