First record and description of three new species in the land snail genus Diplommatina Benson, 1849 (Caenogastropoda, Diplommatinidae) from Satun Province, Thailand

The micro land snail genus Diplommatina (family Diplommatinidae) is widely distributed in Southeast Asia and includes many endemic species. Three new species of Diplommatina are described from Satun Province in southern Thailand. Diplommatina bu-lonensis sp. nov. , D. laemsonensis sp. nov. and D. prakaiphetensis sp. nov. are distinguished from other species in the genus by their shell size and shape, the number of radial ribs on the penultimate whorl, the number of whorls, and features of the peristome. The agreement between phylogenetic tree based on analyses of COI and 16S sequences and comparative morphology support the delineation of these new species which, when compared to related species, belong to well-differentiated clades. The K2P distance between any of the three new species and other Diplommatina species included in the molecular phylogenetic analysis was at least 5.5% in COI and 3.9% in 16S. Two of the three new species ( D. prakaiphetensis sp. nov. and D. bulonensis sp. nov. ) are apparently endemic to Prakaiphet Hill and Bulon Pai Island, respectively. Additionally, we documented a new regional record for D. naiyanetri in Satun Province. These new species and records contribute to the knowledge of Thailand’s land snail biodiversity and highlight the need of conservation protections for regional karst habitats.


Introduction
Southern Thailand is located in the transition zone between two major biodiversity hotspots, the Indo-Burma and Sundaland -both of which have numerous endemic species (Myers et al. 2000). Satun Province in southern Thailand is characterized by karst features, with limestone mountains, foothills, and coastal islands. Satun's limestone hills support high biodiversity and endemism in micro land snails (Panha and Burch 2005). The taxonomy of micro land snails in Thailand's other limestone areas has been studied comprehensively over the previous two decades, resulting in descriptions of several new species (Panha and Burch 2005). However, Satun's microsnail fauna remains largely unexplored.
The shell characters used to identify Diplommatina species (Kobelt 1902;Panha and Burch 2005;Yamazaki et al. 2015;Nurinsiyah and Hausdorf 2017) may display adaptation to environmental characteristics, including the presence of predators (Schilthuizen et al. 2006). As a consequence, morphological features may be confusing and molecular phylogenetic techniques can be powerful tools for resolving the taxonomy and additionally inform phylogenetic rela-tionships among snail taxa (Douris et al. 1998;Chiba 1999;Thacker and Hadfield 2000;Holland and Hadfield 2002;Steinke et al. 2004;Desouky and Busais 2012). The DNA barcoding markers of cytochrome c oxidase subunit 1 mitochondrial gene (COI) and 16S ribosomal DNA (16S rDNA) have been used in discriminating land snail species, including those within Diplommatinidae, and to investigate phylogenetic relationships (Webster et al. 2012;Liew et al. 2014).
Within a larger study of the microsnails of karst areas in Saturn Province, we especially targeted the genus Diplommatina. After conducting an extensive field survey, we used both morphological and molecular analyses to identify known species and describe new species.

Ethical statements
We followed the guidelines for animal care in the International Guiding Principles of Biomedical Research Involving Animals (Council for International Organizations of Medical Sciences: CIOMS) including the relevant document (U1-03304-2559 to NN.).

Specimen sampling
Karst areas throughout Satun Province, Thailand ( Fig. 1) were surveyed for land snails during the rainy seasons in December 2020 through January 2022. Specimens of Diplommatina were collected by visual searching with one to three searchers and up to three hours of searching per site. Snails were found on soil surfaces, on limestone, stones, wood, moss and under leaf litter. Micro land snails were especially common in limestone crevices and on moist surfaces. Both live snails and empty shells were collected. All live specimens were drowned in water, preserved in 95% ethanol and processed in the Biology Department, Faculty of Science, in Chiang Mai University.

Morphological study
All Diplommatina specimens were identified by comparing shell morphology with type specimens and the original descriptions. Specimens of Diplommatina were examined using a Leica MZ16 microscope and photographs were taken using the Leica Application Suite Version 3.4.0 program. Shell morphological characteristics, including shell height and width and aperture height and width, were measured from digital images using ImageJ (Schneider et al. 2012). Shell morphological measurements and ratios among measurements (e.g., shell height to width) of each morphospecies were analyzed using ANOVA (α = 0.05) using IBM SPSS Statistics ver. 22.0.
The outer wall of the body whorl of at least two shells of each species was removed to reveal the internal lamellar features (Vermeulen 1993;Panha and Burch 2005;Neubert and Bouchet 2015;Budha et al. 2017), which were photographed. Shells were cleaned by using sonication prior to viewing with a scanning electron microscope (SEM). Dried shells were sputter-coated with gold and examined with a JSM 5910 LV scanning electron microscope at the Electron Microscope Research and Service, Faculty of Science, Chiang Mai University.
Using a dissecting microscope, the radulae of microsnails were extracted from the buccal cavity, cleared in 1% sodium hydroxide overnight and then washed with distilled water. Radulae were dehydrated by immersion in increasing alcohol concentrations (10%, 30%, 50%, 70%, 80% and 95%) (Nantarat et al. 2014). The dehydrated radulae were fixed onto SEM stubs with carbon tape and coated with gold (Franklin et al. 2007) and viewed on the scanning electron microscope.

Molecular study and phylogenetic analyses
This study included data from GenBank for fourteen species and three unidentified species of Diplommatina (totalling 20 records) (Rundell 2008;Webster et al. 2012), and twelve records of species from other genera in the family that were used as outgroups to root the tree (Rundell 2008; Webster et al. 2012). Sequences of ten individuals of Diplommatina spp. collected during our surveys were included in our molecular phylogenetic analysis.
The foot tissue of the ethanol-preserved snails was removed for DNA extraction. The samples were incu-bated with 150 μl Chelex 100 and 3 μl Proteinase K for 1 hour at 55 °C, followed by 30 minutes at 95 °C (de Lamballerie et al. 1992). All DNA samples were stored at -20 °C for later use. The COI gene was amplified using primers LCO1490 (5'-GGTCAACAA ATCATA AAGA-TATTGG -3') and HCO2198 (5'-TAAACTTCAGGGT-GACCAAAA AATCA -3') (Folmer et al. 1994). PCR reactions were performed with cycle parameters of 94 °C for 3 min, followed by 36 cycles of 94 °C for 30 s, 50 °C for 60 s, and 72 °C for 1 min, and a final extension step at 72 °C for 5 min. The 16S rRNA gene was amplified by using primers 16Sar (5'-CGCCTGTTTATCAAAAA-CAT-3') and 16Sbr (5'-CCGGTCTGAACTCAGAT-CACGT-3') (Palumbi 1996). PCR amplification was performed at 95 °C for 3 min, followed by 35 cycles of 94 °C for 30 s, 45 °C for 1 min, and 72 °C for 2 min, and then a final 72 °C for 5 min. Primer PCR products were checked using 1% (w/v) agarose gel electrophoresis with 1X TBE buffer. Purification and sequencing were performed using Barcode-Tagged Sequencing (BTSeq) Services. After sequencing, the sequences were proofread on chromatograms, and the coding was aligned using Clustal W (Thompson et al. 1994) and then manually edited with MEGA X (Kumar et al. 2018). A total of 42 sequences were used in this study. New sequences have been deposited in GenBank (https://www.ncbi.nlm.nih.gov/genbank) and are shown in Table 2. Phylogenetic trees were constructed using Maximum likelihood (ML) and Bayesian inference (BI) methods. Maximum likelihood analyses were performed based on the TIM3+F+I+G4 model for the COI gene and GTR+F+I+G4 model for the 16S gene by using IQ-TREE on XSEDE and CIPRES (Miller et al. 2010;Nguyen et al. 2015) with 1000 bootstrap repeats (Hoang et al. 2018). The Bayesian Inference analysis was performed using MrBayes (Ronquist et al. 2012) version 3.2.7. Parameter settings were 4 nchains and 4 nruns in a Markov Chain Monte Carlo algorithm (MCMC). The BI analysis was performed for 10,000,000 generations, with a temperature parameter for heating = 0.8, sampled every 100 generations, and setting burn-in at 25% of the run (Ronquist and Huelsenbeck 2003 Etymology. The specific epithet bulonensis is an adjective referring to the type locality (Bulon Pai Island in Satun Province, Thailand). Differential diagnosis. Among approximately 400 species of Diplommatina, the new species is most similar to D. conditioria Maassen, 2007, D. baliana Fulton, 1899, D. maduana Laidlaw, 1949, D gomantongensis Smith, 1894and D. antheae Vermeulen, 1993 (Table 1). Typical characteristics shared by D. bulonensis with all these species are shell dextral with a slender fusiform shape with rather evenly rounded sides, radial ribs not sinuous, constriction situated halfway on the parietal side of the peristome and aperture round. However, D. bulonensis sp. nov. differs from D. gomantongensis and D. antheae by the presence of a transverse palatalis and an inconspicuous columellar denticle in the aperture (Fig. 4A). D. bulonensis sp. nov dif-fers in the combination of shell height, shell width, aperture height and aperture width from D. maduana and D. antheae (Table 1). D. bulonensis sp. nov differs from D. conditioria and D. maduana in the number of whorls (Table 1) and differs from D. maduana, D. gomantongensis, D. antheae and D. baliana in the number of radial ribs/0.5 mm on the penultimate whorl (Table 1). D. bulonensis sp. nov has fewer radial ribs on body whorls above the aperture than D. conditioria (12-13 vs. 16 ribs/1 mm) (Maassen 2007). Moreover, it differs from D. baliana in having a columella fold.
Description. Shell dextral, fusiform, convex and red rushes or red-orange in color (Figs 2A, 3A). Penultimate whorl slightly wider than body whorl, side flat. Spire conical with slightly convex sides. Suture impressed. Constriction level with the middle parietal side of the peristome, with three lamellae: one parietalis very distinct, one distinct and long transversal palatalis and one distinct columellaris. (Fig. 4A, B). Protoconch smooth with dimples and about 2 1/2 whorls (Fig. 3B). Radial ribs straight, inconspicuous but distinct, moderately spaced about 6 1/2-7 ribs/0.5 mm on the penultimate whorl (Table 1). The aperture slightly tilted against the coiling axis, columellar denticle slightly weak in the aperture. Peristome double, rather expanding, palatal side not sinuous, basal side and columella side weakly sinuous. Outer peristome expanded less than inner peristome, inner peristome expanded at parietalis side, with a palatal lip. Umbilicus closed. Operculum multispiral, flat, corneous, transparent, slightly concave, outer surface smooth, inner surface smooth with raised peripheral circular margin (Fig. 3C). The radula of the taenioglossate type (Fig. 5B). The central tooth round and flat with seven cusps of similar size. The basal plate of the central tooth, cuspate and small. The lateral teeth with five cusps. The marginal teeth with inner and outer teeth, the inner teeth with seven small cusps and the outer teeth with five cusps and larger than the inner teeth (Fig. 5B). Etymology. The specific epithet laemsonensis is an adjective referring to the type locality (Laem Son subdistrict, Lan-gu district, Satun Province, Thailand). Differential diagnosis. Among the sinistral species of Diplommatina, Diplommatina laemsonensis sp. nov. is most similar to D. diminuta Möllendorff, 1891, D. sinistra Tomlin, 1938and D. acme Laidlaw, 1949 (Table 1). All these species share a moderately slender fusiform shape, rounded periphery, shell height range and number of whorls (Table 1). However, Diplommatina laemsonensis differs from D. acme, D. diminuta and D. sinistra in the number of radial ribs in the penultimate whorl (Table 1). It differs from D. diminuta in shell width and the ratio of AH/AW (Table 1). Moreover, it differs from D. diminuta by the absence of teeth on the basal margin of the aperture and differs from D. sinistra in having a distinct columellar denticle in the aperture.

Diplommatina laemsonensis
Description. Shell sinistral, fusiform, convex and honey brown in color (Figs 2B, 3D). The penultimate whorl widest. Spire conical with slightly convex sides. Suture impressed. Constriction level with the edge between the parietal and columellar side of the peristome, with three lamellae: one distinct parietalis which starts at the constriction, one transversal palatalis, one distinct columellaris which continues into the tuba and visible in the aperture (Fig. 4C, D). Protoconch sculpture smooth with fine pitting and about 1 whorl (Fig. 3E). Radial ribs straight, distinct, rather dense on top of the teleoconch, suddenly changing to a moderately spaced about 8-9 ribs/0.5 mm on the penultimate whorl (Table 1). The aperture tilted up to 30 °C in relation to the coiling axis, columellar denticle visible in the aperture, deflected downwards. Peristome double, expanding, palatal side rounded with slightly edge, basal side with edge slightly protruding. Outer peristome expands beyond the inner peristome with about four layers, inner peristome with distinct a palatal, basal and columellar lips. Umbilicus closed. Operculum multispiral, flat, corneous, transparent, slightly concave, outer surface smooth with small pits distributed over whole surface, inner surface smooth with raised peripheral circular margin (Fig. 3F). The radula of the taenioglossate type (Fig.  5A). Central tooth strong with a large central cusp, with 2 pairs of developed lateral cusps. Basal plate of central tooth prominent with 2 small cusps. The lateral teeth with six cusps, the third cusp longest. In the marginal teeth, the inner teeth larger than the outer, with six cusps, the third cusp longest. The outer tooth with four cusps (Fig. 5A). Type locality. Thailand, Satun Province, Thung Wa District, Prakaiphet Hill, 7°00'00.1"N, 99°46'08.7"E, 19 January 2022, coll. T. Boonmachai.
Etymology. The specific epithet prakaiphetensis is an adjective referring to the type locality (Prakaiphet Hill in Na Thon subdistrict, Thung Wa District, Satun Province, Thailand).
Differential diagnosis. Among sinistral diplommatinids, Diplommatina prakaiphetensis sp. nov. most closely resembles D. krabiensis Panha & J. B. Burch, 1998 from Thailand, and D. karoensis Maassen, 2002 from Sumatra, Indonesia as all these species share a minute size, an ovate shell shape with rather flat whorls, radial ribs being distinct and comparatively widely spaced. However, D. prakaiphetensis sp. nov. differs from both other species in the number of whorls (Table 1). It also differs from D. karoensisi in the number of radial ribs/0.5 mm on the penultimate whorl and ratio of SH/SW (Table 1). Moreover, the absence of radial ribs on the body whorl on the parietal side and slightly wavy radial ribs distinguishes it from D. krabiensis.
Description. Shell sinistral, fusiform, thin, convex, color Aztec gold, (Figs 2C, 3G). Penultimate whorl width slightly smaller than the body whorl, suture impressed. Constriction level with the middle parietal side of the peristome, with three lamellae: one parietalis, one short transverse palatalis and one prominent columellaris that continues into the tuba and is visible in the aperture (Fig. 4E, F). Protoconch smooth, with very fine pits, about 1 1/4 whorl (Fig. 3H). Radial ribs are very fine and closely spaced on top of the teleoconch; the following ones are more widely spaced, and become prominent on third to last whorl and are wavy, inconspicuous or almost absent on the body whorl on the parietal side of peristome with about 3-4 ribs/0.5 mm on the penultimate whorl (Table 1). Aperture rounded, columellar denticle distinct in the aperture. Peristome double and expanding. The outer peristome expanding beyond the inner, with palatal and columellar side protruding, basal side not protruding, inner peristome expanding with a weak parietal lip. Umbilicus closed. Operculum multispiral, flat, corneous, transparent, slightly concave, outer surface smooth, inner surface smooth with a large ridge and raised peripheral circular edge (Fig. 3I). The radula of the taenioglossate type (Fig. 5C). The central tooth strong and pointed cusps with a large central cusp, two pairs of developed lateral cusps. The basal plate of the central tooth prominent with two small cusps. The lateral teeth with five cusps; the longest is the third cusp. In the marginal teeth, the inner teeth larger than the outer, with four cusps, and the longest is the second cusp. The outer marginal teeth with three cusps (Fig. 5C).

Molecular phylogenetic analyses
Sequences from a total of 30 individuals of Diplommatina spp. and 12 individuals from four additional diplommatinid genera (Hungerfordia Beddome, 1889, Palaina Semper, 1865, Opisthoporus Manter, 1947and Opisthostoma W. T. Blanford & H. F. Blanford, 1860; the out groups) were used for phylogenetic reconstruction ( Table 2). The concatenated alignment of COI and 16S sequences had a total length of 996 base pairs (COI = 594, 16S = 402). The phylogenetic tree based on the analyses of the concatenated COI and 16S sequences supported Diplommatina as a monophyletic group. Within Diplommatina, the phylogenetic tree was divided into three main clades. Clade A was strongly supported and included all dextral species from Boneo, Peninsular Malaysia, and southern Thailand (Fig. 6). Within this clade, D. bulonensis sp. nov. formed the sister group contain-  ing D. gomantongensis, Diplommatina sp. from Borneo and D. canaliculata (Fig. 6). Clade B consisted of most Diplommatina species from Borneo, such as D. centralis, D. rubra, D. rubicunda, D. electa and two Diplommatina spp., plus D. laidlawi from Peninsular Malaysia. Clade C contained the sister pair of D. prakaiphetensis sp. nov. and D. laemsonensis sp. nov. The pair's sister group was D. naiyanetri (Fig. 6).

Discussion
Both morphological and molecular evidence support the recognition of the three new species Diplommatina laemsonensis sp. nov, Diplommatina bulonensis sp. nov. and Diplommatina prakaiphetensis sp. nov. Two of the species are presumed to be endemic to the region: D. bulonensis sp. nov. has been found only on Bulon Pai Island and D. prakaiphetensis sp. nov. has been found only on Prakaiphet Hill on the mainland. In addition, we found an additional regional record of D. naiyanetri (Figs 1-3).
One species, D. canaliculata, had previously been reported from Satun Province. This species was also found in other areas of Thailand, such as Trang, Nakhon Si Thammarat and Krabi Provinces (Panha and Burch 2005), as well as in Malaysia (Peninsular and Borneo) and Indonesia (Sumatra, Java and Borneo) (Laidlaw 1949;Vermeulen 1993;Maassen 2001aMaassen , 2002. A second species, D. naiyanetri, is a new Satun Province record and was found on Tarutao Island. Previously, this species had been reported in three other provinces in Thailand; namely, Trang, Phatthalung and Nakhon Si Thammarat (Panha and Burch 2005). All of these provinces share a similar origin of Paleozoic limestones on the continental Shan-Thai fragment, which may explain the observed present-day pattern of distribution (Agematsu et al. 2006a, b).
Several shell morphological characters can be used to distinguish the new species from other Diplommatina species; these include shell height, shell width, aperture height, aperture width, the ratio of SH/SW, the ratio of AH/AW, the internal lamellar system, radial ribs on the penultimate whorl, the number of whorls and the structure of the peristome. Moreover, scanning electron microscopy (SEM) showed additional details in the sculpturing and number of whorls of the protoconch in the three new species (Fig. 3). These protoconch features have previously been used as criteria for identification in Diplommatina species (Panha and Burch 2001;Maassen 2002;). Furthermore, Table 1. Comparison of shell morphological characters of closely related Diplommatina species with new species. Shell morphological characters were tested using one-way ANOVA (P < 0.05).

Species
Characters ( this is the first study of radular morphology of Diplommatina. Differences in the shape of basal plate, and the shape and number of cusps on central tooth were observed in the three new species (Fig. 3), indicating that radular characters are useful for identification within this genus. In contrast, the opercula are very similar, being paucispiral, flat, transparent, with a smooth outer and inner surface with a raised peripheral circular margin. The operculum is infrequently used in taxonomy among diplommatinids because it has few characters and is very similar among species (Neubert and Bouchet 2015).
Molecular phylogenetic techniques were used to verify taxonomy and indicate classification, and are especially useful where morphological characters alone do not provide clear delineations (Douris et al. 1998;Chiba 1999;Thacker and Hadfield 2000;Holland and Hadfield 2002;Steinke et al. 2004;Desouky and Busais 2012). The phylogenetic tree revealed three main clades with-in Diplommatina and species within these clades shared the same sinistral or dextral shell spire and had similar distribution patterns. Clade A was a strongly supported clade consisting of dextral species from Borneo and Peninsular Malaysia, southern Thailand and Taiwan (Fig. 6). D. bulonensis sp. nov. had high support as a sister group of D. gomantongensis and D. canaliculata and was also placed in the same clade as D. hidagai from Thailand (Fig. 6). The phylogenetic tree was consistent with the pattern of difference in shell morphology of D. bulonensis sp. nov. versus D. canaliculata and D. hidagai in the ratio of SH/SW and the number of whorls, and the difference from D. gomantongensis in the radial ribs/0.5 mm on the penultimate whorl (Table 1).
All sinistral species were placed in Clade B and Clade C, which were geographically distinct. All species in Clade B were from Borneo, Malaysia and all species in Clade C were from Peninsular Malaysia and southern Thailand. The close placement of D. laemsonensis sp. nov. and D. prakaiphetensis sp. nov. with their sister species D. naiyanetri in Clade C was strongly supported (Fig. 6). The phylogenetic tree agreed with the significant differences in shell morphology of D. laemsonensis sp. nov. and D. prakaiphetensis sp. nov. versus D. naiyanetri in the ratio of SH/SW, the number of whorls, and the number of radial ribs/0.5 mm on the penultimate whorl (Table 1). In addition, the genetic K2P distance based on COI and 16S genes demonstrated that the distances of the three new species were greater than the overall mean of the species in this genus. Thus, the morphological char-acters and molecular analysis both indicate that the three described taxa of Diplommatina in this study are, indeed, new species. Many tropic and subtropic karst regions support high biodiversity and endemic species of land snails, especially microsnails (Panha and Burch 2005). A variety of human activities impact limestone outcrop habitats in Southeast Asia (Sodhi et al. 2010;Hughes 2017). The destruction of limestone outcrop habitats leads to the extinction of species, especially endemic species (Sodhi et al. 2010). Hence, it is necessary to understand unique aspects of biodiversity for these small 'hotspots' Figure 6. Phylogenetic tree of Diplommatina species based on COI and 16S genes. The number on each clade are statistic supports based on ML and BI methods, respectively. The ML exceed less than 60% of the Boorstrap support and the posterior probability of BI is greater than 0.70. for planning and implementing conservation strategies, especially for threatened endemic species, including land snails.