Integrative systematics reveals the new land-snail genus Taphrenalla (Eupulmonata: Ariophantidae) with a description of nine new species from Thailand


The endemic terrestrial Crown Snail from Southern Thailand formerly in Macrochlamys Gray, 1847 is now described as Taphrenalla Pholyotha & Panha gen. nov., based on comparative morphology and molecular data. Overall, eleven Taphrenalla species are now recognized, including two nominal species: T. asamurai and T. diadema. A total of nine new species are proposed: T. alba sp. nov., T. conformis sp. nov., T. corona sp. nov., T. dalli sp. nov., T. incilis sp. nov., T. macrosulcata sp. nov., T. parversa sp. nov., T. pygmaea sp. nov. and T. zemia sp. nov. The molecular phylogeny constructed from the mitochondrial cytochrome oxidase c subunit I (COI) and 16S rRNA gene fragments plus the nuclear 28S rDNA gene fragments revealed that Taphrenalla gen. nov. is monophyletic with a well-supported clade. The diagnostic characters of Taphrenalla gen. nov. are the shell sculpture with several radial grooves, body with well-developed colourful stripes running from the head to tail, and genitalia similar to Macrochamys but with an un-coiling epiphallic caecum. The spermatophore has one or two spines near the sperm sac and a spineless tail.


Introduction
Thailand, especially the southern part, is rich in biodiversity of malacofauna and is recognized as a biodiversity hotspot (Myers et al., 2000). The topography of Southern Thailand is a peninsula between the Gulf of Thailand and the Andaman Sea. The long ridge of two mountain ranges (Phuket Range and Nakhon Si Thammarat Range) separated by low-lying areas of less than 200 m above mean sea level (amsl) extend to this part. The Phuket Range runs parallel to the west coast of upper Southern Thailand which extends northwards to the Tenasserim Range, whereas the Nakhon Si Thammarat Ranges runs from Surat Thai southwards to the Malaysian border (Gupta, 2005;Ridd et al., 2011;Gardner et al., 2015;Department of Mineral Resources, 2018).
The diversity of land snails in Southern Thailand is due to several interrelated factors. There is a high relative humidity, a short dry season (2-4 months) and a great amount of rainfall (Thai Meteorological Department, 2015). According to the key zoogeographical position of Thailand, the snail fauna has dispersed from Indo-Himalayan, Indo-Chinese, and Malaysian regions . In addition, there are many extensive ranges of limestone karsts and outcrops scattered along this area. Limestones in Southern Thailand are formed from the Permian Ratburi Lime stone and, to a lesser extent, the Ordovician-Devonian Satun Group limestone (Gupta, 2005;Ridd et al., 2011;Gardner et al., 2015;Department of Mineral Resources, 2018). Many documents show that land snail diversity is associated with limestone karsts and outcrops (Clements et al., 2006;Naggs et al., 2006). The larger karsts probably contain a higher number and diversity of microhabitats that are conducive for land snail speciation and endemism (Clements et al., 2008;Foon et al., 2017).
The land snail family Ariophantidae Godwin-Austen, 1888 is recognized as one of the most diverse families and contains a remarkably high number of species with various shell characteristics from snails to semi-slugs (Blanford & Godwin-Austen, 1908;Schileyko, 2002Schileyko, , 2003. Ariophantid taxa are only characterized by the absence of the penial nerve fibres running through the cerebral ganglion (Hausdorf, 1998). Later, the definitions of Ariophantidae are given by Schileyko (2002), who made extensive changes to the classification of Limacoidea s.l. The characters of Ariophantidae are shell helicoid to plate-like, animal has no frontal organ, caudal foss and caudal horn well-developed, genitalia with various length flagellum, a straight, spirally coiled or missing epiphallic caecum attached by penial retractor, penis with or without verge, dart apparatus absent or present (when present, it is vaginal, with apical retractor). Ariophantidae is comprised of three subfamilies (Bouchet et al., 2017). Among these, the Macrochlamydinae is widely distributed throughout Asia, especially South and Southeast Asia (Blanford & Godwin-Austen, 1908). Due to the similarity in their shell morphology (glossy, thin, and semi-transparent), early malacologists attributed most Asian species to Macrochlamys (Godwin-Austen, 1883, 1907Blanford & Godwin-Austen, 1908). The type genus of this subfamily is Macrochla mys Gray, 1847, which is recognized as the most species-rich genus in Ariophantidae (Blanford & Godwin-Austen, 1908;Pholyotha et al., 2018Pholyotha et al., , 2020Sajan et al., 2019). Subsequently, many genera, such as Taphrospi ra Blanford, 1905, Sarika Godwin-Austen, 1907, Sakiella Godwin-Austen, 1908, Syama Godwin-Austen, 1908, and Rasama Laidlaw, 1932, have been described as different genera on the basis of their reproductive anatomy or some unique characters of their shells (Blanford, 1905;Blanford & Godwin-Austen, 1908;Laidlaw, 1932).
Taxonomic studies of the two Crown Snails, Nanina (Macrochlamys) diadema Dall, 1897 and Macrochlamys asamurai (Panha, 1997), are still problematic in their generic placement because of the striking appearance of many radial grooves on their shell sculpture, leading to their name of "Crown Snails", and their remarkable body colouration. Early authors placed them in Macrochlamys (e.g., Dall, 1897;Panha, 1996Panha, , 1997Maneevong, 2000;Hemmen & Hemmen, 2001), while they were transferred to Sarika by later authors (e.g., Maassen, 2001;Sutcharit & Panha, 2008;Pholyotha et al., 2018Pholyotha et al., , 2020. Although Maassen (2001) suggested that a new genus should be established for these two species, Sutcharit & Panha (2008) still included them in Sarika. During a re-examination of all specimens having remarkable radial grooves in our collection, many peculiar specimens were found that were distinct in their shell sculpture, animal colouration, and genital anatomy.
Moreover, molecular analysis suggested that they formed a monophyletic group of a different lineage to Sarika. Therefore, Maassen's suggestion was agreed upon.
In this present study, several newly collected specimens of crown snails from Southern Thailand were examined. The new genus is proposed based on the distinct morphological characters and molecular data. The descriptions of nine new species are included herein and the status of two previously described species is updated. Additionally, phylogenetic trees constructed from concatenated sequences of three gene fragments (COI, 16S, and 28S) were used to infer the evolutionary relationship and distribution patterns among members of this genus in Southern Thailand.

Sampling and morphological studies
This study is based on voucher specimens deposited in Chulalongkorn University Museum Downloaded from Brill.com06/14/2020 12:57:50AM via free access of Zoology (CUMZ) and new samples collected throughout Southern Thailand from 2015 to 2019 ( fig. 1). The living snails were photographed and euthanised (American Veterinary Medical Association, 2013). Then, specimens from each locality were fixed and preserved in 70% (v/v) ethanol for anatomical studies and duplicate specimens and/or tissues were preserved in 95% (v/v) ethanol for molecular analysis. The genitalia of 3-10 specimens of each species were dissected and examined under an Olympus SZX2-TR30 stereoscopic light microscope. Shells and genitalia were imaged using a digital camera (DSLR D850 -Nikon) and a stereo microscope with Cell'D Imaging Software. Radulae were imaged by scanning electron microscopy (SEM; JEOL, JSM-6610 LV). Species identification followed Dall (1897), Blanford & Godwin-Austen (1908), Panha (1997), and Sutcharit & Panha (2008), and was subsequently compared to the relevant type specimens. In the examined material sections, shells refer to empty shells, while specimens refer to specimens preserved in ethanol. Descriptions of a new genus and all new species herein are attributed to the first and fifth authors, Pholyotha & Panha, respectively. The general vouchers and type specimens were deposited in the CUMZ and the Natural History Museum, London, United Kingdom (NHM).

Molecular studies
DNA extraction, amplification, and sequencing We extracted DNA from a total of 41 individuals of 11 Taphrenalla species. Our outgroup sampling included a total of 21 specimens representing six genera, including five ariophantid genera: Macrochlamys, Sarika, Hemiplecta Albers, 1850, Cryptozona Mörch, 1872and Megaustenia Cockerell, 1912, and one helicarionid genus Durgella Blanford, 1863 ( table 1). We choose these genera because their distributions are in Thailand and some members of the genera Macrochlamys and Sarika had been hypothesized previously to be closely related genera (Blanford & Godwin-Austen, 1908;Solem, 1966;Sutcharit & Panha, 2008) as well as to the new genus. Whole genomic DNA was extracted from a small piece of the foot muscle tissue using the NucleoSpin® Tissue Kit (Macherey-Nagel, Germany), following the manufacturer's standard instructions. The two partial mitochondrial genes (COI and 16S rRNA) were amplified using the LCO1491 and HCO2198 universal primers for COI (Folmer et al., 1994) and the 16Sar and 16Sbr universal primers for 16S (Palumbi et al., 1991), while the 28S rDNA was amplified using the LSU2 and LSU4 primers (Wade & Mordan, 2000). The PCR thermal cycling was performed under the following conditions: (i) COI: initial 94°C for 2 min followed by 36 cycles of 94°C for 30 s, 50 or 52°C for 120 s, and 72°C for 120 s, and then followed by a final 72°C for 5 min; (ii) 16S rRNA: 94°C for 2 min, followed by 40 cycles of 94°C for 30 s, 50°C for 60 s, and 72°C for 120 s, and then followed by a final 72 °C for 5 min; (iii) 28S rDNA: 94°C for 1 min, followed by 36 cycles of 94°C for 30 s, 62°C for 90 s, and 72°C for 120 s, and then followed by a final 72°C for 5 min. The size of the PCR products was checked under UV transillumination after gel electrophoresis. The PCR products were sent for commercial sequencing at Bioneer Co., Korea.
Sequence alignment and phylogenetic analyses Chromatograms were manually corrected for misreads using MEGA v. 7.0 (Kumar et al., 2016). To achieve a single consensus sequence, individual forward and reverse sequence traces were aligned and edited using MUSCLE, as implemented in MEGA v. 7.0. The newly generated sequences have been deposited in the GenBank databases with accession numbers: MT364934-MT364995 and MT365667-MT365778 (table 1).
The sequence alignments of the COI, 16S rRNA, and 28S rDNA gene fragments were performed separately using MAFFT v. 7 in the MAFFT online service (https://mafft.cbrc.jp/ alignment/server/index.html) with the default options (Katoh et al., 2017). The regions of ambiguous alignment were checked and cleaned with Gblocks v. 0.91b using relaxed parameters and allowing all gap positions (Castresana, 2000;Talavera & Castresana, 2007). To check for substitution saturation, we performed the test implemented in DAMBE (Xia, 2013). The phylogenetic trees were then constructed using maximum likelihood (ML) and Bayesian inference (BI). Prior to the ML and BI analyses, the best-fit models of nucleotide substitution were identified for each gene separately with five partitions (16S rRNA, 28S rDNA and the three codon positions of COI) using Kakusan4 (Tanabe, 2007(Tanabe, , 2011. The ML phylogenetic analysis was performed by applying the GTRGAMMA model to all three genes at the default settings of RAxML-HPC2 on XSEDE v. 8.2.10 (Stamatakis, 2014) in the CIPRES Science Gateway (Miller, Pfeiffer & Schwartz, 2010). Nodal support values of ML were assessed with 1000 bootstrap (BS) replicates.
The BI analysis was performed using MrBayes on XSEDE v. 3.2.6 (Ronquist et al., 2012) in the CIPRES Science Gateway. The analysis was performed using two simultaneous runs consisted of running four simultaneous chains for 10 million generations. Every 500 generations were sampled and the first 50% of sampled trees were discarded as burnin. The other settings of the analysis followed the developer's recommendation (Ronquist et al., 2012). For ML, BS support values of ≥70% were considered highly supported and BI posterior probabilities (PP) of ≥ 0.95 were considered statistically significant (Hillis & Bull, 1993;Felsenstein, 2004;Huelsenbeck & Rannala, 2004;Mauro & Agorreta, 2010;Hirano et al., 2018). Pairwise genetic distances based on the COI and 16S sequences were calculated using Kimura's two-parameter model (K2P) in MEGA v. 7.

Molecular species delimitation
We conducted species delimitation analyses using two methods: (i) the Automatic Barcode Gap Discovery (ABGD) method (Puillandre et al., 2012) as a distance-based method and (ii) the Poisson Tree Processes (PTP) method (Zhang et al., 2013;Kapli et al., 2017) as a phylogeny-based method. We applied two approaches only to the COI dataset because COI-based DNA barcoding can delimit diverse animal species (Hebert et al., 2003). To apply ABGD, the sequence alignment of the COI gene was uploaded at https://bioinfo.mnhn. fr/abi/public/abgd/abgdweb.html (last modification date: 7 December 2018) and then the K2P model was chosen as the distance type with the following settings: P min = 0.001, P max = 0.1, Steps = 40, X (relative gap width) = 1, and Nb bins (for distance distribution) = 20. The analysis with Jukes-Cantor distance (JC69) was employed with the same settings, except for the relative gap width (X = 0.8). The PTP species delimitation analyses using the singlerate PTP (sPTP) were processed thorough the online PTP web server (http://mptp.h-its.org). A phylogenetic input tree was obtained from the ML analysis, which was conducted based on an alignment of the 41 COI sequences from all Taphrenalla species with an additional two sequences of Sarika resplendens as the outgroup using RAxML-HPC2 on XSEDE v. 8.2.10. The molecular operational taxonomic units (MOTUs) delimited by the two species delimitation analyses were regarded as preliminary hypothesis, then the distinction based on morphological and anatomical characters were observed to treat them as either distinct species or conspecific lineages.

Collections
CUMZ, Chulalongkorn University Museum of Zoology, Bangkok; NHM, The Natural History Museum, London; SMF, Forschungsinstitut und Naturmuseum Senckenberg, Frankfurt am Main; USNM, National Museum of Natural History, Smithsonian Institution, Washington, DC.

Molecular phylogeny
From a total of 56 individuals used (including outgroups), we obtained COI, 16S, and 28S sequences for 35 specimens from all Taphrenal la species. The final concatenated dataset of the aligned sequences of the three gene fragments had a total length of 1672 bp (COI: 655 bp, 16S: 478 bp, and 28S: 539 bp), after 41 bp were excluded as ambiguously aligned by Gblocks (COI: 1 bp, 16S: 40 bp). Using DAMBE, no substitution saturation was detected in all datasets (Iss < Iss.c with p < 0.01). The best-fit models of evolution for each gene fragment (1st COI; 2nd COI; 3rd COI; 16S; 28S) based on the Bayesian Information Criterion (BIC) test were the Kimura 1980 model with gamma distribution (K80+G) for the 1st COI codon, the Felsenstein (1981) model with gamma distribution (F81+G) for the 2nd COI codon, and the general time reversible model with time-reversible gamma distribution (GTR+G) for the 3rd COI codon, the 16S gene, and the 28S gene.

Phylogenetic relationships
The phylogenetic trees ( fig. 2) based on the concatenated datasets using ML and BI methods recovered 11 species of Taphrenalla forming a well-supported clade (BS = 89%, PP = 1). Focusing on the sister-group relationships among genera, M. tanymentula Pholyotha & Panha, 2018 was retrieved as the closest sister group to Taphrenalla, but with poor support (BS = 67%, PP = 0.88). The two other Macro chlamys species in this analysis, which are endemic in Thailand and Myanmar, formed a sister clade to the Taphrenalla + M. tanymen tula clade with strong support (BS = 96%, PP = 0.99). The genera Sarika and the Cryptozona were retrieved as monophyletic with very strong support (BS = 100%, PP = 1). The Cryp tozona + Sarika clade was also a sister group to the Taphrenalla + Macrochlamys clade with high support (BS = 97%, PP = 1). In addition, compared with the other outgroups, the large land snail genus Hemiplecta and the semi-slug genus Megaustenia positioned as basal clades with strong support (BS = 94%, PP = 1).
With respect to the monophyly of all Taphrenalla species, the phylogenetic trees (figs 2, 3) were clearly divided into two wellseparated clades (clade A and B). Clade A contained T. diadema and eight new species with good support (BS = 85%, PP = 0.99), while clade B contained T. macrosulcata and T. asamurai with very strong support (BS = 100%, PP = 1). Although the species relationships among clade A were not well resolved,  Results of species delimitation analyses based on the COI gene using ABGD and PTP. Black circles represent nodes with both ML (when ≥ 70%) and BI (when ≥ 0.95) support. White circles represent nodes with ML BS support but no BI PP support (when < 0.95). Each clade colour refers to the distribution range in fig. 1. this clade was further divided into five subclades (A1 to A5). Four new species (T. par versa, T. corona, T. zemia, and T. conformis) formed clade A1 with good support (BS = 78%, PP = 1). Moreover, within clade A1, T. parversa was very closely related to T. corona with high support (clade A1/1; BS = 94%, PP = 0.99). Interestingly, clade A1 showed various degrees of the radial grooves on shell sculpture, ranging from distinct to absent. The strongly supported clade A2 (BS = 100%, PP = 1) consisted of one species, T. dalli. Within clade A3, the type species T. diadema was the sister lineage of T. incilis with moderate support (BS = 79%, PP = 0.93). Clades A4 and A5 consisted of T. pygmaea and T. alba, respectively, both of which were highly supported (BS = 100%, PP = 1). Taxa with a similar shell morphology were not always retrieved as monophyletic, e.g. T. dalli (clade A2) and T. diadema (clade A3).

Pairwise genetic distances
The aligned COI sequence data matrix of 62 specimens, including 41 specimens of Ta phrenalla species, and the 16S data matrix of 56 specimens, including 35 specimens of Ta phrenalla species, were used to calculate the mean sequence divergences. The mean genetic divergence observed among the genera of the family Ariophantidae ranged between 10.0-16.7% in COI and 12.2-19.5% in 16S ( table 2), while the interspecific divergences among the 11 species of Taphrenalla ranged from 3.4-7.7% for COI and from 1.0-9.2% for 16S (table 3). The Taphrenalla intraspecific divergences ranged from 0-2.4% for COI and from 0-1.5% for 16S (table 4). The highest intraspecific mean sequence divergence of COI and 16S within Taphrenalla was observed in T. alba. No variation in the COI was observed in T. parversa. and no variation in the 16S was observed in T. parversa, T. incilis, and T. conformis.

Species delimitation
A total of 41 COI sequences, representing all Taphrenalla species were assembled for the molecular species delimitation to identify species boundaries. The results of the ABGD species delimitation approach using the JC69 model and the K2P model recovered 11 and 12 MOTUs, respectively ( fig. 3). The other method, the single-rate PTP analysis recovered 13 MOTUs ( fig. 3). Therefore, the two species delimitation analyses (ABGD and PTP) suggested there were between 11 and 13 MOTUs in Taphrenalla gen. nov. By comparing them with morphology-based delimitation, we concluded that there were 11 confirmed candidate species being treated as distinct species, and the two remaining MOTUs were treated as the conspecific lineages of T. asamurai and T. alba sp. nov., respectively. In general, the results from the ABGD and PTP approaches recognized higher number of species than morphological distinctiveness (Jirapatrasilp et al., 2019;Nantarat et al., 2019). The ABGD analysis using the K2P model also yielded higher numbers of MOTUs than the JC69 model (Jirapatrasilp et al., 2019), which is similar to this study. Therefore, the K2P model, which is popular for COI barcoding may yield biased results (Collins et al., 2012;Srivathsan & Meier, 2012;Jirapatrasilp et al., 2019). Also similar to our study, in the recent analysis of the operculated land snails, Nantarat et al. (2019) reported that the PTP species delimitation revealed additional MOTUs relative to the ABGD method.

Phylogeny and taxonomic implications
Based on two mitochondrial and one nuclear gene fragments, the phylogenetic analyses supported that both Taphrenalla gen. nov. and all the recognized species were monophyletic ( fig. 2). Previously, the two species of the Crown Snail (T. asamurai and T. diadema) were placed in the genus Macrochlamys s.l. because of their resemblance in having a depressed and thin shell with a shiny shell surface (Dall, 1897;Panha, 1996). Generally, taxonomic confusion in ariophantid snails has arisen when principally relying on shell morphology, since this is highly similar between Table 3 Mean intra-and inter-specific genetic divergences among species within Taphrenalla gen. nov. from the mitochondrial COI (lower-left) and 16S (upper-right)   species. The snail's anatomy is now required in later taxonomic works, especially the reproductive organs that show a significant difference among genera and species (Blanford & Godwin-Austen, 1908;Schileyko, 2002Schileyko, , 2003. For example, Macrochlamys has a spirally coiled epiphallic caecum, Sarika has a straight epiphallic caecum, Taphrenalla gen. nov. has a straight or slightly bent epiphallic caecum and Rasama has no epiphallic caecum (Blanford & Godwin-Austen, 1908). Thus, T. asamu rai and T. diadema were then transferred to Sarika based on their straight epiphallic caecum (Sutcharit & Panha, 2008).
However, Taphrenalla gen. nov. and Sari ka did not appear to be closely related in this study ( fig. 2). Two anatomical features supported that this new genus is closer to the Indochinese Macrochlamys even though they have a straight epiphallic caecum. The shared characters between them are a penial verge and penial caecum in the male genitalia. Interestingly, T. corona sp. nov. and T. parversa sp. nov. do not develop the penial caecum as well as M. tanymentula (Pholyotha et al., 2018). On the other hand, Cryptozona siamensis is closer to Sarika in the molecular phylogeny. Table 5 Comparison of the shell sculpture and genitalia of Taphrenalla gen. nov. and the related genera. Data are taken from the original descriptions and additional references: Blanford & Godwin-Austen (1908) and Schileyko (2002Schileyko ( , 2003  The genitalia without penial verge and penial caecum of C. siamensis showed a strong similarity with Sarika, even though the shell with a reticulated sculpture was an obvious difference (Godwin-Austen, 1907;Pholyotha et al., 2020). Several reports (e.g., Hausdorf, 1998;Hirano et al., 2014;Hyman et al., 2017) have suggested that genital traits are more likely to reflect the actual phylogenetic relationships than morphological appearances. On one hand, phenotypic plasticity in snail shells is a well-documented phenomenon and is probably a response to environmental variation (Emberton, 1991(Emberton, , 1995Gustafson et al., 2014;Proćków et al., 2017). The other is convergence and parallelism in the character states of the shell morphology and other anatomical structures that are generally known in land snails (Hirano et al., 2014(Hirano et al., , 2015Köhler & Criscione, 2015;Hyman et al., 2017). On the other hand, if they are sister relationships based on molecular data, the result of the similarity in shell morphology in the close species groups is best explained by retention of the ancestral characters (Köhler & Criscione, 2015).
In addition, our results suggested that the shell features with radial grooves on the shell sculpture of Taphrenalla gen. nov., appeared to be suitable for differentiating them from ariophantid genera. Moreover, the anatomical characters of (i) having a penial verge and straight to bent epiphallic caecum, (ii) one or two spines on tail filament of the spermatophore, and (iii) well-developed colourful lateral stripes are the important diagnostic characters found in this new genus (table 6). Even though T. zemia sp. nov. and T. alba sp. nov. have no radial grooves, they have the other unique features of this new genus. The comparative information of genitalia of these genera is mentioned in the remarks of the genus.
The spermatophore morphology is unique in this new genus. Variation can only be found in the tail filament close to the sperm sac bearing one or two spines, whereas the other parts and head filament are spineless. In contrast, the spermatophore of the Sarika, Macro chlamys, and Cryptozona species have a series of complex spine arrangements on the tail filament (Blanford & Godwin-Austen, 1908;Maneevong, 2000;Pholyotha et al., 2018;Pholyotha, unpubl. data).
In the case of the soft-body colouration, most of the ariophantid snails generally have a black or dark grey to milky colour even though some species have a bright (attractive) colour, such as red, orange, or yellow (Blanford & Godwin-Austen, 1908; Pholyotha et al., 2018;Inkhavilay et al., 2019;Sajan et al., 2019). For example, the Indochina M. aurantia Pholyotha & Panha, 2018 and Hemiplecta pluto (Pfeiffer, 1863) have a bright reddish-orange soft-body colour (Pholyotha et al., 2018;Inkhavilay et al., 2019). Remarkably, colourful stripes on the body only occurrs in Taphrenal la species.

Endemism and biogeographical patterns
Interestingly, Taphrenalla nov. gen. is one of the high endemism genera and is found in limestone areas along the two main mountain ranges of Southern Thailand. Our results also found that (i) the species distributed near the Phuket and Nakhon Si Thammarat mountain ranges are likely to present strong radial grooves (T. asamurai, T. diadema, T. macrosul cata sp. nov., and T. dalli sp. nov.), while (ii) the species distributed further away or near the coastal areas may be prone to independent reduction (T. parversa sp. nov., T. confor mis sp. nov., T. incilis sp. nov., and T. pygmaea sp. nov.) or loss (T. zemia sp. nov. and T. alba sp. nov.) of the radial grooves on the shell sculpture. However, all Taphrenalla species are conspicuous in this hypothesis presented here except for T. corona sp. nov. This new species has strong radial grooves but can be found In support, firstly the populations of T. diadema, found on limestone outcrops in Phatthalung close to the Gulf of Thailand, have shallower radial grooves on the shell sculpture and are smaller than the other populations distributed along the Nakhon Si Thammarat Range. Secondly, T. incilis sp. nov. is distributed near the southern part of Nakhon Si Thammarat Range but its localities are close (within 7 km) to coastal areas. Thirdly, the isolated limestone outcrops of the southern part of the Phuket Range, not far from the Andaman sea, contain three allopatric species with a different form of radial groove; two species with a weak radial groove (T. parversa sp. nov. and T. conformis sp. nov.) and one species without a radial groove (T. zemia sp. nov.). Fourthly, the smooth-shelled species T. alba sp. nov. was found in the limestone hills along the shore of the Andaman sea at Krabi.
In tropical landmasses, limestone forests are considered de facto habitat islands because of their isolation from one another by habitats without limestone substrata, and this spatial configuration limits gene flow and induces high levels of species endemism (Clements et al., 2008;Foon et al., 2017). However, it is not clear whether the high level of endemic species or the degree of radial grooves on the sculpture of Taphrenalla shells are also caused by adaptation to these factors because insufficient environmental data was obtained in the present work. To fill this gap, further analyses incorporating the habitat condition of these Taphrenalla species are needed. alongkorn University for their kind help during field trips. We are indebted to J. Ablett and F. Naggs (NHM, London) and E.E. Strong (NMNH, Washington, D.C.) for allowing us to examine the collections and photographs. We express our sincere gratitude to Dr. Frank Köhler, Dr. Bernhard Hausdorf, and an anonymous reviewer for valuable comments. We also thank R. Radula. Teeth arranged in U-shape with very wide angle. Central tooth symmetrical tricuspid; mesocone lanceolated-shaped; ectocones short and located at middle of tooth height. Lateral teeth asymmetrical tricuspid; mesocone triangular shaped; endocone smaller than ectocone; ectocone located below endocone. Marginal teeth with obliquely bicuspid, elongate and narrower teeth, and outermost teeth shorter and smaller than inner teeth ( fig. 4H).
External features. Living snails usually covered with reticulated skin; three colourful stripes running from head to tail (one at middle of body and two on each side of body near foot sole). Eye stalks and head usually with dark colour; tail region usually with lighter colour than head region. Pallial cavity and some internal organs visible through translucent shell (figs 4F, G). Mantle collar large, well developed and composed of two shell lobes and three dorsal lobes (figs 4E-G); shell lobes usually slender and longer than dorsal lobes. Right shell lobe placed on upper shell surface and longer than left shell lobe placed below periphery. Right dorsal lobe larger than left dorsal lobe; left dorsal lobe divided into anterior left dorsal lobe and posterior left dorsal lobe. Foot sole tripartite; caudal foss long and narrow; caudal horn raised and large. Colouration of soft body usually variable from reddish, orange, yellow, milky-white to greyish, and colour disappeared in preserved specimens. Remarks. Taphrenalla diadema is the type species of the new genus because this species somewhat represents the morphology in general among congeneric species and is also the oldest valid species. Although the absence of radial grooves is evolved in T. zemia sp. nov. and T. alba sp. nov., they present the other characteristics of yellowish stripes on the body and a simple spermatophore. The spermatophore of T. alba sp. nov. has two spines near the sperm sac but is spineless in other regions. In addition, the molecular analysis grouped them within the Taphrenalla clade with strong support.
For comparison with the other closely related ariophantid genera, Taphrenalla gen. nov. can be distinguished from Sarika, Macrochlamys, Taphro spira, Syama, and Rasama by the shell sculpture having radial grooves, while the others have a smooth shell without radial grooves (table 5). The shell morphology of Hemiplecta and Cryptozona are clearly different from the new genus in being much larger, with a smooth shell surface and without a radial groove. Regarding the genitalia anatomy, Taphrenalla gen. nov. have a penial verge, dart apparatus, and straight or slightly bent epiphallic caecum. Compared to the other genera, Macro chlamys and Syama both have a spirally coiled epiphallic caecum, but only Syama has no dart apparatus. The genus Taphrospira also has a straight epiphallic caecum and is without a dart apparatus, while the genus Rasama has neither an epiphallic caecum nor a dart apparatus (Blanford & Godwin-Austen, 1908;Schileyko, 2002Schileyko, , 2003. Thus, the genitalia of the genus Sarika is similar to Taphrenalla gen. nov. However, all species of the genus Sarika, except for S. consepta (Benson, 1860), have no penial verge (Pholyotha et al., 2020), while this character is present in all Taphrenalla species. The taxonomic status of S. consepta is needed to be investigated in the future using a molecular approach.
In addition, most Taphrenalla species have bright body colouration patterns of the left, middle, and right stripes, either restricted to the head section or running from head to tail, while all Sari ka species have a monochrome greyish to blackish body colour (Pholyotha et al., 2020).
Taphrenalla asamurai (Panha, 1997)  Remarks. Taphrenalla asamurai had been redescribed and illustrated by Sutcharit & Panha (2008). The unique characters are a depressed conic shell of medium size (width up to 21.1 mm; height up to 11.7 mm), deep and narrow impressed suture, and long radial grooves that appeared on    Spermatophore with one spine on tail filament close to sperm sac.
Description Shell ( fig. 6B). Shell comparatively depressed conic, medium-sized (width up to 18.7 mm; height up to 9.8 mm) and pale brown. Whorls 5-6; suture deep and narrow impressed. Upper shell surface with strong and long radial grooves continuing from suture, gradually inconspicuous and then disappear below periphery. Radial grooves starting between whorl 3 to 4 and with 42-58 grooves at last whorl (n = 24; fig. 5). Spire depressed conic; apex raised with a spire angle of about 143-164°. Last whorl shouldered and well-rounded at periphery. Aperture lip simple; umbilicus narrowly opened.
Genital organs (figs 7D-F). Atrium short. Penis elongate cylindrical and same length as total vagina length; penial caecum present. Inner sculpture of proximal penial wall has very fine wrinkled, longitudinal penial pilasters, with thick and oblique longitudinal folds in middle, and then transformed to a thin, transverse-folded, surrounded penial verge. Epiphallus slender tube and about two times total penis length. Epiphallic caecum very short; thick penial retractor muscle attached at tip. Flagellum large and irregular slender tube. Vas deferens thin tube. Vagina long and cylindrical tube. Dart apparatus located on vagina away from penis joining to atrium. Spermatophore long and needle-shaped. Tail filament very long tube; region close to sperm sac bearing a very small spine.
External features (fig. 3). Animal dark gray mixed with pale yellow on head and body and monochrome bright yellowish on tail.
Remarks. Taphrenalla macrosulcata sp. nov. is similar to T. asamurai in shell morphology but can be differentiated by a paler brown shell and a pale yellowish soft body. Whereas, T. asamurai has a dark brown shell and reddish to dark red soft body.
Regarding the genital anatomy, this new species has a short flagellum, straight epiphallic caecum, and dart apparatus connecting to the vagina away from the penis junction and without thick connective tissue surrounding the proximal end. For comparison, T. asamurai has a long flagellum, bent epiphallic caecum, and dart apparatus located opposite the penis junction and with thick connective tissue at the proximal end.
The presence of one spine on the tail filament of the spermatophore is the remarkable character of T. macrosulcata sp. nov. and T. asamurai, while other Taphrenalla species have two spines. (Dall, 1897)   Remarks. Taphrenalla diadema had been recently re-described and illustrated by Sutcharit & Panha (2008). The distinguishing characters are a depressed conic shell, medium-sized (width up to 17.8 mm; height up to 10.6 mm), a wide and deep channel-shaped suture, and short radial grooves appeared on last about 24-43 grooves (n = 39; figs 5, 8A-C). The genitalia have a short and straight epiphallic caecum, and dart apparatus located on vagina away from vagina and penis junction (figs 9A, B). Spermatophore has one short spine on tail filament close to sperm sac ( fig. 9C). Living snail has yellowish stripes on head and tail ( fig. 3). The specimens from Khao Ok Talu, Phatthalung Province ( fig. 8B) tended to have a smaller diameter and shallower radial grooves than the other populations (figs 8A, C). However, all the genital characters and spermatophores from all localities appear to be identical. In addition, the molecular data also confirmed that the shell variations were all grouped within the T. diadema clade. Pholyotha & Panha,sp. nov. (figs 1,3,5,8D,E, . 8D). Paratypes. Same data as holotype: CUMZ 7184 (31 shells and nine specimens in ethanol), CUMZ 7188 (one shell), NHMUK (two shells) and SMF (two shells). Type locality. Limestone outcrop at Tham Lord, Nopphitam District, Nakhon Si Thammarat, Thailand (8°47'16.7"N 99°38'29.9"E).

Taphrenalla dalli
Etymology. This species is named after William Healey Dall, an American malacologist, who discovered the first species of Crown Snail, T. diadema.
Diagnosis. Shell with a very wide channel-shaped suture and with deep and short radial grooves. Epiphallic caecum straight. Penial caecum very short. Inner sculpture of proximal penis with very small, irregular and longitudinal folds, and then with obliquely and wrinkled to very fine pilasters distally. Dart apparatus connected on vagina.
Spermatophore with two spines on tail filament close to sperm sac.

Description
Shell (figs 8D, E). Shell depressed conic to globosely depressed, medium-sized (width up to 18.3 mm; height up to 11.3 mm) and pale to dark brown. Whorls 5-6; suture very wide, deep and channeled-shape. Upper shell surface with strong and short radial grooves continuing from suture to inconspicuous at periphery and then disappear below periphery. Radial grooves beginning between whorl 3 to 4 and having 23-37 grooves at last whorl (n = 33; fig. 5). Spire depressed conic; apex raised with a spire angle of about 135-155°. Last whorl shouldered and well-rounded at periphery. Aperture lip simple; umbilicus narrowly opened.
Genital organs (figs 9D-F). Atrium slightly short. Penis elongate cylindrical and nearly two times that of total vagina length; penial caecum present. Inner sculpture of proximal penial wall supported by penial pilasters starting proximally with very small irregular longitudinal folds, then gradually, obliquely, wrinkled pilasters in middle, to very fine pilasters surrounding penial verge. Epiphallus slender tube, approximately one and a half times total penis length. Epiphallic caecum slightly prolonged, and attached with thick penial retractor muscle (prm). Flagellum enlarged at base and tapering with irregular shape to tip. Vas deferens slender tube. Vagina long and cylindrical tube. Dart apparatus located on proximal vagina away from penis joining to atrium. Spermatophore long and needle-shaped. Tail filament very long tube; region close to sperm sac bearing two spines; spine close to sperm sac shorter than the other.
External features (fig. 3). Animal dark gray in body colour with three pale orange stripes running from head to caudal horn. One stripe at middle of the body and others two stripes on side of body close to foot sole; stripes on tail wider than anterior.
Remarks. Shell morphology of this new species looks similar to T. diadema, but can be distinguished by having a wider base of the suture than in T. diadema. In comparison, T. dalli sp. nov. shows a small penial caecum, shorter flagellum, and large and coarse penial sculpture pilaster at the middle of penis. Whereas, T. diadema has a large penial caecum, longer flagellum, and inner wall of penis is sculptured with very fine wrinkled pilasters. In addition, the penial sculpture at the middle of the penis of T. dalli sp. nov. has larger, more obliquely wrinkled pilasters than T. diadema.
This new species shows variation in size and shell sculpture, the like of which also appears in T. diadema. However, the shell suture and genitalia of all variants are identical, and are also clustered together in the T. dalli clade in the molecular analysis. Pholyotha & Panha,sp. nov. (figs 1,3,5  Etymology. The specific epithet "corona" is from the Latin word "corona" meaning "crown" and is in reference to the characteristic radial grooves on the shell sculpture of this genus.

Taphrenalla corona
Diagnosis. Shell with wide channel-shaped suture, and slightly deep and short radial grooves. Penis with thick penial sheath. Penial caecum absent. Epiphallic caecum straight. Inner sculpture of proximal penis with fine irregular longitudinal folds and then with obliquely longitudinal folds distally. Dart apparatus connected on vagina. Spermatophore with two spines on tail filament close to sperm sac.
Description Shell ( fig. 10A). Shell depressed conic, mediumsized (width up to 16.0 mm; height up to 9.3 mm) and slightly milky to brownish. Whorl 5-6; suture wide and deep groove, channel-shaped. Upper shell surface with rather strong and short radial grooves continuing from suture to inconspicuous at periphery and disappears below periphery. Radial grooves starting between whorl 3 to 4 and having 26-47 grooves at last whorl (n = 26; fig. 5). Spire slightly depressed conic; apex raised with a spire angle of about 140-155°. Last whorl barely shouldered and well-rounded at periphery. Aperture lip simple; umbilicus narrowly opened.
Genital organs (figs 11A-C). Atrium short. Penis enlarge cylindrical and same length as vagina; penial caecum absent. Inner sculpture of proximal penis with fine irregularly longitudinal penial pilasters, gradually transforming to obliquely longitudinal folds surrounding penial verge. Epiphallus slender tube, about two and a half times total penis length and smaller diameter than penis. Epiphallic caecum short, and attached with thick penial retractor muscle. Flagellum prolonged with irregular cylindrical tip. Vas deferens slim tube. Vagina prolonged, cylindrical-shaped and distal part larger diameter than proximal part. Dart apparatus located on proximal vagina away from penis joining to atrium. Spermatophore long and needle-shaped. Tail filament very long tube; region near sperm sac bearing two spines, spine close to sperm sac very small and shorter than the other.
External features ( fig. 3). Animal very dark gray in body colour with one creamy to pale orange stripe in middle of body running from head to caudal horn; stripe on tail wider than on body to head.
Remarks. Although the shell morphology of this new species is close to those of T. diadema and T. dalli sp. nov., its genitalia is unique. The absence of a penial caecum and presence of a thick penial sheath covering a proximal portion of penis distinguished T. corona sp. nov. from the other Ta phrenalla species.
Taphrenalla parversa Pholyotha & Panha,sp. nov. (figs 1,3,10B,  Genital organs (figs 11D-F). Atrium very short. Penis stout cylindrical and about same length as vagina; penial caecum absent. Inner sculpture of proximal penial wall with fine irregular longitudinal penial pilasters and then gradually irregular, zigzag, longitudinal folds in middle and surrounded penial verge. Epiphallus slender tube, around two times total penis length, and smaller diameter than penis; proximal epiphallus larger than distal portion. Epiphallic caecum short, same diameter as proximal epiphallus, and attached with thick penial retractor muscle. Flagellum short with irregular shape. Vas deferens small tube. Vagina very enlarged, cylindrical-shaped, and distal part enlarged. Dart apparatus located on proximal vagina away from penis joining to atrium. Spermatophore elongate and needle-shaped. Tail filament very long tube; region close to sperm sac bearing two spines; spine located close to sperm sac smaller and shorter than the other.
External features (fig. 3). Animal dark gray colour on head to body with three bright orange stripes running from head to body (two stripes near foot sole and one at middle of body), and then gradually only bright orange stripes on body to tail.
Remarks. Taphrenalla  Genital organs (figs 12A-C). Atrium slightly prolonged. Penis cylindrical and nearly three times total vagina length; penial caecum present. Inner sculpture of proximal penis with small longitudinal folds; distal portion with narrow obliquely cuboidal pilasters, then reduced in size and surrounding penial verge. Epiphallus slender tube, around two times total penis length and smaller diameter than penis. Epiphallic caecum short and bent at the tip; thick penial retractor muscle connecting near the tip. Flagellum short, tapering and irregular slender tube. Vas deferens thin tube. Vagina long and cylindrical-shaped. Dart apparatus located near proximal vagina away from penis join to atrium. Spermatophore long and needle-shaped. Tail filament very long tube; region close to sperm sac bearing two spines; spine close to sperm sac small and shorter than the other.
External features (fig. 3). Animal dark gray in body colour with three pale milky yellow stripes running from head to caudal horn. One stripe at middle of the body and other two stripes above foot sole. Etymology. The specific epithet "incilis" is from the Latin word "incile" meaning "ditch, trench", which referrs to the characteristic small radial grooves in the shell sculpture.  13A). Paratypes. Same data as holotype: CUMZ 7211 (19 shells and 12 specimens in ethanol), NHMUK (two shells), and SMF (two shells).
Etymology. The specific epithet 'pygmaea' is from the Latin word 'pygmaeus' meaning 'dwarf' , which referrs to it being the smallest sized snail in this genus.
Diagnosis. Shell small to medium-sized, narrow and deep channel-shaped suture, and weak, diminutive radial grooves. Epiphallic caecum straight. Penial caecum short. Inner sculpture of proximal penis with very fine longitudinal folds and then transition to oblique and transverse folds distally. Dart apparatus located at junction of vagina and penis.
Description Shell ( fig. 13A). Shell moderately depressed conic, small to nearly medium-sized (width up to 12.3 mm; height up to 6. 8 mm) and pale brown. Whorls 5-6; suture narrow, deep, and channel-shaped. Upper shell surface with weak and diminutive radial grooves that appear only near suture. Spire de ressed conic; apex raised with a spire angle of about 131-151°. Last whorl barely shouldered and periphery almost rounded. Aperture lip simple; umbilicus narrowly opened.
Genital organs (figs 14A, B). Atrium short. Penis funnel-shaped and slightly longer than vagina; penial caecum present. Inner sculpture of proximal Etymology. The specific epithet is from the Greek word 'zemia' meaning 'loss' , which referrs to the shell being without radial grooves on the upper surface.
Diagnosis. Shell moderate to large-sized, shallowly impressed suture, and without radial groove. Epiphallic caecum straight. Dart apparatus located on vagina.
Description Shell ( fig. 13B). Shell globosely depressed, moderate to nearly large (width up to 21.3 mm; height up to 11.9 mm) and yellowish-brown. Whorls 5-6; suture shallowly impressed. Upper shell surface smooth without radial grooves. Spire moderately elevated; apex raised with a spire angle of about 137-151°. Last whorl slightly shouldered and almost rounded periphery. Aperture lip simple; umbilicus narrowly opened.
Genital organs ( fig. 14C). Atrium long. Penis slender tube and slightly longer than vagina. Epiphallus cylindrical tube, nearly one and a half times total penis length. Epiphallic caecum short, and attached with penial retractor muscle. Flagellum large and slender tube. Vas deferens thin tube connected between distal end of epiphallus and free oviduct. Vagina long and cylindrical-shaped. Dart apparatus located near proximal vagina away from penis joining to atrium.
External features ( fig. 3). Animal dark gray in body colour with three pale milky yellow stripes running from head to caudal horn. One stripe at middle of the body and two stripes close to foot sole.
Remarks. Taphrenalla zemia sp. nov. is distinguished from all other Taphrenalla species by the shell having a shallowly impressed suture without a subsutural band and no radial grooves on shell sculpture. Although we surveyed three localities during the wet season, only juvenile specimens were found for the examination and illustrated herein. Pholyotha & Panha,sp. nov. (figs 1,3,13C,15,  Etymology. The specific epithet 'alba' is from the Greek word 'albus' meaning 'white, whitish' , and referrs to the characteristics of the shell having a white line or white subsutural band below the suture.

Taphrenalla alba
Diagnosis. Suture shallowly impressed and with white subsutural band. Epiphallic caecum short and bent at tip. Penial caecum short. Inner sculpture of proximal penis with very soft longitudinal folds then transition to small cuboidal pilasters distally. Dart apparatus located at vagina and penis junction. Spermatophore with two spines on tail filament close to sperm. Figure 15 Genitalia and spermatophore of Taphrenalla alba sp. nov. specimen CUMZ 7223. A. whole reproductive system. B. internal wall of penis with penial verge. C. spermatophore; inset showing two spines located close to the sperm sac.