Five new species of the land snail genus Landouria Godwin-Austen, 1918 (Gastropoda, Camaenidae) from northeastern Thailand, with note on genitalia and radula morphology of Landouria diplogramma (Möllendorff, 1902)

1,2,3 Department of Biology, Faculty of Science, Mahasarakham University, Kantharawichai District, Maha Sarakham, 44150 Thailand. 1 Present address: Educational Research Development and Demonstration Institute, Srinakharinwirot University, Ongkharak District, Nakhon Nayok, 26120 Thailand. 2 Palaeontological Research and Education Centre, Mahasarakham University, Kantharawichai District, Maha Sarakham, 44150 Thailand.

In the present study, we describe five new species of Landouria from northeastern Thailand based on their shell morphology, genital anatomy, radular structure, and a phylogenetic analysis of mtDNA sequence data.

Material and methods
Empty shells and living specimens were collected from limestone and sandstone hills in northeastern Thailand. Three living adult specimens of each species were drowned in water for 24 hours. Foot muscle tissue of each specimen was cut and preserved in 80% ethanol, and keep in a freezer at -20ºC. The remaining parts were preserved in 70% ethanol for anatomical study.

Morphological studies
A digital vernier calipers was used to take the following shell measurements (Table 1): shell height (SH), shell width (SW), aperture height (AH), aperture width (AW), and umbilicus width (UW). The number of whorls of adult shells was counted by drawing straight lines to separate the whorls and then the whorls were counted as indicated in Fig. 1. Adult snails were dissected to examine their genitalia under a stereo microscope. Radulae were extracted from the buccal mass and examined under a scanning electron microscope, following the methods of Geiger et al. (2007). The material studied is deposited in the land snail collection of the Natural History Museum, Mahasarakham University (NHMSU-00023 to 00034).

DNA sequence analyses
DNA was extracted from foot muscle tissue using the GF-1 Nucleic Acid Extraction Kit (Vivantis Technologies Sdn. Bhd, Malaysia) following the manufacturer's protocol. Fragments of the cytochrome c oxidase subunit 1 (COI) and mitochondrial 16S rRNA (16S) genes were used to construct a phylogenetic tree of species of Landouria. COI was also used to evaluate genetic divergences (p-distance) between species. The fragments of COI and 16S were amplified by PCR using the primer pairs L1490 and H2198 (Folmer et al. 1994), and 16Scs1 (Chiba 1999) and 16S_MN3R (Neiber et al. 2017) respectively. Reaction conditions were as follows: an initial denaturation step at 94ºC for 2 min; 36 cycles of 94ºC for 30 s, 50ºC for 45 s and 72ºC for 45 s; and a final extension step at 72ºC for 5 min for COI, and an initial denaturation step at 94ºC for 2 min; 36 cycles of 94ºC for 30 s, 50ºC for 30 s and 72ºC for 30 s; and a final extension step at 72ºC for 5 min for 16S. The PCR products were checked with 1% agarose gel electrophoresis. Successful PCR products were sent for NGS-based sequencing at Celamics DNA Sequencing Services (Seoul, Korea) and 1 st BASE DNA Sequencing Services (Selangor, Malaysia).

Phylogenetic analysis
The quality of the mtDNA sequences was checked manually with Bioedit ver. 7.0.9 (Hall 1999(Hall , 2001. Sequences were aligned using the Clustal W algorithm in MEGA X (Kumar et al. 2018). New sequences were deposited in GenBank under the accession numbers MN449400-MN449404, MN449408 and MN449411 for COI, and MZ435745-MZ435749, MZ435751 and MZ435752 for 16S. We included sequences of Landouria from Timor-Leste, China, and Sumatra, Indonesia (Köhler et al. 2018) in our analyses, and used sequences from Euhadra peliomphala (Pfeiffer, 1850) as outgroup following Hirano et al. (2014) and Nurinsiyah et al. (2019) (Table 2).
The program jModelTest ver. 2. 1.10 (Posada 2008;Darriba et al. 2012) was used to determine the best fitting model of DNA substitution using the Akaike Information Criterion (AIC) (Akaike 1974). Sequence divergences were calculated with pairwise p-distances in MEGA X. Phylogenetic trees were constructed using neighbor-joining (NJ), maximum likelihood (ML) and Bayesian inference (BI).
The NJ tree was estimated in MEGA X and node support was calculated using the bootstrapping with 1000 replicates. The ML analysis was conducted with RAxML ver. 8.0.0 (Stamatakis 2014) applying the GTR+F0+G substitution model and 1000 bootstrap replicates to assess branch support. The concatenated sequence alignment was partitioned by gene, allowing different evolution rates for COI and 16S rRNA. The BI analysis was performed in MrBayes ver. 3.2.6 (Ronquist et al. 2012) and run for 2 million generations, with a sampling frequency of 100 generations. The first 2000 generations of each run were discarded as burn-in, the final consensus tree was built using the last 8002 trees. Convergence was confirmed by verifying that the standard deviations of split frequencies were below 0.01. Support for nodes was defined as posterior probabilities. Finally, a 50% majority-rule consensus tree with posterior probabilities as node support was constructed. Phylogenetic trees were displayed and edited using FigTree ver. 1.4.0 (Rambaut 2012).
Following Hirano et al. (2014), nodal support values were considered to be meaningful if ≥ 70% for bootstrapping or ≥ 0.95 for posterior probabilities.

Molecular phylogeny
The COI dataset included 655 bp, while the 16S rRNA dataset included 460 bp. The concatenated dataset therefore comprised 1115 bp.
The COI uncorrected p-distances within Thai species of Landouria ranged from 0.073 to 0.249, whereas interspecific distances among species from Thailand, Timor-Leste, China, and Sumatra, Indonesia ranged from 0.073 to 0.249 (Table 3). The BI, ML and NJ phylogenetic trees reconstructed by combined analyses of the partial COI and 16S rRNA were congruent (Fig. 2). The phylogenetic trees divided Landouria into two major clades which were strongly supported by a BI posterior probability of 1.00, and 100% for ML and NJ bootstrapping (Fig. 2). These clades were the Mainland Southeast Asia clade and the Archipelago clade. The Mainland Southeast Asia clade contains Chinese and Thai taxa (8 species), while the Archipelago clade contains Timor-Leste and Sumatran taxa (4 species). The 8 species of Landouria from the Mainland Southeast Asia clade (i.e., L. strobiloides C. Tumpeesuwan & S. Tumpeesuwan, 2019, L. circinata sp. nov., L. tuberculata sp. nov., L. trochomorphoides sp. nov., L. chloritoides sp. nov., L. elegans sp. nov., L. diplogramma (Möllendorff, 1902)

Diagnosis
Shell brownish-corneous, periphery angular, sharply keeled. Flagellum circinate; penis cylindrical and longer than vagina; base of gametolytic sac thick and stout. Radula with unicuspid short tongue-shaped central teeth.

Etymology
The Latin word 'circinata' refers to the coiled or curled flagellum which resembles the circinate vernation of a fern shoot.  Table 1). Dextral, medium sized, conical-lenticular, with six slightly convex whorls. Suture shallow. Apex obtuse, depressed and low-conical. Light brown at 4 early whorls, dark brown at 2 last whorls. Sharply keeled, with pale brown band on the peripheral keel. Umbilicus very deep and wide. Aperture lip somewhat thickened, rounded rhombic.
Genital SyStem (n = 3) (Fig. 5A, Table 4). Penis very long, cylindrical, and curled, divided into two portions of equal length, internally with parallel, transverse folds, containing a short, rather rounded verge. Epiphallus short cylindrical, thinner than the penis. Flagellum slightly crenated and curled up, resembling the circinate vernation of a fern shoot, with thin grooves internally and tubercles on its surface at the end, internally with transverse robust folds. Vas deferens is a slender, cylindrical tube, thicker at the end where it connects to free oviduct; entering flagellum basally. Vagina less than half as long as penis, internally with four thin longitudinal pilasters. Free oviduct shorter than vagina. Gametolytic sac thickened at base, stout and gradually tapering distally to form a slender distal tube ending in a balloonshaped, medium-sized sac. Prostate gland very long, uterus long and swollen.

Diagnosis
Shell moderately keeled, with numerous tiny tubercles on shell surface (Fig. 4B). Flagellum like a short protrusion with a pointed tip, penis swollen, base of gametolytic sac narrow. Radula with slender, lanceolate central teeth.

Etymology
The specific epithet 'tuberculata' refers to the shell sculpture consisting of numerous tiny tubercles (Fig. 4B).
Genital SyStem (n = 3) (Fig. 5B, Table 4). Penis cylindrical, swollen in the middle part, internally with rather wavy, corrugated longitudinal pilasters and a short, hollow verge with 2-3 lobes around its opening. Epiphallus evenly cylindrical, as long as penis. Flagellum like a short protrusion with a pointed tip, without nodes, internally with longitudinal slender pilasters. Vas deferens cylindrical, narrow, entering in the lower half of the flagellum. Vagina relatively short, internally with five corrugated longitudinal pilasters. Free oviduct very short. Gametolytic sac slightly thicker at base, with a long, narrow and thin cylindrical tube, and at its distal end a small, swollen spherical sac. Prostate gland very long. Uterus long and thin.

Remarks
Landouria tuberculata sp. nov. differs from other Thai species of Landouria, by the shell sculpture of numerous tiny tubercles (Fig. 4B). Lateral teeth of radula bicuspid and lanceolate (Fig. 6D-E).

Distribution
Landouria tuberculata sp. nov. is currently only known from the area surrounding Phu Rua National Park, Phu Rua District, Loei Province, Thailand.

Etymology
The specific epithet 'trochomorphoides' refers to the conchological similarity between the shell of the new species and that of the land snail genus Trochomorpha Albers, 1850 (Trochomorphidae Möllendorf, 1890).  Table 1). Dextral, relatively large-sized. Whorls six, suture shallow, spire only slightly elevated. Protoconch with radially elongated scars. Body whorl sharply keeled, brownish-corneous with a brown zone at periphery, scaly processes all over shell surface (Fig. 4C). Umbilicus moderately deep and very wide. Aperture lip rhombic, peristome reflexed, and expanded.

Holotype
Genital SyStem (n = 3) (Fig. 5C, Table 4). Penis slightly longer than vagina, slender cylindrical, internally with parallel, transverse robust folds; verge absent. Epiphallus slender cylindrical with thickened end. Flagellum as long as epiphallus, basal portion thick and progressively tapering towards tip, without nodes, inner surface smooth. Vas deferens is a thin cylindrical tube, entering epiphallus apically. Vagina thinner than penis, internally with parallel, transverse robust folds. Free oviduct very short. Gametolytic sac slightly narrower at base, with a long narrow cylindrical tube, and a small, oval sac at the distal end. Prostate gland and uterus very long.

Diagnosis
Shell shape similar to that of the genus Chloritis Beck, 1837 (Camaenidae), but with last whorl stout and usually slightly angular, peripheral keel blunt, suture deep, aperture oval and oblique. Flagellum small ovate, penis very large and stout compared to vagina; vagina and free oviduct short (Fig. 5D). Radula with lanceolate teeth.

Etymology
The specific epithet 'chloritoides' refers to the conchological similarity between the shell of the new species and that of the camaenid genus Chloritis.
Genital SyStem (n = 3) (Fig. 5D, Table 4). Penis very large and stout, longer than vagina, cylindrical, gradually tapering towards epiphallus, internally with six rather thick, corrugated longitudinal pilasters; opening of the short, grooved verge surrounded by 2-3 lobes. Epiphallus short and thick. Flagellum short, elongate ovate, without nodes, internally with three large longitudinal pilasters. Vas deferens long and slender, entering in the lower half of the flagellum. Vagina short, as long as free oviduct, internally with seven, corrugated longitudinal pilasteres varying in size. Gametolytic sac thickened at base, with a long narrow cylindrical tube, and a small oval sac at distal end. Uterus swollen, prostate gland long.

Diagnosis
Shell similar to Landouria circinata sp. nov., but with a sharper and more prominent keel at the periphery and dark brown zones on the periphery, just above and below the keel. Flagellum droplet shaped, penis long and cylindrical, vagina and free oviduct very short. Radula with elongate tongue-shaped central and lateral teeth.

Etymology
The specific epithet 'elegans' (Latin for 'magnificent') refers to the beauty of the shell.

Description
Shell (Fig. 3E, Table 1). Dextral, conical, relatively large-sized. Whorls 6. Protoconch with radially elongated wrinkles. Suture rather shallow, apex obtuse depressed, with rather high conical spire. Last whorl with a sharp keel that is distinctly bent downwards. Colour brownish-corneous with a dark brown zone bordering the keel at the periphery. Umbilicus very deep and wide. Apertural lip thickened, rounded rhombic.
Genital SyStem (n = 3) (Fig. 5E, Table 4). Penis longer than vagina, cylindrical, internally with four corrugated, longitudinal pilasters; verge absent. Epiphallus shorter than penis, distal part (ep2) small, whereas proximal part (ep1) is swollen. Flagellum droplet-shaped, without nodes, internally with four corrugated, longitudinal pilasters. Vas deferens long, cylindrical, proximal part that connects to free oviduct somewhat thicker than distal part, entering the flagellum at its base. Vagina very short, internally with five corrugated longitudinal pilasters. Free oviduct shorter than vagina. Proximal part of gametolytic sac rather stout, but tapering to become a slender tube with large oval sac at distal end. Uterus and prostate gland very long and large.

Remarks
Landouria elegans sp. nov. is conchologically quite similar to L. circinata sp. nov. and L. strobiloides, from which it differs by its sharper and more distinct keel with its downward bent rim (Fig. 3A vs Fig. 3E). The radula of L. elegans sp. nov. resembles that of L. circinata sp. nov., but central tooth and lateral teeth of L. elegans sp. nov. are longer and more slender than in L. circinata sp. nov. (Fig. 6A-C vs Fig. 6M-O). Epiphallus of L. elegans sp. nov. is similar to that of L. chloritoides sp. nov., but in L. elegans sp. nov. it is larger and connects with the vas deferens near the flagellum base ( Fig. 5D vs Fig. 5E).

Diagnosis
Shell small, low-conical, weakly keeled, with a slightly elevated spire, whitish corneous with a pale brown band above periphery. Flagellum like a short protrusion, with rounded tip. Penis short swollen. Central and the first few lateral radular teeth triangular. Description Shell (Fig. 3F, Table 1). Dextral, small, depressed conical. Whorls 5½, suture rather shallow, apex obtuse and depressed, whitish corneous, with a pale brown peripheral band. Protoconch almost smooth. Umbilicus moderately deep and wide. Aperture oval oblique, with thin, solid, and weakly reflected lip.
Genital SyStem (n = 3) (Fig. 5F, Table 4). Penis divided into two short portions, the portion that connects to epiphallus is swollen and the portion that connects to atrium is constricted; internally with three corrugated longitudinal pilasters; with short, rather rounded verge. Flagellum like a short protrusion, internally with two narrow longitudinal pilasters. Vas deferens long, cylindrical, entering the epiphallus at base. Free oviduct very short. Vagina twice as long as free oviduct, internally with five thick, corrugated longitudinal pilasters. Proximal part of gametolytic sac slightly thicker than the cylindrical tube, with a medium-sized oval sax at distal end. Uterus and prostate gland long.

Remarks
Specimens deposited in the Leiden Museum, The Netherlands, RMNH.MOL.309867 and RMNH. MOL.309851 (https://images.app.goo.gl/PgrpqKaWuSrgjdvN9), are identified as Plectotropis diplogramme Möllendorff, 1902. These specimens appear identical to specimens from Khao See Siad Ah in Nakhon Ratchasima Province, therefore we used the name "Landouria diplogramma (Möllendorff, 1902)" for this species in the present study. Landouria diplogramma differs from other Thai species of Landouria by its relatively small lustrous shell without peripheral keel, but with a pale brown band present above periphery (Figs 3F, 4F). Its radular teeth are relatively short (Fig. 6P-R). Its penis and flagellum are relatively short (Fig. 5F).

Discussion
Previously, Thai land snails with small, depressed conical shells were usually identified as Aegista Albers, 1850 (Bradybaenidae), based on their shell morphology only. Yet, recent studies of the genital system of these species showed that they lacked a dart apparatus and mucus glands, indicating that they are not species of Aegista, but rather belong in the genus Landouria (Camaenidae) (e.g., Tumpeesuwan & Tumpeesuwan 2019).
Phylogenetic analysis divided Landouria into two clades in accordance with the geographical boundaries between the Mainland Southeast Asia, Sumatra, and Timor-Leste. In Thailand the species form a monophyletic group, with four species endemic to Loei Province. These four species were discovered on different isolated limestone and sandstone hills or mountains, so their distribution ranges do not overlap. Furthermore, each hill or mountain was surrounded by geographic barriers, such as floodplains or other unsuitable habitats. The present study illustrates that in northeastern Thailand there remains a substantial amount of overlooked malacological diversity to be discovered. Hence further taxonomic research on the malacofauna of this region will undoubtedly uncover still many more undescribed terrestrial snail species.