Zoosyst. Evol. 99 (1) 2023, 195-207 | DOI 10.3897/zse.99.99030

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BERLIN

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

Tuangthong Boonmachai’:*°, Elizabeth A. Bergey*, Nattawadee Nantarat?>°

Ph.D.’s Degree Program in Biology (International Program), Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand
Department of Biology, Faculty of Science, Chiang Mai University 50200, Thailand

Applied Parasitology Research Laboratory, Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand
Oklahoma Biological Survey, University of Oklahoma, Norman, OK 73019, USA

Research Center in Bioresources for Agriculture, Industry and Medicine, Faculty of Science, Chiang Mai University, Chiang Mai, 50200, Thailand

oF WN PF

https://zoobank. org/E28 DD35A-30EA-4C53-B5A6-F0AS80CA0CFCO

Corresponding author: Nattawadee Nantarat (n_nantarat@yahoo.com)

Academic editor: Frank Kéhler # Received 20 December 2022 @ Accepted 15 February 2023 Published 7 March 2023

Abstract

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.

Key Words

endemic, limestone, micro land snail, phylogeny, taxonomy

Introduction

Southern Thailand is located in the transition zone be-
tween 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 lime-
stone mountains, foothills, and coastal islands. Satun’s
limestone hills support high biodiversity and endemism
in micro land snails (Panha and Burch 2005). The taxon-
omy of micro land snails in Thailand’s other limestone

areas has been studied comprehensively over the previ-
ous two decades, resulting in descriptions of several new
species (Panha and Burch 2005). However, Satun’s mi-
crosnail fauna remains largely unexplored.

Recent studies suggest that there are only five genera
of micro land snails in Satun Province; namely, Gyliotra-
chela Tomlin, 1930, Hypselostoma Benson, 1856, Aula-
cospira Mollendorff, 1890, Sinoennea Kobelt, 1904 and
Diplommatina Benson, 1849 (Panha and Burch 2005;
Dumrongrojwattana and Womgkamhaeng 2013; Dum-
rongrojwattana and Tanmuangpak 2020). Among these

Copyright Boonmachai, T. et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use,
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196 Boonmachai, T. et al.: First record and description of three new species genus Diplommatina

five genera, the genus Diplommatina is very diverse and
is widely distributed in Asia; including India, Myanmar,
Laos, Thailand, Malaysia, Indonesia, Japan and northern
Australia (Kobelt 1902; Thiele 1929; Wenz 1939: Ver-
meulen 1993). The large number of species are usually di-
agnosed solely by shell characters (Neubert and Bouchet
2015). The shells of Diplommatina are generally conical
in shape, with radial ribs, a lipped aperture, and an opercu-
lum (Kobelt 1902; Panha and Burch 2005; Yamazaki et al.
2015; Nurinstyah and Hausdorf 2017). Of the more than
A400 described species of Diplommatina (Kobelt 1902;
Thiele 1929; Wenz 1939; Vermeulen 1993), 22 species
that are known to occur in Thailand and about 28 addition-
al species are known from the neighboring countries of
Malaysia, Singapore and Indonesia (van Benthem Jutting
1959; Maassen 2001la; Maassen 2002; Panha and Burch
2005; Tongkerd et al. 2013; Foon et al. 2017; Inkhavilay et
al. 2019; Dumrongrojwattana et al. 2020). Among the 10
species reported from southern Thailand (Maassen 2001b;
Panha and Burch 2005; Tongkerd et al. 2013; Inkhavilay
et al. 2019; Dumrongrojwattana et al. 2020), only a single
species, D. canaliculata, has previously been documented
in Satun province (Panha and Burch 2005).

The shell characters used to identify Diplommatina spe-
cies (Kobelt 1902; Panha and Burch 2005; Yamazaki et al.
2015; Nurinsiyah and Hausdorf 2017) may display adapta-
tion 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-

99.00 99.25

® D. akron

© D. angulifera umpangensis
e D. doichiangdao

e D. kewlom

@ D. inthanon

@ D. crispata khaochamoensis
A D. krabiensis

A D. hidagai

4 D. insularis

4. D, jirasaki

A D. prakayangensis

* D. laemsonensis sp. nov.
»: D. prakaiphetensis sp. nov.

99.50

S

HOSOOOOE

CAMBODIA

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 mito-
chondrial gene (COI) and 16S ribosomal DNA (16S rDNA)
have been used in discriminating land snail species, includ-
ing those within Diplommatinidae, and to investigate phylo-
genetic relationships (Webster et al. 2012; Liew et al. 2014).

Within a larger study of the microsnails of karst ar-
eas in Saturn Province, we especially targeted the genus
Diplommatina. After conducting an extensive field sur-
vey, we used both morphological and molecular analyses
to identify known species and describe new species.

Materials and methods

Ethical statements

We followed the guidelines for animal care in the Interna-
tional Guiding Principles of Biomedical Research Involv-
ing Animals (Council for International Organizations of
Medical Sciences: CIOMS) including the relevant docu-
ment (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

99.75 100.00 100.25

= D. nimanandhi
® D. naiyanetri
© D. pongrati

Z D. suratensis

canaliculata
burapha
chadathongae

D.

D.

D.

D. chantaburiensis

D. fusiformis

D. khwantongae

D. samuiana

D. bulonensis sp. nov.

Figure 1. Localities of micro land snail sampling in Satun Province, Thailand. A. Bulon Pai Island; B. Talutao Island; C. Khao Yai
Island; D. Laem Son Hill; E. Prakaiphet Hill; F. Tharn Pliew Waterfall; G. Phu Pha Phet Cave. The symbols without letters show
previous records for different species of Diplommatina that are known from Thailand (Maassen 2001b; Panha and Burch 2005;
Tongkerd et al. 2013; Inkhavilay et al. 2019; Dumrongrojwattana et al. 2020).

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Zoosyst. Evol. 99 (1) 2023, 195-207

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, pre-
served in 95% ethanol and processed in the Biology De-
partment, Faculty of Science, in Chiang Mai University.

Morphological study

All Diplommatina specimens were identified by com-
paring shell morphology with type specimens and the
original descriptions. Specimens of Diplommatina were
examined using a Leica MZ16 microscope and photo-
graphs were taken using the Leica Application Suite Ver-
sion 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 measure-
ments and ratios among measurements (e.g., shell height
to width) of each morphospecies were analyzed using
ANOVA (a. = 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 lamel-
lar 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 view-
ing 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 Elec-
tron Microscope Research and Service, Faculty of Sci-
ence, Chiang Mai University.

Using a dissecting microscope, the radulae of micros-
nails were extracted from the buccal cavity, cleared in 1%
sodium hydroxide overnight and then washed with dis-
tilled 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 spe-
cies and three unidentified species of Diplommatina (to-
talling 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 (Run-
dell 2008; Webster et al. 2012). Sequences of ten individ-
uals 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-

197

bated with 150 ul Chelex 100 and 3 pl 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 per-
formed 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 per-
formed using Barcode-Tagged Sequencing (BTSeq) Ser-
vices. 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 depos-
ited in GenBank (https://www.ncbi.nlm.nih.gov/genbank)
and are shown in Table 2. Phylogenetic trees were con-
structed 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).

Morphological abbreviations

AH aperture height

AH/AW aperture height/ aperture width
AW aperture width

cd columellar denticle

col columellaris

con constriction

NI ribs/0.5 mm on the penultimate whorl
N2 total ribs on the penultimate whorl
prt parietalis

SH shell height

SH/SW shell height/ shell width

SW shell width

tp transversal palatalis

Ww number of whorls.

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198 Boonmachai, T. et al.: First record and description of three new species genus Diplommatina

Results

Land snails from 41 survey sites in Satun Province were
identified based on morphological characters combined
with molecular analysis. Five species of Diplommatina
were found. One species, D. canaliculata, had previously
been reported from Satun Province and a second species,
D. naiyanetri, is a new provincial record and was found
on Tarutao Island. The three remaining species are new
species and are described below.

Systematics

Family Diplommatinidae Pfeiffer, 1856

Genus Diplommatina Benson, 1849

Type species. Diplommatina folliculus Pfeifter, 1846.

Diplommatina bulonensis Boonmachai & Nantarat,
sp. nov.
https://zoobank.org/94CA 70AE-54F |-4B9D-8604-29766A ESFB80
Figs 2A, 3A—C

Type material. Holotype CMUZ 9050001 (Fig. 2A);
Shell measurements: SH = 2.72 mm, SW = 1.27 mm,
AH = 0.94 mm, AW = 0.91 mm, W = 6 1/2. Paratypes
CMUZ 9050002-9050006 (5 shells); Shell measurements:
SH = 2.58-2.82 mm, SW = 1.26—1.34 mm, AH = 0.97—
1.04 mm, AW = 0.92—1.03 mm, W = 6 1/2-7 1/2.

Type locality. Thailand, Satun Province, La-ngu Dis-
trict, Bulon Pai Island on limestone rocks and under
leaves, 6°49'53.3"N, 99°35'19.5"E, 22 December 2020,
coll. T. Booonmachai.

Other material examined. Thailand, Satun Prov-
ince; La-ngu District, Bulon Pai Island, 6°49'53.3"N,
99°35'19.5"E, 22 December 2020: CMUZ 9050007-
9050016 (10 shells); La-ngu District, Bulon Pai Island,
6749153. 3°N, 9953 9'19.5 Eo 11S: Febmary 2022: 26 MZ
9050017-9050018 (2 shells).

Etymology. The specific epithet bu/onensis is an ad-
jective referring to the type locality (Bulon Pai Island in
Satun Province, Thailand).

Differential diagnosis. Among approximately 400 spe-
cies of Diplommatina, the new species is most similar to
D. conditioria Maassen, 2007, D. baliana Fulton, 1899,
D. maduana Laidlaw, 1949, D gomantongensis Smith, 1894
and D. antheae Vermeulen, 1993 (Table 1). Typical char-
acteristics 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-

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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 dif-
fers from D. maduana, D. gomantongensis, D. antheae and
D. baliana in the number of radial ribs/0.5 mm on the pen-
ultimate whorl (Table 1). D. bulonensis sp. nov has fewer
radial ribs on body whorls above the aperture than D. condi-
tioria (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 coni-
cal with slightly convex sides. Suture impressed. Constric-
tion level with the middle parietal side of the peristome,
with three lamellae: one parietalis very distinct, one dis-
tinct and long transversal palatalis and one distinct colu-
mellaris. (Fig. 4A, B). Protoconch smooth with dimples
and about 2 1/2 whorls (Fig. 3B). Radial ribs straight, in-
conspicuous but distinct, moderately spaced about 6 1/2—7
ribs/0.5 mm on the penultimate whorl (Table 1). The ap-
erture 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 expand-
ed less than inner peristome, inner peristome expanded at
parietalis side, with a palatal lip. Umbilicus closed. Oper-
culum 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. SB).

Diplommatina laemsonensis Boonmachai & Nantarat,
Sp. nov.
https://zoobank.org/A 1F06C5D-F 1EC-41FC-8345-D9BA 7693D8E2
Figs 2B, 3D-F

Type material. Holotype CMUZ 9050019 (Fig. 2B);
Shell measurements: SH = 2.26 mm, SW = 1.17 mm,
AH =0.77 mm, AW = 0.83 mm, W = 7. Paratypes CMUZ
9050020-9050024 (5 shells); Shell measurements:
SH = 2.14-2.49 mm, SW = 1.08—1.21 mm, AH = 0.76—
0.91 mm, AW = 0.79-0.86 mm, W = 6 1/2-7.

Type locality. Thailand, Satun Province, La-ngu Dis-
trict, Limestone Hill, 6°54'43.7"N, 99°41'59.0"E, 25 De-
cember 2020, coll. T. Booonmachai.

Other material examined. Thailand, Satun
Province; La-ngu District, Limestone Hill, 6°54'43.7"N,
99°41'59.0"E, 25 December 2020: CMUZ 9050025-
9050047 (23 shells); La-ngu District, Khoa Yai Island,
6°83'08.6"N, 99°69'70.1"E, 21 December 2022: CMUZ
9050048-9050065 (18 shells).

Zoosyst. Evol. 99 (1) 2023, 195-207

Etymology. The specific epithet /aemsonensis is an
adjective referring to the type locality (Laem Son subdis-
trict, Lan-gu district, Satun Province, Thailand).

Differential diagnosis. Among the sinistral species of
Diplommatina, Diplommatina laemsonensis sp. nov. 1S
most similar to D. diminuta Mollendorff, 1891, D. sinis-
tra Tomlin, 1938 and 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 laemson-
ensis 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.

Description. Shell sinistral, fusiform, convex and hon-
ey 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 con-
striction, one transversal palatalis, one distinct columel-
laris which continues into the tuba and visible in the aper-
ture (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 peri-
stome 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 mar-
gin (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).

Diplommatina prakaiphetensis Boonmachai &
Nantarat, sp. nov.

https://zoobank. org/C468B471-1728-4C9C-ABC3-4C1530C644D8
Figs 2C, 3G-I

Type material. Holotype CMUZ 9050066 (Fig. 2C);
Shell measurements: SH = 1.76 mm, SW = 0.89 mm,
AH = 0.66 mm, AW = 0.64 mm, W = 6 1/2. Paratypes
CMUZ 9050067-9050071 (5 shells); Shell measurements:

199

SH = 1.62—1.75 mm, SW = 0.83-0.90 mm, AH = 0.59-—
0.69 mm, AW = 0.61—0.72 mm, W = 6-7.

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.

Other material examined. Thailand, Satun Prov-
ince, Thung Wa District, Prakaiphet Hill, 7°00'00.1"N,
99°46'08.7"E, 19 January 2022: CMUZ 9050072-
9050088 (17 shells).

Etymology. The specific epithet prakaiphetensis is an
adjective referring to the type locality (Prakaiphet Hill
in Na Thon subdistrict, Thung Wa District, Satun Prov-
ince, Thailand).

Differential diagnosis. Among sinistral diplommati-
nids, Diplommatina prakaiphetensis sp. nov. most close-
ly resembles D. krabiensis Panha & J. B. Burch, 1998
from Thailand, and D. karoensis Maassen, 2002 from Su-
matra, 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. How-
ever, 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 distinguish-
es 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 im-
pressed. Constriction level with the middle parietal side
of the peristome, with three lamellae: one parietalis, one
short transverse palatalis and one prominent columellar-
is that continues into the tuba and is visible in the aper-
ture (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 protrud-
ing, 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. 31). The radula of the taenioglossate
type (Fig. 5C). The central tooth strong and pointed cusps
with a large central cusp, two pairs of developed later-
al 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).

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200 Boonmachai, T. et al.: First record and description of three new species genus Diplommatina

Figure 2. Shell morphology of Diplommatina from Satun Province. A. D. bulonensis sp. nov. (Holotype CMUZ 9050001; Bulon Pai Is-
land); B. D. Jaemsonensis sp. nov. (Holotype CMUZ 9050019; Laem Son Hill); C. D. prakaiphetensis sp. nov. (Holotype CMUZ 9050066;
Prakaiphet Hill); D. D. naiyanetri (CMUZ 905090; Tarutao Island); E. D. canaliculata (CMUZ 9050158; Tharn Pliew Waterfall).

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Zoosyst. Evol. 99 (1) 2023, 195-207 201

Figure 3. Scanning electron microscope of shell morphology, protoconch and operculum of Diplommatina new species. A-C, D. bu-
lonensis sp. nov. (Bulon Pai Island); D—F. D. /aemsonensis sp. nov. (Laem Son Hill); G-I. D. prakaiphetensis sp. nov. (Prakaiphet Hill).

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202 Boonmachai, T. et al.: First record and description of three new species genus Diplommatina

Figure 4. Shell internal terminology of new species genus Diplommatina from Satun Province under the stereo microscope and the
drawing. A, B. D. bulonensis sp. nov. (Bulon Pai Island); C, D. D. /aemsonensis sp. nov. (Laem Son Hill); E, F. D. prakaiphetensis
sp. nov. (Prakaiphet Hill).

Figure 5. Radular morphology of Diplommatina. A. Diplommatina laemsonensis sp. nov. (Laem Son Hill); B. Diplommatina bu-

lonensis sp. nov. (Bulon Pai Island); C. Diplommatina prakaiphetensis sp. nov. (Prakaiphet Hill). Colours show the position of the
teeth of the radula; blue: central tooth, yellow: the basal plate of the central tooth, green: lateral teeth, pink: inner marginal teeth,

and red: outer marginal teeth.
Molecular phylogenetic analyses

Sequences from a total of 30 individuals of Diplom-
matina spp. and 12 individuals from four additional
diplommatinid genera (Hungerfordia Beddome, 1889,
Palaina Semper, 1865, Opisthoporus Manter, 1947 and
Opisthostoma W. T. Blanford & H. F. Blanford, 1860;
the out groups) were used for phylogenetic reconstruc-
tion (Table 2). The concatenated alignment of COI and

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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. With-
in Diplommatina, the phylogenetic tree was divided into
three main clades. Clade A was strongly supported and
included all dextral species from Boneo, Peninsular Ma-
laysia, and southern Thailand (Fig. 6). Within this clade,
D. bulonensis sp. nov. formed the sister group contain-

Zoosyst. Evol. 99 (1) 2023, 195-207

203

Table 1. Comparison of shell morphological characters of closely related Diplommatina species with new species. Shell morpho-

logical characters were tested using one-way ANOVA (P < 0.05).

Species

Characters (mm)

(SH) (SW) (AH) (AW) SH/SW AH/AW N2

D. bulonensis sp. | 2.58-2.82 | 1.26-1.34 | 0.94-1.04 | 0.91-1.03 1.93-2.15 | 0.96-1.10 6 1/2-7 6-7 44-51

nov. (2.71+0.08)* |(1.31+0.04)* |(1.00+0.03)* |(0.97+0.04)* | (2.07+0.07) |(1.03+0.05)* | (6.95+0.27) | (6.33+0.52)* | (47.33+3.51)*

D. laemsonensis 2.14-2.49 1.08-1.21 | 0.76-0.91 | 0.79-0.86 1.94-2.14 0.93-1.09 6-7 8-9 36-44

Sp. nov. (2.34+0.10)* |(1.15+0.04)* |(0.82+0.05)* |(0.81+0.02)* |(2.04 +0.08)* | (1.00+0.05) | (6.86+0.31) | (8.50+0.55)* | (39.33+4.16)*

D. prakaiphetensis | 1.62-1.76 | 0.83-0.90 | 0.59-0.69 | 0.61-0.72 1.92-2.06 | 0.89-1.05 6-7 3-4 15-17

Sp. nov. (1.69+0.04)* | (0.86+0.02)* |(0.64+0.03)* | (0.66+0.03* | (1.97+0.04)* |(0.97+0.06)* | (6.88+0.30) | (3.50+0.58)* | (16.00+1.00)*

D. karoensis 0.90-1.10 - - 1.73-1.78 - 5-5 1/2 -
(1.75+0.21)* |(1.00+0.14)* (1.76+0.04)* (5.20+3.54)*

D. krabiensis 0.90* z = 1.89-2.39 bs a pe -
(1.65+0.71)* (2.14+0.35)

D. canaliculata 3.20-4.95 | 1.30-2.07 | 0.60-1.48 | 0.50-1.67 | 2.23-2.63 | 0.89-1.20 7-9 1/8 3-5 -
(3.93+0.81)* |(1.62+0.33)* |(0.97+0.40)* |(0.97+0.51)* | (2.43+0.17)* |(1.05+40.13)* |(8.03+0.96)* | (4.25+0.96)*

D. hidagai 2.20-2.70 | 1.20-1.40 - - 1.83-1.93 - 8* - -
(2.45+0.35)* |(1.30+0.14)* (1.88+0.07)*

D. naiyanetri 1.90-2.63 | 1.00-1.13 | 0.70-0.75 | 0.70-0.80 1.90-2.35 | 0.94-1.00 6-8 1/2 6-7 29-36
(2.41+0.31)* |(1.10+0.06)* |(0.73+0.03)* | (0.76+0.06) | (2.18+0.19)* | (0.96+0.03) |(7.88+1.25)* | (6.33+0.58)* | (32.67+3.51)*

D. acme 2:20* 1.00* - - 2.20* ~ 7 2 - 26*

D. diminuta 2.00* 0.90* - 22a RB 24*

D. sinistra 1.90* 1.00* - 1.91* 7 2 24*

D. conditoria 2.50* 1.20* - 2.08 6* -

D. maduana 1.3-1.7 0.70-0.90 | 0.30-0.40 | 0.30-0.40 | 1.86-1.89 5-6 1/2 7-12 -
(1.50+0.28)* |(0.80+0.14)* |(0.35+0.07)* |(0.35+0.07)* | (1.88+0.02)* (5.75+1.06)*| (9.50+3.54)*

D. gomantongensis} 2.50-3.50 | 1.20-1.60 | 0.50-0.60 | 0.50-0.60 | 2.08-2.19 6 1/4-7 1/2 3-5 —
(3.00+0.71)* |(1.40+0.28)* |(0.55+0.07)* |(0.55+0.07)*} (2.14+0.08) (6.88+0.88) | (4.00+1.41)*

D. antheae 1.60-2.10 | 0.80-1.00 | 0.30-0.40 | 0.30-0.40 | 2.00-2.20 5 3/4-6 8/5 7-13 -
(1.85+0.35)* | (0.90+0.14)* |(0.35+0.07)* |(0.35+0.07)* |} (2.10+0.14) (6.68+1.31) |(10.00+4.24)*

D. baliana 1.90-2.10 - 5 3/4-6 3/4 12-13 36-60
(2.52+0.33)* |(1.27+0.23)* (2.00+1.00) (6.3340.52) |(12.50+0.71)* | (48.00+16.97)*

* Significant difference in shell morphology between Diplommatina species and new species at P < 0.05.

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. /aidlawi 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).

Pairwise K2P genetic distances between Diplommatina
species ranged from 3.9-21.8% for COI and 3.2—21.3%
for 16S. Intraspecific K2P distances ranged from 0—1.9%
and 0 to 1.2% for COI and 16S, respectively. The new spe-
cles were separated from other species by K2P distances
of at least 5.5% in COI and 3.9% in 16S. The intraspecific
K2P distances were minuscule (Suppl. material 1).

Discussion

Both morphological and molecular evidence support the
recognition of the three new species Diplommatina laem-
sonensis sp. nov, Diplommatina bulonensis sp. nov. and
Diplommatina prakaiphetensis sp. nov. Two of the spe-
cles are presumed to be endemic to the region: D. bulon-
ensis 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 2001a, 2002). A second
species, D. naiyanetri, 1s anew Satun Province record and
was found on Tarutao Island. Previously, this species had
been reported in three other provinces in Thailand; name-
ly, 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 pres-
ent-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 struc-
ture of the peristome. Moreover, scanning electron mi-
croscopy (SEM) showed additional details in the sculp-
turing 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; Maas-
sen 2002; Dumrongrojwattana et al. 2020). Furthermore,

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204

Boonmachai, T. et al.: First record and description of three new species genus Diplommatina

Table 2. List of sampling from GenBank for use in phylogenetic analysis.

Species Voucher codes Locality Accession number Reference
col 16S
Diplommatina ventriculus Mollendorff, 1891 MOL119823 Malaysia, Perak HM753324 HM753495 Webster et al. 2012
Diplommatina centralis Vermeulen, 1993 MOL119785 Malaysia, Borneo HM753339 HM753505 Webster et al. 2012
Diplommatina electa Fulton, 1905 MOL119815 Malaysia, Borneo HM753359 HM753503 Webster et al. 2012
MOL119816 Malaysia, Borneo HM753360 HM753517 Webster et al. 2012
Diplommatina plecta Fulton, 1901 MOL119818 Malaysia, Borneo HM753362 HM753519 Webster et al. 2012
Diplommatina rubicunda Martens, 1864 MOL119819 Malaysia, Borneo HM753363 HM753520 Webster et al. 2012
Diplommatina rubra Godwin-Austen, 1889 MOL119797 Malaysia, Borneo HM753346 HM753514 Webster et al. 2012
MOL119814 Malaysia, Borneo HM753358 HM753516 Webster et al. 2012
Diplommatina laidlawi Sykes, 1903 MOL119821 Malaysia, Kelanta HM753364 HM753522 Webster et al. 2012
Diplommatina canaliculata Mollendorff, 1887 MOL119783 Malaysia, Pahang HM753338 HM753504 Webster et al. 2012
Diplommatina demorgani Laidlaw, 1949 MOL119787 Malaysia, Pahang HM753340 HM753507 Webster et al. 2012
Diplommatina suratensis Panha & Burch, 1998 MOL119827 Thailand, Krabi HM753328 HM753499 Webster et al. 2012
Diplommatina hidagai Panha, 1998 MOL119791 Thailand, Trang HM753327 HM753498 Webster et al. 2012
Diplommatina hidagai Panha, 1998 MOL119826 Thailand, Trang HM753343 HM753510 Webster et al. 2012
Diplommatina naiyanetri Panha, 1997 MOL119794 Thailand, Trang HM753344 HM753512 Webster et al. 2012
Diplommatina prava Pilsbry & Hirase, 1905 MOL119796 Taiwan HM753345 HM753513 Webster et al. 2012
Diplommatina gomantongensis Smith, 1894 MOL119800 Malaysia, Borneo HM753342 HM753509 Webster et al. 2012
Diplommatina sp. MOL119822 Malaysia, Borneo HM753365 HM753523 Webster et al. 2012
MOL119780 Malaysia, Borneo HM753337 HM753501 Webster et al. 2012
MOL119813 Malaysia, Borneo HM753357 HM753515 Webster et al. 2012
Diplommatina laemsonensis sp. nov. MCMU 0401 Thailand, Satun ON568751 OP857482 This study
MCMU 0409 Thailand, Satun 0P825126 OP857486 This study
Diplommatina canaliculata Mollendorff, 1887 MCMU 0402 Thailand, Satun ON568749 OP857480 This study
Diplommatina bulonensis sp. nov. MCMU 0403 Thailand, Satun ON568752 OP857483 This study
MCMU 0410 Thailand, Satun OP825127 OP857487 This study
MCMU 0411 Thailand, Satun OP825128 OP857488 This study
Diplommatina naiyanetri Panha, 1997 MCMU 0406 Thailand, Satun ON568750 OP857481 This study
MCMU 0408 Thailand, Satun OP825125 OP857485 This study
Diplommatina prakaiphetensis sp. nov. MCMU 0407 Thailand, Satun ON568753 OP857484 This study
MCMU 0412 Thailand, Satun OP825129 OP857489 This study
Hungerfordia sp. FMNH310545 Belau EU742109 EU742028 Rundell 2008
FMNH310544 Belau EU742107 EU742026 Rundell 2008
= Belau HM753351 HM753526 Webster et al. 2012
FMNH310550 Belau EU742113 EU742032 Rundell 2008
Palaina moussoni Crosse, 1866 FMNH310574 Belau EU742089 EU742008 Rundell 2008
MOL 119811 Belau HM753355 HM753532 Webster et al. 2012
Palaina rubella Beddome, 1889 FMNH310572 Belau EU742090 EU742009 Rundell 2008
FMNH310573 Belau EU742091 EU742010 Rundell 2008
Palaina striolata Crosse, 1866 MOL 119812 Belau HM753356 HM753533 Webster et al. 2012
Opisthostoma fraternum Smith, 1905 MOL 119824 Malaysia, Borneo HM753325 HM753496 Webster et al. 2012
Opisthostoma mirabile Smith, 1893 MOL 119807 Malaysia, Borneo HM753353 HM753529 Webster et al. 2012
Opisthoporus birostris Pfeiffer, 1854 MOL.119772 Malaysia, Borneo HM753333 HM753488 Webster et al. 2012

this is the first study of radular morphology of Diplomma-
tina. 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 char-
acters 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 ver-
ify taxonomy and indicate classification, and are espe-
cially 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-

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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 Pen-
insular 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. bulonen-
sis Sp. nov. versus D. canaliculata and D. hidagai in the
ratio of SH/SW and the number of whorls, and the differ-
ence from D. gomantongensis in the radial ribs/O.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

Zoosyst. Evol. 99 (1) 2023, 195-207

iplommatina canaliculata
fommatina canaliculata
Diplommatina sp.

ommatina bulonensis sp. nov.
atina bulonensis sp. nov.
atina bulonensis sp. nov.

93/1

tina naiyanetri

tina naiyanetri

ina naiyanetri

fommatina prakaiphetensis sp. nov.
ymmatina prakaiphetensis sp. nov.
atina laemsonensis sp. nov.

/ommatina laemsonensis sp. nov.

_ Diplommatina gomantongensis

205

AM

0.5mm

Hungerfordia sp.

Hungerfordia sp.

91/1 Hungerfordia sp.

Hungerfordia sp.
100/1 ; Palaina moussoni
93/1 10.84 97/1 Palaina moussoni
| 98/1 96/1 Palaina rubella

Palaina rubella
Palaina striolata
4100/1 Opisthostoma fraternum

100/1

Opisthostoma mirabile

Opisthostoma birostris

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

Bl] is greater than 0.70.

Thailand. The close placement of D. /aemsonensis 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. /aemsonensis sp.
nov. and D. prakaiphetensis sp. nov. versus D. naiyane-
tri 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, espe-
cially 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’

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206 Boonmachai, T. et al.: First record and description of three new species genus Diplommatina

for planning and implementing conservation strategies,
especially for threatened endemic species, including
land snails.

Acknowledgements

This study was mainly supported by the Thailand Re-
search Fund (TRF) through a Royal Golden Jubilee (RGJ)
Ph.D. Scholarship to TB (PHD/0248/2560). This research
work also was partially supported by Chiang Mai Uni-
versity. Grateful thanks are extended to the Parasitology
Research Laboratory for the use of instruments.

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Supplementary material 1

Linked data table for pirmary biodiversity
data of Diplommatina

Authors: Tuangthong Boonmachai, Elizabeth A. Bergey,
Nattawadee Nantarat

Data type: occurences and phylogenetic (word document)

Copyright notice: This dataset is made available under
the Open Database License (http://opendatacommons.
org/licenses/odbl/1.0/). The Open Database License
(ODbL) is a license agreement intended to allow us-
ers to freely share, modify, and use this Dataset while
maintaining this same freedom for others, provided
that the original source and author(s) are credited.

Link: https://doi.org/10.3897/zse.99.99030.suppl 1

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