Accepted Manuscript

Phylogenetic Relationships Of The Operculate Land Snail Genus Cyclopho-rus Montfort, 1810 In Thailand

Nattawadee Nantarat, Piyoros Tongkerd, Chirasak Sutcharit, Christopher M.Wade, Fred Naggs, Somsak Panha

PII: S1055-7903(13)00367-9DOI: http://dx.doi.org/10.1016/j.ympev.2013.09.013Reference: YMPEV 4714

To appear in: Molecular Phylogenetics and Evolution

Received Date: 6 April 2013Revised Date: 13 September 2013Accepted Date: 14 September 2013

Please cite this article as: Nantarat, N., Tongkerd, P., Sutcharit, C., Wade, C.M., Naggs, F., Panha, S., PhylogeneticRelationships Of The Operculate Land Snail Genus Cyclophorus Montfort, 1810 In Thailand, MolecularPhylogenetics and Evolution (2013), doi: http://dx.doi.org/10.1016/j.ympev.2013.09.013

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PHYLOGENETIC RELATIONSHIPS OF THE OPERCULATE LAND SNAIL 1 GENUS CYCLOPHORUS MONTFORT, 1810 IN THAILAND 2

3

Nattawadee Nantarata,b 4

a Biological Sciences Program, Faculty of Science, Chulalongkorn University, Bangkok 5

10330, Thailand 6 b Animal Systematics Research Unit, Department of Biology, Faculty of Science, 7

Chulalongkorn University, Bangkok 10330, Thailand 8 Email: n_nantarat@yahoo.com 9

10

Piyoros Tongkerdb 11

b Animal Systematics Research Unit, Department of Biology, Faculty of Science, 12

Chulalongkorn University, Bangkok 10330, Thailand 13

14 Chirasak Sutcharit

b 15

b Animal Systematics Research Unit, Department of Biology, Faculty of Science, 16

Chulalongkorn University, Bangkok 10330, Thailand 17

18

Christopher M. Wadec 19

c School of Biology, University of Nottingham, University Park, Nottingham, NG7 2RD, UK 20

21 Fred Naggs

d 22

d Department of Zoology, The Natural History Museum, London SW7 5BD, UK 23

24

Somsak Panhab,*

25 b Animal Systematics Research Unit, Department of Biology, Faculty of Science, 26 Chulalongkorn University, Bangkok 10330, Thailand (*Corresponding author) 27

Email: somsak.pan@chula.ac.th 28 29 30

31 32 33

--------------------------- 34 35

*Corresponding author. Tel./fax: +66 2 2185273 36 E-mail addresses: somsak.pan@chula.ac.th (S. Panha). 37

38 39 40 41

42 43 44

45 46

47

ABSTRACT. - Operculate land snails of the genus Cyclophorus are distributed widely in 48 sub-tropical and tropical Asia. Shell morphology is traditionally used for species 49 identification in Cyclophorus but their shells exhibit considerable variation both within and 50 between populations; species limits have been extremely difficult to determine and are poorly 51 understood. Many currently recognized species have discontinuous distributions over large 52

ranges but geographical barriers and low mobility of snails are likely to have led to long 53 periods of isolation resulting in cryptic speciation of allopatric populations. As a contribution 54 towards solving these problems, we reconstructed the molecular phylogeny of 87 55 Cyclophorus specimens, representing 29 nominal species (of which one was represented by 56 four subspecies), plus three related out-group species. Molecular phylogenetic analyses were 57

used to investigate geographic limits and speciation scenarios. The analyses of COI, 16S 58

rRNA and 28S rRNA gene fragments were performed using neighbour-joining (NJ), 59

maximum likelihood (ML), and Bayesian inference (BI) methods. All the obtained 60 phylogenetic trees were congruent with each other and in most cases confirmed the species 61 level classification. However, at least three nominate species were polyphyletic. Both C. 62 fulguratus and C. volvulus appear to be species complexes, suggesting that populations of 63 these species from different geographical areas of Thailand are cryptic species. C. 64

aurantiacus pernobilis is distinct and likely to be a different species from the other members 65 of the C. aurantiacus species complex. 66 67 68

KEY WORDS.- Gastropoda, Cyclophoridae, Taxonomy, Systematics, Cryptic species 69

70

1. Introduction 71 72

The Cyclophoridae are dioecious terrestrial caeonogastropod snails with a long fossil 73 record extending from the Mesozoic era (Gordon and Olson, 1995; Kongim et al., 2006) and 74 with a wide current geographical distribution: Southern Europe, Central America, Asia, 75

Africa, various Pacific islands and Australia (Kobelt, 1902; Solem, 1959). The most broadly 76 used classification for the Cyclophoridae is that of Kobelt (1902). He classified the 77 cyclophorids based on shell, opercular and radular characters, but whilst this undoubtedly 78 reflects some of the broad relationships, it is of little value below the subfamily level (Solem, 79 1956). Currently, the Cyclophoridae is comprised of about 870 species arranged in three 80

subfamilies and 35 genera (Kobelt, 1902, 1908; Wenz, 1938; Vaught, 1989; Bouchet and 81

Rocroi, 2005; Lee et al., 2008b). The genus Cyclophorus Montfort, 1810 is the most species 82

rich genus in the family Cyclophoridae with over 100 described species distributed from 83 South Asia to Southeast Asia and including the south of China, Korea and Japan (Reeve, 84 1862; Kobelt, 1902, 1908; Gude, 1921; Pilsbry, 1916, 1926; Benthem Jutting, 1948, 1949; 85 Solem, 1959, 1966; Minato and Habe, 1982). Members of Cyclophorus have a distinctive 86 large solid, low conical shell form with a thin and multispiral operculum. They are “ground-87

dwelling” in leaf litter, under logs etc. and occur in a wide range of forest habitats from 88 evergreen rainforest to monsoon deciduous forest. In Thailand, the highest densities occur in 89 limestone forest (Kobelt, 1902, 1908; Gude, 1921; Solem, 1959). 90

The validity of the nominal Cyclophorus species level classification is not clear 91

because of the degree of shell variation within and between nominated species and the limited 92 number of characters available in Cyclophorus shells. Kobelt (1902) classified Cyclophorus 93

into eight subgenera while Vaught (1989) rearranged Cyclophorus into five subgenera. Few 94 descriptions of the internal anatomy, including reproductive organs have been published for 95 Cyclophorus (Tielecke, 1940; Kasinathan, 1975). Available information demonstrates a high 96 degree of similarity for internal anatomy within Cyclophorus that does not provide robust 97 characters for recognizing species level categories (Welber, 1925; Kongim et al., 2006). Thus 98

there has been little advance on the use of characters based on shell morphology, including 99 shell size, shape, colour pattern and peristome morphology (e.g. Reeve, 1862; Kobelt, 1902, 100 1908; Gude, 1921). Environmental factors can greatly affect shell morphology (Uit de Weerd 101 et al. 2004; Lee et al. 2008 a, 2008b; Elejalde et al., 2009) and homoplasy confounds the 102 recognition of biological species of Cyclophorus. Species limits in Cyclophorus are 103

notoriously difficult to establish with numerous geographically isolated populations 104

exhibiting seemingly minor differences in their morphology. 105

There is evidence to suggest that Cyclophorus was important in the diet of Stone Age 106

cave dwellers in Oriental regions (Rabett et al., 2011). In the present day, these edible snails 107

have been utilized for food in many parts of Thailand, Laos and Vietnam (Oakley, 1964; Paz 108

and Solheim, 2004). However, the number of Cyclophorus snails seems to have dropped 109

(Hildyard, 2001) most likely due to a range of contributing factors such as changes in the 110

environmental conditions around snails‟ habitats especially in limestone areas and improper 111

snail harvesting (Clements et al., 2006). Recent work has focused on systematic research and 112

development of conservation strategies (Clements et al., 2008). In addition to its intrinsic 113

scientific interest, a reliable taxonomy is important for avoiding the mistaken treatment of 114

multi-species genera as a single taxon that may fail to effectively regulate their conservation. 115

There is therefore practical conservation value in the recognition of Cyclophorus cryptic 116

species (Kongim et al., 2006; Prasankok et al., 2009). 117

Over the past decade, sequence data has been used to help clarify problems in 118 systematics and evolution where morphological and physiological characters have proven to 119 be ambiguous (Vogler and Monaghan, 2007). Studies on land snail phylogeny using 120 molecular DNA sequences have suggested that such approaches are potentially useful (e.g. 121 Harasewych, 1998; Wade et al., 2006; Colgan et al., 2007; Lee et al., 2008a, 2008b). The 122

genes most commonly used for land snail systematics, at various taxonomic levels are the 123 mitochondrial cytochrome oxidase subunit I (COI) and16S rRNA genes and the nuclear 124 ribosomal RNA genes (e.g. Harasewych, 1998; Wade et al., 2006; Colgan et al. 2007; Liew et 125 al., 2009). They provide a highly effective tool to resolve taxonomic problems and identify 126 cryptic species (Paquin and Hedin, 2004). These genes have been found to be suitable for use 127

at the generic level in cyclophorids, revealing that the evolution of morphological and 128

ecological traits occurs at extremely high rates during adaptive radiation, especially in 129

fragmented environments (Sanders et al., 2006; Lee et al., 2008a, 2008b). The work reported 130 here is the first molecular phylogenetic study of the genus Cyclophorus in Thailand. 131

132 133 2. Material and Methods 134 135 2.1 Taxon sampling and identification 136 137

Eighty seven specimens of Cyclophorus, attributed to 29 nominal species (Figure 1), 138

including the type species Cyclophorus volvulus (Müller, 1774) and, for one of these, four 139 nominal subspecies, were collected from 67 localities in Thailand and 7 additional localities 140

in Laos, Vietnam, Malaysia and Japan (Figure 2 and Table 1) representing approximately 141 30% of the total nominal species of the genus. Tissue samples were fixed and preserved in 142 95% (v/v) ethanol. Ethanol was changed at least twice to eliminate water content of the 143 samples. Vouchers were preserved in 70% (v/v) ethanol for anatomical study. Provisional 144 identification of species was based upon shell morphology, making use the literature (Reeve, 145

1862; Kobelt, 1902, 1908; Benthem Jutting, 1948, 1949) and by examination of reference 146 collections, including type material in the following museums: The Natural History Museum, 147 London (NHMUK); Senckenberg Museum, Frankfurt (SMF); The Royal Belgian Institute of 148 Natural Sciences (RBINS); Zoological Museum University of Copenhagen, Denmark 149 (ZMUC) and the Chulalongkorn University Museum of Zoology (CUMZ), Bangkok, 150

Thailand. 151

152 2.2 DNA extraction, PCR amplification and sequencing 153

154 Total genomic DNA was extracted from approximately 3–5 mm

3 of foot tissue from 155

each individual using a DNAeasy Tissue Kit (QIAGEN Inc.), and was then stored at –20 ºC 156 until use. The COI and 16S rRNA mitochondrial genes and 28S rRNA nuclear gene were 157 amplified by PCR using the primers LCOI490 (5‟-158

GGTCAACAAATCATAAAGATATTGG-3‟) and HCO2198 (5‟-159 TAAACTTCAGGGTGACCAAAAAATCA-3‟) for the COI gene (Folmer et al., 1994), 16sar 160 (5'-CGCCTGTTTATCAAAAACAT-3') and 16sbr (5'-CCGGTCTGAACTCAGATCACGT-161 3') for the16S rRNA gene (Kessing et al., 1989) and 28SF4 (5'- 162

AGTACCGTGAGGGAAAGTTG-3') and 28SR5 (5'- ACGGGACGGGCCGGTGGTGC-3') 163

for the 28S rRNA gene (Morgan et al., 2002). For all genes PCR reactions were undertaken 164 in a 50 µl final volume using 25 µl of 2xIllustra hot starts master mix (GE Healthcare), 10µM 165 of each primer and about 10 ng of DNA template. For COI, thermal cycling was at 94 °C for 166 2 min, followed by 36 cycles of 94 °C for 30 s, 42 °C for 2 min, and 72 °C for 2 min, and 167

then a final extension step of 72 °C for 5 min. For 16S rRNA and 28S rRNA, thermal cycling 168 was performed in the same way with the exception that the annealing temperature was 169 changed to 50 °C for 30 s and the extension time to 90 s. The amplified products were 170 checked following 1% (w/v) agarose gel electrophoresis resolution in 0.5x TBE buffer and 171 visualized with SYBR Safe and UV Transillumination. The PCR products were purified 172

using a QIAquick purification Kit protocol (QIAGEN Inc.). The amplified PCR products 173 were directly cycle-sequenced using the original amplification primers with the sequencing 174 reaction products run on an Applied Biosystems automatic sequencer (ABI 3730XL) at 175 Macrogen, Inc. (Korea). The DNA sequences were compared with sequences from the 176 GenBank database by using the BLASTn algorithm to confirm the homology of the 177

amplification products to targeted genes. 178

179

2.3 Sequence alignment and phylogenetic analysis 180 181

Sequences were edited with reference to the trace data and aligned using MUSCLE 182 version 3.6 (Edgar, 2004). The alignments were improved manually where necessary by 183 using MEGA 5.0 (Tamura et al., 2011). Ambiguous regions and gaps in the alignment were 184

excluded from the dataset. 660 nucleotide sites were unambiguously aligned across all taxa 185 for the COI fragment, with 396 unambiguously aligned nucleotide sites for 16S rRNA and 186 585 unambiguously aligned nucleotide sites for 28S rRNA. 187

The COI, 16S rRNA and 28S rRNA sequences were checked for saturation and 188

phylogenetic signal using DAMBE v. 4.5.33 (Xia and Xie, 2001) and through plotting 189 uncorrected pairwise transition and transversion distances against total uncorrected distances 190

in order to visualize saturation and identify the taxa responsible. For COI, saturation tests 191 were performed using all codon positions and each codon position individually. All base 192 frequencies and molecular character statistics were calculated using MEGA 5.0 (Tamura et 193 al., 2011). 194 Phylogenetic analyses were undertaken using the following datasets; all codon 195

positions of COI (660 bp), first and second codon positions of COI (440 bp), 16S rRNA (396 196 bp), 28S rRNA (585 bp), concatenated COI (all codon positions) and 16S rRNA (1056 bp), 197 concatenated COI (1

st and 2

nd codon positions only) and 16S rRNA (839 bp) and 198

concatenated COI (all codon positions), 16S rRNA and 28S rRNA (1641 bp). Details are 199 shown in Table 3. Heterogeneity in base composition between sequences is known to affect 200

phylogenetic inference (Lockhart et al. 1994), so we tested the variation in base pair 201

composition among sequences for the datasets using a χ2 analysis, as implemented in PAUP* 202

v4.0b10 (Swofford, 2003). In order to assess the validity of combining datasets a partition 203 homogeneity test (Farris et al. 1994) was undertaken in PAUP 4.0b10, using 100 replicates 204 (Swofford, 2003). 205

Phylogenetic trees of all 32 taxa (including 3 outgroup taxa) were constructed using 206 neighbor-joining (NJ), maximum likelihood (ML), and Bayesian inference (BI). The 207 phylogenetic trees were rooted on Cyclotus, Leptopoma and Rhiostoma genera that have been 208

suggested to be close relatives of the genus Cyclophorus and fall within the same family 209 (Colgan et al., 2007; Lee et al., 2008a, b). For all methods, correction for multiple hits was 210 made using the general time-reversible (GTR) model (Lanave et al., 1984), with between-site 211 rate heterogeneity taken into account by combining gamma correction (G) into the model (Gu 212

et al., 1995). This model was found to be the most appropriate substitution model for all 213

datasets using jModeltest 0.1.1 (Posada, 2008). NJ analysis was undertaken using PAUP* 214 v4.0b10 (Swofford, 2003) with model parameters, including the rate matrix, base frequencies 215

and gamma shape parameter () of the gamma distribution (based on 16 rate categories) 216 estimated using likelihood based on iteration from an initial NJ tree. The iteration process 217

was repeated with estimated parameters used for subsequent rounds of tree building until no 218 further improvement in likelihood score was obtained. Bootstrap resampling (Felsenstein, 219 1985) with 1000 replicates was undertaken to assign support to branches in the NJ tree. ML 220 analysis was undertaken using RAxML v7.2.6 (Stamatakis, 2006). Bootstrap resampling 221 (Felsenstein, 1985) via the rapid bootstrap procedure of Stamatakis et al. (2008) was 222

undertaken to assign support to branches in the ML tree. BI analysis was performed using 223 MrBayes version 3.1.2 (Huelsenbeck and Ronquist, 2001), with the tree space explored using 224 four chains of a Markov chain Monte Carlo algorithm (MCMC). The Bayesian analysis was 225 run for 3 million generations (heating parameter = 0.03), sampling every 100 generations. 226 The first 58002 trees were discarded as burnin, such that the final consensus tree was built 227

using the last 2000 trees. In addition, we also analyzed our concatenated COI (all codon 228

positions), 16S rRNA and 28S rRNA (1641 bp) dataset under the phylogenetic mixture model 229

approach (Ronquist and Huelsenbeck, 2003). The analyses under the various assumed 230 partitions were run using HKY+I+G for COI, GTR+G for 16S rRNA and GTR+G for 28S 231 rRNA. 232 233 2.4 Phylogenetic networks 234

235 In order to evaluate the structure and possible reticulating relationships Neighbour 236 Net analysis (Huson and Bryant, 2006) were undertaken for the widespread species, C. 237 fulguratus and C. volvulus using concatenated COI (all codon position), 16S rRNA and 28S 238

rRNA sequences. This allowed additional sites to be recruited into the analysis with 1739 239 unambiguously aligned sites for C. fulguratus and 1745 sites for C. volvulus. Neighbour Net 240

alaysis was performed using the GTR+G model computed using SplitsTree version 4.11.3 241 (Huson and Bryant, 2006). Bootstrap support for splits was conducted with 1000 replicates. 242 The dataset was also evaluated for homoplasy using the homoplasy index (PHI) statistic 243 (Bruen et al., 2006) using SplitsTree version 4.11.3 to test for recombination within the 244 sequences. 245

246 2.5 Supplemental analyses of the COI gene incorporating Genbank data 247 248

An additional 232 COI sequences (630 bp) of Cyclophorus from Genbank 249 (HM753719.1-HM753950.1) enabled the inclusion of an additional 10 Cyclophorus sp. and 4 250

subspecies from three new countries (Japan, Taiwan and China) into our analyses. 251

Phylogenetic analyses were undertaken as above (section 2.3) using NJ, ML and BI methods 252

and incorporating a GTR+G model. 253 254

2.6 GenBank Accession Numbers 255 256

Nucleotide sequences of this study have been deposited in GenBank under accession 257 numbers JX474562 - JX474651 for COI, JX474652 - JX474741 for the 16S rRNA fragment 258

and KF319126-KF319215 for the 28S rRNA, and are shown in Table 1. 259

260 261

3. Results 262

263 264 3.1 DNA sequence variation and distance analysis 265

266

The aligned 660 bp sequence of the COI dataset comprising all codon positions had 267 38.3% GC content [range 36.97% GC to 42.48% GC, χ2 test=141.7596 d.f.=267, P=1.000], 268 292 (16.6%) parsimony informative and 310 variable sites (46.97%). The uncorrected p-269 distance between the taxa ranged from 0.000 to 0.264 [inter/intraspecific p-distances = 0.142 270 and 0.052, respectively]. Stop codons were absent in all codon positions of COI sequences. 271

Therefore, we assumed that no pseudogenes were accidentally sequenced. Transition, 272 transversion and p-distances estimated from MEGA 5.0 were used in the saturation tests. The 273 results of the saturation test using DAMBE v. 4.5.33 (Xia and Xie, 2001) did not show 274 evidence for saturation in a symmetrical tree of the dataset, with an index of substitution 275 saturations (ISS) value of 0.411, which was significantly lower than the critical saturation 276

value (ISS.C) of 0.719, P<0.0001. However, the ISS value was higher than the ISS.C value of 277

0.392 for an asymmetrical tree although not significantly so P=0.4669 (N=32) and such 278

asymmetrical trees are possibly not realistic for the dataset. This suggests that there is still 279 sufficient phylogenetic information in the all codon positions of COI. Moreover, the 280 saturation plots suggested no evidence of saturation for the ingroup data though some 281 evidence for some saturation when outgroups were included. 282

The aligned 16S rRNA gene fragment (396 bp), after exclusion of ambiguously 283

aligned nucleotide positions, had 39.6% GC content [range 37.63% GC to 42.42% GC, χ2 284 test = 44.042137 d.f.=267, P=1.000], with 147 (37.1%) parsimony informative and 182 285 (46.0%) variable sites. The result of the DAMBE test did not detect saturation in the 286 sequences. The results showed an ISS value of 0.146, which was significantly lower than the 287

critical saturation value (ISS.C) of 0.691 for a symmetrical and 0.362 for an asymmetrical tree 288 topology, P<0.0001. Moreover, the saturation plot did not show evidence of saturation. The 289

uncorrected p- distance between the taxa ranged from 0.000 to 0.230 [inter/intraspecific p-290 distances = 0.098 and 0.0171, respectively]. 291

The aligned 28S rRNA gene fragment (585 bp), after exclusion of ambiguously 292 aligned nucleotide positions, had 59.8% GC content [range 58.97% GC to 61.20% GC, χ2 293 test = 17.138669 d.f.=267, P=1.000], with 97 (16.6%) parsimony informative and 197 294

(33.7%) variable sites. The result of the DAMBE test did not detect saturation in the 295 sequences. The results showed an ISS value of 0.083, which was significantly lower than the 296 critical saturation value (ISS.C) of 0.711 for a symmetrical and 0.383 for an asymmetrical tree 297 topology, P<0.0001. Moreover, the saturation plot did not show evidence of saturation. The 298 uncorrected p- distance between the taxa ranged from 0.000 to 0.214 [inter/intraspecific p-299

distances = 0.022 and 0.004, respectively]. 300

The concatenated data set of all codons of COI, 16S rRNA and 28S rRNA (1641 bp) 301

had 46.3% GC content [range 44.79% GC to 48.87% GC, χ2 test = 87.342328 d.f.=267, 302 P=1.000], with 535 (32.6%) parsimony informative and 689 (41.9%) variable sites. The 303 results of a partition homogeneity test by PAUP 4.0b10, using 100 replicates (Swofford, 304 2003) showed no significant differences were found between markers (P = 0.095). The result 305 of the DAMBE test did not detect saturation in the sequences. The results showed an ISS 306 value of 0.298, which was significantly lower than the critical saturation value (ISS.C) of 0.779 307

for a symmetrical and 0.501 for an asymmetrical tree topology, P<0.0001. Moreover, the 308 saturation plot did not show evidence of saturation. The uncorrected p-distance between the 309 taxa ranged from 0.000 to 0.217 [inter/intraspecific p-distances = 0.082 and 0.025, 310 respectively]. A summary of the molecular data is shown in Table 2 and 3. 311

312

3.2. Phylogenetic analyses 313 314 The phylogenetic trees reconstructed using the NJ, ML and BI methods were highly 315 congruent, with almost identical topologies and with the same supported nodes for all major 316

clades (data not shown), and with all datasets. The ML phylogenetic tree based on the 317 concatenated dataset of all codons of COI, 16S rRNA and 28S rRNA (1641 nucleotide sites) 318 is shown in Figure 3. 319

Cyclophorus was separated into two principal clades (clades 1 and 2, Figure 3) with 320 strong support (100% NJ bootstraps, 100% ML bootstraps and posterior probability (PP) =1 321

in BI for clade 1 and 99% NJ, 100% ML, 1 PP for clade 2). Clade 1 comprised the sole 322 species C. perdix tuba from Malaysia, whilst clade 2 comprised the remainder of the 323 Cyclophorus species. Clade 2 was further subdivided into two major subclades; clade 2a that 324 includes C. semisulcatus, C. jourdyi, C. herklotsi and C. turgidus (95% NJ, 97% ML, 1 PP) 325 and clade 2b that includes all of the Thai Cyclophorus as well as C. songmaensis from 326

Vietnam and C. bensoni from Laos (90% NJ, 94% ML, 0.96 PP). 327

Of the 29 nominal species (Figure 1), 25 were monophyletic and strongly supported in 328

greater than 77% of NJ and ML bootstrap replicates and PP>0.91 in BI (in Figure 3). The 329 individual genes generally supported the monophyly of most Cyclophorus species, but 330 yielded poor resolution (data not shown). The molecular trees provided strong support 331 indicating that 3 species, C. fulguratus, C. volvulus, and C. aurantiacus, were not 332 monophyletic. Cyclophorus fulguratus fell in 4 well-supported locations in the trees (labeled 333

1f, 2f, 3f and 4f in Figure 3). The first group (1f) from West and Central Thailand was most 334 closely related to C. volvulus (99% NJ, 100% ML, 1 PP) also from West Thailand. The 335 second group (2f) from upper Northeastern Thailand was most closely related to C. 336 consociatus (100% NJ, 100% ML, 1 PP). A single individual (3f) from lower Northeastern 337

Thailand fell immediately outside the C. consociatus and C. fulguratus 2f cluster (92% NJ, 338 96% ML, 1 PP). A further individual (4f) from East Thailand clustered with C. speciosus 339

(99% NJ, 100% ML, 1 PP). Cyclophorus volvulus fell in 3 well-supported locations in the 340 trees (labeled 1v, 2v and 3v in Figure 3). The first group (1v) from West Thailand was most 341 closely related to C. fulguratus 1f (99% NJ, 100% ML, 1 PP) also from West Thailand. 342 Group 2 (2v) from East and Central Thailand was most closely related to C. labiosus (94% 343 NJ, 96% ML, 0.99 PP). A single individual (3v) from North Thailand clustered with C. 344

subfloridus (99% NJ, 98% ML, 1 PP). Finally, C. aurantiacus was not monophyletic in our 345 trees with C. a. pernobilis falling separately to the other subspecies (87% NJ, 89% ML 346 bootstrap support and 1 PP in the concatenated COI/16S/28S dataset, see Figure 3). Within 347 the main C. aurantiacus group the subspecies, C. a. aurantiacus, C. a. nevilli and C. a. 348 andersoni fell as monophyletic units (Figure 3). The monophyly of a 4

th species C. 349

consociatus proved equivocal in all analyses. C. consociatus was poorly resolved in the 350

concatenated COI, 16S rRNA and 28S rRNA (Figure 3) and the individual genes analyses. 351

For supplementary analysis, C. pfeifferi appeared to be paraphyletic in the mtDNA tree 352 (Figure S1) though with low support: 50% NJ, 53% ML, 0.59 PP but was recovered as a 353 monophyletic unit in trees based on concatenated COI, 16S rRNA and 28S rRNA (93% NJ, 354 97% ML, 1 PP, Figure 3). C. malayanus was poorly resolved in the mtDNA tree (Figure S1) 355 and COI tree but were recovered as a monophyletic unit in trees based on concatenated COI, 356 16S rRNA and 28S rRNA (91% NJ, 97% ML, 1 PP for C. malayanus). C. consociatus was 357

poorly resolved in all analysis. C. bensoni appeared to be polyphyletic in the COI analyses 358 (62% NJ, 62% ML, 0.77 PP, Supplemental analyses –Figure S3). 359

360 3.3. Phylogenetic networks of C. fulguratus and C. volvulus 361

362

Neighbour Net networks of C. fulguratus and C. volvulus were consistent with the phylogeny 363 (Figure 4). C. fulguratus (Figure 4A) was dispersed in four splits that correspond to Western-364 Central Thailand (split I in Fig 4, 1f in Fig 3), upper Northeastern Thailand (split II in Fig 4, 365 2f in Fig 3), lower Northeastern Thailand (split III in Fig 4, 3f in Fig 3) and Eastern Thailand 366

(split IV in Fig 4; 4f in Fig 3). For C. volvulus (Figure 4B), the sequences were distributed in 367 3 main splits with Western Thailand (split I in Fig 4, 1v in Fig 3), Eastern-Central Thailand 368 (split II in Fig 4, 2v in Fig 3), and Northern Thailand (split III in Fig 4, 3v in Fig 3). The PHI 369 test for recombination did not find statistically significant evidence for recombination in 370 either C. fulguratus (PHI test; P = 0.5681) or C. volvulus (PHI test; P = 0.3464). 371

372 3.4 Supplemental analyses of the COI gene incorporating Genbank data 373 374

The inclusion of an additional 232 COI sequences of Cyclophorus from Genbank 375 (HM753719.1-HM753950.1) enabled the incorporation of an additional 10 Cyclophorus sp. 376

and 4 subspecies from 3 countries into our analyses (Supplemental analyses –Figure S3). 377

Consistent with our concatenated COI, 16S and 28S rRNA tree (Figure 3), the Cyclophorus 378

sequences fell into 2 principal clades: clade 1, comprising the sole species C. perdix tuba 379 from Malaysia, and clade 2, comprising the remainder of the Cyclophorus species. Clade 2 380 was further subdivided into two major subclades; clade 2a that includes sequences from 381 China, Vietnam, Malaysia and Taiwan and clade 2b that includes all of the Thai Cyclophorus 382 as well as C. songmaensis from Vietnam and C. bensoni from Laos. The tree generally 383

supported the monophyly of Cyclophorus species with 35 of 43 species appearing to be 384 monophyletic in the COI gene tree, though 8 of 43 species appeared to be polyphyletic based 385 on analyses of just the COI gene (Figure S3). Interestingly, C. jourdyi from Vietnam and C. 386 semisulcatus from Malaysia appeared to be related with the Cyclophorus sp. from China. 387

Overall the results of the analyses of the COI gene (including sequences from Genbank) 388 showed consistent information in the placement of the Thai clade (clade 2b) in the broad 389

Cyclophorus phylogeny. 390 391 392

4. Discussion 393

394 Land snails are powerful research tools for investigating evolutionary processes. 395

However, their traditional classification is based on morphological characters that are liable 396 to extensive homoplasy. Cyclophorus shows some interesting questions on convergence and 397 polymorphism of shell color, patterns and shapes. Critical studies of stylommatophoran land 398 snail reproductive systems have probably led to a much better understanding of species limits 399

in these pulmonate snails than is the case with terrestrial Caenogastropoda where the basis of 400

morphospecies recognition has largely been dependent on variation in shell characters. 401

Conversely, conservative shell forms may conceal cryptic species; these two trends combine 402 to make species limits based on morphology difficult to recognize. The present study is the 403 first investigation of the relationships of members of Cyclophorus in Thailand using DNA 404 sequence based systematics. The phylogenetic trees showed good resolution in clarifying the 405 taxonomy and relationships in Cyclophorus with consistent results obtained with different 406 genes (COI, 16S rRNA and 28S rRNA) and with different analytical methods (NJ, ML and 407

BI). Most (25/29) but not all nominate species appeared to be monophyletic. 408 409 4.1 Evolutionary relationships within Cyclophorus 410

411

The phylogenetic tree presented in Figure 3 (ML analysis of the combined all codon 412

positions of COI, 16S rRNA and 28S rRNA sequences) does not conflict with the analysis of 413 the three genes separately or by the analysis with ML, NJ or BI (data not shown). For some 414 species (e.g. C. pfeifferi, C. malayanus, C. consociatus and C. bensoni) for which the 415 relationships inferred in the concatenated COI, 16S rRNA and 28S rRNA tree (Figure 3) 416

differed from those inferred in analyses based on other datasets. These differences and/or lack 417 of resolution are consistent with the pattern we would expect if many or all genes shared the 418 same history, but some of them individually contained too little information relative to 419 stochastic noise to support clades. This supports the idea that concatenation can overcome 420 misleading homoplasy (Townsend et al., 2011). We note also the possibility that 421

mitochondrial introgression can mislead phylogenetic analyses, but as our analyses were 422 based on both nuclear and mitochondrial markers, and analyses of the nuclear 28S rRNA 423 gene (Supplemental analyses – Figure S2) gave identical results with mtDNA (Supplemental 424 analyses – Figure S1) and concatenated datasets (Figure 3), it seems improbable that 425 introgression has had any major effect on our phylogenies. 426

In the molecular tree (Figure 3), Cyclophorus divides into two major clades (clades 1 427

and 2), with C. perdix tuba from Malaysia the sole species in clade 1 and with the remaining 428

Cyclophorus species in clade 2. The sole species of C. perdix tuba (Clade 1, Figure 3) also 429 showed some distinctive morphological characters (unique colour patterns, more prominent 430 keel, a conical shape but with peculiar trumpet shaped dilatation of the ultimate whorl 431 towards the aperture and it also possesses a unique line around the suture.) when compared to 432 the rest of the Cyclophorus group. Moreover, the p-distance between C. perdix tuba and those 433

species in clade 2 was 16% and 19% in the 16S rRNA and COI genes, respectively. We note 434 similar genetic distances have been inferred to be indicative of distinct genera in studies of 435 other land snails (Köhler, 2011). Clade 2 was further divided into two subgroups, the minor 436 clade 2a comprised the non-Thai species, and the main clade 2b comprised all of the Thai 437

species plus one species from each of Laos and Vietnam. Thus both Laos and Vietnam have 438 close relatives to Thai Cyclophorus. The inferred phylogenetic tree suggests that similar 439

morphologies appeared independently and rapidly in different lineages. Shells of 440 Cyclophorus commonly reveal complicated dark brown colour patterns that likely function to 441 disguise them from predators and are therefore likely to be homoplasious. 442

The supplemental analysis (Supplemental analyses –Figure S3), in which we 443 incorporated additional COI sequences from Genbank, allowed us to explore Cyclophorus 444

relationships in greater detail both through the inclusion of additional Cyclophorus species 445 and the inclusion of samples from over a wider geographical range. On the whole, the 446 monophyly of Cyclophorus species was generally supported, though 8 of 43 species appeared 447 to be polyphyletic based on analyses of just the COI gene (Figure S3). Moreover, the wider 448 geographic sampling has revealed that clade 2a includes sequences from China and Taiwan, 449

as well as sequences from Malaysia, Japan and Vietnam. Clade 2b remains unchanged with 450

the inclusion of all of the Thai Cyclophorus as well as C. songmaensis from Vietnam and C. 451

bensoni from Laos. We note that most Cyclophorus species from clade 2a have a small size 452 (about 2-3 cm) with a simple aperture while snails from clade 2b show various sizes (about 3-453 6.5 cm) with a diverse morphology. 454 455 4.2 Taxonomic implications 456 457

Recent work on Cyclophorus has indicated that there are likely to be a number of 458 cryptic species (Kongim et al., 2006; Prasankok et al., 2009). Whilst most species of 459 Cyclophorus sampled here correspond with molecular tree placements, some polyphyletic 460 groups can be identified. Three morphological species are not monophyletic on molecular 461

criteria and most likely represent cryptic species or complexes. (1) C. fulguratus in groups 1f, 462

2f, 3f and 4f. (2) C. volvulus in groups 1v, 2v and 3v and (3) C. aurantiacus in group 1a and 463 C. aurantiacus pernobilis in group 2a. 464

In the case of C. fulguratus, the specimens divided into four well supported clades; 1f 465 from Western Thailand, 2f from upper Northeastern Thailand, 3f from lower Northeastern 466

Thailand and 4f from Eastern Thailand. Neighbour Net networks were also consistent with 467 the phylogenetic trees with individuals distributed in four splits (Figure 4A) and with high 468 bootstrap support. Furthermore, PHI analysis did not detect evidence of recombination 469 (p>0.05) between the four subpopulations. Our molecular findings are consistent with 470 previous observations of variation in karyotypes (Kongim et al., 2006) and allozymes 471

(Prasankok et al., 2009). Based on analysis of karyotype variation, Kongim et al. (2006) 472 separated C. fulguratus into two distinct groups (species) with populations from Central 473 Thailand having a 12m + 2sm karyotype and those from Northeastern Thailand having a 13m 474 + 1sm karyotype, consistent with the principal division of C. fulguratus haplotypes in our 475 tree. Moreover, allozyme analysis revealed large genetic divergences between populations 476

from Central Thailand and those from Northeastern Thailand as well as large genetic 477

divergences between populations from Central and Eastern Thailand (Prasankok et al., 2009), 478

congruent with the separate phylogenetic placement of C. fulguratus from Western-Central 479 Thailand versus Northeastern and Eastern parts of Thailand, observed here. Cyclophorus 480 fulguratus thus appears to represent several separate cryptic species that show convergent 481 patterns of morphological evolution. Previous studies have suggested that these distinct 482 clades are allopatric (Kongim et al., 2006; Prasankok et al., 2009). In this regard, the low 483

motility of the snail, coupled with the separation of different geographical populations by 484 geographical barriers, such as fragments of mountain ranges (the Phu Phan range in Northeast 485 Thailand, the Tenasserim range in Western Thailand and the Sankamphaeng and Cardamom 486 ranges in Eastern Thailand) led to allopatric speciation. We propose that increased physical 487

and environmental heterogeneity over time delivered chances for localized allopatry (Mayr, 488 1963). 489

For C. volvulus, individuals are separated into three well-supported clades; group 1v 490 from Western Thailand, group 2v from Eastern-Central Thailand and group 3v from Northern 491 Thailand. Neighbor Net networks were consistent with the phylogenetic trees with 492 individuals distributed in three splits (Figure 4B) with high bootstrap support. Moreover, the 493 PHI analysis did not detect evidence of recombination (p>0.05) between the three 494

subpopulations. Thus, our findings reveal evidence for the likelihood of allopatric speciation 495 within C. volvulus sensu lato. Allopatric speciation is related geographic isolation most likely 496 due to mountain ranges in Western Thailand (split I; Figure 4B) and Central-Eastern Thailand 497 (split II; Figure 4B). 498

Cyclophorus aurantiacus falls in two separate locations in the phylogenetic tree with 499

the subspecies C. a. pernobilis (1a) falling separately to a group comprising C. a. 500

aurantiacus, C. a. nevilli and C. a. andersoni (2a). It seems likely that geographical isolation 501

between C. a. pernobilis in the West and the other C. aurantiacus subspecies in the South has 502 led to allopatric speciation. Moreover, C. a. pernobilis exhibits a significantly different 503 morphology when compared to the subspecies in the South. Cyclophorus aurantiacus 504 pernobilis was originally identified as Cyclostoma pernobilis by Gould in 1843 and 505 transferred to the genus Cyclophorus by Hanley and Theobald, 1870. Kobelt (1902) placed 506 this nominal species as a subspecies of C. aurantiacus. However, our analyses support Gould 507

(1843) and Hanley and Theobald (1870) who recognized C. pernobilis (Gould, 1843) as a 508 distinct species. 509

510 511

5. Conclusion 512

513 This work is the first molecular phylogenetic study of Cyclophorus in Thailand; 87 514

individuals of 29 nominal species (and four subspecies for one of these) were included with 515 three outgroup genera. Phylogenetic placement of most (25/29) Cyclophorus species 516

corresponded with traditional shell-based morphospecies. However, C. fulguratus and C. 517 volvulus are resolved as cryptic species complexes, supporting previous conclusions based on 518 karyotype and allozyme variation (Kongim et al., 2006; Prasankok et al., 2009). Cyclophorus 519 aurantiacus perbobilis fell in a distinct lineage from the other members of the C. a. 520 aurantiacus group, which supports its original classification as a separate species (C. 521

pernobilis) by Gould (1843) rather than the subsequent subspecies classification of C. a. 522 perbobilis by Kobelt (1902). The monophyly of C. consociatus proved equivocal. 523

Cyclophorus is one of the most diverse land snail groups both morphologically and 524 taxonomically (Kobelt, 1902). The mtDNA and nucDNA provided a powerful tool for 525 investigating relationships within the genus and for recognizing genetically divergent and 526

morphologically cryptic lineages. The phylogenetic results raise many questions about the 527

species complexes that still remain to be answered. Allopatric cryptic species are likely to be 528

widespread throughout the range of Cyclophorus and in other terrestrial caeonogastropod 529 land snails where systematic studies have largely been restricted to shell-based 530 morphospecies. This has important consequences for conservation because of the possible 531 existence of a large number of what might be highly localized and endangered species that 532 have so far not even been recognized. 533

534 535

ACKNOWLEDGMENTS 536 537 The main funding source for this project analyses was from The Thailand Research Fund 538 (TRF) to SP under TRF-Senior Scholar Research Grant (2012-2014) RTA5580001, and also 539 to the supply for a graduate student (NN) through The Royal Golden Jubilee Ph.D. Program 540

(PHD/0315/2550). The first funding for basic collecting specimens and taxonomic work were 541 provided by the Commission on Higher Education under The National Research University 542 Project of Thailand (FW646A). We would express our sincere gratitude to The Plant Genetic 543 Conservation Project under the Initiative of Her Royal Highness Princess Maha Chakri 544 Sirindhorn for providing our great opportunity of collecting specimens in many restricted 545

areas especially on several islands in Andaman Sea. And also thanks to UNITAS 546

Malacologica for providing Student Research Awards 2011. We thank members of the 547

Animal Systematics Research Unit, Chulalongkorn University for assistance in collecting 548 material. We further extend our thanks to Dr. Peter Foster (NHM) for invaluable help with 549 our analysis. We thank Prof. Takahiro Asami for providing contributory funding to visit 550 Japan and to Dr. Kiyonori Tomiyama for field collecting arrangements at Kagoshima, 551 Kyushu, Japan. 552

553 554

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707

Table 1. Samples (species, number of specimen and locality) and GenBank accession

numbers for all the sequences. Map numbers correspond to the locality number in

Figure 2.

Species

No.

of

sam

-ples

Map

No.

Localities Name GenBank accessesion No.

COI 16S rRNA 28S rRNA

C. affinis 3 10. Chiang Dao, CHIANG

MAI, Thailand

JX474587-9 JX474678-

80

KF319151-

3

1 48. Pla cave, MAE HONG

SON, Thailand

JX474590 JX474681 KF319154

C. amoenus 1 59. Thepsatit temple,

NAKHON SAWAN,

Thailand

JX474595 JX474660

KF319159

1 66. Tam Rakang temple,

SUKHOTHAI,

Thailand

JX474597 JX474662 KF319161

3 25. Khao Nor, NAKHON

SAWAN, Thailand

JX474596,J

X474598-9

JX474661,

JX474663-4

KF319160,

KF319162-

3

C. a. aurantiacus 1 23. Khao Lak Lam Lu,

PHANG-NGA,

Thailand

JX474642

JX474723 KF319206

1 31. Khiri Rat Nikhom,

SURAT THANI,

Thailand

JX474641 JX474724 KF319205

1 35. Kob cave, PHANG-

NGA, Thailand

JX474640 JX474725 KF319204

C. a. andersoni 1 27. Khao Poo Khao Ya,

PHATTHALUNG,

Thailand

JX474637 JX474730 KF319201

1 37. Koo Ha Sa Wan cave,

PHATTHALUNG,

Thailand

JX474636 JX474729 KF319200

1 9. Charoenkooha temple,

SONGKHLA, Thailand

JX474638 JX474731 KF319202

1 61. To Poo view point,

SATUN, Thailand

JX474639

JX474732 KF319203

C. a. nevilli 1 14. Hin Lad waterfall,

SURAT THANI,

Thailand

JX474634

JX474728 KF319198

1 56. Sramorakod, KRABI,

Thailand

JX474633 JX474726 KF319197

1 40. Mae Koh, SURAT

THANI, Thailand

JX474635 JX474727 KF319199

C. a. pernobilis 1 38. Kra Teng Jeng

waterfall,

KANCHANABURI,

Thailand

JX474623 JX474722 KF319187

C. bensoni 1 52. Sa Pan waterfall, NAN,

Thailand JX474574 JX474670 KF319138

1 43. Nam Min waterfall,

PHAYAO, Thailand

JX474572 JX474671 KF319136

Tables

1 68. Pha Hom, WIANG

JUN, Laos

JX474573 JX474669 KF319137

C. cantori 1 31. Khiri Rat Nikhom,

SURAT THANI,

Thailand

JX474629 JX474718 KF319193

1 47. Phuket Marine

Biological Center,

PHUKET, Thailand

JX474628 JX474717 KF319192

C. consociatus 1 39. Lampahang school,

SAKON NAKHON,

Thailand

JX474621 JX474702 KF319185

1 64. Tam Pa Num Pok

temple, KHON KAEN,

Thailand

JX474620 JX474703 KF319184

C. courbeti 2 45. Pang Sri Da waterfall,

SA KAEO, Thailand

JX474611-2 JX474693-4 KF319175-

6

1 29. Khao Sib Ha Chan

waterfall,

CHANTHABURI,

Thailand

JX474613 JX474695 KF319177

C.

cryptomphalus

1 57. Sri U Thum Porn

temple,

NAKHONSAWAN,

Thailand

JX474594 JX474665 KF319158

C. diplochilus 1 54. Sa Tit Kee Ree Rom

temple, SURAT

THANI, Thailand

JX474627 JX474714 KF319191

1 36. Koh Yao Yai,

PHANG-NGA,

Thailand

JX474626 JX474713 KF319190

1 14. Hin Lad waterfall,

SURAT THANI,

Thailand

JX474625 JX474716 KF319189

1 24. Khao Nam Lak,

NAKHON SI

THAMMARAT,

Thailand

JX474624 JX474715 KF319188

C. expansus 1 46. Pha Ka Yang cave,

RANONG, Thailand

JX474630 JX474719 KF319194

1 26. Khao Pho way-service,

PRACHUAP KHIRI

KHAN, Thailand

JX474631 JX474720 KF319195

C. fulguratus 1 60. Thep Muang Thong

temple, UTHAI

THANI, Thailand

JX474579 JX474705 KF319143

1 32. Klong Lan waterfall,

KAMPHAENGPHET,

Thailand

JX474582 JX474708 KF319146

1 53. Sa Pan Hin waterfall,

TRAT, Thailand

JX474577 JX474657 KF319141

1 17. Kang Lam Duan

waterfall,

UBONRATCHATHA

NI, Thailand

JX474622 JX474704 KF319186

1 63. Tam Nam Thip temple,

ROI- ET, Thailand

JX474619 JX474701 KF319183

1 50. Phu Phan cave,

SAKON NAKHON,

Thailand

JX474618 JX474700 KF319182

1 5. A.Na Kae road,

NAKHON PHANOM

to Dong Luang,

MUKDAHAN,

Thailand

JX474617 JX474699 KF319181

1 12. Doi Hau Mod

mountain, TAK,

Thailand

JX474580 JX474706 KF319144

1 18. Khao Bin cave,

RATCHABURI,

Thailand

JX474581 JX474707 KF319145

C. haughtoni 1 20. Khao Cha Mao,

RAYONG, Thailand

JX474615 JX474697

KF319179

1 29. Khao Sib Ha Chan

waterfall,

CHANTHABURI,

Thailand

JX474616 JX474698 KF319180

1 34. Klong Pla Kang

waterfall, RAYONG,

Thailand

JX474614 JX474696 KF319178

C. herklotsi 1 69. Yamatanishi,

KAGOSHIMA, Japan

JX474644 JX474734 KF319208

C. jourdyi 1 30. Khe Sanh river,

QUảNG TRị, Vietnam

JX474645 JX474735 KF319209

C. labiosus 1 22. Khao Luk Chang,

NAKHON

RATCHASIMA,

Thailand

JX474610 JX474692 KF319174

C. malayanus 1 21. Khao Loy, RAYONG,

Thailand

JX474568 JX474653 KF319132

1 58. Tad Kham waterfall,

NAKHONPHANOM,

Thailand

JX474571 JX474659 KF319135

1 62. Dao Wa Deung cave,

KANCHANABURI,

Thailand

JX474569 JX474652 KF319133

1 8. Chaloem Ratana Kosin,

KANCHANABURI,

Thailand

JX474570 JX474654 KF319134

C. perdix tuba 1 7. Bukit Chintamanis,

PAHANG, Malaysia

JX474648 JX474738 KF319212

1 49. Pulau Besar, JOHOR,

Malaysia

JX474647 JX474737 KF319211

C. pfeifferi 2 2. The 90th km., road

No.105, TAK, Thailand

JX474591-2 JX474683-4 KF319155-

6

1 12. Doi Hau Mod

mountain, TAK,

Thailand

JX474593 JX474682 KF319157

C. saturnus 2 16. Jun cave,

UTTARADIT,

JX474566-7 JX474674-5 KF319130-

1

Thailand

2 15. Huay Rong waterfall,

PHRAE, Thailand

JX474564-5 JX474672-3 KF319128-

9

1 6. Bor Ri Jin Da cave,

CHIANG MAI,

Thailand

JX474562 JX474676 KF319126

1 65. Tam Pha Lom temple,

LOEI, Thailand

JX474563 JX474677 KF319127

C. semisulcatus 1 7. Bukit Chintamanis,

PAHANG, Malaysia

JX474646 JX474736 KF319210

C. songmaensis 1 11. Cuc Phoung, NINH

BINH, Veitnam

JX474578 JX474658 KF319142

C. speciosus 1 33. Klong Na Rai

waterfall,

CHANTHABURI,

Thailand

JX474575

JX474655 KF319139

1 41. Ma Kok waterfall,

CHANTHABURI,

Thailand

JX474576 JX474656 KF319140

C. subfloridus 1 44. Pa Ma Muang temple,

PHITSANULOK,

Thailand

JX474600 JX474666 KF319164

1 55. Som But cave,

PHETCHABUN,

Thailand

JX474601 JX474667 KF319165

C. turgidus 1 42. Naha, Nishinara,

OKINAWA, Japan

JX474643 JX474733 KF319207

C. volvulus 1 66. Tam Ra Kang temple,

SUKHOTHAI,

Thailand

JX474602

JX474668 KF319166

1 4. Ban Ta Sao community

forest, SARABURI,

Thailand

JX474609 JX474691 KF319173

1 1. 1 km. before Dao Khao

Kaeo cave,

SARABURI, Thailand

JX474606 JX474688 KF319170

3 67. Wang Kan Luang

waterfall, LOPBURI,

Thailand

JX474603-5 JX474685-7 KF319167-

9

1 19. Khao Chakun

mountain, SA KAEO,

Thailand

JX474608 JX474689 KF319172

1 51. Sao Wa Lak camp,

NAKHONRATCHASI

MA, Thailand

JX474607 JX474690 KF319171

2 3. Aow Noi temple,

PRACHUAPKHIRI

KHAN, Thailand

JX474583-4 JX474709-

10

KF319147-

8

1 28. Khao Rong cave,

PHETCHABURI,

Thailand

JX474585 JX474711 KF319149

1 13. Erawan waterfall,

KANCHANABURI,

Thailand

JX474586 JX474712 KF319150

C. zebrinus 1 38. Kra Teng Jeng

waterfall,

KANCHANABURI,

Thailand

JX474632 JX474721 KF319196

Cyclotus sp. 1 11. Cuc Phoung, NINH

BINH, Veitnam

JX474649 JX474739 KF319213

Leptopoma

vitrium

1 41. Ma Kok waterfall,

CHANTHABURI,

Thailand

JX474650 JX474741 KF319214

Rhiostoma

hainesi

1 41. Ma Kok waterfall,

CHANTHABURI,

Thailand

JX474651 JX474740 KF319215

Table 2. Average base frequencies across COI, 16S rRNA and 28S rRNA genes for

Cyclophorus

Genes Average base frequencies

A C G T

C+O C C+O C C+O C C+O C

COI (All positions) 0.217 0.216 0.171 0.171 0.213 0.213 0.399 0.400

COI (1st and 2

nd positions) 0.188 0.188 0.217 0.217 0.243 0.243 0.352 0.352

COI (3rd

positions) 0.274 0.271 0.079 0.077 0.153 0.154 0.497 0.498

16S rRNA (All positions) 0.326 0.326 0.142 0.142 0.207 0.207 0.325 0.325

28S rRNA 0.206 0.206 0.269 0.268 0.331 0.332 0.194 0.194

16S rRNA (Selected

position) 0.293 0.293 0.168 0.142 0.228 0.227 0.311 0.338

All COI+16S rRNA 0.245 0.245 0.170 0.170 0.218 0.218 0.366 0.366

1st and 2

nd COI+16S rRNA 0.238 0.238 0.194 0.194 0.235 0.235 0.333 0.333

All COI+16S rRNA+28S

rRNA 0.231 0.231 0.205 0.205 0.259 0.259 0.305 0.305

C = Cyclophorus, C+O = Cyclophorus with outgroup

Table 3. Summary of molecular data across COI, 16S rRNA and 28S rRNA genes for

Cyclophorus

Genes Length

(bp)

Variable sites (%) Range of distances Parsimony

informative site

C+O C C+O C C+O C

COI (All positions) 660 310 (46.97) 304 (46.06) 0.000-0.264 0.000-0.220 292 287

COI (1st and 2

nd positions) 440 90 (20.45) 84 (19.09) 0.000-0.107 0.000-0.084 73 70

COI (3rd

positions) 220 220 (100) 217 (98.64) 0.000-0.623 0.000-0.523 219 217

16S rRNA Selected

position 396 182 (45.96) 152 (38.38) 0.000-0.230 0.00-0.179 147 131

28S rRNA 585 197(33.68) 97 (16.58) 0.000-0.214 0.000-0.072 97 74

All COI+16S rRNA 1056 492 (46.59) 456 (43.18) 0.000-0.214 0.000-0.200 438 418

1st and 2

nd COI+16S

rRNA 836 272 (32.54) 236 (28.23) 0.000-0.156 0.000-0.120 219 201

All COI+16S rRNA+28S

rRNA 1641 689 (41.99) 553 (33.70) 0.000-0.217 0.00-0.149 535 492

C = Cyclophorus, C+O = Cyclophorus with outgroup

FIGURE LEGENDS

Figure1. Shell pictures of all Cyclophorus species examined in this study; (A) C. affinis, (B) C.

amoenus, (Ci) C. aurantiacus aurantiacus, (Cii) C. a. nevilli, (Ciii) C. a. andersoni, (Civ) C.

a. pernobilis, (D) C. bensoni, (E) C. cantori, (F) C. consociatus, (G) C. courbeti, (H) C.

cryptomphalus, (I) C. diplochilus, (J) C. expansus, (K) C. fulguratus, (L) C. haughtoni, (M)

C. herklotsi, (N) C. jourdyi, (O) C. labiosus, (P) C. malayanus, (Q) C. perdix tuba, (R) C.

zebrinus, (S) C. pfeifferi, (T) C. saturnus, (U) C. semisulcatus, (V) C. songmaensis, (W) C.

speciosus, (X) C. subfloridus, (Y) C. turgidus and (Z) C. volvulus

Figure 2. Sampling sites of the Cyclophorus specimens from Thailand and also in some parts of

Laos, Vietnam, Malaysia and Japan. The numbered sample sites are detailed in Table 1 (Map

Number).

Figure 3. Phylogenetic tree of the genus Cyclophorus reconstructed using maximum-likelihood

analysis of 1641 nucleotide sites of the mtDNA and ncDNA (concatenate genes of all codon

position of COI, 16s rRNA and 28S rRNA) using the GTR+G model. Bootstrap support

values for individual nodes are shown on the tree (based on NJ/ML/BI method). The

phylogeny is rooted on Cyclotus, Leptopoma and Rhiostoma. The species names are shaded

according to species complex: dark grey C. fulguratus, grey: C. volvulus, light grey: C.

aurantiacus. (1f) C. fulguratus from West and Central Thailand, (2f) C. fulguratus from

upper Northeast Thailand, (3f) C. fulguratus from lower Northeast Thailand, (4f) C.

fulguratus from East Thailand (1v) C. volvulus from West Thailand, (2v) C. volvulus from

East and Central Thailand, (3v) C. volvulus from North Thailand. Numbers in round brackets

refer to collection localities (shown on map, Figure 2).

Figure 4. Phylogenetic networks of the concatenated all codon position of COI, 16s rRNA and 28S

rRNA sequences using GTR+G (A) Phylogenetic networks and distribution map of C.

fulguratus (B) Phylogenetic networks and distribution map of C. volvulus (groups coloured

by geographic range and labels in round brackets reflect groups in the phylogenetic tree,

Figure 2)

Figure

SUPPLEMENTARY MATERIAL LEGENDS

Supplementary material S1 Phylogenetic relationships of Cyclophorus inferred from the

concatenated mtDNA datasets (all condon positions of COI and 16S rRNA; 1056 bp).

Supplementary material S2 Phylogenetic relationships of Cyclophorus inferred from the nucDNA

dataset (28S rRNA; 585 bp)

Supplementary material S3 Phylogenetic relationships of Cyclophorus inferred from all codon

positions of the COI gene (including Cyclophorus sequences available on Genbank; 630 bp)

Figure

Figure

Figure

Figure

Figure

Figure

Figure

Highlights 1. This work is the first molecular phylogenetic study of Cyclophorus in Thailand.

2. The phylogenetic trees obtained in general confirmed the species level classification.

3. We found cryptic species of Cyclophorus in Thailand.