Phylogenetic relationships of the operculate land snail genus Cyclophorus Montfort, 1810 in Thailand

Accepted Manuscript Phylogenetic Relationships Of The Operculate Land Snail Genus Cyclophorus Montfort, 1810 In Thailand Nattawadee Nantarat, Piyoros Tongkerd, Chirasak Sutcharit, Christopher M. Wade, Fred Naggs, Somsak Panha PII: DOI: Reference:

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

To appear in:

Molecular Phylogenetics and Evolution

Received Date: Revised Date: Accepted Date:

6 April 2013 13 September 2013 14 September 2013

Please cite this article as: Nantarat, N., Tongkerd, P., Sutcharit, C., Wade, C.M., Naggs, F., Panha, S., Phylogenetic Relationships Of The Operculate Land Snail Genus Cyclophorus Montfort, 1810 In Thailand, Molecular Phylogenetics 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 GENUS CYCLOPHORUS MONTFORT, 1810 IN THAILAND

a

Nattawadee Nantarata,b Biological Sciences Program, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand b Animal Systematics Research Unit, Department of Biology, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand Email: [email protected]

b

Piyoros Tongkerdb Animal Systematics Research Unit, Department of Biology, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand

Chirasak Sutcharitb b Animal Systematics Research Unit, Department of Biology, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand Christopher M. Wadec c School of Biology, University of Nottingham, University Park, Nottingham, NG7 2RD, UK Fred Naggsd d Department of Zoology, The Natural History Museum, London SW7 5BD, UK Somsak Panhab,* b Animal Systematics Research Unit, Department of Biology, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand (*Corresponding author) Email: [email protected]

--------------------------*Corresponding author. Tel./fax: +66 2 2185273

E-mail addresses: [email protected] (S. Panha).

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ABSTRACT. - Operculate land snails of the genus Cyclophorus are distributed widely in sub-tropical and tropical Asia. Shell morphology is traditionally used for species identification in Cyclophorus but their shells exhibit considerable variation both within and between populations; species limits have been extremely difficult to determine and are poorly understood. Many currently recognized species have discontinuous distributions over large ranges but geographical barriers and low mobility of snails are likely to have led to long periods of isolation resulting in cryptic speciation of allopatric populations. As a contribution towards solving these problems, we reconstructed the molecular phylogeny of 87 Cyclophorus specimens, representing 29 nominal species (of which one was represented by four subspecies), plus three related out-group species. Molecular phylogenetic analyses were used to investigate geographic limits and speciation scenarios. The analyses of COI, 16S rRNA and 28S rRNA gene fragments were performed using neighbour-joining (NJ), maximum likelihood (ML), and Bayesian inference (BI) methods. All the obtained phylogenetic trees were congruent with each other and in most cases confirmed the species level classification. However, at least three nominate species were polyphyletic. Both C. fulguratus and C. volvulus appear to be species complexes, suggesting that populations of these species from different geographical areas of Thailand are cryptic species. C. aurantiacus pernobilis is distinct and likely to be a different species from the other members of the C. aurantiacus species complex.

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

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1. Introduction The Cyclophoridae are dioecious terrestrial caeonogastropod snails with a long fossil record extending from the Mesozoic era (Gordon and Olson, 1995; Kongim et al., 2006) and with a wide current geographical distribution: Southern Europe, Central America, Asia, Africa, various Pacific islands and Australia (Kobelt, 1902; Solem, 1959). The most broadly used classification for the Cyclophoridae is that of Kobelt (1902). He classified the cyclophorids based on shell, opercular and radular characters, but whilst this undoubtedly reflects some of the broad relationships, it is of little value below the subfamily level (Solem, 1956). Currently, the Cyclophoridae is comprised of about 870 species arranged in three subfamilies and 35 genera (Kobelt, 1902, 1908; Wenz, 1938; Vaught, 1989; Bouchet and Rocroi, 2005; Lee et al., 2008b). The genus Cyclophorus Montfort, 1810 is the most species rich genus in the family Cyclophoridae with over 100 described species distributed from South Asia to Southeast Asia and including the south of China, Korea and Japan (Reeve, 1862; Kobelt, 1902, 1908; Gude, 1921; Pilsbry, 1916, 1926; Benthem Jutting, 1948, 1949; Solem, 1959, 1966; Minato and Habe, 1982). Members of Cyclophorus have a distinctive large solid, low conical shell form with a thin and multispiral operculum. They are “grounddwelling” in leaf litter, under logs etc. and occur in a wide range of forest habitats from evergreen rainforest to monsoon deciduous forest. In Thailand, the highest densities occur in limestone forest (Kobelt, 1902, 1908; Gude, 1921; Solem, 1959). The validity of the nominal Cyclophorus species level classification is not clear because of the degree of shell variation within and between nominated species and the limited number of characters available in Cyclophorus shells. Kobelt (1902) classified Cyclophorus into eight subgenera while Vaught (1989) rearranged Cyclophorus into five subgenera. Few descriptions of the internal anatomy, including reproductive organs have been published for Cyclophorus (Tielecke, 1940; Kasinathan, 1975). Available information demonstrates a high degree of similarity for internal anatomy within Cyclophorus that does not provide robust characters for recognizing species level categories (Welber, 1925; Kongim et al., 2006). Thus there has been little advance on the use of characters based on shell morphology, including shell size, shape, colour pattern and peristome morphology (e.g. Reeve, 1862; Kobelt, 1902, 1908; Gude, 1921). Environmental factors can greatly affect shell morphology (Uit de Weerd et al. 2004; Lee et al. 2008 a, 2008b; Elejalde et al., 2009) and homoplasy confounds the recognition of biological species of Cyclophorus. Species limits in Cyclophorus are notoriously difficult to establish with numerous geographically isolated populations exhibiting seemingly minor differences in their morphology. There is evidence to suggest that Cyclophorus was important in the diet of Stone Age cave dwellers in Oriental regions (Rabett et al., 2011). In the present day, these edible snails have been utilized for food in many parts of Thailand, Laos and Vietnam (Oakley, 1964; Paz and Solheim, 2004). However, the number of Cyclophorus snails seems to have dropped (Hildyard, 2001) most likely due to a range of contributing factors such as changes in the environmental conditions around snails‟ habitats especially in limestone areas and improper snail harvesting (Clements et al., 2006). Recent work has focused on systematic research and development of conservation strategies (Clements et al., 2008). In addition to its intrinsic scientific interest, a reliable taxonomy is important for avoiding the mistaken treatment of multi-species genera as a single taxon that may fail to effectively regulate their conservation. There is therefore practical conservation value in the recognition of Cyclophorus cryptic species (Kongim et al., 2006; Prasankok et al., 2009).

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Over the past decade, sequence data has been used to help clarify problems in systematics and evolution where morphological and physiological characters have proven to be ambiguous (Vogler and Monaghan, 2007). Studies on land snail phylogeny using molecular DNA sequences have suggested that such approaches are potentially useful (e.g. Harasewych, 1998; Wade et al., 2006; Colgan et al., 2007; Lee et al., 2008a, 2008b). The genes most commonly used for land snail systematics, at various taxonomic levels are the mitochondrial cytochrome oxidase subunit I (COI) and16S rRNA genes and the nuclear ribosomal RNA genes (e.g. Harasewych, 1998; Wade et al., 2006; Colgan et al. 2007; Liew et al., 2009). They provide a highly effective tool to resolve taxonomic problems and identify cryptic species (Paquin and Hedin, 2004). These genes have been found to be suitable for use at the generic level in cyclophorids, revealing that the evolution of morphological and ecological traits occurs at extremely high rates during adaptive radiation, especially in fragmented environments (Sanders et al., 2006; Lee et al., 2008a, 2008b). The work reported here is the first molecular phylogenetic study of the genus Cyclophorus in Thailand.

2. Material and Methods 2.1 Taxon sampling and identification Eighty seven specimens of Cyclophorus, attributed to 29 nominal species (Figure 1), including the type species Cyclophorus volvulus (Müller, 1774) and, for one of these, four nominal subspecies, were collected from 67 localities in Thailand and 7 additional localities in Laos, Vietnam, Malaysia and Japan (Figure 2 and Table 1) representing approximately 30% of the total nominal species of the genus. Tissue samples were fixed and preserved in 95% (v/v) ethanol. Ethanol was changed at least twice to eliminate water content of the samples. Vouchers were preserved in 70% (v/v) ethanol for anatomical study. Provisional identification of species was based upon shell morphology, making use the literature (Reeve, 1862; Kobelt, 1902, 1908; Benthem Jutting, 1948, 1949) and by examination of reference collections, including type material in the following museums: The Natural History Museum, London (NHMUK); Senckenberg Museum, Frankfurt (SMF); The Royal Belgian Institute of Natural Sciences (RBINS); Zoological Museum University of Copenhagen, Denmark (ZMUC) and the Chulalongkorn University Museum of Zoology (CUMZ), Bangkok, Thailand. 2.2 DNA extraction, PCR amplification and sequencing Total genomic DNA was extracted from approximately 3–5 mm3 of foot tissue from each individual using a DNAeasy Tissue Kit (QIAGEN Inc.), and was then stored at –20 ºC until use. The COI and 16S rRNA mitochondrial genes and 28S rRNA nuclear gene were amplified by PCR using the primers LCOI490 (5‟GGTCAACAAATCATAAAGATATTGG-3‟) and HCO2198 (5‟TAAACTTCAGGGTGACCAAAAAATCA-3‟) for the COI gene (Folmer et al., 1994), 16sar (5'-CGCCTGTTTATCAAAAACAT-3') and 16sbr (5'-CCGGTCTGAACTCAGATCACGT3') for the16S rRNA gene (Kessing et al., 1989) and 28SF4 (5'AGTACCGTGAGGGAAAGTTG-3') and 28SR5 (5'- ACGGGACGGGCCGGTGGTGC-3') for the 28S rRNA gene (Morgan et al., 2002). For all genes PCR reactions were undertaken in a 50 µl final volume using 25 µl of 2xIllustra hot starts master mix (GE Healthcare), 10µM of each primer and about 10 ng of DNA template. For COI, thermal cycling was at 94 °C for 2 min, followed by 36 cycles of 94 °C for 30 s, 42 °C for 2 min, and 72 °C for 2 min, and

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then a final extension step of 72 °C for 5 min. For 16S rRNA and 28S rRNA, thermal cycling was performed in the same way with the exception that the annealing temperature was changed to 50 °C for 30 s and the extension time to 90 s. The amplified products were checked following 1% (w/v) agarose gel electrophoresis resolution in 0.5x TBE buffer and visualized with SYBR Safe and UV Transillumination. The PCR products were purified using a QIAquick purification Kit protocol (QIAGEN Inc.). The amplified PCR products were directly cycle-sequenced using the original amplification primers with the sequencing reaction products run on an Applied Biosystems automatic sequencer (ABI 3730XL) at Macrogen, Inc. (Korea). The DNA sequences were compared with sequences from the GenBank database by using the BLASTn algorithm to confirm the homology of the amplification products to targeted genes. 2.3 Sequence alignment and phylogenetic analysis Sequences were edited with reference to the trace data and aligned using MUSCLE version 3.6 (Edgar, 2004). The alignments were improved manually where necessary by using MEGA 5.0 (Tamura et al., 2011). Ambiguous regions and gaps in the alignment were excluded from the dataset. 660 nucleotide sites were unambiguously aligned across all taxa for the COI fragment, with 396 unambiguously aligned nucleotide sites for 16S rRNA and 585 unambiguously aligned nucleotide sites for 28S rRNA. The COI, 16S rRNA and 28S rRNA sequences were checked for saturation and phylogenetic signal using DAMBE v. 4.5.33 (Xia and Xie, 2001) and through plotting uncorrected pairwise transition and transversion distances against total uncorrected distances in order to visualize saturation and identify the taxa responsible. For COI, saturation tests were performed using all codon positions and each codon position individually. All base frequencies and molecular character statistics were calculated using MEGA 5.0 (Tamura et al., 2011). Phylogenetic analyses were undertaken using the following datasets; all codon positions of COI (660 bp), first and second codon positions of COI (440 bp), 16S rRNA (396 bp), 28S rRNA (585 bp), concatenated COI (all codon positions) and 16S rRNA (1056 bp), concatenated COI (1st and 2nd codon positions only) and 16S rRNA (839 bp) and concatenated COI (all codon positions), 16S rRNA and 28S rRNA (1641 bp). Details are shown in Table 3. Heterogeneity in base composition between sequences is known to affect phylogenetic inference (Lockhart et al. 1994), so we tested the variation in base pair composition among sequences for the datasets using a χ2 analysis, as implemented in PAUP* v4.0b10 (Swofford, 2003). In order to assess the validity of combining datasets a partition homogeneity test (Farris et al. 1994) was undertaken in PAUP 4.0b10, using 100 replicates (Swofford, 2003). Phylogenetic trees of all 32 taxa (including 3 outgroup taxa) were constructed using neighbor-joining (NJ), maximum likelihood (ML), and Bayesian inference (BI). The phylogenetic trees were rooted on Cyclotus, Leptopoma and Rhiostoma genera that have been suggested to be close relatives of the genus Cyclophorus and fall within the same family (Colgan et al., 2007; Lee et al., 2008a, b). For all methods, correction for multiple hits was made using the general time-reversible (GTR) model (Lanave et al., 1984), with between-site rate heterogeneity taken into account by combining gamma correction (G) into the model (Gu et al., 1995). This model was found to be the most appropriate substitution model for all datasets using jModeltest 0.1.1 (Posada, 2008). NJ analysis was undertaken using PAUP* v4.0b10 (Swofford, 2003) with model parameters, including the rate matrix, base frequencies and gamma shape parameter () of the gamma distribution (based on 16 rate categories) estimated using likelihood based on iteration from an initial NJ tree. The iteration process

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was repeated with estimated parameters used for subsequent rounds of tree building until no further improvement in likelihood score was obtained. Bootstrap resampling (Felsenstein, 1985) with 1000 replicates was undertaken to assign support to branches in the NJ tree. ML analysis was undertaken using RAxML v7.2.6 (Stamatakis, 2006). Bootstrap resampling (Felsenstein, 1985) via the rapid bootstrap procedure of Stamatakis et al. (2008) was undertaken to assign support to branches in the ML tree. BI analysis was performed using MrBayes version 3.1.2 (Huelsenbeck and Ronquist, 2001), with the tree space explored using four chains of a Markov chain Monte Carlo algorithm (MCMC). The Bayesian analysis was run for 3 million generations (heating parameter = 0.03), sampling every 100 generations. The first 58002 trees were discarded as burnin, such that the final consensus tree was built using the last 2000 trees. In addition, we also analyzed our concatenated COI (all codon positions), 16S rRNA and 28S rRNA (1641 bp) dataset under the phylogenetic mixture model approach (Ronquist and Huelsenbeck, 2003). The analyses under the various assumed partitions were run using HKY+I+G for COI, GTR+G for 16S rRNA and GTR+G for 28S rRNA. 2.4 Phylogenetic networks In order to evaluate the structure and possible reticulating relationships Neighbour Net analysis (Huson and Bryant, 2006) were undertaken for the widespread species, C. fulguratus and C. volvulus using concatenated COI (all codon position), 16S rRNA and 28S rRNA sequences. This allowed additional sites to be recruited into the analysis with 1739 unambiguously aligned sites for C. fulguratus and 1745 sites for C. volvulus. Neighbour Net alaysis was performed using the GTR+G model computed using SplitsTree version 4.11.3 (Huson and Bryant, 2006). Bootstrap support for splits was conducted with 1000 replicates. The dataset was also evaluated for homoplasy using the homoplasy index (PHI) statistic (Bruen et al., 2006) using SplitsTree version 4.11.3 to test for recombination within the sequences. 2.5 Supplemental analyses of the COI gene incorporating Genbank data An additional 232 COI sequences (630 bp) of Cyclophorus from Genbank (HM753719.1-HM753950.1) enabled the inclusion of an additional 10 Cyclophorus sp. and 4 subspecies from three new countries (Japan, Taiwan and China) into our analyses. Phylogenetic analyses were undertaken as above (section 2.3) using NJ, ML and BI methods and incorporating a GTR+G model. 2.6 GenBank Accession Numbers Nucleotide sequences of this study have been deposited in GenBank under accession numbers JX474562 - JX474651 for COI, JX474652 - JX474741 for the 16S rRNA fragment and KF319126-KF319215 for the 28S rRNA, and are shown in Table 1.

3. Results

3.1 DNA sequence variation and distance analysis

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The aligned 660 bp sequence of the COI dataset comprising all codon positions had 38.3% GC content [range 36.97% GC to 42.48% GC, χ2 test=141.7596 d.f.=267, P=1.000], 292 (16.6%) parsimony informative and 310 variable sites (46.97%). The uncorrected pdistance between the taxa ranged from 0.000 to 0.264 [inter/intraspecific p-distances = 0.142 and 0.052, respectively]. Stop codons were absent in all codon positions of COI sequences. Therefore, we assumed that no pseudogenes were accidentally sequenced. Transition, transversion and p-distances estimated from MEGA 5.0 were used in the saturation tests. The results of the saturation test using DAMBE v. 4.5.33 (Xia and Xie, 2001) did not show evidence for saturation in a symmetrical tree of the dataset, with an index of substitution saturations (ISS) value of 0.411, which was significantly lower than the critical saturation value (ISS.C) of 0.719, P<0.0001. However, the ISS value was higher than the ISS.C value of 0.392 for an asymmetrical tree although not significantly so P=0.4669 (N=32) and such asymmetrical trees are possibly not realistic for the dataset. This suggests that there is still sufficient phylogenetic information in the all codon positions of COI. Moreover, the saturation plots suggested no evidence of saturation for the ingroup data though some evidence for some saturation when outgroups were included. The aligned 16S rRNA gene fragment (396 bp), after exclusion of ambiguously aligned nucleotide positions, had 39.6% GC content [range 37.63% GC to 42.42% GC, χ2 test = 44.042137 d.f.=267, P=1.000], with 147 (37.1%) parsimony informative and 182 (46.0%) variable sites. The result of the DAMBE test did not detect saturation in the sequences. The results showed an ISS value of 0.146, which was significantly lower than the critical saturation value (ISS.C) of 0.691 for a symmetrical and 0.362 for an asymmetrical tree topology, P<0.0001. Moreover, the saturation plot did not show evidence of saturation. The uncorrected p- distance between the taxa ranged from 0.000 to 0.230 [inter/intraspecific pdistances = 0.098 and 0.0171, respectively]. The aligned 28S rRNA gene fragment (585 bp), after exclusion of ambiguously aligned nucleotide positions, had 59.8% GC content [range 58.97% GC to 61.20% GC, χ2 test = 17.138669 d.f.=267, P=1.000], with 97 (16.6%) parsimony informative and 197 (33.7%) variable sites. The result of the DAMBE test did not detect saturation in the sequences. The results showed an ISS value of 0.083, which was significantly lower than the critical saturation value (ISS.C) of 0.711 for a symmetrical and 0.383 for an asymmetrical tree topology, P<0.0001. Moreover, the saturation plot did not show evidence of saturation. The uncorrected p- distance between the taxa ranged from 0.000 to 0.214 [inter/intraspecific pdistances = 0.022 and 0.004, respectively]. The concatenated data set of all codons of COI, 16S rRNA and 28S rRNA (1641 bp) had 46.3% GC content [range 44.79% GC to 48.87% GC, χ2 test = 87.342328 d.f.=267, P=1.000], with 535 (32.6%) parsimony informative and 689 (41.9%) variable sites. The results of a partition homogeneity test by PAUP 4.0b10, using 100 replicates (Swofford, 2003) showed no significant differences were found between markers (P = 0.095). The result of the DAMBE test did not detect saturation in the sequences. The results showed an ISS value of 0.298, which was significantly lower than the critical saturation value (ISS.C) of 0.779 for a symmetrical and 0.501 for an asymmetrical tree topology, P<0.0001. Moreover, the saturation plot did not show evidence of saturation. The uncorrected p-distance between the taxa ranged from 0.000 to 0.217 [inter/intraspecific p-distances = 0.082 and 0.025, respectively]. A summary of the molecular data is shown in Table 2 and 3. 3.2. Phylogenetic analyses The phylogenetic trees reconstructed using the NJ, ML and BI methods were highly congruent, with almost identical topologies and with the same supported nodes for all major

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clades (data not shown), and with all datasets. The ML phylogenetic tree based on the concatenated dataset of all codons of COI, 16S rRNA and 28S rRNA (1641 nucleotide sites) is shown in Figure 3. Cyclophorus was separated into two principal clades (clades 1 and 2, Figure 3) with strong support (100% NJ bootstraps, 100% ML bootstraps and posterior probability (PP) =1 in BI for clade 1 and 99% NJ, 100% ML, 1 PP for clade 2). Clade 1 comprised the sole species C. perdix tuba from Malaysia, whilst clade 2 comprised the remainder of the Cyclophorus species. Clade 2 was further subdivided into two major subclades; clade 2a that includes C. semisulcatus, C. jourdyi, C. herklotsi and C. turgidus (95% NJ, 97% ML, 1 PP) and clade 2b that includes all of the Thai Cyclophorus as well as C. songmaensis from Vietnam and C. bensoni from Laos (90% NJ, 94% ML, 0.96 PP). Of the 29 nominal species (Figure 1), 25 were monophyletic and strongly supported in greater than 77% of NJ and ML bootstrap replicates and PP>0.91 in BI (in Figure 3). The individual genes generally supported the monophyly of most Cyclophorus species, but yielded poor resolution (data not shown). The molecular trees provided strong support indicating that 3 species, C. fulguratus, C. volvulus, and C. aurantiacus, were not monophyletic. Cyclophorus fulguratus fell in 4 well-supported locations in the trees (labeled 1f, 2f, 3f and 4f in Figure 3). The first group (1f) from West and Central Thailand was most closely related to C. volvulus (99% NJ, 100% ML, 1 PP) also from West Thailand. The second group (2f) from upper Northeastern Thailand was most closely related to C. consociatus (100% NJ, 100% ML, 1 PP). A single individual (3f) from lower Northeastern Thailand fell immediately outside the C. consociatus and C. fulguratus 2f cluster (92% NJ, 96% ML, 1 PP). A further individual (4f) from East Thailand clustered with C. speciosus (99% NJ, 100% ML, 1 PP). Cyclophorus volvulus fell in 3 well-supported locations in the trees (labeled 1v, 2v and 3v in Figure 3). The first group (1v) from West Thailand was most closely related to C. fulguratus 1f (99% NJ, 100% ML, 1 PP) also from West Thailand. Group 2 (2v) from East and Central Thailand was most closely related to C. labiosus (94% NJ, 96% ML, 0.99 PP). A single individual (3v) from North Thailand clustered with C. subfloridus (99% NJ, 98% ML, 1 PP). Finally, C. aurantiacus was not monophyletic in our trees with C. a. pernobilis falling separately to the other subspecies (87% NJ, 89% ML bootstrap support and 1 PP in the concatenated COI/16S/28S dataset, see Figure 3). Within the main C. aurantiacus group the subspecies, C. a. aurantiacus, C. a. nevilli and C. a. andersoni fell as monophyletic units (Figure 3). The monophyly of a 4th species C. consociatus proved equivocal in all analyses. C. consociatus was poorly resolved in the concatenated COI, 16S rRNA and 28S rRNA (Figure 3) and the individual genes analyses. For supplementary analysis, C. pfeifferi appeared to be paraphyletic in the mtDNA tree (Figure S1) though with low support: 50% NJ, 53% ML, 0.59 PP but was recovered as a monophyletic unit in trees based on concatenated COI, 16S rRNA and 28S rRNA (93% NJ, 97% ML, 1 PP, Figure 3). C. malayanus was poorly resolved in the mtDNA tree (Figure S1) and COI tree but were recovered as a monophyletic unit in trees based on concatenated COI, 16S rRNA and 28S rRNA (91% NJ, 97% ML, 1 PP for C. malayanus). C. consociatus was poorly resolved in all analysis. C. bensoni appeared to be polyphyletic in the COI analyses (62% NJ, 62% ML, 0.77 PP, Supplemental analyses –Figure S3). 3.3. Phylogenetic networks of C. fulguratus and C. volvulus Neighbour Net networks of C. fulguratus and C. volvulus were consistent with the phylogeny (Figure 4). C. fulguratus (Figure 4A) was dispersed in four splits that correspond to WesternCentral Thailand (split I in Fig 4, 1f in Fig 3), upper Northeastern Thailand (split II in Fig 4, 2f in Fig 3), lower Northeastern Thailand (split III in Fig 4, 3f in Fig 3) and Eastern Thailand

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(split IV in Fig 4; 4f in Fig 3). For C. volvulus (Figure 4B), the sequences were distributed in 3 main splits with Western Thailand (split I in Fig 4, 1v in Fig 3), Eastern-Central Thailand (split II in Fig 4, 2v in Fig 3), and Northern Thailand (split III in Fig 4, 3v in Fig 3). The PHI test for recombination did not find statistically significant evidence for recombination in either C. fulguratus (PHI test; P = 0.5681) or C. volvulus (PHI test; P = 0.3464). 3.4 Supplemental analyses of the COI gene incorporating Genbank data The inclusion of an additional 232 COI sequences of Cyclophorus from Genbank (HM753719.1-HM753950.1) enabled the incorporation of an additional 10 Cyclophorus sp. and 4 subspecies from 3 countries into our analyses (Supplemental analyses –Figure S3). Consistent with our concatenated COI, 16S and 28S rRNA tree (Figure 3), the Cyclophorus sequences fell into 2 principal clades: clade 1, comprising the sole species C. perdix tuba from Malaysia, and clade 2, comprising the remainder of the Cyclophorus species. Clade 2 was further subdivided into two major subclades; clade 2a that includes sequences from China, Vietnam, Malaysia and Taiwan and clade 2b that includes all of the Thai Cyclophorus as well as C. songmaensis from Vietnam and C. bensoni from Laos. The tree generally supported the monophyly of Cyclophorus species with 35 of 43 species appearing to be monophyletic in the COI gene tree, though 8 of 43 species appeared to be polyphyletic based on analyses of just the COI gene (Figure S3). Interestingly, C. jourdyi from Vietnam and C. semisulcatus from Malaysia appeared to be related with the Cyclophorus sp. from China. Overall the results of the analyses of the COI gene (including sequences from Genbank) showed consistent information in the placement of the Thai clade (clade 2b) in the broad Cyclophorus phylogeny.

4. Discussion Land snails are powerful research tools for investigating evolutionary processes. However, their traditional classification is based on morphological characters that are liable to extensive homoplasy. Cyclophorus shows some interesting questions on convergence and polymorphism of shell color, patterns and shapes. Critical studies of stylommatophoran land snail reproductive systems have probably led to a much better understanding of species limits in these pulmonate snails than is the case with terrestrial Caenogastropoda where the basis of morphospecies recognition has largely been dependent on variation in shell characters. Conversely, conservative shell forms may conceal cryptic species; these two trends combine to make species limits based on morphology difficult to recognize. The present study is the first investigation of the relationships of members of Cyclophorus in Thailand using DNA sequence based systematics. The phylogenetic trees showed good resolution in clarifying the taxonomy and relationships in Cyclophorus with consistent results obtained with different genes (COI, 16S rRNA and 28S rRNA) and with different analytical methods (NJ, ML and BI). Most (25/29) but not all nominate species appeared to be monophyletic. 4.1 Evolutionary relationships within Cyclophorus The phylogenetic tree presented in Figure 3 (ML analysis of the combined all codon positions of COI, 16S rRNA and 28S rRNA sequences) does not conflict with the analysis of the three genes separately or by the analysis with ML, NJ or BI (data not shown). For some species (e.g. C. pfeifferi, C. malayanus, C. consociatus and C. bensoni) for which the relationships inferred in the concatenated COI, 16S rRNA and 28S rRNA tree (Figure 3)

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differed from those inferred in analyses based on other datasets. These differences and/or lack of resolution are consistent with the pattern we would expect if many or all genes shared the same history, but some of them individually contained too little information relative to stochastic noise to support clades. This supports the idea that concatenation can overcome misleading homoplasy (Townsend et al., 2011). We note also the possibility that mitochondrial introgression can mislead phylogenetic analyses, but as our analyses were based on both nuclear and mitochondrial markers, and analyses of the nuclear 28S rRNA gene (Supplemental analyses – Figure S2) gave identical results with mtDNA (Supplemental analyses – Figure S1) and concatenated datasets (Figure 3), it seems improbable that introgression has had any major effect on our phylogenies. In the molecular tree (Figure 3), Cyclophorus divides into two major clades (clades 1 and 2), with C. perdix tuba from Malaysia the sole species in clade 1 and with the remaining Cyclophorus species in clade 2. The sole species of C. perdix tuba (Clade 1, Figure 3) also showed some distinctive morphological characters (unique colour patterns, more prominent keel, a conical shape but with peculiar trumpet shaped dilatation of the ultimate whorl towards the aperture and it also possesses a unique line around the suture.) when compared to the rest of the Cyclophorus group. Moreover, the p-distance between C. perdix tuba and those species in clade 2 was 16% and 19% in the 16S rRNA and COI genes, respectively. We note similar genetic distances have been inferred to be indicative of distinct genera in studies of other land snails (Köhler, 2011). Clade 2 was further divided into two subgroups, the minor clade 2a comprised the non-Thai species, and the main clade 2b comprised all of the Thai species plus one species from each of Laos and Vietnam. Thus both Laos and Vietnam have close relatives to Thai Cyclophorus. The inferred phylogenetic tree suggests that similar morphologies appeared independently and rapidly in different lineages. Shells of Cyclophorus commonly reveal complicated dark brown colour patterns that likely function to disguise them from predators and are therefore likely to be homoplasious. The supplemental analysis (Supplemental analyses –Figure S3), in which we incorporated additional COI sequences from Genbank, allowed us to explore Cyclophorus relationships in greater detail both through the inclusion of additional Cyclophorus species and the inclusion of samples from over a wider geographical range. On the whole, the monophyly of Cyclophorus species was generally supported, though 8 of 43 species appeared to be polyphyletic based on analyses of just the COI gene (Figure S3). Moreover, the wider geographic sampling has revealed that clade 2a includes sequences from China and Taiwan, as well as sequences from Malaysia, Japan and Vietnam. Clade 2b remains unchanged with the inclusion of all of the Thai Cyclophorus as well as C. songmaensis from Vietnam and C. bensoni from Laos. We note that most Cyclophorus species from clade 2a have a small size (about 2-3 cm) with a simple aperture while snails from clade 2b show various sizes (about 36.5 cm) with a diverse morphology. 4.2 Taxonomic implications Recent work on Cyclophorus has indicated that there are likely to be a number of cryptic species (Kongim et al., 2006; Prasankok et al., 2009). Whilst most species of Cyclophorus sampled here correspond with molecular tree placements, some polyphyletic groups can be identified. Three morphological species are not monophyletic on molecular criteria and most likely represent cryptic species or complexes. (1) C. fulguratus in groups 1f, 2f, 3f and 4f. (2) C. volvulus in groups 1v, 2v and 3v and (3) C. aurantiacus in group 1a and C. aurantiacus pernobilis in group 2a. In the case of C. fulguratus, the specimens divided into four well supported clades; 1f from Western Thailand, 2f from upper Northeastern Thailand, 3f from lower Northeastern

467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516

Thailand and 4f from Eastern Thailand. Neighbour Net networks were also consistent with the phylogenetic trees with individuals distributed in four splits (Figure 4A) and with high bootstrap support. Furthermore, PHI analysis did not detect evidence of recombination (p>0.05) between the four subpopulations. Our molecular findings are consistent with previous observations of variation in karyotypes (Kongim et al., 2006) and allozymes (Prasankok et al., 2009). Based on analysis of karyotype variation, Kongim et al. (2006) separated C. fulguratus into two distinct groups (species) with populations from Central Thailand having a 12m + 2sm karyotype and those from Northeastern Thailand having a 13m + 1sm karyotype, consistent with the principal division of C. fulguratus haplotypes in our tree. Moreover, allozyme analysis revealed large genetic divergences between populations from Central Thailand and those from Northeastern Thailand as well as large genetic divergences between populations from Central and Eastern Thailand (Prasankok et al., 2009), congruent with the separate phylogenetic placement of C. fulguratus from Western-Central Thailand versus Northeastern and Eastern parts of Thailand, observed here. Cyclophorus fulguratus thus appears to represent several separate cryptic species that show convergent patterns of morphological evolution. Previous studies have suggested that these distinct clades are allopatric (Kongim et al., 2006; Prasankok et al., 2009). In this regard, the low motility of the snail, coupled with the separation of different geographical populations by geographical barriers, such as fragments of mountain ranges (the Phu Phan range in Northeast Thailand, the Tenasserim range in Western Thailand and the Sankamphaeng and Cardamom ranges in Eastern Thailand) led to allopatric speciation. We propose that increased physical and environmental heterogeneity over time delivered chances for localized allopatry (Mayr, 1963). For C. volvulus, individuals are separated into three well-supported clades; group 1v from Western Thailand, group 2v from Eastern-Central Thailand and group 3v from Northern Thailand. Neighbor Net networks were consistent with the phylogenetic trees with individuals distributed in three splits (Figure 4B) with high bootstrap support. Moreover, the PHI analysis did not detect evidence of recombination (p>0.05) between the three subpopulations. Thus, our findings reveal evidence for the likelihood of allopatric speciation within C. volvulus sensu lato. Allopatric speciation is related geographic isolation most likely due to mountain ranges in Western Thailand (split I; Figure 4B) and Central-Eastern Thailand (split II; Figure 4B). Cyclophorus aurantiacus falls in two separate locations in the phylogenetic tree with the subspecies C. a. pernobilis (1a) falling separately to a group comprising C. a. aurantiacus, C. a. nevilli and C. a. andersoni (2a). It seems likely that geographical isolation between C. a. pernobilis in the West and the other C. aurantiacus subspecies in the South has led to allopatric speciation. Moreover, C. a. pernobilis exhibits a significantly different morphology when compared to the subspecies in the South. Cyclophorus aurantiacus pernobilis was originally identified as Cyclostoma pernobilis by Gould in 1843 and transferred to the genus Cyclophorus by Hanley and Theobald, 1870. Kobelt (1902) placed this nominal species as a subspecies of C. aurantiacus. However, our analyses support Gould (1843) and Hanley and Theobald (1870) who recognized C. pernobilis (Gould, 1843) as a distinct species.

5. Conclusion This work is the first molecular phylogenetic study of Cyclophorus in Thailand; 87 individuals of 29 nominal species (and four subspecies for one of these) were included with three outgroup genera. Phylogenetic placement of most (25/29) Cyclophorus species

517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535

corresponded with traditional shell-based morphospecies. However, C. fulguratus and C. volvulus are resolved as cryptic species complexes, supporting previous conclusions based on karyotype and allozyme variation (Kongim et al., 2006; Prasankok et al., 2009). Cyclophorus aurantiacus perbobilis fell in a distinct lineage from the other members of the C. a. aurantiacus group, which supports its original classification as a separate species (C. pernobilis) by Gould (1843) rather than the subsequent subspecies classification of C. a. perbobilis by Kobelt (1902). The monophyly of C. consociatus proved equivocal. Cyclophorus is one of the most diverse land snail groups both morphologically and taxonomically (Kobelt, 1902). The mtDNA and nucDNA provided a powerful tool for investigating relationships within the genus and for recognizing genetically divergent and morphologically cryptic lineages. The phylogenetic results raise many questions about the species complexes that still remain to be answered. Allopatric cryptic species are likely to be widespread throughout the range of Cyclophorus and in other terrestrial caeonogastropod land snails where systematic studies have largely been restricted to shell-based morphospecies. This has important consequences for conservation because of the possible existence of a large number of what might be highly localized and endangered species that have so far not even been recognized.

536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584

ACKNOWLEDGMENTS The main funding source for this project analyses was from The Thailand Research Fund (TRF) to SP under TRF-Senior Scholar Research Grant (2012-2014) RTA5580001, and also to the supply for a graduate student (NN) through The Royal Golden Jubilee Ph.D. Program (PHD/0315/2550). The first funding for basic collecting specimens and taxonomic work were provided by the Commission on Higher Education under The National Research University Project of Thailand (FW646A). We would express our sincere gratitude to The Plant Genetic Conservation Project under the Initiative of Her Royal Highness Princess Maha Chakri Sirindhorn for providing our great opportunity of collecting specimens in many restricted areas especially on several islands in Andaman Sea. And also thanks to UNITAS Malacologica for providing Student Research Awards 2011. We thank members of the Animal Systematics Research Unit, Chulalongkorn University for assistance in collecting material. We further extend our thanks to Dr. Peter Foster (NHM) for invaluable help with our analysis. We thank Prof. Takahiro Asami for providing contributory funding to visit Japan and to Dr. Kiyonori Tomiyama for field collecting arrangements at Kagoshima, Kyushu, Japan.

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Tables

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 C. affinis

No. of Map sam No. -ples 3 10. 1

48.

1

59.

1

66.

3

25.

1

23.

1

31.

1

35.

1

27.

1

37.

1

9.

1

61.

1

14.

1

56.

1

40.

C. a. pernobilis

1

38.

C. bensoni

1

52.

1

43.

C. amoenus

C. a. aurantiacus

C. a. andersoni

C. a. nevilli

Localities Name

GenBank accessesion No. COI 16S rRNA 28S rRNA

Chiang Dao, CHIANG MAI, Thailand Pla cave, MAE HONG SON, Thailand Thepsatit temple, NAKHON SAWAN, Thailand Tam Rakang temple, SUKHOTHAI, Thailand Khao Nor, NAKHON SAWAN, Thailand

JX474587-9

Khao Lak Lam Lu, PHANG-NGA, Thailand Khiri Rat Nikhom, SURAT THANI, Thailand Kob cave, PHANGNGA, Thailand Khao Poo Khao Ya, PHATTHALUNG, Thailand Koo Ha Sa Wan cave, PHATTHALUNG, Thailand Charoenkooha temple, SONGKHLA, Thailand To Poo view point, SATUN, Thailand Hin Lad waterfall, SURAT THANI, Thailand Sramorakod, KRABI, Thailand Mae Koh, SURAT THANI, Thailand Kra Teng Jeng waterfall, KANCHANABURI, Thailand Sa Pan waterfall, NAN, Thailand Nam Min waterfall, PHAYAO, Thailand

JX474590

JX47467880 JX474681

KF3191513 KF319154

JX474595

JX474660

KF319159

JX474597

JX474662

KF319161

JX474596,J X474598-9

JX474661, JX474663-4

JX474642

JX474723

KF319160, KF3191623 KF319206

JX474641

JX474724

KF319205

JX474640

JX474725

KF319204

JX474637

JX474730

KF319201

JX474636

JX474729

KF319200

JX474638

JX474731

KF319202

JX474639

JX474732

KF319203

JX474634

JX474728

KF319198

JX474633

JX474726

KF319197

JX474635

JX474727

KF319199

JX474623

JX474722

KF319187

JX474574

JX474670

KF319138

JX474572

JX474671

KF319136

1

68.

1

31.

1

47.

1

39.

1

64.

2

45.

1

29.

C. cryptomphalus

1

57.

C. diplochilus

1

54.

1

36.

1

14.

1

24.

1

46.

1

26.

1

60.

1

32.

1

53.

1

17.

C. cantori

C. consociatus

C. courbeti

C. expansus

C. fulguratus

Pha Hom, WIANG JUN, Laos Khiri Rat Nikhom, SURAT THANI, Thailand Phuket Marine Biological Center, PHUKET, Thailand Lampahang school, SAKON NAKHON, Thailand Tam Pa Num Pok temple, KHON KAEN, Thailand Pang Sri Da waterfall, SA KAEO, Thailand Khao Sib Ha Chan waterfall, CHANTHABURI, Thailand Sri U Thum Porn temple, NAKHONSAWAN, Thailand Sa Tit Kee Ree Rom temple, SURAT THANI, Thailand Koh Yao Yai, PHANG-NGA, Thailand Hin Lad waterfall, SURAT THANI, Thailand Khao Nam Lak, NAKHON SI THAMMARAT, Thailand Pha Ka Yang cave, RANONG, Thailand Khao Pho way-service, PRACHUAP KHIRI KHAN, Thailand Thep Muang Thong temple, UTHAI THANI, Thailand Klong Lan waterfall, KAMPHAENGPHET, Thailand Sa Pan Hin waterfall, TRAT, Thailand Kang Lam Duan waterfall, UBONRATCHATHA NI, Thailand

JX474573

JX474669

KF319137

JX474629

JX474718

KF319193

JX474628

JX474717

KF319192

JX474621

JX474702

KF319185

JX474620

JX474703

KF319184

JX474611-2

JX474693-4

JX474613

JX474695

KF3191756 KF319177

JX474594

JX474665

KF319158

JX474627

JX474714

KF319191

JX474626

JX474713

KF319190

JX474625

JX474716

KF319189

JX474624

JX474715

KF319188

JX474630

JX474719

KF319194

JX474631

JX474720

KF319195

JX474579

JX474705

KF319143

JX474582

JX474708

KF319146

JX474577

JX474657

KF319141

JX474622

JX474704

KF319186

1

63.

1

50.

1

5.

1

12.

1

18.

1

20.

1

29.

1

34.

C. herklotsi

1

69.

C. jourdyi

1

30.

C. labiosus

1

22.

C. malayanus

1

21.

1

58.

1

62.

1

8.

1

7.

1

49.

2

2.

1

12.

2

16.

C. haughtoni

C. perdix tuba

C. pfeifferi

C. saturnus

Tam Nam Thip temple, ROI- ET, Thailand Phu Phan cave, SAKON NAKHON, Thailand A.Na Kae road, NAKHON PHANOM to Dong Luang, MUKDAHAN, Thailand Doi Hau Mod mountain, TAK, Thailand Khao Bin cave, RATCHABURI, Thailand Khao Cha Mao, RAYONG, Thailand Khao Sib Ha Chan waterfall, CHANTHABURI, Thailand Klong Pla Kang waterfall, RAYONG, Thailand Yamatanishi, KAGOSHIMA, Japan Khe Sanh river, QUảNG TRị, Vietnam Khao Luk Chang, NAKHON RATCHASIMA, Thailand Khao Loy, RAYONG, Thailand Tad Kham waterfall, NAKHONPHANOM, Thailand Dao Wa Deung cave, KANCHANABURI, Thailand Chaloem Ratana Kosin, KANCHANABURI, Thailand Bukit Chintamanis, PAHANG, Malaysia Pulau Besar, JOHOR, Malaysia The 90th km., road No.105, TAK, Thailand Doi Hau Mod mountain, TAK, Thailand Jun cave, UTTARADIT,

JX474619

JX474701

KF319183

JX474618

JX474700

KF319182

JX474617

JX474699

KF319181

JX474580

JX474706

KF319144

JX474581

JX474707

KF319145

JX474615

JX474697

KF319179

JX474616

JX474698

KF319180

JX474614

JX474696

KF319178

JX474644

JX474734

KF319208

JX474645

JX474735

KF319209

JX474610

JX474692

KF319174

JX474568

JX474653

KF319132

JX474571

JX474659

KF319135

JX474569

JX474652

KF319133

JX474570

JX474654

KF319134

JX474648

JX474738

KF319212

JX474647

JX474737

KF319211

JX474591-2

JX474683-4

JX474593

JX474682

KF3191556 KF319157

JX474566-7

JX474674-5

KF3191301

2

15.

1

6.

1

65.

C. semisulcatus

1

7.

C. songmaensis

1

11.

C. speciosus

1

33.

1

41.

1

44.

1

55.

C. turgidus

1

42.

C. volvulus

1

66.

1

4.

1

1.

3

67.

1

19.

1

51.

2

3.

1

28.

1

13.

C. subfloridus

Thailand Huay Rong waterfall, PHRAE, Thailand Bor Ri Jin Da cave, CHIANG MAI, Thailand Tam Pha Lom temple, LOEI, Thailand Bukit Chintamanis, PAHANG, Malaysia Cuc Phoung, NINH BINH, Veitnam Klong Na Rai waterfall, CHANTHABURI, Thailand Ma Kok waterfall, CHANTHABURI, Thailand Pa Ma Muang temple, PHITSANULOK, Thailand Som But cave, PHETCHABUN, Thailand Naha, Nishinara, OKINAWA, Japan Tam Ra Kang temple, SUKHOTHAI, Thailand Ban Ta Sao community forest, SARABURI, Thailand 1 km. before Dao Khao Kaeo cave, SARABURI, Thailand Wang Kan Luang waterfall, LOPBURI, Thailand Khao Chakun mountain, SA KAEO, Thailand Sao Wa Lak camp, NAKHONRATCHASI MA, Thailand Aow Noi temple, PRACHUAPKHIRI KHAN, Thailand Khao Rong cave, PHETCHABURI, Thailand Erawan waterfall, KANCHANABURI, Thailand

JX474564-5

JX474672-3

JX474562

JX474676

KF3191289 KF319126

JX474563

JX474677

KF319127

JX474646

JX474736

KF319210

JX474578

JX474658

KF319142

JX474575

JX474655

KF319139

JX474576

JX474656

KF319140

JX474600

JX474666

KF319164

JX474601

JX474667

KF319165

JX474643

JX474733

KF319207

JX474602

JX474668

KF319166

JX474609

JX474691

KF319173

JX474606

JX474688

KF319170

JX474603-5

JX474685-7

KF3191679

JX474608

JX474689

KF319172

JX474607

JX474690

KF319171

JX474583-4

JX47470910

KF3191478

JX474585

JX474711

KF319149

JX474586

JX474712

KF319150

C. zebrinus

1

38.

Cyclotus sp.

1

11.

Leptopoma vitrium

1

41.

Rhiostoma hainesi

1

41.

Kra Teng Jeng waterfall, KANCHANABURI, Thailand Cuc Phoung, NINH BINH, Veitnam Ma Kok waterfall, CHANTHABURI, Thailand Ma Kok waterfall, CHANTHABURI, Thailand

JX474632

JX474721

KF319196

JX474649

JX474739

KF319213

JX474650

JX474741

KF319214

JX474651

JX474740

KF319215

Table 2. Average base frequencies across COI, 16S rRNA and 28S rRNA genes for Cyclophorus Genes A C+O

C

Average base frequencies C G C+O C C+O C

0.217 0.216 0.171 COI (All positions) 0.188 0.188 0.217 COI (1st and 2nd positions) 0.274 0.271 0.079 COI (3rd positions) 0.326 0.326 0.142 16S rRNA (All positions) 0.206 0.206 0.269 28S rRNA 16S rRNA (Selected 0.293 0.293 0.168 position) 0.245 0.245 0.170 All COI+16S rRNA st nd 0.238 0.238 0.194 1 and 2 COI+16S rRNA All COI+16S rRNA+28S 0.231 0.231 0.205 rRNA C = Cyclophorus, C+O = Cyclophorus with outgroup

T C+O

C

0.171 0.217 0.077 0.142 0.268

0.213 0.243 0.153 0.207 0.331

0.213 0.243 0.154 0.207 0.332

0.399 0.352 0.497 0.325 0.194

0.400 0.352 0.498 0.325 0.194

0.142

0.228

0.227

0.311

0.338

0.170 0.194

0.218 0.235

0.218 0.235

0.366 0.333

0.366 0.333

0.205

0.259

0.259

0.305

0.305

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

Length (bp)

Parsimony informative site C+O C C+O C C+O C 310 (46.97) 304 (46.06) 0.000-0.2640.000-0.220 292 287 90 (20.45) 84 (19.09) 0.000-0.1070.000-0.084 73 70 220 (100) 217 (98.64) 0.000-0.6230.000-0.523 219 217 Variable sites (%)

COI (All positions) 660 st nd COI (1 and 2 positions) 440 COI (3rd positions) 220 16S rRNA Selected 396 182 (45.96) 152 (38.38) position 28S rRNA 585 197(33.68) 97 (16.58) All COI+16S rRNA 1056 492 (46.59) 456 (43.18) 1st and 2nd COI+16S 836 272 (32.54) 236 (28.23) rRNA All COI+16S rRNA+28S 1641 689 (41.99) 553 (33.70) rRNA C = Cyclophorus, C+O = Cyclophorus with outgroup

Range of distances

0.000-0.230 0.00-0.179

147

131

0.000-0.2140.000-0.072 0.000-0.2140.000-0.200

97 438

74 418

0.000-0.1560.000-0.120

219

201

0.000-0.217 0.00-0.149

535

492

Figure

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)

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.