Cyclophorus and Cyclotus Taivanus Ssp

國立臺灣師範大學生命科學系博士論文

大山蝸牛屬及台灣山蝸牛屬之種化事件與山蝸牛科之系統發育學研究 Cyclophoridae phylogeny and the speciation events of Cyclophorus and Cyclotus taivanus ssp.

研究生：李彥錚 Yen-Chen Lee

指導教授：呂光洋 Kuang-Yang Lue 巫文隆 Wen-Lung Wu

中華民國九十七年六月 96.11. 版學位論文授權書

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論文題目：大山蝸牛屬及台灣山蝸牛屬之種化事件與山蝸牛科之系統發育學

研究

指導教授：呂光洋‧巫文隆

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中華民國 97 年 6 月 27 日

Content

Abstract………………………………………………………………………....………..III

Chapter 1 General introduction ……………………………………...…………..………..1

Chapter 2 Molecular phylogeny of the family Cyclophoridae (Gastropoda: Architaenioglossa) in East Asia…………………………………..……………3

Chapter 3 Ring speciation and morphological adaptations of genus Cyclophorus in Taiwan………………………………………………………………………...37

Chapter 4 The phylogenetic evolution and morphological adaptations in Cyclotus taivanus ssp.………………………………………...…………..…………….63

Chapter 5 The cyclophorids fauna of Taiwan……………..……..………………………91

Chapter 6 Summary and conclusion……………………………………………………151

References………………………………………………………………………………155

Acknowledgment…………………………………………………………...…………..163

II Abstract

Cyclophoridae consists of four subfamilies and about 300 species currently arranged in 38 genera, occuping varies habitats, with great morphological diversity allover the world. However, the relationship among Cyclophoridae is thus far not clear. In order to investigate this, I sampled cyclophorid snails around Taiwan and its adjacent areas, and then sequenced part of the mitochondrial COI (cytochrome oxidase subunit I) and the 16S rRNA gene from 32 species of 10 cyclophorid genera to establish a phylogenetic tree of Cyclophoridae. Phylogenetic relationships based on mtDNA sequences suggest that Cyclophorus, Cyclotus, Leptopoma, and Platyrhaphe are monophyletic while the traditional genus Japonia is polyphyletic, and the previous J. zebra should be placed into a new genus Pilosphaera. In addition, Pilosphaera yentoensis n. sp. and Japonia boonkioensis n. sp. will also be described as new species. Members of Cyathopoma are tiny white cyclophorid snails occurring in East Asia, Madagascar and the Seychelles. Phylogenetic relationships of Cyathopoma are uncertain. Combined with COI and radular data, I conclude that Cyathopoma and Cyclotus are only distantly related. Cyathopoma iota has been considered to be a controversial member of this group. Through molecular and radular data, I found C. iota to be closer to C. taiwanicum than to C. micron, and concluded that C. micron, C. ogaitoi, C. iota and C. taiwanicum all belong to Cyathopoma. There are 10 genera and 29 cyclophorid species in Taiwan. Among them, the most interesting taxa are Cyclophorus and Cyclotus, both sharing similar ecological niches and representing by a north and south form in morphology. In order to clarify their relationship, I have to find out their sister group as out groups to compare with the members among Cyclophorus and Cyclotus. The gene trees of Cyclophoridae indicate that Japonia and Pterocyclus are sister group of Cyclophorus and Cyclotus, respectively. The former two will be used as the out groups of Cyclophorus and Cyclotus in their phylogenetic studies. Both COI and 16S rRNA gene trees of Taiwan Cyclophorus show prominent geographic structure. The Mantel test showed significant positive correlation between fixation index (FST) and cumulative geographic anti-clockwise distance (origin in the region around Tainan, anti-clockwise pass through south cape, Taidung, Hualian, Iran, Taipei, Taichung and meet the original populations in Jia-yi). There are finite gene flew between adjacent populations. And there are series of clines around the Central Range. Cyclophorus of Taiwan is a proposed “ring species”. In the morphology and environmental variables correlation study, I found the currently shell morphology may

III be caused by the adaptations of recent long term climate. In traditional classification, Cyclotus taivanus consists of five subspecies, with clear morphological diversity. The molecular phylogenetic relationships of this group have never been discussed before. I sequenced part of the mitochondrial COI (cytochrome oxidase subunit I) and the 16S rRNA gene from 27 sampling sites. I also measured 9 shell traits to investigate the relationships between C. taivanus ssp. Even though the morphology PCA revealed a more or less continuous distribution of individuals in morph-space, the two highly divergent haplotype clades in COI and 16S rRNA analysis indicated the presence of two independently evolving lineages. The sequence divergence between two clades was almost as high as between other Cyclophoridae species. Therefore C. adamsi should be a considered valid species. For the environmental analysis, temperature may be a limiting element to the distribution of C. adamsi and C. taivanus group. The ecological divergence probably is the ruling force of speciation in my case. The PLS analysis results indicate, that phenotypic plasticity may be a key element of variable shell in C. taivanus group. The ecological divergence probably appears rule of speciation in C. taivanus ssp. case. The speciation process may be incomplete among C. t. dilatus, C. t. diminutus, C. t. peraffinis, and C. t. taivanus, and the adaptation of climatic pressure continue being a rule of speciation process. This study provides an opportunity to understand that no matter how similar two taxonomic groups are, occupying similar niche, undergoing the same geology history, with morphological adaptation to the same long term climate, they may have different speciation model.

IV Chapter 1

General introduction

Kobelt (1902) had been introduced cyclophorids in 1902, there were only 7 genera and 13 species in Taiwan. After Kobelt, Pilsbry and Hirase (1906) reported 7 genera and 21 cyclophorid species of Taiwan. There were 9 genera and 19 Taiwan cyclophorid species in Kuroda’s report of 1941 (Kuroda 1941). After these pioneers, Lee and Wu reported 9 genera and 29 species of Cyclophoridae in Taiwan (Lee & Wu, 2001). However, the phylogeny of cyclophorid is not clear. Besides, some new species and new records were found recently. Cyclophorids are very common snails in Taiwan, the genus Cyclophorus and Cyclotus are the most mysterious groups. The Cyclophorus moellendorffi found in southern Taiwan was considered a subspecies of C. formosaensis found in northern Taiwan by some authors (Kuroda, 1941; Chang, 1984; Lai, 1990; Higo & Goto, 1993). Besides, in the eastern side of Central Mountain Range, C. friesianus and C. latus were also believed to share a subspecies relationship by some authors (Kuroda, 1941; Chang, 1984; Lai, 1990; Higo & Goto, 1993; Hsieh et al., 2006). It is interesting that all the southern Cyclophorus species possess keeled shells, but the northern species possess round shells. Cyclotus taivanus ssp. are another interesting group. Pilsbry and Hirase (1905, 1906) reported C. t. peraffinis and C. t. adamsi as a subspecies of C. taivanus by their glossy surface and tall spire, respectively. Lee & Wu (2001) reported C. t. dilatus and Cyclotus t. diminutus as subspecies of C. taivanus by their wide out lip and very small shell, respectively. Like Cyclophorus, Cyclotus taivanus ssp. could divide north form (C. t. adamsi) and south from (C. t. dilatus, C. t. diminutus and C. t. peraffinis), roughly. The north form possess tall spire, the south form possess low spire. It is interesting that both Cyclophorus and Cyclotus taivanus ssp. occupy similar habitat, have two morphologic forms. Do Cyclophorus and Cyclotus taivanus ssp. undergo similar speciation process? The objectives of this study were (1) to establish the Molecular phylogeny of the family Cyclophoridae, (2) to understand the speciation model morphological adaptations of genus Cyclophorus, (3) to understand the speciation model morphological adaptations of genus Cyclotus, (4) to discuss the biogeography and morphology of Taiwan Cyclophoridae.

2 Chapter 2

Molecular phylogeny of the family Cyclophoridae

(Gastropoda: Architaenioglossa) in East Asia

Abstract Cyclophoridae consists of four subfamilies and about 300 species currently arranged in 38 genera, occupying varies habitats, with great morphological diversity. The molecular phylogenetic relationships of this group have never been discussed before. In order to investigate the relationships between cyclophorids, I sequenced part of the mitochondrial COI (cytochrome oxidase subunit I) and the 16S rRNA gene from 32 species of 10 genera of cyclophorid (including Cyclophoridae of China and Japan). I constructed phylogenetic trees using neighbor joining, Bayesian and maximum likelihood analyses on part of COI and 16S rRNA gene data set comprising. Phylogenetic relationships based on mtDNA sequences suggest that Cyclophorus, Cyclotus, Leptopoma, and Platyrhaphe are monophyletic while the traditional genus Japonia is polyphyletic and the previous J. zebra should be placed into a new genus Pilosphaera. In addition, Pilosphaera yentoensis n. sp. and Japonia boonkioensis n. sp. will also be described as new species. Besides, members of Cyathopoma are tiny white cyclophorid snails occurring in East Asia, Madagascar and the Seychelles. Phylogenetic relationships of Cyathopoma are uncertain. Combined with COI and radular data, I conclude that Cyathopoma and Cyclotus are only distantly related. Cyathopoma iota has been considered to be a controversial member of this group. Through molecular and radular data, I found C. iota to be closer to C. taiwanicum than to C. micron, and concluded that C. micron, C. ogaitoi, C. iota and C. taiwanicum belong to Cyathopoma. In addition we provide the first report of C. ogaitoi from Guei-Jou Province.

Introduction Recent studies on phylogenetic relationships within the molluscan class Gastropoda have involved morphological (Kay et al. 1998), ultrastructural (Healy 1996), anatomical (Kantor 1996) and molecular (e.g., McArthur & Koop 1999, Lydeard et al.

3 2002, Remigio & Hebert 2003) approaches. These investigations have provided new insights into gastropod affinities and classification and have enabled a rigorous testing of taxonomic schemes for the group. While gastropod phylogeny has received much recent attention (Tillier et al. 1992, Ponder & Lindberg 1997, Rosenberg et al. 1997, Thollesson 1999, Remigio & Hebert 2003), relationships within some land snail clades are still poorly understood. One of this is Cyclophoridae which consists of 4 subfamilies and about 300 species currently arranged in 34 genera. The most generally accepted system of classification today, partitioning the diverse Cyclophoridae into four subfamilies (Vaught 1989; Millard 1996). The Cyclophorinae are extremely diverse. By contrast, the other three, Spirotomatinae, Alycaeinae, and Pterocyclinae are less diverse. Although some investigators treat Spirotomatinae and Alycaeinae as independent families (Higo & Goto 1993, Azuma 1982), their subordinate taxa are uncontentious. However, the traditional classification is based on shell morphology, but part of the shell morphology may be subject to convergent evolution and thus may hamper a straightforward taxonomy (Lee et al. 2008). The phylogenetic knowledge of Cyclophoridae is limited and thus there is great interest in resolving their phylogenetic issues. Furthermore, the classification of tiny white shell cyclophorids, genus Cyathopoma W. & H. Blanford, 1861 and Nakadaella Ancey, 1904 have long been controversial. Cyathopoma micron (Pilsbry, 1900) was the first record of this tiny cyclophorid from East Asia. Cyathopoma iota (Pilsbry & Hirase 1904), C. taiwanicum Pilsbry & Hirase, 1906, C. taiwanicum degeneratum Pilsbry & Hirase, 1906, C. nishinoi Minato, 1980, C. ogaitoi (Minato, 1988) were subsequently reported but their generic placements were doubtful. For example, C. micron was initially placed in Cyclotus (Pilsbry 1900) before transferring to Nakadaella by Ancey (1904) and Cyathopoma (Pilsbry & Hirase, 1906) but the generic status of C. micron remained controversial. Afterward, whether this species should belong to Cyathopoma, Cyclotus, or Nakadaella was a huge controversy. It is interesting to understand the relationship within this group and with other cyclophorid genus. Dentition of the radula has long been established as providing informative characters for the taxonomy of gastropods (Trochel 1856-1863, Habe 1942, Ponder & Lindberg 1996). Radulae differ fundamentally among different gastropod groups. However, radular morphology must be used with care when inferring relationships because there is marked convergence in the radulae of taxa that belong to different clades but occupy similar adaptive zones (Lindberg & McLean 1981). Cuspidal features, number of teeth as well as teeth shape play an important role in classification and have proved to be of particular value at generic level (Kilburn 1988). Traditional classification of these tiny cyclophorids was based on shell and operculum morphology, while little attention has been paid to the radular morphology of cyclophorids, and the

4 Cyathopoma radula has not been previously described. Over the past decade molecular approaches have proven their value not only in clearing up relationships among taxa, but also in providing a sense of the time scales of evolutionary divergence. Among the 13 protein-coding genes within the mitochondrial genome, COI has gained particular popularity for resolving affinities among species (Hebert et al. 2004a, b; Hogg & Hebert 2004; Barrett & Hebert 2005; Smith et al. 2006). In the case of possible cryptic species identification and identifying morphologically difficult species (Paquin & Hedin 2004), COI provided a high degree of taxonomic resolution. Besides, 16SrRNA the more rapidly evolving mitochondrial gene has generally been employed to infer relationships among groups with a more recent ancestry i.e. at the species and population level (e.g. Chiba 1999; Pinceel et al. 2004). However, 16SrRNA gene can also provide insights concerning deeper divergences (e.g. Thollesson 1999; Remigio & Hebert 2003). COI and 16S data could be suitable to infer the affinities at the genus level of Cyclophoridae. The objectives of this study were (1) to test whether the genus of Cyclophoridae is monophyletic, (2) if part one is negative, to rearranged its classification or created a new genus, (3) to describe new species and provide its distributional and ecological data, (4) to resolve the relationship of the Cyathopoma species, (5) to understand the affinity between Cyathopoma species and other cyclophorid genus, (6) to provide Cyathopoma radular microstructure data.

Materials and methods DNA preparing and sequencing All samples of 32 species representing 9 genera and including members from 3 major subfamilies (Cyclophorinae, Alycaeinae and Pterocyclinae) were collected from 116 sites (Fig. 2.1) (Table 2.1), and separated their shells and soft parts at library. Shells were well cleaned for identification, soft parts were stored at -80℃, except for very tiny species (e.g. C. micron) which were placed in pure ethanol until DNA extraction. DNA was extracted from columellar muscle; the whole animal was used in the case of tiny species. I extracted DNA from separate individuals using TEK-based protocol (Jiang et al. 1997) with minor modifications. Tissue was placed in TEK buffer (12.5mM Tris-Cl pH 7.3, 2.5mM EDTA, 0.4% KCl), then ground with glass pestle and incubated at 57℃ with 20µl of proteinase K (20mg/ml) more than 2 hrs. The tissue extract was extracted at least twice with phenol and chloroform. 400µl DNA extract was precipitated by adding 1000µl pure ice-cold ethanol, and was then placed in -20℃ for 20 min. DNA was pelleted by centrifugation for 30 min. After 70% ethanol rinse, DNA was resuspended in distilled water and stored at -80℃ for DNA

5 amplification. An approximately 800bp mitochondrial 16S rRNA gene was amplified by PCR using primers 16SRT (5’–ACA TAT CGC CCG TCA CTC TC–3') and 16SL900 (5’–AAA TGA TTA TGC TAC CTT TGC–3'), exactly 531bp COI using primer LCO1490 (5'–GGT CAA CAA ATC ATA AAG ATA TTG G–3') and HCO2198 (5'–TAA ACT TCA GGG TGA CCA AAA AAT CA–3') (Williams et al. 2003). For genus Chamalycaeus, I design primer 16SRPL (5’–TTT TGC ATC ATG GTT TAG CAA G–3') and 16SLS (5’–ATG CTA CCT TAG CAC AGT CA–3') to amplify approximately 530bp 16S rRNA gene. PCR reactions contained template DNA 10–50ng/µl, 10pmol of each primer, 5µl 10× reaction buffer (10mM Tris-HCl, pH9.0,

50Mm KCl, 1.5Mm MgCl2, 0.1% gelatin, 1% Trinton X-100), 0.4µl 25mM/µl dNTP, 0.2µl 50mM Mg2+ and 0.4µl Taq polymerase (5unit/µl) in a total volume of 50µl. Thermal cycling for 16S rRNA was performed with an initial denaturation for 5min at 95℃, followed by 30 cycles of 30 sec. at 95℃, 45 sec. at 57℃, 50 sec. at 72℃ and ultimate extension at 72℃ for 10min, final hold in 4℃. Thermal cycling for COI gene was performed similarly, changing only its annealing temperature to 47℃. PCR products were purified using a purification kit (AMP PCR purification, Beckman) and then sequenced using an ABi 3700 autosequencer.

Phylogenetic analyses The COI sequences were combined with data of the out-groups (Littoraria scabra Linnaeus and Littoraria undulata (Gray) accession No. are AJ488637 & AJ488635 respectively) from GenBank. Sequences were assembled and edited using Bioedit 5.0.9 (Hall 1999). All alignments employed Clustal X (Thompson et al. 1997) and were manually proofread. Codon positions within COI were tested using the incongruence length difference (ILD) test (Farris et al. 1995), as implemented by the partition homogeneity test in PAUP 4.0b10 (Swofford 1998) (100 replicates). COI sequence data were divided into two partitions, first and second codon positions in one and third codon positions in the other. Two parts of COI gene were congruent and all codon positions were combined and used in the following analysis. In 16S rRNA, Truncatella guerinii A. & J.B. Villa was used as out-group. Regions where the alignment was ambiguous were excluded from the analyses. All data sets were subject to Neighbor-joining (NJ) using PAUP 4.0b10, to the Maximum likelihood (ML) analyses using PHYML 3.0 (Guindon & Gascuel 2003), to the Bayesian analysis using MrBayes 3.1.2 (Ronquist & Huelsenbeck 2003). The substitution model used for the COI data set corresponded to the General time reversible model, and included invariable sites, and rate variation among sites (GTR+I+G). The substitution model used for the 16S rRNA data set corresponded to the transitional model, include invariable sites, rate variation among sites (TIM+I+G).

6 These were the best models found using Modeltest 3.06 (Posada & Crandall 1998). Before model fitting, the full-length sequences were tested to confirm that there was no significant heterogeneity in base frequencies across taxa (in COI: X2=237.21, df=249, P=0.6939; in 16S rRNA: X2=97.31, df=165, P=1). NJ bootstraps consisted of 1000 iterations. Reliability of ML trees were estimated by the approximate likelihood ratio test (aLRT) (using custom define model, base frequencies: A = 0.3184, C = 0.1131, G = 0.1239, T = 0.4327 in COI and A =0.3664, C = 0.0720 G = 0.1351, T = 0.4266 in 16S) using PHYML 3.0 (Guindon & Gascuel 2003). In the Bayesian analysis was run for 2,000,000 generations, with a sample frequency of 100. The first 2000 trees were discarded, so that the final consensus was based on 18,000 trees. Support for nodes was expressed as posterior probabilities (calculated by MrBayes). For constraint analyses, I conducted parsimony heuristic searches to find the best trees and using the Kishino-Hasegawa test to evaluate the resulting trees and trees which are consistent with traditional taxonomy.

Environmental Scanning electron microscope (ESEM) microstructure observation The empty shell was glued to the SEM specimen stub for further observation. Radulae were removed from snails and soaked in 0.5% NaOH solution to remove organic tissue adhering to the radula, then fixed using 90% ethanol. The radula was glued to the SEM specimen stub and one side of the marginal teeth was unfolded for further observation. A final step prior to examination with the SEM, the specimens were coated, in vacuum, with gold-palladium. The specimens were then observed and photographed using Environmental Scanning Electron Microscope (FEI Quanta 200).

Results Phylogenetic analysis The aligned 531 bp COI gene data matrix, including 270 variable sites of which 250 (92.59%) were parsimony informative. No length difference from the out-group was detected among members of three subfamilies, the Cyclophorinae, Alycaeinae, and Pterocyclinae. The average p-distance of Cyclophoridae haplotype was =0.197. Sequence divergence among the haplotypes ranged from 0.002 to 0.277, intra generic ranges were from 0.066 to 0.156, and inter generic ranges from 0.193 to 0.268. The alignment of the 16S rRNA gene fragment data yielded 592 characters of which 444 could be unambiguously aligned. Of these 444 positions, 300 (67.6%) were parsimony informative. The average p-distance among haplotypes was 0.239. Sequence divergence among the haplotypes ranged from 0.002 to 0.352. The COI and 16S rRNA sequence divergence among species of the same genus ranged from 0.028

7 to 0.172 and 0.041 to 0.166, respectively (Table 2.2). The inferred phylogenetic trees between the haplotypes of COI gene are shown in Figs. 2.2, 2.3 & 2.4, of 16S rRNA gene are shown in Figs. 2.5, 2.6 & 2.7. Three monophyletic groups, Cyclophorus, Cyclotus, and Leptopoma, are present, with support values higher than 97% using NJ, Bayesian and ML methods. Although with lower support, members of genus Platyrhaphe also had a monophyletic relationship. The relationship between subfamily Cyclophorinae, Alycaeinae and Pterocyclinae were uncertain because of the low bootstrap support.

Cyathopoma Phylogeny It was controversial whether micron, ogaitoi, and iota belong to Cyathopoma, Cyclotus, or Nakadaella in the previous literatures. However, all cladograms in this study indicated these species were all in the same clade as C. taiwanicum instead of genus Cyclotus. Particularly, C. iota was clustered with C. taiwanicum in a subclade, and without resolution. In order to test the position of these species in classification, I performed two likelihood analyses between topologies constrained to match given mutually exclusive hypotheses. The constraints were designed to test these cyclophorid snails as the different contentions described in the previous literatures. I performed the Kishino-Hasegawa likelihood evaluation to test these contentions. Using the constraints option in PAUP, I conducted parsimony heuristic searches (specifics same as below) to find the best trees that were consistent and inconsistent with the monophyly of these clades. The sets of trees consistent and inconsistent with the constraint were then compared using the Kishino-Hasegawa test (Kishino & Hasegawa 1989). For (micron, ogaitoi, iota, and genus Cyclotus) clade issue, the COI data significantly reject the monophyly of this clade, six “best” trees supporting monophyly were significantly worse than the 48 genuinely best trees (P ≤ 0.0008). For (micron, ogaitoi, and genus Cyclotus) clade issue, all 112 “best” trees were significantly worse (P ≤ 0.0025) than the 48 trees which did not match the constraint. Some investigators considered iota is a subspecies of micron (Higo & Goto 1993, Lee & Wu 2001). I also performed the Kishino-Hasegawa likelihood evaluation to test this issue. For this issue, 48 “best” trees were significantly worse (P ≤ 0.0044) than the 48 trees which did not match the constraint. Furthermore, if (micron, ogaitoi) and (iota, taiwanicum) were separated groups, the divergence among these two groups were 0.151 which was moderately lower than among other genus (Table 2.3). I conclude that micron, ogaitoi, and iota had closer relationship with C. taiwanicum than with members of genus Cyclotus, while micron, ogaitoi, iota and taiwanicum belong to genus Cyathopoma.

8 Japonia & Pilosphaera Phylogeny Interestingly, the genus Japonia appeared polyphyletic for both the COI and 16S rRNA data. The zebra group, traditionally placed within the genus Japonia, differs conchologically from all other members of the genus Japonia in having red-brown longitudinal stripes on the shell and a reflected outer lip. In order to compare our results with traditional classifications, we performed two likelihood analyses between topologies constrained to match given mutually exclusive hypotheses. The constraints were designed to test the genus Japonia as described in the literature (Pilsbry & Hirase 1906; Kuroda 1941; Higo & Goto 1993). We performed the Kishino-Hasegawa likelihood evaluation to test the monophyly of the traditional classification of Japonia clades (Japonia formosana Pilsbry & Hirase, 1906, Japonia boonkioensis n. sp., Japonia lanyuensis Lee & Wu, 2001, Pilosphaera zebra (Pilsbry & Hirase, 1906), Pilosphaera yentoensis n. sp.). Using the constraints option in PAUP, we conducted parsimony heuristic searches (specifics same as above) to find the best trees that were consistent and inconsistent with the monophyly of these clades. The sets of trees consistent and inconsistent with the constraint tree were then compared using Kishino-Hasegawa test (Kishino and Hasegawa 1989). COI data significantly rejected the monophyly of Japonia. All 16 “best” trees supporting monophyly were considerably worse than the ten genuinely best trees (P < 0.0001). For 16S rRNA data, one “best” tree supporting monophyly of Japonia was significantly worse (P < 0.0001) than the two genuinely best trees. Accordingly we consider the monophyly of Japonia to be suspected.

Cyathopoma ESEM microstructure Radular morphology was very similar between C. iota and C. taiwanicum. Both possessed the same number of teeth cuspids and general shape. They had a scoop-shaped central tooth, 5 cuspids on the convex side, 7–9 irregular tiny cuspids on the convex side; inner lateral teeth possessed 5 cuspids and outer lateral teeth 6–7 cuspids within the same individual; marginal teeth with 6 small cuspids on the inner side and 3 large cuspids on the outer side (Fig. 2.8D, 2.9D). The radulae of C. micron and C. ogaitoi were similar to the above, but the cuspids on the teeth were shorter and broader (Fig. 2.10D, 2.11D). Further, the outer lateral teeth had fewer cuspids (5–6 in number). The protococh of C. iota and C. taiwanicum exhibited a granular surface (Fig. 2.8B, 2.9B), but C. micron and C. ogaitoi were relatively smooth (Fig. 2.9B, 2.11B). The opercula of C. iota, C. micron, C. ogaitoi and C. taiwanicum were almost the same shape (Fig. 2.8C, 2.9C, 2.10C, 2.11C). In addition, we examined four subspecies of Cyclotus taivanus H. Adams, 1870. The radulae within the Cyclotus we examined were similar to each other. Namely 5

9 cuspids on the central tooth, 4 cuspids on inner lateral teeth, 4 cuspids on outer lateral teeth and 3 cuspids on marginal teeth (Fig. 2.12). The only distinct difference was the shape of marginal teeth. The marginal teeth of Cyclotus taivanus adams Pilsbry et Hirase, 1905 were sickle-shaped, while the other three species exhibited hook shaped marginal teeth.

Discussion Although the molecular relationship between the three major subfamilies of cyclophorids are uncertain, our results show that many taxa in traditional classifications of cyclophorids are nonmonophyletic. Because Cyclophorinae, Spirotomatinae (not included in this study), Alycaeinae and Pterocyclinae in a Linnean system are considered to be equal entities, problems associated with ranks and synonymy arise. I conclude that COI gene is useful in unraveling cyclophorid phylogeny but need to be combined with other data (such as morphological and anatomical data) to fully clarify the evolutionary relationships. Cyathopoma were known as an Indian endemic before 1900 (Pilsbry 1900). After Pilsbry’s report, C. iota, C. nishinoi, C. ogaitoi, and C. taiwanicum were found occurring in Japan and Taiwan. Prior to our discovery of several C. ogaitoi specimens in Lei Gong Shan, Guei-Jou Province in July 2006 there were no reports indicating that Cyathopoma occurs in the area between East Asia and India. This is the first report of Cyathopoma from this area. C. micron was initially placed in the genus Cyclotus (Pilsbry 1900), then placed in the genus Nakadaella by Ancey in 1904. Pilsbry reversed his conclusion and treated micron as genus Cyathopoma with coauthor in 1906 (Pilsbry & Hirase 1906). Subsequently, Kuroda (1941) and Chang (1984) recognized this species as belonging to the genus Cyathopoma, while Lai (1990) and Lee and Wu (2001) placed it in the genus Cyclotus. C. iota was considered a closer relative to C. micron than to C. taiwanicum (Pilsbry & Hirase 1904, Higo & Goto 1993, Lee & Chen 2003). However, based on our Kishino-Hasegawa test result, molecular phylogenetic and radular data, C. iota is closer to C. taiwanicum than to C. micron. Ancey (1904) proposed a genus Nakadaella for micron, but this species does not differ from typical forms of Jerdonia W. & H. Blanford, 1861 except for the absence of spiral striation (Pilsbry & Hirase 1906), and genus Nakadaella was retained as a subgenus for the smooth species (Pilsbry & Hirase 1906). However, our results indicate that the smooth shell may be a plesiomorphic character of these species, and Nakadaella was a synonym for Jerdonia. In all cladograms C. iota and C. taiwanicum were clustered without resolution. All C.

10 iota were reported from mountain regions higher than 1000m altitude (Lee & Chen 2003), except the types, and we suspect C. iota may be an ecotype of C. taiwanicum. Comparison of the radulae between these species and Cyclotus, the serrate cuspids on the convex side of central teeth in Cyathopoma are not present in Cyclotus. In addition, the fine serrate cuspids on the inner side of marginal teeth are not present in Cyclotus. Thus the radulae of Cyathopoma are clearly distinctly from those of Cyclotus. Based on molecular and radula data, I conclude that Cyathopoma and Cyclotus are distant relatives. However, the Indian species were not included in this study. Obviously, further studies are needed to include the Indian species and to understand the phylogenetic relationship of these interesting snails. The genus Japonia was established by Gould in 1859. He indicated that the group characterized by the thin paucispiral opercle with thinned edgeds, the globose conic form, free umbilicus, nearly circular peristome which barely touches the preceding whorl, and the projecting lamellar striae of growth decussating with revolving ridges in some cares furnished with epidermal barbs (Gould 1859). Japonia zebra Pilsbry & Hirase, 1906 was named by Pilsbry and Hirase and placed in genus Japonia. However, the beautiful red brown longitudinal stripes and reflexed outerlip exhibited by the zebra group are not present in the members of genus Japonia. Furthermore, in the molecular characters, the haplotype distance between zebra group and the other members of Japonia is almost twice distance within groups (0.207–0.226 between group; 0.026–0.164. within zebra group, 0.002–0.122 within Japonia group) (Table 2.4). Compared to other genera of Cyclophoridae, including the zebra group in the genus Japonia presents the highest p-distance value (Table 2.5). Base on molecular evidence and shell morphology, zebra and its analogous from riverside of Nan-Xi river in Zhejiang province are polyphyletic relationship with Japonia. I would like to establish a new genus for this group here and will describe it below.

Pilosphaera new genus Type species: Japonia zebra Pilsbry & Hirase, 1906 Diagnosis: Shell small, turbinate, conical-globe shape, with convex shell whorls. Shell is always festucine color with reddish brown longitudinal stripes. Surface is sculptured with some spiral cords and irregular growth lines, above these furnished with several row regular periostracum hairs. Umbilicus opened. The aperture is circular, outer lip reflexed. The operculum is ceratoid, translucent, a little concave, multispiral type with very thin pellucid edge. Etymology: Pilo (grow hairy) + sphaera (ball).

11 Pilosphaera yentoensis n. sp. (Fig. 2.13A–F) Description: Shell small, 5.17–5.27mm in length and 5.27–5.49mm in width. Shell turbinate and conical-globe shape, with moderately convex whorls 5–5.25 in number. Shell is festucine color with reddish brown longitudinal stripes. Surface sculptured with several indistinct spiral cords, covered with festucine color dull periostracum and regular periostracum lamella, interval with irregular fine growth lines. There are 3 row regular periostracum hairs between the sutures on penultimate whorl. There is no periostracum hair and with polish periostracum under the peripheral line. The periostracum hairs are sometimes entirely lost, perhaps in old shells such as holotype. Umbilicus opened. The aperture is circular. The outer lip is reflexed. The operculum is translucent ceratoid, a little concave center, multispiral type with very thin pellucid edge. There is an orange red proboscis between two purplish gray tentacles on the head. Dark gray food covers with two pieces of dark gray lobe, which has pale color edge. The lobes join at the tail and come into being a pale color groove. (Fig. 2.15A–B) Notes: the present species differs from its only know congener Pilosphaera zebra in dark gray soft body color (Fig. 2.15A–C) and fewer periostracum hairs (Fig. 2.13A–I, 2.16A–B). The later has 5–6 rows regular periostracum hairs between the sutures on penultimate whorl which is 3 on the new species. The later shell base with 6–7 rows periostracum hairs, but not present on the new species (Fig. 2.13). The later has 4–7 rows tiny periostracum hairs on the position just under the suture (Fig. 2.16B), which are not present on the new species. In COI gene data, the average distance among this new species and Pilosphaera zebra was = 0.160, which was close to the average distances among cyclophorids species (among species was = 0.198, with in species was = 0.061). Measurement and type depository Holotype: SL: 5.17mm, SW: 5.27mm; APL: 2.82mm, APW: 2.82mm; NMNS5635-001, National Museum of Natural Science, Taiwan. Paratype: SL: 5.27mm, SW: 5.49mm; APL: 3.10mm, APW: 2.93mm; NMNS5635-002, National Museum of Natural Science, Taiwan.

Etymology: The name is after the Yen-To town, primary habitat of this species.

Type locality: Yen-To town near Nan-Xi river in Zhejiang province, China. Gathered from grass slope under leaves.

Japonia boonkioensis n. sp. (Fig. 2.14A–C) Description: Shell small, 4.47mm in length and 4.7mm in width. Shell turbinate and

12 conical-globe shape, with five moderately convex whorls. Shell is red brown color, somewhat pale at peripheral. Surface sculpture of 6–10 indistinct spiral veins crossed by finer growth lines, rendering them somewhat crispate and the interstices minutely plicaulate. Shell surface covers with red brown slight shining periostracum and irregular periostracum lamella. There are 2 row regular periostracum hairs between the sutures, one furnished on the position of shoulder, one on the peripheral site. There are 7 rows periostracum hairs under the peripheral, they longer its length from umbilicus side to the peripheral side. The shoulder and peripheral periostracum hairs are 3–4 times longer than basal periostracum hairs. The periostracum hair is tapering tip. Umbilicus opened. The aperture is circular. The outer lip is not reflexed. The operculum is translucent ceratoid, a little concave center, multispiral type with very thin pellucid edge. There is an orange red proboscis between two bluish gray tentacles on the head, gray food cover with two pieces of dark gray lobe. (Fig. 2.15D) Notes: This new species is similar to its sympatric species Japonia formosana (Fig. 2.14G–I). But the later is festucine color, and having spoon like periostracum hairs (Fig. 2.16D), instead tapering tip (Fig. 2.16C). Japonia lanyuensis (Fig. 2.14D–F) is another analog, but with rougher periostracum and longer basal periostracum hairs.

Measurement and type depository Holotype: SL: 4.47mm, SW: 4.70mm; APL: 2.21mm, APW: 2.33mm; NMNS5636-001, National Museum of Natural Science, Taiwan.

Etymology: The name is after the boonkio (old toponym of Fen-chi-hu), primary habitat of this species.

Type locality: Fen-chi-hu in Jia-i County, central Taiwan, 1400 meters in altitude, gathered from grass slope under leaves.

13 Table 2.1 List of species included in the molecular analysis, sampling sites, and GenBank accession numbers Subfamily GenBank accession Genus and Sampling sites/references number. Species COI 16S Alycaeinae Chamalycaeus C. varius 1. Ming-jyu Shan, Nei-hu, Taipei City EU219770 C. varius 2. Da-chi-jiau, Shin-dian City EU219771 C. varius 3. Yn-her-donq, Taipei County EU219792 C. varius 4. Nei-gou, Nei-hu, Taipei City EU219791 Dioryx D. swinhoei 5. Wu-Lai, Taipei County EU219759 D. swinhoei 6. Jang-hu, Jia-yi County EU219819 D. swinhoei 7. Yue-mei waterful, San-diau-ling, Taipei County EU219760 D. swinhoei 8. Ren-tzer, Iran County EU219820 D. swinhoei 9. Li-jia mountain road, Taidung County EU219758 EU219821 D. swinhoei 10. Gu-lu mountain road, Iran County EU249291 Cyclophorinae Cyathopoma C. iota 11. Lake of Mandarin Ducks, Iran County EU219766 C. iota 12 Fwu-shan, Taipei County EU249284 C. iota 13. Bai-liing, Iran County EU219764 C. iota 14. Bai-liing, Iran County EU249288 C. iota 15. Bai-liing, Iran County EU249289 C. micron 16. li-shyng mountain road, Nan-tou County EU219768 C. micron 17. li-shyng mountain road, Nan-tou County EU249275 C. micron 18. li-shyng mountain road, Nan-tou County EU249276 C. micron 19. Fwu-shan, Taipei County EU249283 C. micron 20. Fwu-shan, Taipei County EU249285 C. micron 21. Gu-lu mountain road, Iran County EU219769 C. ogaitoi 22. Lei Gong Shan, Guei-Jou Province EU249292 C. taiwanicum 23. San-diau-ling, Taipei County EU219793 C. taiwanicum 24. San-diau-ling, Taipei County EU249282 C. taiwanicum 25. San-diau-ling, Taipei County EU219767 C. taiwanicum 26. Chu-shuei-shi mountain road, Iran County EU219765

14 C. taiwanicum 27. Da-chi-jiau, Shin-dian City EU249295 Cyclophorus C. formosaensis 28. Charn-guang Temple, Taroko valley EU219801 C. formosaensis 29. Charn-guang Temple, Taroko valley EU249274 C. formosaensis 30. Sheau-jiau-shi, Iran County EU249278 C. formosaensis 31. Nan-an, Hualian County EU219743 C. formosaensis 32. Nan-an, Hualian County EU219739 C. formosaensis 33. Hong-ye, Hualian County EU219740 EU219799 C. formosaensis 34. Torng-men, Hualian County EU219802 C. formosaensis 35. Chorng-der, Hualian County EU219744 C. formosaensis 36. Hong-ye, Hualian County EU219741 EU219800 C. friesianus 37. Shan-ping, Kaohsiung County EU219745 C. friesianus 38. Provincial Highway No.20, 49k, Nan-huah, EU219808 Tainan City C. friesianus 39. Provincial Highway No.20, 49k, Nan-huah, EU219747 Tainan City C. friesianus 40. San-dih-men, Ping-dung County EU219746 C. friesianus 41. San-dih-men, Ping-dung County EU219809 C. latus 42. County highway No.100, Dung-biann village, EU219737 Taichung County C. latus 43. Dar-Guan Mountain, Tauyuan County EU219742 EU219795 C. latus 44. Gu-guan, Taichung County EU219738 C. latus 45. Li-leeng mountain road, Her-ping township, EU249281 EU219796 Taichung County C. latus 46. Mei-feng, Nan-tou County EU219797 C. latus 47. Sheau-jiau-shi, Iran County EU249287 C. latus 48. Dong-shan, Iran County EU219794 C. martensianus 49. Jing Shan Zoo, Wen Zhou EU219756 EU219813 C. moellendorffi 50. Mu-dan township, Ping-dung County EU219806 C. moellendorffi 51. Shuang-liou Wood Park, Ping-dung County EU219752 C. moellendorffi 52. East Coastal Mountain, Taidung County EU219749 EU219804 C. moellendorffi 53. Taidung County EU249280 C. moellendorffi 54. Chair-shan, Kaohsiung City EU249286 EU219807 C. moellendorffi 55. Chair-shan, Kaohsiung City EU219750 C. moellendorffi 56. Chair-shan, Kaohsiung City EU219748 C. moellendorffi 57. Jy-been, Taidung County EU219803 C. moellendorffi 58. Da-wu, Taidung County EU219751

15 C. moellendorffi 59. Da-wu, Taidung County EU219805 C. pyrostoma 60. Jian Feng Ling, Hainan EU219755 EU219814 C. subcarinatus 61. Hong Kong EU219757 EU219812 C. t. angulatus 62. Okinawa EU219754 C. t. angulatus 63. Okinawa EU219811 C. t. radians 64. Iriomote Island EU219810 C. t. radians 65. Iriomote Island EU219753 C. cf. turgidus 66. Provincial Highway No.149, Yun-lin County EU219798 C. hirasei Lydeard et al. 2002 AY010505 Cyclotus C. adamsi 67. Guan-in Mountain, Taipei County EU219838 C. adamsi 68. Fwu-shan, Taipei County EU219786 C. adamsi 69. The Cao-ling Historic Trail , Iran County EU219837 C. adamsi 70. Ren-tzer, Iran County EU249290 C. taivanus dilatus 71. Charn-guang Temple, Taroko valley EU249273 C. t. dilatus 72. Chorng-der, Hualian County EU219840 C. t. dilatus 73. Chorng-der, Hualian County EU249279 C. t. dilatus 74. Chin-heng Bridge, Taroko valley, Hualian County EU219841 C. t. diminutus 75. Yongsing, Lanyu Island, Taidung County EU219843 C. t. diminutus 76. Yongsing, Lanyu Island, Taidung County EU219790 C. t. peraffinis 77. Iriomote Island EU219789 EU219844 C. t. taivanus 78. Jang-hu, Jia-yi County EU249269 EU219839 C. t. taivanus 79. Shuang-liou Wood Park, Ping-dung County EU249270 C. t. taivanus 80. County highway No. 136, 39.5K, Taichung County EU249271 C. t. taivanus 81. Liou-guei, Kaohsiung County EU249272 EU219842 C. t. taivanus 82. Shan-ping, Kaohsiung County EU219788 C. t. taivanus 83. County Highway No.129, 20.5k, Jia-yi County EU249293 C. t. taivanus 84. Provincial Highway No.20, 79k, Bau-Lai, EU249294 Kaohsiung County C. t. taivanus 85. Provincial Highway No.20, 79k, Bau-Lai, EU219787 Kaohsiung County C. t. taivanus 86. South Cross-Island Highway, Ping-dung County EU249296 Japonia J. boonkioensis 87. Fen-chi-hu in Jia-i County EU219762 EU219815 J. formosana 88. County Highway No. 126, 26k, Miau-li County EU219763 J. lanyuensis 89. Yongsing, Lanyu Island, Taidung County EU219761 Leptopoma

16 L. nitidum 90. Sha-ka-dang pavement, Taroko valley EU219783 L. nitidum 91. Okinawa, Nakagusuku-jo EU249277 L. nitidum 92. Ishigaki Island EU219823 L. nitidum 93. Iran County EU219822 L. tigris 94. Lanyu Island, Tai-dong County EU219784 EU219825 L. tigris 95. Liu-dau, Tai-dong County EU219824 Pilosphaera P. yentoensis 96. Nan-Xi river in Zhejiang province, China EU219772 EU219818 P. zebra 97. Nei-gou, Nei-hu, Taipei City EU219773 EU219816 P. zebra 98. Wu-yu sentry post, Wu-Lai, Taipei County EU219817 Pterocyclinae Platyrhaphe P. lanyuensis 99. Lanyu Island, Tai-dong County EU219781 EU219834 P. lanyuensis 100. Round-the-island highway, 10-14K, Liu-dau, EU219782 Tai-dong County P. m inu tus 101. Bei-Nan, Taidung County EU219830 P. m inu tus 102. Chair-shan, Kaohsiung City EU219833 P. m inu tus 103. Liouciou Township EU219779 EU219832 P. m inu tus 104. Fashing Buddhist temple, Kaohsiung City EU219780 P. sunggangensis 105. Cycas taitungensis nature reserve, Taidung County EU219778 P. s. depressus 106. Dah-dong-shan, Jai-yi County EU219827 P. s win ho e i 107. County highway No.100, Dung-biann village, EU219828 Taichung County P. s win ho e i 108. Jang-hu, Jia-yi County EU219774 EU219826 P. s win ho e i 109. Shin-liau waterfall, Iran County (2) EU219777 EU219831 P. s win ho e i 110. Provincial Highway No.20, 49k, Nan-huah, EU219829 Tainan City P. s win ho e i 111. County highway No.175, 3k, Tainan County EU219775 P. s win ho e i 112. County highway No. 106B, Shih Ding, Taipei County EU219776 Pterocyclos P. wilsoni 113. Frong Stone, Sin-Xu County EU219785 P. wilsoni 114. Frong Stone, Sin-Xu County EU219835 P. wilsoni 115. Wu-yu sentry post, Wu-Lai, Taipei County EU219836 Out group Littoraria scabra Williams et al. 2003 AJ488637 L. undulata Williams et al. 2003 AJ488635 Truncatella guerinii 116. Kaan-ding, Ping-dung County EU233814

17 Table 2.2 The range of p-distance among Cyclophoridae species of the same genus Genus COI 16S rRNA Alycaeinae Chamalycaeus ＃＃ Dioryx ＃＃ Cyclophorinae Cyathopoma 0.028–0.152 ＃ Cyclophorus 0.083–0.162 0.041–0.158 Cyclotus 0.066 0.081 Japonia 0.107–0.121 ＃ Leptopoma 0.148 0.153 Pilosphaera 0.156 0.104 Ptychopoma ＃＃ Pterocyclinae Platyrhaphe 0.103–0.172 0.085–0.166 ＃indicates only one species contained in analysis

Table 2.3 Average pairwise differences between genera of Cyclophoridae of East Asia using COI data set. 1 2 3 4 5 6 7 8 9 10 11 1. Chamalycaeus 2. Dioryx 0.241

3. (micron, ogaitoi) 0.2270.243

4. (iota, taiwanicum) 0.212 0.225 0.151 5. Cyclophorus 0.221 0.218 0.216 0.212 6. Cyclotus 0.220 0.268 0.222 0.221 0.221 7. Japonia 0.224 0.248 0.203 0.204 0.196 0.220 8. Leptopoma 0.236 0.255 0.227 0.218 0.230 0.211 0.213 9. Pilosphaera 0.246 0.266 0.247 0.235 0.234 0.248 0.219 0.231 10. Platyrhaphe 0.207 0.229 0.207 0.190 0.200 0.197 0.193 0.207 0.217 11. Ptychopoma 0.213 0.241 0.235 0.206 0.229 0.206 0.227 0.211 0.238 0.193

18 Table 2.4 Pairwise differences between Japonia and Pilosphaera using COI data set. Species 1 2 3 4 5 6 7 8 9 1. Japonia boonkioensis 0.107 0.113 0.111 0.113 0.111 0.218 0.215 0.218 2. Japonia formosana 57 0.122 0.121 0.119 0.121 0.218 0.220 0.226 3. Japonia lanyuensis 60 65 0.002 0.004 0.002 0.213 0.207 0.224 4. Japonia lanyuensis 59 64 1 0.002 0.004 0.213 0.207 0.222 5. Japonia lanyuensis 60 63 2 1 0.006 0.213 0.207 0.220 6. Japonia lanyuensis 59 64 1 2 3 0.211 0.205 0.222 7. Pilosphaera yentoensis n. sp. 116 116 113 113 113 112 0.026 0.156 8. Pilosphaera yentoensis n. sp. 114 117 110 110 110 109 14 0.164 9. Pilosphaera zebra 116 120 119 118 117 118 83 87 Below diagonal: total character differences. Above diagonal: p-distance. Shade areas indicate pairwise between different genera.

Table 2.5 The average p-distance of haplotype within genus of Cyclophoridae Genus COI 16S rRNA Alycaeinae Chamalycaeus 0.034＃ 0.005＃ Dioryx 0.056＃ 0.035＃ Cyclophorinae Cyathopoma 0.106 0.000＃ Cyclophorus 0.111 0.083 Cyclotus 0.066 0.056 Japonia (include zebra group) 0.181 0.185 Japonia (exclude zebra group) 0.113 0.071 Leptopoma 0.099 0.083 Ptychopoma 0.000＃ 0.019＃ Pterocyclinae Platyrhaphe 0.120 0.087 ＃indicates only one species contained in analysis

Fig. 2.1 Map of East Asia, with sampling sites of Cyclophoridae indicated. Solid square and asterisk indicated the sampling sites of Cyathopoma and Cyclotus respectively. Solid and open triangle indicated the sampling sites of Japonia and Pilosphaera respectively. Open circles indicated sampling sites of the others.

Fig. 2.2 Molecular phylogeny of Cycloporidae produced by Neighbor-joining

(NJ) analysis of individual gene sequence data from COI. Relative branch support indices are given as bootstraps test consisted of 1000 iterations. Different numbers after scientific name indicate sampling site number show in table 2.1 and fig. 2.1.

Fig. 2.3 Molecular phylogeny of Cycloporidae produced by maximum likelihood

(ML) analysis of individual gene sequence data from COI. Relative branch support indices are given as approximate likelihood ratio test. (aLRT). Different numbers after scientific name indicate sampling site number show in table 2.1 and fig. 2.1.

Fig. 2.4 Molecular phylogeny of Cycloporidae produced by Bayesian analysis of

individual gene sequence data from COI. Relative branch support indices are given as Bayesian probability. Different numbers after scientific name indicate sampling site number show in table 2.1 and fig. 2.1.

Fig. 2.5 Molecular phylogeny of Cycloporidae produced by Neighbor-joining

(NJ) analysis of individual gene sequence data from 16S rRNA. Relative branch support indices are given as bootstraps test consisted of 1000 iterations. Different numbers after scientific name indicate sampling site number show in table 2.1 and fig. 2.1.

Fig. 2.6 Molecular phylogeny of Cycloporidae produced by maximum likelihood

(ML) analysis of individual gene sequence data from 16S rRNA. Relative branch support indices are given as approximate likelihood ratio test. (aLRT). Different numbers after scientific name indicate sampling site number show in table 2.1 and fig. 2.1.

Fig. 2.7 Molecular phylogeny of Cycloporidae produced by Bayesian analysis of

individual gene sequence data from 16S rRNA. Relative branch support indices are given as Bayesian probability. Different numbers after scientific name indicate sampling site number show in table 2.1 and fig. 2.1.

Fig. 2.8 ESEM photograph of Cyathopoma iota (Pilsbry & Hirase, 1904) from Bai-liing, Iran County (24.525722N, 121.516083E), A: shell lateral view; B: Protoconch; C: operculum (outer view); D: radula, cm = central tooth, Ilt = inner lateral teeth, Olt = outer lateral teeth, mt = marginal teeth.

Fig. 2.9 ESEM photograph of Cyathopoma taiwanicum Pilsbry & Hirase, 1906 from Da-chi-jiau, Shin-dian City (24.957972N, 121.571833E), A: shell lateral view; B: Protoconch; C: operculum (outer view); D: radula, cm = central tooth, Ilt = inner lateral teeth, Olt = outer lateral teeth, mt = marginal teeth.

Fig. 2.10 ESEM photograph of Cyathopoma micron (Pilsbry, 1900) from li-shyng mountain road, Nan-tou County (24.067833N, 121.159722E), A: shell lateral view; B: Protoconch; C: operculum (outer view); D: radula, cm = central tooth, Ilt = inner lateral teeth, Olt = outer lateral teeth, mt = marginal teeth.

Fig. 2.11 ESEM photograph of Cyathopoma ogaitoi (Minato, 1988) from Lei Gong Shan, Guei-Jou Province (26.404N, 108.214E), A: shell lateral view; B: Protoconch; C: operculum (outer view); D: radula, cm = central tooth, Ilt = inner lateral teeth, Olt = outer lateral teeth, mt = marginal teeth.

Fig. 2.12 ESEM photograph of Cyclotus species, A1: shell lateral view of Cyclotus taivanus adams Pilsbry et Hirase, 1905, A2: radula of Cyclotus t. adams; B1: shell lateral view of Cyclotus t. dilatus Lee et Wu, 2001, B2: radula of Cyclotus t. dilatus; C1: shell lateral view of Cyclotus t. peraffinis Pilsbry et Hirase, 1905, C2: radula of Cyclotus t. peraffinis; D1: shell lateral view of Cyclotus t. taivanus H. Adams, 1870, D2: radula of Cyclotus t. taivanus.

Fig. 2.13 a-c: Lateral, apex and basal view of Pilosphaera shell. Holotype of Pilosphaera yentoensis n. sp. NMNS5635-001 (28°18’32.7”N; 120°32’30”E); d-f: Paratype of Pilosphaera yentoensis n. sp. NMNS5635-002 (28°18’32.7”N; 120°32’30”E); h-j: Pilosphaera zebra from Wu-Lai, Taipei, Taiwan (24° 50’57.1”N; 121°34’11.5”E). Black bar is 1mm.

Fig. 2.14 a-c: Lateral, apex and basal view of Japonia shell. Holotype of Japonia boonkioensis n. sp. NMNS5636-001 (23°29’31.1N”; 120°41’59.4”E); d-f: Japonia lanyuensis from Lan-Yu, Taiwan (22°1’41.6”N; 121°34’46.8”E); g-h: Japonia formosana from Sin-Xu, Taiwan (24°34’57.1”N; 120°52’30.0”E). Black bar is 1mm.

Fig. 2.15 A-B: Pilosphaera yentoensis n. sp. from Yen-To town near Nan-Xi river in Zhejiang province, China (28°18’32.7”N; 120°32’30”E); C: Pilosphaera zebra from Nei-Hu, Taipei, Taiwan (25°5’19.1”N; 121°37’42.4”E); D: Japonia boonkioensis n. sp.

NMNS5636-001 from Fen-chi-hu in Jia-i County, central Taiwan (23°29’31.1”N; 120° 41’59.4”E).

Fig. 2.16 Environmental Scanning Electron Microscope (FEI Quanta 200) photographs of periostracum hairs on Pilosphaera and Japonia shell. A: Paratype of Pilosphaera yentoensis n. sp. NMNS5635-002 (28°18’32.7”N; 120 °32’30”E); B: Pilosphaera zebra from Nei-Hu, Taipei, Taiwan (25°5’19.1”N; 121°37’42.4”E); C: Holotype of Japonia boonkioensis n. sp.; D: Japonia formosana from in Sin-Xu, Taiwan (24°34’57.1”N; 120°52’30.0”E)

36 Chapter 3

Ring speciation and morphological adaptations of genus

Cyclophorus in Taiwan

Abstract The samples of Cyclophorus span two critical links in the chain of morphologically distinct units: the transition from the round and keeled peripheral shell types from north to south Taiwan. I examined COI and 16S rRNA mitochondrial DNA of the five taxa of Taiwan Cyclophorus (see chapter 1, p10–16) and found both COI and 16S rRNA gene trees show strong geographic structures. The Mantel test show significant

positive correlation between fixation index (FST) and cumulative geographic anti-clockwise distance (origin in the region around Tainan, anti-clockwise pass through south cape, Taidung, Hualian, Iran, Taipei, Taichung and meet the original populations in Jia-yi). There are finite gene flew between adjacent population. And there are series clines around the Central Range. I believe Cyclophorus of Taiwan is a proposed “ring species”. In the morphology and environmental variables correlation study, I found the currently shell morphology may be caused by the adaptation of recent long term climate.

Introduction Cyclophorids are very common snails in Taiwan, the genus Cyclophorus is the most abundant group. Koblet (1902) was the first to revise the world Cyclophorus, but no major review of the Taiwan Cyclophorus has been completed till Lee and Wu’s study (2001). There are five Cyclophorus taxa in Taiwan, C. formosaensis, C. friesianus, C. latus, C. moellendorffi and C. cf. turgidus, all of them have yellowish solid shell, with reddish brown zigzag interlard with several brown bands. It is interesting that all the northern Cyclophorus possess peripheral keeled shells and the southern Cyclophorus possess peripheral round shells. However, the northern and southern terminal forms are connected with gradual geographical variation (Lee & Wu 2001). The evolutionary processes shaping the phenotypic variation among and within relative landsnail have become a focus of research interest recently (Pfenninger & Magnin

37 2001, Teshima et al. 2003, Pfenninger et al. 2003). There are many suggestions on the peripheral keel evolved in previous studies. Gould (1971) suggested that the peripheral keel evolved as the retention of a juvenile character. Cook and Pettitt (1979) considered that keeled shells may be more resistant to cruching than round shell. The other authors suggested the form of shells were associated with habitat types (Solem & Climo 1985, Alonso et al. 1985, Pfenninger & Magnin 2001). But it is hard to test whether an association really exists between phenotype and environment. And I am unaware of any phylogeny controlled comparative studies that have tested whether an association exists. However, since Carles R. Darwin (1859) the idea of the relation between phenotype and environment had been widely described by evolutionists. Ring species are a distinct cline where the geographical distribution is circular in shape, so that the two ends of the cline overlap with one another. The two adjacent populations rarely interbreed due to the cumulative effect of the many changes in phenotype along the cline. The populations elsewhere along the cline interbreed with their geographically adjacent populations as in a standard cline. The ring species teach us about speciation and reconstruct the pathway of speciation. Therefore, finding a potential ring species is a great interest to evolutionists. There are many cases about ring species (Jackman & Wake 1994, McKnight 1995, Irwin 2000, Irwin et al. 2001, Knijff et al. 2001); however few of the cases have characteristics of ideal ring species (Irwin et al. 2001). The topography of Taiwan provides a good chance to find the ring species because of the Central Mountain Chain. In traditional classification, there are five Cyclophorus species around mountain area of Taiwan. However, there are some intermediate forms between these Cyclophorus species (Lee & Wu 2001). These Cyclophorus species are potential ring species. The objectives of this study were using the genetic information from COI and 16S rRNA haplotype analysis as a background to study the historical biogeographical changes and quantitative morphologic differences of all described Cyclophorus taxa in Taiwan. In particular, I focus on five questions: (1) How do currently Cyclophorus biogeographical pattern cause by? (2) Is Taiwan Cyclophorus a “ring species”? (3) Does the observed phenotypic variation correspond to different evolutionary lineages? (4) How is the phenotypic variation distributed within and among populations in relation to the COI and 16S variation? And (5) Can I identify environmental variables that co-vary with population differences in morphology?

Materials and methods Populations sampled

38 In total, 57 populations of Cyclophorus were examined in Taiwan (Fig. 3.1) (Table 3.1). I do not get any living specimen (sampling site 31, 32, 33, 34, 35, 38, 49, 53) and adult individual (sampling site 18, 27, 28, 29, 46) in some sampling sites. Morphological analysis was performed on adult individuals of a subset from 52 locations, molecular analysis using 214 individuals from 46 locations. In molecular analysis, it seems hard to estimate the parameters using cumulative geographic anti-clockwise distance for all populations simultaneously. Therefore, I pooled the populations within 30 km and representing distinct geographical regions in groups. The centeral site of the groups is calculated using ArcGIS 9.2 (2006).

Morphology analysis Morphological analysis was performed on adult individuals of a subset from 52 locations (205 individuals). The data set for morphological and genetic analysis overlapped in 153 individuals for sampled populations. Individuals were photographed through a Canon D1 digital camera from the aperture view. The paper prints were digitalized with a resolution of 300 pixel/inch. Eight shell morphology characters were measured by digital image analysis: W (width), H (height), h (height to periphery keel or mid-point of periphery), AW (aperture width), AH (aperture height), BW (body whorl width), BH (body whorl height) and PW (penultimate whorl width) (Fig. 3.2). H/W, h/H, PW/W, AW/W, AW/AH, BH/BW were calculated as indices of tallness/flatness, keel position, sharp/dull spire, aperture proportion, aperture shape and flat degree of body whorl, respectively. It was not possible to measure accurately the angle of the keel, so I chose three qualitative categories (Fig. 3.3). The digital images were then scored by 10 persons for ‘angularity’ and the mean used as an index of angularity. These shell morphology variables were used to perform a principal component analysis (PCA) using PCA option of the package XLSTAT (2007). Partial least square (PLS) analysis was performed using the PLS option of the package XLSTAT (2007) to assess the correlations between the quantitative shell traits and environmental variables for each population (Lin 1990).

DNA preparing and sequencing The shells and soft parts were separated at laboratory. Shells were well cleaned for identification and measuring its shell characters, soft parts were stored at -80℃ until DNA extraction. DNA was extracted from columellar muscle. I extracted DNA from separate individuals using TEK-based protocol (Jiang et al. 1997) with minor modifications. Tissue was placed in TEK buffer (12.5mM Tris-Cl pH 7.3, 2.5mM EDTA, 0.4% KCl), then ground with glass pestle and incubated at 57℃ with 20µl of

39 proteinase K (20mg/ml) more than 2 hrs. The tissue extract was extracted at least twice with phenol and chloroform. 400µl DNA extract was precipitated by adding 1000µl pure ice-cold ethanol, and was then placed in -20℃ for 20 min. DNA was pelleted by centrifugation for 30 min. After 70% ethanol rinse, DNA was resuspended in distilled water and stored at -80℃ for DNA amplification. An approximately 860bp mitochondrial 16S rRNA gene was amplified by PCR using primers 16SRT (5’– ACA TAT CGC CCG TCA CTC TC–3') and 16SL900 (5’–AAA TGA TTA TGC TAC CTT TGC–3'), exactly 531bp COI using primer LCO1490 (5'–GGT CAA CAA ATC ATA AAG ATA TTG G–3') and HCO2198 (5'–TAA ACT TCA GGG TGA CCA AAA AAT CA–3') (Williams et al. 2003). PCR reactions contained template DNA 10–50ng/µl, 10pmol of each primer, 5µl 10× reaction buffer (10mM Tris-HCl, pH9.0,

Phylogenetic analyses Both COI and 16S rRNA sequences were combined with data of the out-group Japonia formosana Pilsbry et Hirase, 1905. Sequences were assembled and edited using Bioedit 5.0.9 (Hall 1999). All alignments employed Clustal X (Thompson et al. 1997) and were manually proofread. Codon positions within COI were tested using the incongruence length difference (ILD) test (Farris et al. 1995), as implemented by the partition homogeneity test in PAUP 4.0b10 (Swofford 1998) (100 replicates). COI sequence data were divided into two partitions, first and second codon positions in one and third codon positions in the other. Two parts of COI gene were congruent and all codon positions were combined and used in the following analysis. All data sets were subject to Neighbor-joining (NJ) using PAUP 4.0b10, to the maximum likelihood (ML) analyses using PHYML 3.0 (Guindon & Gascuel 2003), to the Maximum parsimony (MP) analysis using MEGA 4.0 (Tamura, Dudley, Nei, and Kumar 2007). The substitution model used for COI data set corresponded to the Tamura-Nei model, and included invariable sites, and rate variation among sites (TrN+I+G); for 16S rRNA data set corresponded to the two transversion-parameters model, and included invariable sites, and rate variation among sites (K81uf+I+G). These were the best models found using Modeltest 3.06 (Posada & Crandall 1998).

40 Before model fitting, the full-length sequences were tested to confirm that there was no significant heterogeneity in base frequencies across taxa (in COI: X2=63.78, df=246, P=1; in 16S rRNA: X2=31.66, df=282, P=1). NJ and MP bootstraps consisted of 1000 iterations. Reliability of ML trees were estimated by the approximate likelihood ratio test (aLRT) (using custom define model, base frequencies: A = 0.2653, C = 0.1357, G = 0.1822, T = 0.4168 in COI and A =0.3779, C = 0.0903 G = 0.1291, T = 0.4027 in 16S) using PHYML 3.0 (Guindon & Gascuel 2003). Gaps (from insertions/deletions) were treated as missing data. Neutrality was tested using Tajima's D (COI: D=-0.40890, P > 0.10; 16S rRNA: D= -1.25632, P > 0.10) and Fu & Li's F statistic (COI: F= -1.06768, P > 0.10; 16S rRNA: F= -2.12474, 0.10 > P > 0.05) (Tajima, 1989; Fu & Li, 1993). There are no selective effects on COI and 16S rRNA gene. The divergent time was estimated using MEGA 4.0 (Tamura, Dudley, Nei, and Kumar 2007). Associations between genetic, morphological, pairwise geographic distance and Cumulative geographic anti-clockwise distance between populations were explored through Mantel-tests in XLSTAT (2007) on the pairwise population fixation indices derived from AMOVA analysis (using Arlequin 3.1) (Excoffier et al. 2005), pairwise shell characters similarity (using Euclidean distance calculated by Primer 5.1.2) (2000), pairwise geographic distance and Cumulative geographic anti-clockwise distance to test for parallel evolution between genetic and morphological data, and isolation by distance, respectively.

Results Morphology analysis The first three principal components account for 34.11%, 22.83% and 17.00 % of the total variation in the matrix. The loadings of characters on the principal components reflect distinct groups of shell traits: sharp/dull spire (PC1), flat degree of body whorl (PC2), and aperture shape (PC3). Most of five taxa are complete overlap (Fig 3.4). However, the north group (sampling sites 22–57) and south group (sampling sites 1–21) are mostly separated, but show partial overlap (Fig 3.5).

Phylogenetic analysis The aligned 531 bp COI gene data matrix, included 225 variable sites of which 187 (83.11%) are parsimony informative. No length difference from the out-group was detected among members of Cyclophorus. The average p-distance among Taiwan and Okinawa haplotypes is = 0.137. Average sequence divergence among Taiwan haplotypes is 0.092, ranged from 0.002 to 0.177. The alignment of the 16S rRNA

41 gene fragment data yielded 852 characters of which 407 are variable sites. Of these 407 positions, 357 (87.7%) are parsimony informative. The average p-distance among Taiwan and Okinawa haplotypes is 0.120. Average sequence divergence among Taiwan haplotypes is 0.077, ranged from 0.001 to 0.192. The inferred phylogenetic trees between the haplotypes of COI gene are shown in Figs. 3.7, 3.8 & 3.9 of 16S rRNA gene are shown in Figs. 3.10, 3.11 & 3.12. Both COI and 16S rRNA cladograms construct using different tree building method present similar topology. The cladograms indicate that the Cyclophorus populations around Tainan (sampling sites 1–11) are the origin, because they are placed nearest the root. All trees show Taichung, Nan-tou and Jia-yi populations (sampling sites 45–57) are latest divergency. The paleogeographical study indicates Taiwan Island was emerged in late Miocene orogeny approximate 6 million years ago (Chen & Wang 1996). I consider the divergent time of Taiwan Cyclophorus would not earlier than 6 million years ago. I use the timing of Taiwan emerged as a calibration point, the COI and 16S rRNA evolutionary rate of Cyclophorus are 2.28% and 2% per million years, respectively.

Mantel test

Pairwise FST increases significantly with Cumulative geographic anti-clockwise distance, despite the strong variation within groups (COI/ Cumulative geographic anti-clockwise distance Mantel test, r = 0.217, P = 0.003; 16S rRNA/ Cumulative geographic anti-clockwise distance Mantel test, r = 0.255, P = 0.00025) (Fig. 3.6). The not particularly high r-value, resulting from a scatter around the linear regression line, might be caused by slightly different rates of increase of FST in different spatial directions because of heterogeneity in population densities. Mantel test was also performed between molecular P-distance and Cumulative geographic anti-clockwise distance matrix. P-distance increases significantly with Cumulative geographic anti-clockwise distance and with high correlation coefficient (COI: r = 0.611, P < 0.0001; 16S rRNA: r = 0.563 P < 0.0001). I also performed Mantel test to explore associations between fixation index, molecular P-distance and pairwise geographic distance. However, the correlation coefficient is relative lower than those associated with cumulative geographic anti-clockwise distance (Table 3.3).

PLS analysis PLS analysis detected significant correlation between shell traits and environmental variables for the respective sampling site (Table 3.2). Populations with a high mean temperature tend to be composed of individuals showing a tall shell with sharp spire

42 and lower keel position. Populations at high altitude tend to have flat shell with dull spire, small aperture and high keel position (Fig. 3.13) (Table 3.2). Shell angularity revealed positive correlation with warm season mean temperature, and negative correlation with cold season amount of precipitation and annual range of monthly mean temperature.

Discussion The gene trees show strong geographic structure, and suggest that the origin of Taiwan Cyclophorus is in the region around Tainan, the divergency pathway anti-clockwise pass through south cape, Taidung, Hualian, Iran, Taipei, Taichung and meet the original populations in Jia-yi. The hypothesis of possible biogeographical history matches that of Taiwan geologic history. Base on COI and 16S rRNA sequences clock and using the timing of Taiwan emerged as a calibration point. The split of east populations occurred in 1.79–2.93 million years ago. Comparing with the geologic history, Coastal Range and East Rift Valley were emerged in late Pleistocene (1.8–2million years ago) (Chen & Wang 1996, Ho 1982). This suggests the Cyclophorus has had a long history in the eastern part of its present range when Coastal Range and East Rift Valley were emerged. It is possible that the phylogeographic structure developed in a continuous isolation by distance model in the absence of any geographic break. However, the alternative, that there was geographical separation of the northern and southern parts of the Cyclophorus from east Taiwan and relatively recent contact between them, cannot presently be rejected. The correlation coefficient between fixation index, molecular P-distance and pairwise geographic distance is relative lower than those associated with cumulative geographic anti-clockwise distance. It suggests that currently populations seem to be connected throughout the ring. However, the correlation coefficient between shell traits and pairwise geographic distance is relative higher than associated with cumulative geographic anti-clockwise distance. It is because that some shell traits (e.g. peripheral keel) of Jia-yi populations are similar to unrelated, but not related taxa. The topology of the mitochondrial DNA tree also indicates that there is little association between shell peripheral type (angular or circular) and mitochondria1 DNA divergence and strongly supports the hypothesis of multiple origins of angularity. For instance, morphologically distinct angular form from Jia-yi County (sampling sites 54–57) clustered together with circular form from Taichung and Nan-tou County (sampling sites 45–52). The mitochondrial DNA evidence implies that this shell peripheral type may have arisen by parallel or convergent evolution. There are several explanations as to why shells might be keeled. One is paedomorphy.

43 As juvenile snails sometimes have a keel, it is easy to imagine keeled shell is paedomorphy (Gould 1971). However, there is no direct evidence that the keel evolved via a paedomorphy, especially since the juvenile of globular Cyclophorus do not usually have a keel. Another hypothesis is that keeled shells are an adaptation to limestone substrates (Alonso et al. 1985), such as Ainohelix editha, Euhadra murayamai, and Helicigona lapicida predominantly found on limestone (Teshima et al. 2003). Some Taiwan Cyclophorus are rest on limestone, such as Taroko and south cape populations. However, south cape individuals are keeled shell but circular in Taroko populations. The main alternative explanation is a single evolution of keeled snails, where some populations have acquired their mtDNA sequences from adjacent populations of different shape snails by hybridization and introgression (Davison 2002). However, the overall shell morphology of Jia-yi population does not look like an intermediate of its parapatric populations (Taichung and Nan-tou). By the evidence of high correlated between shell traits and pairwise geographic distance, I doubt the shell traits may cause by geographic and climatic features of sampling sites geographical position. The results from PLS analysis showed that differences between populations in some shell traits co-varied significantly with long term climatic conditions and altitude. I found climatically warmer and stable temperature tend to have keeled shell. The warm and stable climate tends to have luxuriant vegetation. The keeled shell would be at an advantage when roam over the ground in the luxuriant vegetation. Ring species should be respect (1) with two distinctively coexistent forms, (2) with currently gene flow between distinctive forms through a chain of population, (3) populations chain from a true geography ring without gaps, (4) the terminal forms connected by gradual geographical variation (Irwin et al. 2001). In my case, there are two distinctive forms of Cyclophorus in southwest Taiwan, one with flat shell and sculpture with spiral cords on shell surface (round mountain region of Tainan and Kaohsiung County, site 1–11), one with tall shell and without spiral cords (round mountain region of Jia-yi County, site 54–57). The molecular data show strong geographic structure (Fig. 3.7 – 3.12) and current gene flow between close populations (Fig. 3.6). The population chain origin in the region around Tainan, anti-clockwise pass through south cape, Taidung, Hualian, Iran, Taipei, Taichung and meet the original populations in Jia-yi. And as I know Cyclophorus snails are widely distributed over Taiwan. There are gradual shell variations between these populations (Fig. 3.4). The shell traits were significant correlated with environmental variables (Table 3.2). The most reasonable hypothesis is, Cyclophorus snails in Taiwan were cumulated

44 their DNA variation by the way of isolation-by-distance in the early time when they colonized in Taiwan. Undergoing Penglai Orogeny (began at 6 million years ago), the Cenral Range arisen and separated west and east populations. Then the west populations were extinction in the Last Glacial Maximum because non-forest occurred in the site of them (Harrison et al. 2001). The currently west populations were followed by recent expansion. The shell morphology may cause by the adaptation of recent long term climate. For taxonomists, this case creates a plain paradox; two sympatric forms can be considered as separate species. However they can also be considered as the same species because of the chain of intermediate forms. I suggest, it need further study to investigate more sampling site, to identify the clear distribution range of each form and the five taxa of Taiwan Cyclophorus may treat as subspecies each other.

45 Table 3.1 List of Cyclophorus sampling sites and the group numbers of pooled populations

Goup Items Location Position No.

1 1 Provincial Highway No.159A, 36k, Jia-yi County 23.469750N, 120.659250E 2 1 Guan-tzy-liing, Tainan County 23.339306N, 120.503111E 3 1 Provincial Highway No.3, 334k 23.328917N, 120.499250E 4 2 Provincial Highway No.20, 79k, Bau-Lai, Kaohsiung County 23.108639N, 120.699722E 5 2 Provincial Highway No.20, 49k, Nan-huah, Tainan City 23.100389N, 120.481556E 6 2 Provincial Highway No.20, 65.5k, Tainan City 23.078111N, 120.582667E 7 3 Shan-ping, Kaohsiung County 22.965917N, 120.683944E 8 3 Liou-guei, Kaohsiung County 22.925222N, 120.652833E 9 3 Mau-lin, Kaohsiung County 22.894056N, 120.649028E 10 3 Hae-shern Temple, San-dih-men, Ping-dung County 22.819972N, 120.640444E 11 3 San-dih-men, Ping-dung County 22.807361N, 120.648250E 12 4 Chair-shan, Kaohsiung City 22.650694N, 120.259000E 13 5 Mu-dan township, Ping-dung County 22.112139N, 120.761472E 14 5 Kaan-ding, Ping-dung County 21.965917N, 120.814500E 15 5 Shuang-liou Wood Park, Ping-dung County 22.217889N, 120.803000E 16 6 Da-wu, Taidung County 22.360639N, 120.879528E 17 6 Taidung County 22.504278N, 120.958111E 18 6 Jy-been, Taidung County 22.530472N, 120.941583E 19 7 Bei-Nan, Taidung County 22.865972N, 121.098333E 20 7 Dung-he, Taidung County 23.074778N, 121.301028E 21 7 East Coastal Mountain, Taidung County 23.074778N, 121.301028E 22 8 Torng-men, Hualian County 23.955556N, 121.503472E 23 9 Charn-guang Temple, Taroko valley 24.159250N, 121.606111E 24 9 Sha-ka-dang pavement, Taroko valley 24.168722N, 121.609111E 25 9 Chorng-der, Hualian County 24.189278N, 121.660889E 26 10 Dung-aw-li, Su-aw, Iran County 24.521194N, 121.824889E 27 10 Gu-lu mountain road, Iran County 24.592556N, 121.680611E 28 10 Shin-liau waterfall, Iran County (1) 24.602056N, 121.745000E 29 10 Shin-liau waterfall, Iran County (2) 24.604139N, 121.744028E 30 10 An-ping-keng, Iran County 24.615000N, 121.777806E 31 – Guan-in Mountain, Taipei County 25.132721N, 121.423889E 32 – Yn-her-donq, Taipei County 24.958417N, 121.574306E 33 – San-shya, Taipei County 24.868222N, 121.410111E

46 34 – Fwu-shan, Taipei County 24.795750N, 121.492944E 35 – Ching-ua-shyr, Shin-zu County 24.686917N, 121.228778E 36 11 Dong-shan, Iran County 24.624056N, 121.762778E 37 11 Harn-chi, Iran County 24.583417N, 121.691167E 38 12 Wu-Lai, Taipei County 24.847028N, 121.539278E 39 12 Shinn-shyan, Wu-Lai, Taipei County 24.839861N, 121.525778E 40 12 Sheau-jiau-shi, Iran County 24.805833N, 121.704417E 41 12 Dah-jiau-shi, Iran County 24.792778N, 121.655889E 42 13 Dar-Guan Mountain, Tauyuan County 24.709250N, 121.431833E 43 13 Ren-tzer, Iran County 24.545611N, 121.507278E 44 13 Bai-liing, Iran County 24.525722N, 121.516083E 45 14 Gu-guan, Taichung County 24.199278N, 120.999417E 46 14 Ba-shian-shan, Taichung County 24.179861N, 121.016639E 47 14 Bai-luh, Her-ping township, Taichung County 24.175333N, 120.909861E 48 14 Li-leeng mountain road, Her-ping township, Taichung County 24.164528N, 120.958083E 49 14 Her-ping township, Taichung County 24.147638N, 120.772111E 50 14 County highway No.100, Dung-biann village, Taichung County 24.118333N, 120.797250E 51 14 County highway No. 136, 39.5K, Taichung County 24.099361N, 120.790389E 52 15 Mei-feng, Nan-tou County 24.088139N, 121.171556E 53 15 Ren-ji-Guan, Nan-tou County 24.006472N, 121.111972E 54 16 Jang-hu, Jia-yi County 23.611944N, 120.64125E 55 16 Provincial Highway No.149, Yun-lin County 23.589639N, 120.569306E 56 16 Provincial Highway No. 162A, 32.5k, Yun-lin County 23.560944N, 120.655583E 57 17 County Highway No.129, 20.5k, Jia-yi County 23.342500N, 120.691861E –the sampling sites where no live specimens were collected, they were excluded from molecular analysis and the analysis of correlation between morphology and molecular data.

47 Table 3.2 Correlations between each shell traits and the corresponding environmental variables Correlation Warm season Cold season Annual Warm season Cold season Annual range of Shell traits Annual days of daily Annual days of daily mean mean mean amount of amount of monthly mean Altitude precipitation≧25mm precipitation≧10mm temperature temperature temperature precipitation precipitation temperature sharp/dull spire 0.402* 0.549* 0.485* 0.055 -0.282 0.003 -0.176 -0.444** -0.560** (PW/W) tallness/flatness 0.225* 0.295** 0.255* -0.065 -0.053 -0.035 -0.105 -0.178 -0.516** (H/W) keel position (h/H) -0.273 -0.422* -0.350* 0.098 0.183 0.143 0.269 0.247 0.442* flat degree of body -0.409 -0.401 -0.421 0.215 0.267 0.226 0.324 0.067 0.093** whorl (BH/BW) aperture proportion -0.158 -0.120 -0.153 -0.048 0.140 0.033 0.078 0.045 -0.154** (AW/W) aperture shape 0.099 0.109 0.115 0.101 -0.028 0.089 0.084 -0.065 0.035 (AW/AH) angularity 0.418* 0.549 0.512 0.387 -0.612** 0.207 -0.124 -0.624** -0.187 *Indicates a significant correlation at the 5% level; **Indicates a significant correlation at the 1% level. Warm season: May, June, July, August and September. Cold season: January, February, March, November and December.

48 Table 3.3 the correlation coefficient derived from Mantel test Cumulative geographic P value Pairwise geographic P value anti-clockwise distance distance Fixation index (COI) 0.217 0.00295 0.197 0.00615 Fixation index (16S rRNA) 0.255 0.00025 0.188 0.01035 P-distance (COI) 0.611 < 0.0001 0.574 < 0.0001 P-distance (16S rRNA) 0.563 < 0.0001 0.501 < 0.0001 Shell trait Euclidean distance 0.374 < 0.0001 0.740 < 0.0001

32 38 33 39 40

35 34 41 36 29 42 27 30 43 28 37 26 44

45 24 47 25 49 50 46 48 52 23 51 53 22

54 55 56 1 2 57 3

5 6 4 20 21 8 7 9 19 10 11 12 18

17 16

Fig. 3.1 Geographic range of the Cyclophorus species complex, different colors and symbols illustrate the ranges and sampling sites of five taxa, respectively. Cyan and X symbol: C. cf. turgidus; green and open circle: C. friesianus; yellow and solid circle: C. moellendorffi; pink and red and solid square: C. formosaensis; blue and solid triangle: C. latus. Populations pooled for the analysis are encircled.

Fig. 3.2 The morphological character measurements of Cyclophorus. AH = aperture height, AW = aperture width, BH = body whorl height, BW = body whorl width, H = height, h = height to periphery or mid-point, PW = penultimate whorl width and W = width.

Fig. 3.3 Three degree of angularity of Cyclophorus shells. A: No peripheral angle; B: Slight peripheral angle; C: Clear peripheral angle.

Fig. 3.4 Scatter plot PCA scores on the first two axes of Principal component analysis on Cyclophorus shell morphological variables. Axis 1 accounts for 34.11%, axis 2 for 22.83% of total variance. Different colors illustrate the ranges of sampling sites show in Fig. 3.1 of five taxa.

Observations (axes PC1 and PC2: 56.94%)

4.000

3.000

2.000

1.000

North group 0.000 South group

PC2 (22.83 %) (22.83 PC2 -1.000

-2.000

-3.000

-4.000 -5.000 -4.000 -3.000 -2.000 -1.000 0.000 1.000 2.000 3.000 4.000 PC1 (34.11 %)

Fig. 3.5 Scatter plot PCA scores on the first two axes of Principal component analysis on Cyclophorus shell morphological variables. Axis 1 accounts for 34.11%, axis 2 for 22.83% of total variance. Cyclophorus from north populations (sampling site 22–57) are encircled and from south populations (sampling site 1–21) in square, respectively.

53 1.2 1.2

1 1

0.8 0.8

0.6

0.4

0.4 COI FixationCOI index 0.2 16S rRNA Fixation index Fixation rRNA 16S

0.2 0

-0.2 0 0 100000 200000 300000 400000 500000 600000 700000 800000 0 100000 200000 300000 400000 500000 600000 700000 800000 Geographical anti-clockwise distance Geographical anti-clockwise distance

Fig. 3.6 Relationship between Cumulative geographic anti-clockwise distance and fixation index of COI and 16S rRNA, respectively of Cyclophorus. (COI/ Cumulative geographic anti-clockwise distance Mantel test, r = 0.217, P = 0.003; 16S rRNA/ Cumulative geographic anti-clockwise distance Mantel test, r = 0.255, P = 0.00025)

54 Fig. 3.7 Molecular phylogeny of Cyclophorus produced by maximum parsimony analysis of individual gene sequence data from

COI. Relative branch support indices are given as bootstraps test consisted of 1000 iterations. Branch tip are labeled with the sampling site numbers.

55 Fig. 3.8 Molecular phylogeny of Cyclophorus produced by maximum likelihood (ML) analysis of individual gene sequence data from

COI. Relative branch support indices are given as approximate likelihood ratio test. (aLRT). Branch tip are labeled with the sampling site numbers.

Fig. 3.9 Molecular phylogeny of Cyclophorus produced by Neighbor-joining (NJ) analysis of individual gene sequence data

from COI. Relative branch support indices are given as bootstraps test consisted of 1000 iterations. Branch tip are labeled with the sampling site numbers.

57 Fig. 3.10 Molecular phylogeny of Cyclophorus produced by maximum parsimony analysis of individual gene sequence data from

16S rRNA. Relative branch support indices are given as bootstraps test consisted of 1000 iterations. Branch tip are labeled with the sampling site numbers. K: angular peripheral shell; C: circular peripheral shell.

59 Fig.3.11 Molecular phylogeny of Cyclophorus produced by maximum likelihood

(ML) analysis of individual gene sequence data from 16S rRNA. Relative branch support indices are given as approximate likelihood ratio test. (aLRT). Branch tip are labeled with the sampling site

60 Fig. 3.12 Molecular phylogeny of Cyclophorus produced by Neighbor-joini ng (NJ) analysis of individual gene sequence data from 16S

rRNA. Relative branch support indices are given as bootstraps test consisted of 1000 iterations. Branch tip are labeled with the sampling site numbers.

warm season total 1 precipitation annual days of daily precipitation≧25mm

0.75

annual days of daily precipitation≧10mm

angularity 0.5

altitude

0.25 flat degree of body w horl aperture shape sharp/dull spire

keel position cold season mean temperature 0

aperture proportion tallness/flatness annual mean AXIS 2 AXIS (16.4%) temperature cold season total warm season mean -0.25 precipitation temperature

-0.5

annual range of monthly mean -0.75 temperature

-1 -1 -0.75 -0.5 -0.25 0 0.25 0.5 0.75 1 AXIS 1 (44.8%)

Fig. 3.13 Partial least square anaylsis (PLS) of shell variables against environmental variables for overall Taiwan Cyclophorus.

62 Chapter 4

The phylogenetic evolution and morphological adaptations

in Cyclotus taivanus ssp.

Abstract Cyclotus taivanus consists of five subspecies, with clear morphological diversity. The molecular phylogenetic relationships of this group have never been discussed before. In order to investigate the relationships between C. taivanus ssp., I sequenced part of the mitochondrial COI and the 16S rRNA gene from 27 sampling sites. I also measured 9 shell traits for morphology analysis. Even though the morphology PCA revealed a more or less continuous distribution of individuals in morph-space, the two highly divergent haplotype clades in COI and 16S rRNA analysis indicated the presence of two independently evolving lineages. The sequence divergence between two clades was almost as high as between other Cyclophoridae species(COI: 0.102 in C. taivanus ssp., 0.028–0.172 in other Cyclophoridae species; 16S rRNA: 0.121 in C. taivanus ssp., 0.041–0.166 in other Cyclophoridae species, see chapter 2, table 2.5). Therefore C. adamsi should be a valid species. For the environmental analysis, temperature may be a limited element of the distribution of C. adamsi and C. taivanus group (C. t. dilatus, C. t. diminutus, C. t. peraffinis, and C. t. taivanus). The ecological divergence probably appears rule of speciation in my case. The PLS analysis results indicate, phenotypic plasticity may be a key element of variable shell in C. taivanus group. The speciation process is not complete among C. taivanus group, and the adaptation of climatic pressure continuing be a rule of speciation process.

Introduction Cyclotus taivanus is widely distributed in Taiwan and Okinawa and five subspecies were described. In traditional classification, the north Taiwan subspecies C. t. adamsi were tall spire with uniform yellow or tawny yellow color, few ornamented with reddish brown zigzag pattern. The other four are flat spire. However, C. t. dilatus is wide extended lip, C. t. diminutus is very narrow lip, C. t. taivanus is intermediate, C. t. peraffinis is similar to C. t. diminutus but with very polish periostracum (Lee & Wu

63 2001). The evolutionary processes shaping the phenotypic variation among and within relative landsnails have become a focus of research interest recently (Pfenninger & Magnin 2001, Teshima et al. 2003, Pfenninger et al. 2003). Two basic approaches have been taken to the study of variation in shell shape. One approach, involves the analysis of the distribution of shapes among taxa and hypothesizing the reasons for low or high frequencies of certain shapes. The second approach involves the study of correlations between variation in shape and environmental variation (Goodfriend 1986). Land snails with low vagility, therefore they can not avoid unfavorable conditions by migration but are doomed to extinction (Akcakaya & Baur 1996). It makes land snails are an ideal model to understand the roles of speciation history. Cyclotus and Cyclophorus are large cyclophorids occurring in East Asia. They occupy almost the same niche. However, the biogeographic pattern of Cyclotus is quite different from Cyclophorus. It may cause by different speciation process. Ring speciation had been described in Taiwan Cyclophorus (see chapter 3). The phylogeny and speciation model of C. taivanus ssp. have not been investigated. The objectives of this study were using the genetic information from COI and 16S rRNA haplotype analysis as a background to study quantitative morphologic differences of all described C. taivanus ssp. In particular, I focus on five questions: (1) How do currently biogeographical pattern of C. taivanus ssp. cause by? (2) What is the speciation process due to? (3) Does the observed phenotypic variation correspond to different evolutionary lineages? (4) How is the phenotypic variation distributed within and among populations in relation to the COI and 16S variation? And (5) Can I identify environmental variables that co-vary with population differences in morphology?

Materials and methods Populations sampled In total, 27 populations were examined in Taiwan (Fig. 4.1) (Table 4.1). I do not get any living specimen (sampling site 2) and adult individual (sampling site 5, 18) in some sampling sites. It makes morphological analysis was performed on adult individuals of a subset from 25 locations, molecular analysis using 89 individuals from 26 locations.

Morphology analysis Morphological analysis was performed on adult individuals of a subset from 25 locations (105 individuals). The data set for morphological and genetic analysis overlapped in 71 individuals for sampled populations. Individuals were photographed

64 through a Canon D1 digital camera from the aperture view. The paper prints were digitalized with a resolution of 300 pixel/inch. Eight shell morphology characters were measured by digital image analysis: H (height), W (width), AW (aperture width), AH (aperture height), BW (body whorl width), BH (body whorl height), LW (outer lip width), UW (umbilicus width) and HN (whorl number) (Fig. 4.2). H×W, H/W, AW/W, AW/AH, UW/W, LW/AW, BH/BW and (W－UW－LW)/HN were calculated as indices of size, tallness/flatness, aperture proportion, aperture shape, umbilicus proportion, outer lip proportion, flat degree of body whorl and the degree of whorl increasing, respectively. These shell morphology variables were used to perform a principal component analysis (PCA) using PCA option of the package XLSTAT (2007). Partial least square (PLS) analysis was performed using the PLS option of the package XLSTAT (2007) to assess the correlations between the quantitative shell traits and environmental variables for each population.

DNA preparing and sequencing The shells and soft parts were separated at library. Shells were well cleaned for identification and measuring its shell characters, soft parts were stored at -80℃ until DNA extraction. DNA was extracted from columellar muscle. I extracted DNA from separate individuals using TEK-based protocol (Jiang et al. 1997) with minor modifications. Tissue was placed in TEK buffer (12.5mM Tris-Cl pH 7.3, 2.5mM EDTA, 0.4% KCl), then ground with glass pestle and incubated at 57℃ with 20µl of proteinase K (20mg/ml) more than 2 hrs. The tissue extract was extracted at least twice with phenol and chloroform. 400µl DNA extract was precipitated by adding 1000µl pure ice-cold ethanol, and was then placed in -20℃ for 20 min. DNA was pelleted by centrifugation for 30 min. After 70% ethanol rinse, DNA was resuspended in distilled water and stored at -80℃ for DNA amplification. An approximately 860bp mitochondrial 16S rRNA gene was amplified by PCR using primers 16SRT (5’–ACA TAT CGC CCG TCA CTC TC–3') and 16SL900 (5’–AAA TGA TTA TGC TAC CTT TGC–3'), exactly 531bp COI using primer LCO1490 (5'–GGT CAA CAA ATC ATA AAG ATA TTG G–3') and HCO2198 (5'–TAA ACT TCA GGG TGA CCA AAA AAT CA–3') (Williams et al. 2003). Some individuals got pool PCR products using primer LCO1490 and HCO2198. For those I use primer CoL90 (5’-TAG TGT TAA AAT TAC GAT CAG T-3') and CoR600 (5’-AAG TCT TCT AAT TCG TGC AGA A-3'). PCR reactions contained template DNA 10–50ng/µl, 10pmol of each primer, 5µl 10×

reaction buffer (10mM Tris-HCl, pH9.0, 50Mm KCl, 1.5Mm MgCl2, 0.1% gelatin, 1% Trinton X-100), 0.4µl 25mM/µl dNTP, 0.2µl 50mM Mg2+ and 0.4µl Taq polymerase (5unit/µl) in a total volume of 50µl. Thermal cycling for 16S rRNA was

65 performed with an initial denaturation for 5min at 95℃, followed by 30 cycles of 30 sec. at 95℃, 45 sec. at 57℃, 50 sec. at 72℃ and ultimate extension at 72℃ for 10min, final hold in 4℃. Thermal cycling for COI gene was performed similarly, changing only its annealing temperature to 47℃. PCR products were purified using a purification kit (AMP PCR purification, Beckman) and then sequenced using an ABi 3700 autosequencer.

Phylogenetic analyses Both COI and 16S rRNA sequences were combined with data of the out-group Ptychopoma wilsoni (Pfeiffer, 1865). Sequences were assembled and edited using Bioedit 5.0.9 (Hall 1999). All alignments employed Clustal X (Thompson et al. 1997) and were manually proofread. Codon positions within COI were tested using the incongruence length difference (ILD) test (Farris et al. 1995), as implemented by the partition homogeneity test in PAUP 4.0b10 (Swofford 1998) (100 replicates). COI sequence data were divided into two partitions, first and second codon positions in one and third codon positions in the other. Two parts of COI gene were congruent and all codon positions were combined and used in the following analysis. All data sets were subject to Neighbor-joining (NJ) using PAUP 4.0b10, to the maximum likelihood (ML) analyses using PHYML 3.0 (Guindon & Gascuel 2003), to the Maximum parsimony (MP) analysis using MEGA 4.0 (Tamura, Dudley, Nei, and Kumar 2007). The substitution model used for COI data set corresponded to the Hasegawa, Kishino, Yano 85 model, and included invariable sites, and rate variation among sites (HKY+I+G); for 16S rRNA data set corresponded to the Transversional model, and included invariable sites, and rate variation among sites (TVM+I+G). These were the best models found using Modeltest 3.06 (Posada & Crandall 1998). Before model fitting, the full-length sequences were tested to confirm that there was no significant heterogeneity in base frequencies across taxa (in COI: X2 = 9.47, df = 96, P = 1; in 16S rRNA: X2 = 34.03, df = 117, P = 1). NJ and MP bootstraps consisted of 1000 iterations. Reliability of ML trees were estimated by the approximate likelihood ratio test (aLRT) (using custom define model, base frequencies: A = 0.2800, C = 0.1865, G = 0.1617, T = 0.3719 in COI and A = 0.3425, C = 0.0962 G = 0.1859, T = 0.3754 in 16S) using PHYML 3.0 (Guindon & Gascuel 2003). Gaps (from insertions/deletions) were treated as missing data. Neutrality was tested using Tajima's D (COI: D = -0.51579, P > 0.10; 16S rRNA: D = -0.79116, P > 0.10) and Fu & Li's F statistic (COI: F = 0.13830, P > 0.10; 16S rRNA: F = 0.24269, P > 0.10) (Tajima, 1989; Fu & Li, 1993). There are no selective effects on COI and 16S rRNA gene. Associations between genetic, morphological and geographic distance between

66 populations were explored through Mantel-tests in XLSTAT (2007) on the pairwise genetic p-distance, pairwise shell characters similarity (using Euclidean distance calculated by Primer 5.1.2) and pairwise geographic distance.

Results Morphology analysis The first three principal components account for 49.59%, 22.38% and 9.68 % of the total variation in the matrix. The loadings of characters on the principal components reflect distinct groups of shell traits: umbilicus proportion (PC1), outer lip proportion (PC2), and aperture proportion (PC3). The C. adamsi group (sampling sites 1–8) are fully separated from the other four taxa (sampling sites 9–27) along the two axes, but C. peraffinis, C. dilatus, C. taivanus and C. diminutus show partial overlap (Fig 4.3).

Phylogenetic analysis The aligned 531 bp COI gene data matrix, included 171 variable sites of which 147 (85.96%) were parsimony informative. No length difference from the out-group was detected among members of Cyclotus. The average p-distance was =0.087. Sequence divergence among the haplotypes ranged from 0.002 to 0.128. The alignment of the 16S rRNA gene fragment data yielded 816 characters of which 353 are variable sites. Of these 353 positions, 307 (86.97%) were parsimony informative. The average p-distance among haplotypes was 0.094. Sequence divergence among the haplotypes ranged from 0.001 to 0.129. The inferred phylogenetic trees between the haplotypes of COI gene are shown in Figs. 4.4, 4.5, 4.6 & 4.7 of 16S rRNA gene are shown in Figs. 4.8, 4.9, 4.10 & 4.11. Both COI and 16S rRNA cladograms construct using different tree building method present similar topology. The topology of the COI and 16S rRNA gene trees strongly suggested the presence of two fundamental haplotype clades named A and B. Analysing each clade separately, the average sequence divergence of COI among haplotype clades A and B was 0.102 changes per site compared to 0.009 and 0.057 changes within clade A and clade B, respectively. In contrast, the sequence divergence of 16S rRNA among haplotype clades A and B was 0.121 changes per site compared to 0.011 and 0.049 changes within clade A and clade B, respectively. All C. adamsi cluster in clade A. Clade B comprises C. peraffinis, C. dilatus, C. taivanus and C. diminutus.

Environmental characterization of sites PCA of the sites shows that the sampled populations primarily comprise (axis 1,

67 44.03% of total variation) an environmental gradient from north Taiwan, with low temperature, to south Taiwan, with high temperature (Fig. 4.12). The second most important gradient (axis 2, 32.42%) is a gradient with more changeable temperature (annual range of monthly mean temperature), lower warm season precipitation and higher cold season precipitation from north to south Taiwan. When the results of the molecular species characterization are plotted on the PCA, it becomes clear that the sites inhabited by the two clades species in question are well separated by this analysis, mainly by the second axis. Clade A populations are preferentially situated on changeable temperature, lower warm season precipitation and higher cold season precipitation climate region of the plot. The ecological amplitude of clade A is much larger than that of clade B.

Mantel test There are four subspecies including variable shell morphology in clade B. Therefore, I perform Mantel test to investigate the correlation between molecular data, morphology and geographic distribution, respectively. The Mantel test for association between the geographic distance separating populations and pairwise p-distance was significant (COI p-distance / geographic distance Mantel test, r = 0.347, P = 0.0001; 16S rRNA p-distance / geographic distance Mantel test, r = 0.489, P = 0.0001). By contrast, there were no correlation between molecular pairwise p-distance and overall morphological similarity in both COI and 16S rRNA cases (COI p-distance / morphological similarity Mantel test, r = 0.029, P = 0.330; 16S rRNA p-distance / morphological similarity Mantel test, r = 0.019, P = 0.499). Mantel test was also performed between pairwise geographic distance and overall morphological similarity matrix, and got significant correlation between them.

PLS analysis PLS analysis detected significant correlation between shell traits and environmental variables of clade B for the respective sampling site (Table 4.2). Populations with a few cold season amount of precipitation tend to be composed of individuals showing a broad outer lip, more rapid increasing shell whorls and more whorl number.

Discussion Two evolutionary lineages in C. taivanus ssp. Morphologic and genetic variation within and among populations was detected in the present survey over the range of the taxon that was described as C. taivanus ssp. Even

68 though the PCA revealed a more or less continuous distribution of individuals in morph-space, the two highly divergent haplotype clades in COI and 16S rRNA analysis indicated the presence of two independently evolving lineages. The sequence divergence between two clades was almost as high as between other Cyclophoridae species (COI: 0.102 in C. taivanus ssp., 0.028–0.172 in other Cyclophoridae species; 16S rRNA: 0.121 in C. taivanus ssp., 0.041–0.166 in other Cyclophoridae species, see chapter 2, table 2.5). Despite of an almost continuous morph-space when looking at the overall phenotypic similarity, PCA yielded a highly discrimination model and the genetic clade as classification variable (Fig. 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 4.10, 4.11). This shows that the genetic clade of an individual is a good predictor of its morphology. The presence of two highly divergent genetic clades or evolutionary lineages is therefore in concordance with morphological differences. This led me to the conclusion that there are two separately evolving lineages in C. taivanus ssp. This view is strengthened by the observation that clade A, found only in sampling sites 1–8 from north Taiwan, occurs in parapatry to populations of clade B (Fig. 4.1). The absence of gene-flow between clade A and clade B suggested reproductive isolation of the clades (Table 4.3). The average sequence divergence in COI and 16S rRNA among clade A and B equalled 0.102 and 0.121 changes per site, respectively. Because of lack of calibration point, I have to assume that two clades diverged approximately 4.5–6.1 million years ago, if the Cyclotus species of investigation with similar mutation rate as Taiwan Cyclophorus (see chapter 3, 2.28% and 2% per million years in COI and 16S rRNA, respecitively). Most species concepts (reviewed in Hull 1997) would recognize clade A as separate species in the light of the presented evidence. However, additional evidence, like crossbreeding experiments and analyses of habitat differences, are needed to confirm the species status of the inferred clades. In order to easy describe, and by comparing genetic clade and shell traits PCA data, I will refer to all individuals with haplotype of clade A as C. adamsi, with haplotype of clade B as C. taivanus group (C. t. dilatus, C. t. diminutus, C. t. peraffinis, and C. t. taivanus) hereafter. Portions of the molecular and phenotypic variation could be shown to be due to an obviously long separation of different evolutionary lineages that I can consider as separate evolutionary entities – C. adamsi and C. taivanus group. As this was a rather unexpected result, I could not adjust the sampling scheme towards the inclusion of more populations of C. adamsi into this study. This lack of data prevented me from exploring further the spatial distribution of phenotypic variation in this taxon and to compare it to the organization in C. taivanus ssp. An initial survey of the geographical distribution of taxon, that there are no distinct differences in habitat preference between the two lineages. However, C. adamsi and C. taivanus group are always

69 parapatric but never synpatric. In the scatter plot of environmental variables PC2 and shell traits PC1, C. adamsi seems to have a good adaptation to unstable, arid warm season, moist cold season and cold temperature, but not in C. taivanus group (Fig. 4.13). Temperature may be a limited element of the distribution of C. adamsi and C. taivanus group. Opposite to C. adamsi, C. taivanus group with flatter shell inhabit in warmer and stable temperature environment. The warm and stable climate tends to have luxuriant vegetation. The flat shell would be at an advantage when roam over the ground in the luxuriant vegetation as keeled shell Cyclophorus species case (see chapter 3).

Hypothetic of C. taivanus ssp. speciation model It appears that a climatic gradient is responsible for the distribution pattern of species (Fig. 4.12). Along this cline, C. adamsi occupies only the sites with the moist winter and arid summer climate. This type of climate seems to exclude C. taivanus group. The actual level of water stress at a particular site depends strongly on the local microclimate, which may account for the observed intermingled pattern in the contact zone. Even though, the sampling population is few and limited, there is probably a cline in umbilicus proportion between populations near contact zone (Fig. 4.14). Likewise, there are more or less clines in the other eight shell traits between populations near contact zone (Fig. 4.15). Even though premating or postmating reproductive isolation may have evolved as a byproduct of ecological divergence, the ecological divergence appears sufficient to prevent immediate contact and therefore acts as an effective barrier to mating. The pattern of distribution along a climatic gradient suggests an ecotone divergence. Clines or ecotones are thought to promote the evolution of specialized adaptations in populations on either side of the border (Pfenninger et al. 2003). The speciation between C. adamsi and taivanus group may due to ecological speciation. The alternative hypothesis is due to vicariance events and secondary contact. The alternative model of speciation offer much less likely scenarios. There was no evidence indicate presenting an obstacle for dispersal around the contact zone in historical geology.

Associations between molecular and morphological variability within C. taivanus group In morphological PCA scatter plots of C. taivanus group, all of its members are partial overlap (Fig. 4.3). In particular, the external of C. t. diminutus and C. t. peraffinis are similar, and as expectation their shell trait PCA scatter plot s are overlap (Fig. 4.3). However, it is limited in gene-flow between them (Table 4.3). The results from the Mantel-tests indicated that the null hypothesis of no association between molecular

70 p-distance and overall morphological similarity in shell traits could not be rejected within C. taivanus group. This suggested that the observed shell traits differences have not evolved in concordance with the mitochondrial genome and resulting phenotypic population structure does not reflect the phylogenetic history of the populations. The significant phenotypic divergence among populations must therefore have other explanations.

Associations between the environment and morphological variability within C. taivanus group The results from PLS analysis showed that differences between populations in some traits co-varied significantly with long-term climatic conditions of the sampling site. I found precipitation is worth to affect some shell traits of C. taivanus group. Cold season precipitation and out lip proportion are significantly negative correlation. By contrast, warm season precipitation and out lip proportion are significantly positive correlation. It is the same in whorl number, sampling sites with more annual amount precipitation were associated with fewer whorl numbers. This was astonishing at first sight because a humid climate allows more feeding activity and snails should therefore attain more whorl numbers. A similar tendency of smaller snail occurs in more rainfall habitat was reviewed by Goodfriend (1986), and possible explained as a potential adaptation of sexual maturity. There is no further shell growth when Cyclotus reaches sexual maturity. To reproduce earlier in wetter cold season would offer the next generation the possibility to reach a bigger size before summer. Early reproduction could thus constitute a selective advantage because small size juveniles of most snails are high mortality in dry summer of Taiwan (experience of field work). The ecological divergence probably appears rule of speciation in C. taivanus ssp. case. The speciation process is not complete among C. t. dilatus, C. t. diminutus, C. t. peraffinis, and C. t. taivanus, and the adaptation of climatic pressure continuing is a rule of speciation process. However, further experiments are needed to determine whether the size/precipitation correlation in C. taivanus group is due to phenotypic plasticity in response to the prevailing conditions or whether it has an adaptational significance. In conclusion, I consider the phenotypic and phylogenetic divergence between the two identified lineages as sufficiently large to propose the presence of two distinct evolutionary entities as valid species of C. adamsi and C. taivanus group. The speciation process between C. adamsi and C. taivanus group is complete, as evidenced by a lack of gene-flow (Table 4.3). The reproductive isolation of two taxa may due to divergent selection on traits in different environments. In another word, the speciation model of this case is ecological speciation. However, more sampling

71 sites around the contact zone should be contained in the further study. For the subordinate members of C. taivanus, there is no resolution in phylogeny analysis of the present investigation. To resolve the relationship of the C. taivanus group, more rapid evolved molecular marker is needed in further study. Besides, the hypotheses about correlation among C. taivanus group divergence and environmental variables could be tested in further study.

72 Table 4.1 List of Cyclotus sampling sites Items Location Position 1 Guan-in Mountain, Taipei County 25.135861N, 121.428861E 2 Chih-shan-yin, Taipei City 25.104833N, 121.529889E 3 The Cao-ling Historic Trail , Iran County 24.993194N, 121.925639E 4 Kai-cheng Temple , Iran County 24.813139N, 121.710583E 5 Sheau-jiau-shi, Iran County 24.805833N, 121.704417E 6 Fwu-shan, Taipei County 24.795750N, 121.492944E 7 Ren-tzer, Iran County 24.545611N, 121.507278E 8 Dung-aw-li, Su-aw, Iran County 24.521194N, 121.824889E 9 Iriomote Island 24.349000N, 123.805000E 10 Chorng-der, Hualian County 24.189278N, 121.660889E 11 Chin-heng Bridge, Taroko valley, Hualian County 24.171167N, 121.560417E 12 Charn-guang Temple, Taroko valley, Hualian County 24.159250N, 121.606111E 13 County Highway No. 136, 39.5K, Taichung County 24.099361N, 120.790389E 14 County Highway No.129, 20.5k, Jia-yi County 23.342500N, 120.691861E 15 Tu-di-gong Temple,Guan-tzy-liing, Tainan County 23.339306N, 120.505556E 16 Provincial Highway No.20, 49k, Nan-huah, Tainan City 23.100389N, 120.481556E 17 Shuang-liou Wood Park, Ping-dung County 22.217889N, 120.803000E 18 Jang-hu, Jia-yi County 23.599333N, 120.656472E 19 Provincial Highway No.149, Yun-lin County 23.589639N, 120.569306E 20 Bau-Lai, Kaohsiung County 23.108639N, 120.699722E 21 Shan-ping, Kaohsiung County 22.965917N, 120.683944E 22 Liou-guei, Kaohsiung County 22.925222N, 120.652833E 23 Hae-shern Temple, San-dih-men, Ping-dung County 22.819972N, 120.640444E 24 Wu-tai ,Ping-dung County 22.751528N, 120.727167E 25 Wu-tai ,Ping-dung County 22.743167N, 120.706944E 26 Yongsing, Lanyu Island, Taidung County 22.028222N, 121.579667E 27 Chung-ai Bridge,Lanyu Island, Taidung County 22.009278N, 121.571861E

73 Table 4.2 Correlations between each shell traits and the corresponding environmental variables of clade B Correlation warm season cold season annual range of warm season cold season annual Shell traits annual mean mean mean monthly mean amount of amount of amount of altitude temperature temperature temperature temperature precipitation precipitation precipitation Size (H×W) -0.032 -0.337 -0.198 0.161 0.280 -0.527 -0.388 0.175 tallness/flatness (H/W) 0.026 -0.003 0.005 0.037 0.070* -0.081* -0.053* 0.033 aperture proportion -0.255 -0.454 -0.399 0.118** 0.189** -0.420* -0.359** 0.175 (AW/W) aperture shape 0.247 0.422 0.368 0.084 -0.316 0.463 0.252 -0.161* (AW/AH) umbilicus proportion 0.337 0.423 0.423 0.045 -0.218 0.398 0.275 -0.132 (UW/W) outer lip proportion -0.406 -0.599 -0.569 0.061** 0.265** -0.598** -0.512** 0.181 (LW/AW) flat degree of body 0.025 -0.130* -0.059* -0.102 0.215 -0.212* -0.093* 0.191 whorl (BH/BW) degree of whorl increasing ([W－UW -0.039 -0.307 -0.185 0.085 0.334* -0.487** -0.227** 0.226 －LW]/HN) whorl number (HN) -0.011** -0.373** -0.203* 0.234** 0.150** -0.514** -0.579** 0.107 *Indicates a significant correlation at the 5% level; **Indicates a significant correlation at the 1% level. Warm season: May, June, July, August and September. Cold season: January, February, March, November and December

74 Table 4.3 Population pairwise FSTs derived from AMOVA analysis of Cyclotus using COI and 16S rRNA data, respectively COI C. adamsi C. t. dilatus C. t. peraffinis C. t. taivanus C. t. diminutus C. adamsi (clade A) 0.00000 C. t. dilatus (clade B) 0.71136* 0.00000 C. t. peraffinis (clade B) 0.87074* 0.22362 0.00000 C. t. taivanus (clade B) 0.69307* 0.19160* 0.22257* 0.00000 C. t. diminutus (clade B) 0.95352* 0.44126* 0.78815* 0.39114* 0.00000 16S rRNA C. adamsi (clade A) 0.00000 C. t. dilatus (clade B) 0.78897* 0.00000 C. t. peraffinis (clade B) 0.92744* 0.40061* 0.00000 C. t. taivanus (clade B) 0.81128* 0.24100* 0.29710* 0.00000 C. t. diminutus (clade B) 0.94225* 0.48908* 0.98652* 0.48698* 0.00000 *Indicates a significant correlation at the 5% level.

75 Fig. 4.1 Fig. 3.1 Geographic 2 1 range of the Cyclotus 3 species complex,

6 4 different symbols 5 illustrate the sampling Ishigaki Is. sites of five taxa. Open 7 8 circle: C. taivanus adamsi; solid square: C. 9 t. dilatus; solid circle: C. 11 10 Iriomote Is. t. taivanus; solid 13 12 triangle: C. t. diminutus; X symbol: C. t. peraffinis.

19 18

15 14

16 20 21 22

23 24 25

26 27

Fig. 4.2 A–C The morphological character of Cyclotus measurements. AH = aperture height, AW = aperture width, BH = body whorl, BW = body whorl width, H = height, HN = whorl number, LW = outer lip width, UW = umbilicus width and W = width.

Fig. 4.3 Scatter plot PCA scores on the first two axes of Principal component analysis on Cyclotus shell morphological variables. Axis 1 accounts for 49.59%, axis 2 for 22.38% of total variance. Different symbols illustrate the sampling sites of five taxa show in Fig. 4.1.

15 7 14 1 36 6 Clade A

82 8 4

99 85 4 91 4 3 70 3

44 11 48 12

45 13 9, 10 7 33 22 9 1 99 23 23

2 34 20 B Clade 99 20

23 20 29 26

98 17 93 17

99 18, 22 22

95 14 7 67 15 23 24 95 25 21 99 21 Ptychopoma wilsoni 99 Ptychopoma wilsoni

Fig. 4.4 Molecular phylogeny of Cyclotus produced by maximum parsimony analysis of individual gene sequence data from COI. Relative branch support indices are given as bootstraps test consisted of 1000 iterations. Branch tip are labeled with the sampling site numbers and five taxa. Open circle: C. adamsi; solid square: C. t. dilatus; solid circle: C. t. taivanus; solid triangle: C. t. diminutus; X symbol: C. t. peraffinis.

Fig. 4.5 Molecular phylogeny of Cyclotus produced by Neighbor-joining (NJ) analysis of individual gene sequence data from COI. Relative branch support indices are given as bootstraps test consisted of 1000 iterations. Branch tip are labeled with the sampling site numbers and five taxa. Open circle: C. adamsi; solid square: C. t. dilatus; solid circle: C. t. taivanus; solid triangle: C. t. diminutus; X symbol: C. t. peraffinis.

1 4 83 Clade A Clade 63 4 85 4 6

78 7 8 3 3 11 67

79 12

96 13 9, 10 72 22 38 20 71 20 100 20 84 100 23 B Clade 23 17 87 47 17 100 26

90 14 15 90 75 24 96 25 9 87 18, 20 99 22 21 97 21

100 Ptychopoma wilsoni Ptychopoma wilsoni

0.05

Fig. 4.6 Molecular phylogeny of Cyclotus produced by maximum likelihood (ML) analysis of individual gene sequence data from COI. Relative branch support indices are given as approximate likelihood ratio test. (aLRT). Branch tip are labeled with the sampling site numbers and five taxa. Open circle: C. adamsi; solid square: C. t. dilatus; solid circle: C. t. taivanus; solid triangle: C. t. diminutus; X symbol: C. t. peraffinis.

81 1 6

8 A Clade 57 7 4 100 96 4 84 4 3 61 3 17 90 100 17 26 13 70 12 52 11 85 22 98 9, 10 18, 22 Clade B Clade 22 24 95 25 87 23 100 23 20 20 100 20 14 89 15 21 100 21 9 Ptychopoma wilsoni 100 Ptychopoma wilsoni

0.02

Fig. 4.7 Molecular phylogeny of Cyclotus produced by Bayesian analysis of individual gene sequence data from COI. Relative branch support indices are given as Bayesian probability. Branch tip are labeled with the sampling site numbers and five taxa. Open circle: C. adamsi; solid square: C. t. dilatus; solid circle: C. t. taivanus; solid triangle: C. t. diminutus; X symbol: C. t. peraffinis.

82 1 6 70 24 34 8 Clade A 79 8 7 65 99 96 3 3 5 28 22 4 82 4, 5 4

96 11 12 78 13

32 98 19 18 22 86 99 10 10

99 9 9 15 97 15 98

14 B Clade

8 16

37 23 5 21 20 28 4 99 23 23 42 24 99 51 25 99 25

99 22 22

58 17 17 99 26 97 36 26 26

99 Ptychopoma wilsoni Ptychopoma wilsoni

Fig. 4.8 Molecular phylogeny of Cyclotus produced by maximum parsimony analysis of individual gene sequence data from 16S rRNA. Relative branch support indices are given as bootstraps test consisted of 1000 iterations. Branch tip are labeled with the sampling site numbers and five taxa. Open circle: C. adamsi; solid square: C. t. dilatus; solid circle: C. t. taivanus; solid triangle: C. t. diminutus; X symbol: C. t. peraffinis.

83 1 77 8 77 6 50 8 Clade A 21 7 98 49 3 96 3 5 51 4 75 91 4, 5 4

97 11

68 12 13 71 96 19 18 87 10 98 10

99 9 9 9

99 15 98 14 Clade B Clade 16

4 51 23 21 15 23 14 99 23 55 24 99 25 91 25 22 94 99 22 17 68 17 96 26 97 26 68 26 20

100 Ptychopoma wilsoni Ptychopoma wilsoni

0.05

Fig. 4.9 Molecular phylogeny of Cyclotus produced by Neighbor-joining (NJ) analysis of individual gene sequence data from 16S rRNA. Relative branch support indices are given as bootstraps test consisted of 1000 iterations. Branch tip are labeled with the sampling site numbers and five taxa. Open circle: C. adamsi; solid square: C. t. dilatus; solid circle: C. t. taivanus; solid triangle: C. t. diminutus; X symbol: C. t. peraffinis.

84 1 8 61 8

88 6 Clade A 7 99 77 3 93 3 5

94 4, 5

76 4 4

99 9 9

96 11 12 19 96 95 18 96 93 10 25100 10 13 21 92 23 Clade B Clade 14 81 96 7 15 99 16 20 67 100 23 23 70 25 69 90 25 100 24

100 22 22 19 17

99 17

51 26

99 26 26

100 Ptychopoma wilsoni Ptychopoma wilsoni

0.05

Fig. 4.10 Molecular phylogeny of Cyclotus produced by maximum likelihood (ML) analysis of individual gene sequence data from 16S rRNA. Relative branch support indices are given as approximate likelihood ratio test. (aLRT). Branch tip are labeled with the sampling site numbers and five taxa. Open circle: C. adamsi; solid square: C. t. dilatus; solid circle: C. t. taivanus; solid triangle: C. t. diminutus; X symbol: C. t. peraffinis.

85 3 99 79 3 7

73 8 Clade A 6 8 90 1 4, 5 4 98 5 4

100 9 9 19 100 18 100 10 100 10 70 11 98 12 13 81

99 14 15 98 Clade B Clade 16 21 51 23 23 100 23 57 25 88 73 25 100 24 20 22 100 22 17

100 17 26

82 26 26 Ptychopoma wilsoni 100 Ptychopoma wilsoni

0.02

Fig. 4.11 Molecular phylogeny of Cyclotus produced by Bayesian analysis of individual gene sequence data from 16S rRNA. Relative branch support indices are given as Bayesian probability. Branch tip are labeled with the sampling site numbers and five taxa. Open circle: C. adamsi; solid square: C. t. dilatus; solid circle: C. t. taivanus; solid triangle: C. t. diminutus; X symbol: C. t. peraffinis.

Fig. 4.12 Scatter plot PCA scores on the first two axes of Principal component analysis on environmental variables for 27 sites. Correlation of variables with PCA axes is indicated by red line vectors. Sites where clade A populations were found are indicated with blue, clade B populations are indicated with brown and series green color. Axis 1 accounts for 44.03%, axis 2 for 32.42% of total variance. Different symbols illustrate the sampling sites of five taxa show in Fig. 4.1.

Fig. 4.13 Scatter plot of environmental variables PC2 and shell traits PC1 averages (± SD). Open and solid circular indicated clade A and B, respectively. Trend line is 4th degree multinomial.

Fig. 4.14 The change in umbilicus proportion of C. adamsi (blue rhombus) and C. taivanus group. (pink square) along north to south population. Spots between dotted lines are measured from individuals around the contact zone.

Fig. 4.15 The change in eight shell traits of C. adamsi (blue rhombus) and C. taivanus group. (pink square) along north to south population. Spots between dotted lines are measured from individuals around the contact zone.

90 Chapter 5

The cyclophorids fauna of Taiwan

Introduction Cyclophoridae are the dominant group of operculated terrestrial snails living in tropical and warm temperate areas of Asia, Indo-Pacific islands, Africa, Australia and Melanesia (Hyman 1967, Abbott 1989). The group consists of four subfamilies and about 300 species currently arranged in 38 genera. They occupy a range of habitats and exhibit considerable morphological diversity. Their shells are small to medium size, turbinate to depressed spire, surface smooth or with axial and spiral sculpture, umbilicus always opened, peristome duplex or simple, with ceratoid or calcareous operculum. The inner edge of the aperture may be notched respiratory purposes or the body whorl shortly behind the aperture may bear a little respiratory tube (Hyman 1967). In the previous study, there are 9 genera and 29 species in Taiwan (Lee & Wu 2001). For the cyclophorids in the adjacent area of Taiwan, there are 11 genera and 78 species in Japan (Higo & Goto 1993), 12 genera and 109 species in China (Kobelt 1902, Yen 1939). 8 genera and 42 species in Malaysia (Kobelt 1902), 7 genera and 165 species in Philippines (Kobelt 1902).

Key to genera of Cyclophoridae from Taiwan 1. The operculum is ceratoid………………………………………………...……….2 The operculum is calcareous………………………...………………………….…8 2. With periostracum hairs……………………………………..………………….…3 Without periostracum hairs…………………………...…..……………….…...….4 3. The outer lip is reflexed…………………………...…..….. Pilosphaera new genus The outer lip is not reflexed………………………....……………………. Japonia 4. With sutural tube…………………..……………………...…………………….....5 Without sutural tube……………………………...…..………………………..…..6 5. Shell high/shell breadth ≧ 1…………………………..………….…..……. Dioryx Shell high/shell breadth ＜ 1………..……………...…………….. Chamalycaeus 6. With sutural sinus………….……………………...………...…………Ptychopoma Without sutural sinus…………………………………...….………………………7 7. Shell is fragile and semitranslucent…………………...………...………Leptopoma

91 Shell is solid, thick, not translucent………………...…...…….………Cyclophorus 8. The central tooth of radula is sierras on the convex side…………...…Cyathopoma The central tooth of redula is not sierras on the convex side……………...……....9 9. Surface reticulate with spiral and/or radual cords……………...……...Platyrhaphe Surface is smooth………………….……………………...….……………Cyclotus

Genus Leptopoma Pfeiffer, 1847 1847. Leptopoma Pfeiffer, L. Übersicht aller bekannten Arten von Cyclostomaceen. Zeitschrift für Malakozoologie, 4: 47, 101-112. Type species: Cyclostoma vitrea Lesson, 1830 Diagnosis: Shell medium size, rather thin, translucent. Spire conical, apex pointed. The umbilicus is narrowly opened. The aperture is circular with extended reflex outer lip. The operculum is translucent yellow, ceratoid, subcircular, with moderately wide growing edge. Distribution: India Peninsula, Malay Peninsula, New Guinea, Taiwan, and Japan. Species included: 62 species worldwide (Kobelt 1902, Goto & Poppe 1996), 2 species in Taiwan. Key to species of Leptopoma from Taiwan 1. Having a periphery keel, with brown pattern…………………………..……L. tigris Round peripheral, white shell, without brown pattern...... …… L. nitidum

1. Leptopoma nitidum (Sowerby, 1843) (Fig. 1.1 A–C; 1.17 A) Records and synonym: 1843. Cyclostoma nitidum Sowerby. Sowerby, G.B. Proc. Zool. Soc. Lond., S: p59. 1883. Leptopoma taivanum Moellendorff. Moellendorff, O.F. Jahrbucher der Deutschen Malakozoologischen Gesellschaft Vol. 10, p287. pl. 10, fig. 4. 1890. Leptopoma taivanum Moellendorff. Schmacker, V.B. & Boettger, O. Nachrichtsblatt der Deutschen Malakozoologischen Gesellschaft, Jahrg, Vol. 22, p125. 1891. Leptopoma vitreum var. lacteal Kob. Schmacker, V.B. & Boettger, O. Nachrichtsblatt der Deutschen Malakozoologischen Gesellschaft, Jahrg, Vol. 23, p190. 1905. Leptopoma vitreum taivanum (Moellendorff). Pilsbry, H.A. & Hirase, Y. Proceedings of Academy of Natural Sciences of Philadelphia, Vol. 57, p723. 1941. Leptopoma nitidum taivanum (Moellendorff). Kuroda, T. Memoirs of the Faculty of Science and Agriculture Taihoku Imperial University, Vol. 22(4), p79, No.125. 1951. Leptopoma vitreum taivanum (Moellendorff). Hirase, S. A handbook of

92 illustrated shells in natural colors from Japanese Islands and their adjacent territories – revised and enlarged edition of “A collection of Japanese shells”. Bunkyokaku. Tokyo, Japan. pl. 78, fig. 11. 1956. Leptopoma nitidum taivanum (Moellendorff). Kuroda, T. Venus, Vol. 19(2), p135. 1963. Leptopoma perlucidum taivanum (Moellendorff). Kuroda, T. A Catalogue of the non-marine mollusks of Japan, including the Okinawa and Ogasawara Island. The Malacological Society of Japan, Tokyo. P9, No. 36. 1984. Leptopoma taivanum Moellendorff. Chang, K. M. & al. Pei-yo. Vol. 9, pl. I, fig. 1. 1988. Leptopoma taivanum Moellendorff. Lai, K.Y. The shells. Holiday Publisher. P13, fig. 5A, 5B. 1990. Leptopoma taivanum Moellendorff. Lai, K.Y. World of landsnail. Taiwan Museum, Taipei. p40. No. 4, pl. 1, fig.8. 1993. Leptopoma nitidum Sowerby. Higo, S. and Goto, Y. A Systematic List of Molluscan Shells from the Japanese Is. and the Adjacent Area. Elle Shell Publications, Japan, p.60, No. 706. 1995. Leptopoma taivanum Moellendorff. Azuma, M. Colored illustrations of the land snails of Japan – Enlarged revised edition. Hoikusha Publishing Co., Ltd. Osaka. P67, No. 9, pl. 1, fig. 9. 2001. Leptopoma nitidum taivanum Moellendorff. Lee, Y.C. & Wu, W.L. Bulletin of Malacolgy Taiwan, Vol. 25, p47-48, pl. 1, fig. 1. 2003. Leptopoma nitidum Sowerby. Lee, Y.C. & Chen, W.D. Natural observational illustrations 3－Land snail. Chin-Chin Publications Ltd. Taipei, p41. 2003. Leptopoma nitidum taivanum Moellendorff. Hsieh, B.C. Landsnails of Taiwan. Council of Agriculture Executive Yuan, p62. 2004. Leptopoma nitidum taivanum Moellendorff. Hsieh, B.C. Incredible snails, Yuan-Liou Publishing Co., Ltd., p92. 2006. Leptopoma nitidum taivanum Moellendorff. Hsieh, B.C. & al. Landsnails of Taiwan. Forestry Bureau Council of Agriculture Executive Yuan, p56. Shell: shell width 14-16mm, height 13.5-16mm, 5.25-6 whorls, rather rapidly increasing, having a rounded periphery, white and translucent shell, rather thin, but thinker in population of Taroko valley region. It looks green when it is alive, because of the green soft part. Spire conical, apex pointed. Surface is sculptured with irregular growth lines. The umbilicus is narrowly opened. The aperture is circular with extended reflex outer lip. The operculum is translucent yellow, ceratoid, subcircular, with moderately wide growing edge. Radula: radula is taenioglossa. Radula format is 1-2-1-2-1. The central tooth is

93 scoop-shaped, 5 plate cuspids, central cuspid is the largest, approximately 1/2 width of central tooth; and the marginal cuspids are the smallest. The lateral teeth are the same shape, scoop-shape, 4 plate cuspids, the inner 2 cuspids are small and equal size, the 3rd cuspid is the largest, approximately 1/2 width of lateral tooth, the most outer cuspid is the smallest. The marginal teeth are sickle-like shaped, 3 plate cuspids, the central cuspid is the largest, semicircle in shape. Distribution: Range north from Tokunoshima, Japan south to Philippines. North Taiwan: Taipei county, Da-shi, Iran County; South Taiwan: Shan-Hua, Tainan County; Lung-chi, Tainan City; Kaohsiung; Liou-guei, Kaohsiung County; Chau-jou, Ping-dung County; Heng-Chueng, Ping-dung County; South cape; East Taiwan: Hualian County; Taroko valley, Hualian County. Habitat: it is arboreal snail, always attach to trunks and leaves.

2. Leptopoma tigris Lee & Wu, 2001 (Fig. 1.1 D–L; 1.17 B) 1932. Leptopoma (Trocholeptopoma) tigris Kuroda & Kano. Kuroda, T. Bulletin of the Biogeographical Society of Japan. Vol. 3(1), p3, No. 1. 1932. Leptopoma vitreum taivanum (Moellendorff). Kuroda, T. Venus, Vol. 3(4), p188. 1941. Leptopoma (Trocholeptopoma) tigris Kuroda & Kano. Kuroda, T. Memoirs of the Faculty of Science and Agriculture Taihoku Imperial University, Vol. 22(4), p80, No.126. 1941. Leptopoma tigris var. callizona, decolorata, millepuncata Kuroda & Kano. Kuroda, T. Memoirs of the Faculty of Science and Agriculture Taihoku Imperial University, Vol. 22(4), p80, No.126. 1956. Leptopoma tigris Kuroda & Kano. Kuroda. T. Venus, Vol. 19 (2). P135. 1974. Leptopoma (Serlucidum) nitidum taivanum Moellendorff. Lin. C.C. Bulletin of the Malacological Society of China. Vol. I, p45, No. 49. 1974. Leptopoma (Trocholeptopoma) tigris Kuroda & Kano, Lin. C.C. Bulletin of the Malacological Society of China. Vol. I, p45, No. 50, 1984. Leptopoma tigris Kliroda & Kano. Chang, K.M. & al. Pei-yo. Vol. 9, p3. No. 5. 1990. Leptopoma tigris Kuroda & Kano. Lai, K.Y. World of landsnail. Taiwan Museum, Taipei. p40. No. 5. 1993. Leptopoma (Trocholeptopoma) tigris Kuroda & Kano. Higo, S. & Goto, Y. A Systematic List of Molluscan Shells from the Japanese Is. and the Adjacent Area. Elle Shell Publications, Japan, p60, No. 707. 2001. Leptopoma tigris Lee & Wu. Lee, Y.C. & Wu, W.L. Bulletin of Malacolgy Taiwan, Vol. 25, p48-49, pl. 1, fig. 2. 2003. Leptopoma tigris Lee & Wu. Lee, Y.C. & Chen, W.D. Natural observational illustrations 3－Land snail. Chin-Chin Publications Ltd. Taipei, p42.

94 2003. Leptopoma tigris Kuroda & Kano. Hsieh, B.C. Landsnails of Taiwan. Council of Agriculture Executive Yuan, p63. 2004. Leptopoma tigris Kuroda & Kano. Hsieh, B.C. Incredible snails, Yuan-Liou Publishing Co., Ltd., p93. 2006. Leptopoma tigris Lee & Wu. Hsieh, B.C. & al. Landsnails of Taiwan. Forestry Bureau Council of Agriculture Executive Yuan, p57. Shell: shell width 10.3mm, height 10.3mm, about 5 whorls, rather rapidly increasing, having a periphery keel surface reticulate with irregular spiral and axial cords, the spiral cords are rougher than the axial one, white and translucent shell, with various reddish to pale brown spiral bands or zigzag pattern on the shell or without any pattern, rather thin, spire conical, apex pointed. Surface is sculptured with irregular growth lines. The umbilicus is narrowly opened. The aperture is subcircular with reflex outer lip. The operculum is translucent yellow, ceratoid, subcircular, with moderately wide growing edge. Radula: radula is taenioglossa. Radula format is 1-2-1-2-1. The central tooth is scoop-shaped (more concave than L. nitidum), 5 plate cuspids, central cuspid is the largest, approximately 1/2 width of central tooth; and the marginal cuspids are the smallest. The lateral teeth are the same, scoop-shape, 4 plate cuspids, the inner 2 cuspids are small and equal size, the 3rd cuspid is the largest, approximately 1/2 width of lateral tooth, the most outer cuspid is the smallest. The marginal teeth are sickle-like shaped, 3 plate cuspids, the central cuspid is the largest, elongated semicircle in shape. Distribution: Endemic to Taiwan. East Taiwan: Lan-yu, Liu-dau, Tai-dong County. Habitat: animals always attach to trunks.

Genus Japonia Gould, 1859 1859. Japonia Gould, Descriptions of shells collected in the North Pacific Exploring Expedition under Captains Ringgold and Rodgers. Proceeding of Boston Society of Natural History, 6: 422–426. Type species: Cyclostoma barbata Gould, 1859 Diagnosis: Shell tiny, turbinate. Shell is always festucine to reddish brown in color. Surface is sculptured with some spiral cords and irregular growth line. And cover with regular periostracum hairs. Umbilicus opened. The aperture is circular. The lip is not reflex. The operculum is a rnultispiral type, ceratoid, translucent, a little concave. Distribution: from Japan, Taiwan, Mainland China, the Philippines to India. Species included: 33 species worldwide (Kobelt 1902, Goto & Poppe 1996), 4 species in Taiwan.

95 Key to species of Japonia from Taiwan 1. The periostracum hairs are spoon like…………………...……………J. formosana The periostracum hairs are hair like……………...……………………………..….2 2. Peripheral periostracum hairs are 3–4 times longer than basal periostracum hairs…………………………………………………...……………J. boonkioensis Peripheral periostracum hairs are 2–3 times longer than basal periostracum hairs……………………………………………………………………J. lanyuensis

3. Japonia formosana Pilsbry & Hirase, 1906 (Fig. 1.2 A–C; 1.17 C) Records and Synonym: 1905. Japonia formosana Pilsbry & Hirase. Pilsbry, H.A. & Hirase, Y. Proceedings of Academy of Natural Sciences of Philadelphia, Vol. 57. p723. 1941. Japonia formosana Pilsbry & Hirase. Kuroda, T. Memoirs of the Faculty of Science and Agriculture Taihoku Imperial University, Vol. 22(4). p80. No. 127. 1956. Japonia formosana Pilsbry & Hirase. Kuroda, T. Venus, Vol. 19(2). p136. 1984. Japoniclfoi'mosclftti Pilsbry & Hirase. Chang, K.M. & al. Pei-yo. Vol. 9, p3. No. 6. 1990. Japonia formosana Pilsbry & Hirase. Lai. K.Y. World of landsnail. Taiwan Museum, Taipei. p40, No. 6. 1993. Japonia formosana Pilsbry & Hirase. Higo. S. & Goto. Y. A Systematic List of Molluscan Shells from the Japanese Is. and the Adjacent Area. Elle Shell Publications, Japan, p60. No. 708. 2001. Japonia formosana Pilsbry & Hirase. Lee, Y.C. & Wu, W.L. Bulletin of Malacolgy Taiwan, Vol. 25, p49-50, pl. 1, fig. 3. 2003. Japonia formosana Pilsbry & Hirase. Lee, Y.C. & Chen, W.D. Natural observational illustrations 3－Land snail. Chin-Chin Publications Ltd. Taipei, p43. Shell: shell width 5-5.5mm, height 4.5-5mm, 5 whorls, turbinate, round periphery. Shell is reddish brown to dark brown in color. Surface is sculptured with irregular growth lines and spiral discontinuous threads, which are 9-10 each whorl and 10-14 on base. There are regular spoon like periostracum hairs at periphery Umbilicus opened, about 1/7.5 of the shell diameter. The aperture is circular. The lip is not reflex. The operculum is translucent ceratoid, a little concave center, multispiral type with very thin pellucid edge. Radula: radula is taenioglossa. Radula format is 1-2-1-2-1. The central tooth is scoop-shaped, 5-7 cuspids, they gently reduce their size from inner to outer site. The lateral teeth are almost the same, scoop-shape, 4 cuspids, the inner cuspid are the smallest, the 3rd cuspid is the largest, approximately 1/2 width of lateral tooth,

96 the 2nd and 4th cuspids are equal size. The marginal teeth are sickle-like shaped, 3 cuspids, the central cuspid is the largest, the inner is the smallest. Distribution: Endemic to Taiwan. North Taiwan: Ju-dung, Shin-ju County; Miau-li County. South Taiwan: Jia-Siang Kaohsiung County; Heng-chueng, Ping-dung County. Habitat: live under the defoliation or on the ground.

4. Japonia lanyuensis Lee & Wu, 2001 (Fig. 1.2 D–F; 1.17 D) Records and Synonym: 1941. Japonia formosana Pilsbry & Hirase. Kuroda, T. Memoirs of the Faculty of Science and Agriculture Taihoku Imperial University, Vol. 22(4). p80. No. 127. (miss identification) 1963. Japonia formosana Pilsbry & Hirase. Reigle, N.J. Quarterly Journal of the Taiwan Museum, Vol. 16 (1, 2), p85. (miss identification) 1974. Japonia formosana Pilsbry & Hirase. Lin. C.C. Bulletin of the Malacological Society of China. Vol. I, p45, No. 51. (miss identification) 2001. Japonia lanyuensis Lee & Wu. Lee, Y.C. & Wu, W.L. Bulletin of Malacolgy Taiwan, Vol. 25, p50-51, pl. 1, fig. 5. 2003. Japonia lanyuensis Lee & Wu. Lee, Y.C. & Chen, W.D. Natural observational illustrations 3－Land snail. Chin-Chin Publications Ltd. Taipei, p44. 2006. Japonia lanyuensis Lee & Wu. Hsieh, B.C. & al. Landsnails of Taiwan. Forestry Bureau Council of Agriculture Executive Yuan, p60. Shell: shell width 4-4.5mm, height 3.7-4mm, 4.25-4.5 whorls, turbinate. Shell is festucine or reddish color. Surface is sculptured with spiral cords, two distinct ones at periphery. There are periostracum hairs on each cord. They are longer at periphery than others. There are several growth lines between two axial periostracum threads. Umbilicus opened, about 1/7 of the shell diameter. The aperture is circular. The lip is not reflex. The operculum is a multispiral type, ceratoid, translucent, cream color, a little concave. Radula: radula is taenioglossa. Radula format is 1-2-1-2-1. The central tooth is scoop-shaped, 5-11 cuspids, they gently reduce their size from inner to outer site. The lateral teeth are scoop-shape, 4 major cuspids, few with minor cuspid between the majors. The inner lateral teeth with a very large cuspid place at the central site, but not present on outer lateral teeth. The marginal teeth are sickle-like shaped, 3 cuspids, the central cuspid is the largest, the inner is the smallest. Distribution: South East Taiwan: Lan-Yu. Habitat: live under the defoliation or on the ground. Remark: This species probably misidentified as Japonia formosana by Kuroda in

97 1941

5. Japonia boonkioensis Lee, Lue et Wu, 2008 (Fig. 1.2 G–I; 1.17 E) 2003. Japonia formosana Pilsbry & Hirase. Hsieh, B.C. Landsnails of Taiwan. Council of Agriculture Executive Yuan, p64. 2004. Japonia formosana Pilsbry & Hirase. Hsieh, B.C. Incredible snails, Yuan-Liou Publishing Co., Ltd., p94. 2006. Japonia formosana Pilsbry & Hirase. Hsieh, B.C. & al. Landsnails of Taiwan. Forestry Bureau Council of Agriculture Executive Yuan, p58. 2008. Japonia boonkioensis Lee, Lue et Wu. Lee, Y.C., Lue, K.Y. & Wu, W.L. Zootaxa, 1972: 22-38. Shell: Shell small, 4.47mm in length and 4.7mm in width. Shell turbinate and conical-globe shape, with five moderately convex whorls. Shell is red brown color, somewhat pale at peripheral. Surface sculpture of 6–10 indistinct spiral veins crossed by finer growth lines, rendering them somewhat crispate and the interstices minutely plicaulate. Shell surface covers with red brown slight shining periostracum and irregular periostracum lamella. There are 2 row regular periostracum hairs between the sutures, one furnished on the position of shoulder, one on the peripheral site. There are 7 rows periostracum hairs under the peripheral, they longer its length from umbilicus side to the peripheral side. The shoulder and peripheral periostracum hairs are 3–4 times longer than basal periostracum hairs. The periostracum hair is tapering tip. Umbilicus opened. The aperture is circular. The outer lip is not reflexed. The operculum is translucent ceratoid, a little concave center, multispiral type with very thin pellucid edge. There is an orange red proboscis between two bluish gray tentacles on the head, gray food cover with two pieces of dark gray lobe. (Fig. 7D) Radula: radula is taenioglossa. Radula format is 1-2-1-2-1. The central tooth is scoop-shaped, 5-7 cuspids, they gently reduce their size from inner to outer site. The lateral teeth are scoop-shape, 4 major cuspids, few with minor cuspid between the majors. The inner lateral teeth with a very large cuspid place at the central site, but not present on outer lateral teeth. The marginal teeth are sickle-like shaped, 3 cuspids, the central cuspid is the largest, the inner is the smallest. Distribution: Fen-chi-hu in Jia-i County, central Taiwan, 1400 meters in altitude, gathered from grass slope under leaves. Habitat: live under the defoliation or on the ground.

Genus Pilosphaera Lee, Lue et Wu, 2008 2008. Pilosphaera Lee, Lue & Wu, Molecular evidence for a polyphyletic genus

98 Japonia (Architaenioglossa: Cyclophoridae) and with the description of a new genus and two new species. Zootaxa, 1972: 22-38. Type species: Japonia zebra Pilsbry & Hirase, 1906 Diagnosis: Shell small, turbinate, conical-globe shape, with convex shell whorls. Shell is always festucine color with reddish brown longitudinal stripes. Surface is sculptured with some spiral cords and irregular growth lines, above these furnished with several row regular periostracum hairs. Umbilicus opened. The aperture is circular, outer lip reflexed. The operculum is ceratoid, translucent, a little concave, multispiral type with very thin pellucid edge. Distribution: East China and Taiwan. Species included: 2 species worldwide (Lee et al. 2008), 1 species in Taiwan.

6. Pilosphaera zebra (Pilsbry & Hirase, 1906) (Fig. 1.2 H–J; 1.17 F) Records and Synonym: 1905. Japonia zebra Pilsbry & Hirase. Pilsbry, H.A. & Hirase, Y. Proceedings of Academy of Natural Sciences of Philadelphia, Vol. 57. p722. 1941. Japonia zebra Pilsbry & Hirase. Kuroda, T. Memoirs of the Faculty of Science and Agriculture Taihoku Imperial University, Vol. 22(4). p80. No. 128. 1956. Japonia zebra Pilsbry & Hirase. Kuroda, T. Venus, Vol. 19 (2). P136. 1984. Japonia zebra Pilsbry & Hirase. Chang, K.M. & al. Pei-yo. Vol. 9. p3, No. 7. 1990. Japonia zebra Pilsbry & Hirase. Lai. K.Y. World of landsnail. Taiwan Museum, Taipei. p40. No. 7. 1993. Japonia zebra Pilsbry & Hirase. Higo. S. & Goto. Y. A Systematic List of Molluscan Shells from the Japanese Is. and the Adjacent Area. Elle Shell Publications, Japan, p.60, No. 709. 2001. Japonia zebra Pilsbry & Hirase. Lee, Y.C. & Wu, W.L. Bulletin of Malacolgy Taiwan, Vol. 25, p51, pl. 1, fig. 6. 2003. Japonia zebra Pilsbry & Hirase. Lee, Y.C. & Chen, W.D. Natural observational illustrations 3－Land snail. Chin-Chin Publications Ltd. Taipei, p45. 2003. Japonia zebra Pilsbry & Hirase. Hsieh, B.C. Landsnails of Taiwan. Council of Agriculture Executive Yuan, p65. 2004. Japonia zebra Pilsbry & Hirase. Hsieh, B.C. Incredible snails, Yuan-Liou Publishing Co., Ltd., p95. 2006. Japonia zebra Pilsbry & Hirase. Hsieh, B.C. & al. Landsnails of Taiwan. Forestry Bureau Council of Agriculture Executive Yuan, p59. 2008. Pilosphaera zebra (Pilsbry & Hirase, 1906). Lee, Y.C., Lue, K.Y. & Wu, W.L. Zootaxa, 1972: 22-38. Shell: shell width 6.2-7.5mm, height 6.5-7.8mm, 4.7 whorls, turbinate. Shell is

99 festucine with reddish brown zigzag radial stripes and maculates. Surface sculptured with 5-6 major spiral cords per whorl and 3-4 secondary spiral cords between major spiral cords. There regular periostracum hairs on each cord. Umbilicus opened. The aperture is circular. The lip is gently reflex. The operculum is multispiral type, ceratoid, translucent, cream color, a little concave. Radula: radula is taenioglossa. Radula format is 1-2-1-2-1. The central tooth is scoop-shaped, 5-7 cuspids, they gently reduce their size from inner to outer site. The lateral teeth are scoop-shape, 4 cuspids. The inner lateral teeth with a very large 3rd cuspid place at the central site, approximately 1/2 width of lateral tooth. The 2nd and 3rd cuspids of lateral teeth are largest and almost equal shape, the other 2 are smaller. The marginal teeth are sickle-like shaped, 3 cuspids, the central cuspid is the largest, the inner is the smallest. Distribution: North Taiwan: Nei-Hu, Taipei City; Wai-Cha-Jiau, San-Shia, Taipei County; Ju-dung, Shin-ju County. Central Taiwan: Tian-Luen, Her-Ping township, Taichung County; Ruei-lung waterfall, Ju-shan, Nan-tou County. Habitat: live under the defoliation or on the ground.

Genus Ptychopoma Moellendorff, 1885 1885. Ptychopoma Moellendorff, O. Materialien zur Fauna von China. Die Auriculaceen. Jahrbücher der Deutschen Malakozoologischen Gesellschaft. Vol. 12, p362 Type species: Cyclophorus chinensis Moellendorff, 1875 Diagnosis: Shell medium to large size, rather rapidly increasing. The suture is deep. Yellowish brown, with reddish brown zigzag radial stripes, usually with an interrupted reddish brown band at the peripheral of body whorl. Spire is very depressed. The body whorl close to the aperture is sharply descending and greatly inclined. The umbilicus is wide, perspective showing the upper whorls. The aperture is circular with duplicate peristome. The ceratoid operculum is a multispiral type, dark brown, with spiral lamina and reflex edge, with a nipple inside. Distribution: China, Cambodia and Taiwan. Species included: 24 species worldwide (Kobelt 1902, Goto & Poppe 1996), 1 species in Taiwan.

7. Ptychopoma wilsoni (Pfeiffer, 1865) (Fig. 1.3 A–F; 1.18 A) Records and Synonym: 1865. Pterocyclos wilsoni Pfeiffer. Pfeiffer, L. Proc. Zool. Sci. London. P831, No 13, Pl. XLVI, Fig. 12.

100 1905. Ptychopoma wilsoni (Pfeiffer). Pilsbry, H.A. & Hirase, Y. Proceedings of Academy of Natural Sciences of Philadelphia, Vol. 57. p725. 1941. Ptychopoma wilsoni (Pfeiffer). Kuroda, T. Memoirs of the Faculty of Science and Agriculture Taihoku Imperial University, Vol. 22(4). p80. No. 129. 1956. Ptychopoma wilsoni (Pfeiffer). Kuroda, T. Venus, Vol. 19 (2). P136. 1984. Pterocyclos wilsoni Pfeiffer. Chang, K.M. & al. Pei-yo. Vol. 9. p21, PI. I, Fig. 4. 1988. Pterocyclos wilsoni Pfeiffer. Lai, K.Y. The shells. Holiday Publisher. PI 2. Fig. 4A. 4B. 1990. Pterocyclos wilsoni Pfeiffer. Lai. K.Y. World of landsnail. Taiwan Museum, Taipei. p40. No. 12, PI. 1. Fig. 5, 6. 1993 Ptychopoma wilsoni (Pfeiffer). Higo. S. & Goto. Y. A Systematic List of Molluscan Shells from the Japanese Is. and the Adjacent Area. Elle Shell Publications, Japan, p.61, No. 722. 2001. Ptychopoma wilsoni (Pfeiffer). Lee, Y.C. & Wu, W.L. Bulletin of Malacolgy Taiwan, Vol. 25, p52, pl. 1, fig. 7. 2003. Pterocyclos wilsoni Pfeiffer. Lee, Y.C. & Chen, W.D. Natural observational illustrations 3－Land snail. Chin-Chin Publications Ltd. Taipei, p46. 2003. Pterocyclos wilsoni Pfeiffer. Hsieh, B.C. Landsnails of Taiwan. Council of Agriculture Executive Yuan, p70-71. 2004. Pterocyclos wilsoni Pfeiffer. Hsieh, B.C. Incredible snails, Yuan-Liou Publishing Co., Ltd., p98. 2006. Pterocyclos wilsoni Pfeiffer. Hsieh, B.C. & al. Landsnails of Taiwan. Forestry Bureau Council of Agriculture Executive Yuan, p68. Shell: shell width 21-22mm. height 9-10mm, 4.7 whorls, rather rapidly increasing. The suture is deep. Yellowish brown, with reddish brown zigzag radial stripes, usually with an interrupted reddish brown bend at the peripheral of body whorl. Spire is very depressed. The body whorl close to the aperture is sharply descending and greatly inclined. The umbilicus is wide, 3/7 of shell width approximate and perspective showing the upper whorls. The aperture is circular with duplicate peristome. The outer lip, which has reflex and evolute edge is notched near suture, the inner has a thick edge and is slightly reflex. The parietal lip is attached to the last whorl. The ceratoid operculum is a multispiral type, dark brown, with spiral lamina and a reflex edge, with a nipple inside. The albino individual is frequently found in some population of this species. Radula: radula is taenioglossa. Radula format is 1-2-1-2-1. The central tooth is scoop-shaped, 5 cuspids, central cuspid is the largest, approximately 1/3 width of central tooth; the other cuspids gently reduce their size from inner to outer site. The lateral teeth are the same, scoop-shape, 4 cuspids, the inner and outer cuspids

101 are small and equal size, the 3rd cuspid is the largest. The marginal teeth are sickle-like shaped, 3 cuspids, the central cuspid is the largest. Distribution: Endemic to Taiwan. North Taiwan: Guan-shi, Shin-ju County; Ju-dung. Shin-ju County; Niau-tzuel Mountain, Miau-li County; Alishan, Jia-yi County. Habitat: live under the defoliation or on the ground.

Genus Cyclophorus Montfort, 1810 1810. Cyclophorus Montfort, P.D. de. Conchyliologie systematique & classification méthodique des coquilles, etc., 2 Bd., Couch. Syst. 2: 290 Type species: Helix volvulus Müller, 1774 Diagnosis: Surface is sculptured with irregular growth lines. Shell is yellowish brown in color, with reddish brown zigzag interlard with several brown bands. The aperture is circular with subduplicate penstome. The lip is reflex in a mature individual. The operculum is a multispiral type, ceratoid, translucent yellow, with an indistinct nipple inside. Distribution: India, Sri Lanka, Rangoon area, Thailand, Vietnam. Malaya Peninsula. Philippines, Sumatra, Taiwan, and Japan. Species included: 185 species worldwide (Kobelt 1902, Goto & Poppe 1996), 5 taxa groups in Taiwan. Key to species of Cyclophorus from Taiwan 1. With peripheral keel……………………...………………..………………………2 Without peripheral keel…………………………...…………………………..……4 2. With spiral cords on the shell surface………………...……….………C. friesianus With spiral cords on the shell surface………………………………………………3 3. With a keel near the umbilicus………………...…………………. C. moellendorffi Without a keel near the umbilicus…………………...………….……C. cf. turgidus 4. The umbilicus is about 1/8 of shell diameter……………...….……C. formosaensis The umbilicus is about 1/5.4–1/5.1 of shell diameter…………………...…. C. latus

8. Cyclophorus formosaensis Nevill, 1881 (Fig. 1.4 A–L; 1.18 B) Records and Synonym: 1881. Cyclophorus formosaensis Nevill. Nevill, G. Journal Asiatic Society of Bengal. Vol. L. Part II, p148. 1882. Cyclophorus formosaensis Nevill. Moellendorff, O.F. Jahrbucher der Deutschen Malakozoologischen Gesellschaft IX, p277. 1905. Cyclophorus formosaensis Nevill. Pilsbry, H.A. & Hirase, Y. Proceedings of Academy of Natural Sciences of Philadelphia, Vol. 57. p722. 1936. Cyclophorus formosaensis Nevill. Taketani, M. Trans. Nat. Hisi. Soc. Formos.,

102 Vol. 26 (154), p275, No. 13. 1941. Cyclophorus formosaensis Nevill. Kuroda, T. Memoirs of the Faculty of Science and Agriculture Taihoku Imperial University, Vol. 22(4). p80. No. 130. 1956. Cyclophorus formosaensis Nevill. Kuroda. T. Venus, Vol. 19 (2), pl37. 1984. Cyclophorus formosensis Nevill. Chang, K.M. & al. Pei-yo. Vol. 9, p21, pl. I, Fig. 2. 1988. Cyclophorus formosensis Nevill. Lai, K.Y. The shells. Holiday Publisher, p12, Fig. 1A, 1B 1990. Cyclophorus formosensis Nevill. Lai, K.Y. World of landsnail. Taiwan Museum, Taipei. p40, No. 8, pl. 1, Fig. 1, 4. 1993. Cyclophorus formosaensis Nevill. Higo. S. & Goto. Y. A Systematic List of Molluscan Shells from the Japanese Is. and the Adjacent Area. Elle Shell Publications, Japan, p.61, No. 723. 2001. Cyclophorus formosensis Nevill. Lee, Y.C. & Wu, W.L. Bulletin of Malacolgy Taiwan, Vol. 25, p53, pl. 2, fig. 8. 2003. Cyclophorus formosensis Nevill. Lee, Y.C. & Chen, W.D. Natural observational illustrations 3－Land snail. Chin-Chin Publications Ltd. Taipei, p47. 2003. Cyclophorus formosensis Nevill. Hsieh, B.C. Landsnails of Taiwan. Council of Agriculture Executive Yuan, p66. 2004. Cyclophorus formosensis Nevill. Hsieh, B.C. Incredible snails, Yuan-Liou Publishing Co., Ltd., p96. 2006. Cyclophorus formosensis Nevill. Hsieh, B.C. & al. Landsnails of Taiwan. Forestry Bureau Council of Agriculture Executive Yuan, p62. Shell: shell width 20-21.2mm, height 17.2-19.3mm, 4.25-4.5 whorls, rapidly increasing. There are 2.5-2.75 purplish to reddish brown protoconch whorls which is sculptured with regular axial cords. The apex is domal. Surface is sculptured with irregular growth lines. It has an obscure, hardly perceptible peripheral keel and very weak angular umbilicus edge at the early part of body whorl, but round periphery at the other part of body whorl, narrow umbilicus, about 1/10 of the shell diameter. Shell is yellowish brown in color, with reddish brown zigzag radial stripe and a dark brown major band just below the periphery. There are also several dark to reddish brown bands on the base or above the periphery of the late part of body whorl. These bands are sometimes interrupted or fuse together to become a wider band. The aperture is circular with subduplicate peristome, and varies from white to reddish tint. The lip is reflex when matured. The operculum is a multispiral type, ceratoid, translucent yellow, a little concave externally, with an indistinct nipple inside.

103 Radula: radula is taenioglossa. Radula format is 1-2-1-2-1. The central tooth is scoop-shaped, 5 cuspids, central cuspid is the largest, approximately 1/3 width of central tooth; the other cuspids gently reduce their size from inner to outer site. The inner lateral teeth are scoop-shape, 4 cuspids, the 3rd cuspid is the largest. The outer lateral teeth are large cuspid on the central site, with 1-2 cuspids on the outer side and 1 cuspid on the inner side. The marginal teeth are sickle-like shaped, 3 cuspids, the central cuspid is the largest. Distribution: Endemic to Taiwan. North Taiwan: Keelung City; Taipei County; Wu-lai, Taipei County; Nei-dung Woods Park; San-shya, Taipei County; Guan-shi, Shin-ju County; Miau-li County; Su-au, Iran County. East Taiwan: Taroko valley, Hualian County; Chorng-der, Hualian County; Torng-men, Hualian County. Habitat: live under the defoliation or on the ground.

9. Cyclophorus friesianus Moellendorff, 1883 (Fig. 1.5 A–L; 1.18 C) Records and Synonym: 1883. Cyclophorus friesianus Moellendorff. Moellendorff, O.F. Jahrbucher der Deutschen Malakozoologischen Gesellschaft Vol. 10, p286. 1891. Cyclophorus friesianus Moellendorff. Schmacker, V.B. & Boettger, O. Nachrichtsblatt der Deutschen Malakozoologischen Gesellschaft, Vol. 23, p191. 1905. Cyclophorus friesianus Moellendorff. Pilsbry, H.A. & Hirase, Y. Proceedings of Academy of Natural Sciences of Philadelphia, Vol. 57. p722. 1936. Cyclophorus friesianus Moellendorff. Taketani, M. Trans. Nat. Hisi. Soc. Formos., Vol. 26 (154), p275, No. 13. 1941. Cyclophorus friesianus Moellendorff. Kuroda, T. Memoirs of the Faculty of Science and Agriculture Taihoku Imperial University, Vol. 22(4). p80. No. 132. 1951. Cyclophorus interioris Pilsbry & Hirase, MS. Hirase, S. A handbook of illustrated shells in natural colors from Japanese Islands and their adjacent territories – revised and enlarged edition of “A collection of Japanese shells”. Bunkyokaku. Tokyo, Japan. pl. 78, fig. 14. 1956. Cyclophorus friesianus Moellendorff. Kuroda. T. Venus, Vol. 19 (2), pl37. 1984. Cyclophorus friesianus Moellendorff. Chang, K.M. & al. Pei-yo. Vol. 9, p4, No. 11. 1990. Cyclophorus friesianus Moellendorff. Lai, K.Y. World of landsnail. Taiwan Museum, Taipei. p40, No. 10. 1993. Cyclophorus friesianus Moellendorff. Higo. S. & Goto. Y. A Systematic List of Molluscan Shells from the Japanese Is. and the Adjacent Area. Elle Shell Publications, Japan, p.61, No. 725. 2001. Cyclophorus friesianus Moellendorff. Lee, Y.C. & Wu, W.L. Bulletin of

104 Malacolgy Taiwan, Vol. 25, p53-54, pl. 2, fig. 9. 2003. Cyclophorus friesianus Moellendorff. Lee, Y.C. & Chen, W.D. Natural observational illustrations 3－Land snail. Chin-Chin Publications Ltd. Taipei, p48. 2003. Cyclophorus friesianus Moellendorff. Hsieh, B.C. Landsnails of Taiwan. Council of Agriculture Executive Yuan, p67. 2006. Cyclophorus friesianus Moellendorff. Hsieh, B.C. & al. Landsnails of Taiwan. Forestry Bureau Council of Agriculture Executive Yuan, p60-61. Shell: shell width 20-23mm, height 17-20mm, 4.5-4.75 whorls. Spire is depressed. There are 2-2.25 purplish to reddish brown Protoconch whorls which is sculptured with obscure axial cords. Surface is sculptured with irregular growth lines and interrupt spiral cords from dorsum to base. It has a distinct peripheral keel and angular umbilicus edge. Umbilicus narrowly opened, about 1/10 of the shell diameter. Shell is yellowish brown in color, with reddish to black brown zigzag radial stripes on dorsum and several interrupted bands around base. The aperture is circular with subduplicated peristome. The lip is reflex in mature individual. The operculum is a multispiral type, ceratoid, translucent yellow, concave externally, with a nipple inside. Radula: radula is similar to Cyclophorus formosaensi. There is no distinct difference between these two species. Distribution: Endemic to Taiwan. South Taiwan: Lung-shi, Tainan City; Nan-huah, Tainan City; Guan-tzy-liing, Tainan County; Chau-jou, Ping-dung County; San-dih-men, Ping-dung County; Kaohsiung; Chi-shan, Kaohsiung County; Bau-Lai, Kaohsiung County; Liou-guei, Kaohsiung County; Mau-lin, Kaohsiung County. Habitat: live under the defoliation or on the ground.

10. Cyclophorus latus (Kuroda, 1941) (Fig. 1.6 A–L, 1.7A–L; 1.18 D–E) Records and Synonym: 1941. Cyclophorus friesianus latus Kuroda. Kuroda, T. Memoirs of the Faculty of Science and Agriculture Taihoku Imperial University, Vol. 22(4), p80, No. 132-a, p177-178. 1956. Cyclophorus friesianus latus Kuroda. Kuroda. T. Venus, Vol. 19 (2), pl37. 1984. Cyclophorus friesianus latus Kuroda. Chang, K.M. & al. Pei-yo. Vol. 9, p4, No. 12. 1990. Cyclophorus friesianus latus Kuroda. Lai, K.Y. World of landsnail. Taiwan Museum, Taipei. p40, No. 11. 1993. Cyclophorus friesianus latus Kuroda. Higo. S. & Goto. Y. A Systematic List of

105 Molluscan Shells from the Japanese Is. and the Adjacent Area. Elle Shell Publications, Japan, p.61, No. 726. 2001. Cyclophorus latus (Kuroda). Lee, Y.C. & Wu, W.L. Bulletin of Malacolgy Taiwan, Vol. 25, p54-55, pl. 2, fig. 10. 2003. Cyclophorus latus (Kuroda). Lee, Y.C. & Chen, W.D. Natural observational illustrations 3－Land snail. Chin-Chin Publications Ltd. Taipei, p49. 2003. Cyclophorus friesianus latus Kuroda. Hsieh, B.C. Landsnails of Taiwan. Council of Agriculture Executive Yuan, p67. 2006. Cyclophorus friesianus latus Kuroda. Hsieh, B.C. & al. Landsnails of Taiwan. Forestry Bureau Council of Agriculture Executive Yuan, p61. Shell: shell width 22-27mm, height 17.3-21.3mm, 4.5-4.8 convex whorls, turbinate and depressed at the top. Spire is depressed, with 2.5 purplish to reddish brown protoconch whorls sculptured with regular axial cords. The wide umbilicus is about 1/8-1/7 of the shell diameter. The peripheral keel is obscure. The aperture is circular with subduplicate peristome in very old individuals. The lip is reflex in mature individuals. The operculum is a multispiral type, ceratoid, translucent yellow, a little concave outside, with an indistinct nipple inside. Radula: radula is similar to Cyclophorus formosaensi. There is no distinct difference between these two species. Distribution: Endemic to Taiwan. North Taiwan: Dar-Guan Mountain, Tau-Yuan County; Wu-lai, Taipei County; Dong-shan, Iran County; Dah-jiau-shi, Iran County. Central Taiwan: Tou-bian-keng, Tai-ping City, Taichung County; Gu-guan, Taichung County; Her-ping township, Taichung County; Shuang-lung waterfall, Di-li, Nan-tou County; Mei-feng, Nan-tou County; Renji-guan, Nan-tou County. East Taiwan: Taroko valley, Hualian County. Habitat: live under the defoliation or on the ground.

11. Cyclophorus moellendorffi Schmacker & Boettger, 1891 (Fig. 1.8 A–L; 1.18 F)) Records and Synonym: 1891. Cyclophorus moellendorffi Schmacker & Boettger. Schmacker, V.B. & Boettger, O. Nachrichtsblatt der Deutschen Malakozoologischen Gesellschaft, Jahrg, Vol. 23, p191, pl. 2, fig. 9. 1891. Cyclophorus moellendorffi var. humicola Schmacker & Boettger. Schmacker, V.B. & Boettger, O. Nachrichtsblatt der Deutschen Malakozoologischen Gesellschaft, Jahrg, Vol. 23, p191, pl. 2, fig. 9. 1905. Cyclophorus moellendorffi Schmacker & Boettger. Pilsbry, H.A. & Hirase, Y. Proceedings of Academy of Natural Sciences of Philadelphia, Vol. 57. p722. 1905. Cyclophorus moellendorffi var. humicola Schmacker & Boettger. Pilsbry, H.A.

106 & Hirase, Y. Proceedings of Academy of Natural Sciences of Philadelphia, Vol. 57. p722. 1936. Cyclophorus moellendorffi Schmacker & Boettger. Taketani, M. Trans. Nat. Hisi. Soc. Formos., Vol. 26 (154), p275, No. 15. 1941. Cyclophorus formosaensis moellendorffi Schmacker & Boettger. Kuroda, T. Memoirs of the Faculty of Science and Agriculture Taihoku Imperial University, Vol. 22(4). p80. No. 131. 1956. Cyclophorus formosaensis moellendorffi (Schmacker & Boettger). Kuroda. T. Venus, Vol. 19 (2), pl37. 1984. Cyclophorus formosensis moellendorffi (Schmacker & Boettger). Chang, K.M. & al. Pei-yo. Vol. 9, p4, No. 10. 1990. Cyclophorus formosensis moellendorffi (Schmacker & Boettger). Lai, K.Y. World of landsnail. Taiwan Museum, Taipei. p40, No. 9; p53, pl. 1, fig. 7. 1993. Cyclophorus formosaensis moellendorffi Schmacker & Boettger. Higo. S. & Goto. Y. A Systematic List of Molluscan Shells from the Japanese Is. and the Adjacent Area. Elle Shell Publications, Japan, p.61, No. 724. 2001. Cyclophorus moellendorffi Schmacker & Boettger. Lee, Y.C. & Wu, W.L. Bulletin of Malacolgy Taiwan, Vol. 25, p55-56, pl. 3, fig. 11. 2003. Cyclophorus moellendorffi Schmacker & Boettger. Lee, Y.C. & Chen, W.D. Natural observational illustrations 3－Land snail. Chin-Chin Publications Ltd. Taipei, p50. 2003. Cyclophorus formosaensis moellendorffi Schmacker & Boettger. Hsieh, B.C. Landsnails of Taiwan. Council of Agriculture Executive Yuan, p66. 2004. Cyclophorus formosaensis moellendorffi Schmacker & Boettger. Hsieh, B.C. Incredible snails, Yuan-Liou Publishing Co., Ltd., p97. 2006. Cyclophorus moellendorffi Schmacker & Boettger. Hsieh, B.C. & al. Landsnails of Taiwan. Forestry Bureau Council of Agriculture Executive Yuan, p63. Shell: shell width 21-23mm, height 17-20mm, 5.5 whorls, convex, very solid. The apex is subconical and differs from domal apex of Cyclophorus formosaensis Nevill, 1881. There are 2.25-2.75 brown protoconch whorls which are sculptured with obscure axial cords. Surface is sculptured with irregular growth lines. The peripheral keel is very distinct and recurved. There is a distinct keel at the base and several indistinct keels near umbilicus. Shell is festucine color, with reddish brown zigzag radial stripes and several interrupted spiral bands on dorsum. The peripheral keel is pale color. On base, there are several interrupted bands between peripheral keel and basal keel, and a dark brown serrate band just above the periphery keel. There is no brown pattern between basal keel and the narrow

107 umbilicus. The aperture is circular with subduplicate peristome which is a trace of peripheral keel on it. The lip is reflex in mature individual. The operculum is multispiral type, ceratoid, translucent yellow, distinct concave outside, with a nipple inside. Radula: radula is taenioglossa. Radula format is 1-2-1-2-1. The central tooth is scoop-shaped, 5 cuspids, central cuspid is the largest, approximately 1/3 width of central tooth; the other cuspids gently reduce their size from inner to outer site. The inner lateral teeth are scoop-shape, 4 cuspids, the 3rd cuspid is the largest. The outer lateral teeth are 3 cuspids, the mid cuspid are the largest, outer and inner side are smaller and equal size. The marginal teeth are sickle-like shaped, 3 cuspids, the central cuspid is the largest, it is relatively longer than Cyclophorus formosaensis, Cyclophorus friesianus and Cyclophorus latus. Distribution: Endemic to Taiwan. South Taiwan: Chair-shan, Kaohsiung City; Jia-siang, Kaohsiung County; Mu-dan, Ping-dung County; Heng-chueng, Ping-dung County; South cape. East Taiwan: Dung-he, Taidung County; Da-wu, Taidung County. Habitat: live under the defoliation or on the ground.

12. Cyclophorus cf. turgidus (Pfeiffer, 1851) (Fig. 1.9 A–L; 1.19 A) 2003. Cyclophorus turgidus (Pfeiffer). Lee, Y.C. & Chen, W.D. Natural observational illustrations 3－Land snail. Chin-Chin Publications Ltd. Taipei, p50, fig. 1 & 4. Shell: shell width 21-25mm, height 19-20mm, 4.75-5 whorls, rapidly increasing. There are 1.75 purplish to reddish brown Protoconch whorls which is sculptured with obscure axial cords. Surface is sculptured with irregular growth lines from dorsum to base, but without spiral cords. It has a distinct peripheral keel. Shell base is convex, without basal keel. Umbilicus narrowly opened, about 1/9 of the shell diameter. Shell is yellowish brown in color, with reddish to black brown zigzag radial stripes on dorsum, several interrupted bands around umbilicus. The aperture is circular. The lip is reflex in mature individual. The operculum is a multispiral type, ceratoid, translucent yellow, concave externally, with a nipple inside. Radula: radula is similar to Cyclophorus formosaensis. There is no distinct difference between these two species. Distribution: South Taiwan: south Yun-lin County; Jia-yi County. Habitat: live under the defoliation or on the ground.

Genus Platyrhaphe Moellendorff, 1890 1890. Platyrhaphe Moellendorff, O. Die Landschnecken Fauna der Insel Cebu. Ber.

108 Senckenb. Naturf. Ges., 267. Type species: Cyclostoma pusilla Sowerby G. B. 1843 Diagnosis: Shell usually small, with depressed spiral. The body whorl where inclined. The umbilicus is wide and perspective showing the upper whorl thinker at the edge of aperture but not reflex in the types. The operculum is multispiral type, calcareous, bone, white. Distribution: Morocco, India, South China, Philippines, Taiwan and Japan. Species included: 47 species worldwide (Kobelt 1902, Goto & Poppe 1996), 4 species in Taiwan. Key to species of Platyrhaphe from Taiwan 1. The axial lamina is throughout the whorls………………...…………….. P. hirasei The axial lamina is not throughout the whorls……………...………………...……2 2. The spiral line of the operculum is very convex...... …….. P. lanyuensis The spiral line of the operculum is very convex…………………...………………3 3. The radial lamina near the suture is not prominent……………...……... P. minutus The radial lamina near the suture is prominent…………………...……………..…4 4. The radial lamina is 1/2 whorl diameter………………...………. P. sunggangensis The radial lamina is <1/2 whorl diameter……………...………………………..…5 5. The spire is very depressed………………………...………. P. swinhoei depressus The spire is not so depressed…………………...……………..P. swinhoei swinhoei

13. Platyrhaphe hirasei (Pilsbry, 1901) (Fig. 1.11 J–L) Records and Synonym: 2001. Platyrhaphe cf. swinhoei (H. Adams, 1866) Lee, Y.C. & Wu, W.L. Bulletin of Malacology Taiwan, Vol. 25, p60. pl. 3, fig. 16. 2003. Platyrhaphe hirasei (Pilsbry). Lee, Y.C. & Chen, W.D. Natural observational illustrations 3－Land snail. Chin-Chin Publications Ltd. Taipei, p52 Shell: shell width 7-11mm, height 4-6.5mm, 4-4.5 whorls, which is moderately convex. Shell is festucine in color. Apex pointed, festucine, succeeding whorls much depressed. Surface is sculptured with axial lamina allover the whorls and some fine spiral cords on basal part. The body whorl close to the aperture is descending and inclined. The umbilicus is wide and perspective showing the upper whorls within. The aperture is thick and gentlely reflex. The operculum is unknown. Distribution: East Taiwan: Hualian. Habitat: habitat is unknown. Remark: only a dead specimen was collected.

109 14. Platyrhaphe lanyuensis Lee & Wu, 2001 (Fig. 1.10 A–F; 1.19 B–C) Records and Synonym: 1941. Platyrhaphe rubrotinctus Kuroda & Kano. Kuroda, T. Memoirs of the Faculty of Science and Agriculture Taihoku Imperial University, Vol. 22(4). p80. No. 135. 1956. Platyrhaphe rubrotinctus Kuroda & Kano. Kuroda. T. Venus, Vol. 19 (2), pl38. 1974. Platyrhaphe rubrotinctus Kuroda & Kano. Lin. C.C. Bulletin of the Malacological Society of China. Vol. I, p45, No. 52. 1984. Platyrhaphe rubrotinctus Kuroda & Kano. Chang, K.M. & al. Pei-yo. Vol. 9, p4, No. 15. 1990. Platyrhaphe rubrotinctus Kuroda & Kano. Lai, K.Y. World of landsnail. Taiwan Museum, Taipei. p41, No. 18. 1993. Platyrhaphe rubrotinctus Kuroda & Kano. Higo. S. & Goto. Y. A Systematic List of Molluscan Shells from the Japanese Is. and the Adjacent Area. Elle Shell Publications, Japan, p.62, No. 738. 2001. Platyrhaphe rubrotinctus Lee & Wu. Lee, Y.C. & Wu, W.L. Bulletin of Malacolgy Taiwan, Vol. 25, p57-58, pl. 3, fig. 15. 2003. Platyrhaphe rubrotinctus Lee & Wu. Lee, Y.C. & Chen, W.D. Natural observational illustrations 3－Land snail. Chin-Chin Publications Ltd. Taipei, p53. 2003. Platyrhaphe rubrotinctus Kuroda & Kano. Hsieh, B.C. Landsnails of Taiwan. Council of Agriculture Executive Yuan, p69. 2004. Platyrhaphe minutus (H. Adams). Hsieh, B.C. Incredible snails, Yuan-Liou Publishing Co., Ltd., p101. 2006. Platyrhaphe rubrotinctus Kuroda & Kano. Hsieh, B.C. & al. Landsnails of Taiwan. Forestry Bureau Council of Agriculture Executive Yuan, p66. Shell: shell width 6.5-9.4mm, height 4.8-5.25mm, 4-4.25 whorls, which is convex. Shell is festucine in color. Apex pointed, festucine or rufous, succeeding whorls much depressed. Surface is sculptured with dense spiral cords, which are reticulated with irregular growth striae. The body whorl close to the aperture is descending and inclined. The umbilicus is wide and perspective showing the upper whorls within. The whorl wall becomes thinker at the edge of aperture but not reflex in the types. The operculum is a multispiral type, calcareous, bone white, sharp concave externally and strongly delve at nucleolus. The spiral line of the operculum is convex and makes the suture deep, which makes the present species different from Platyrhaphe minutus (H. Adams, 1866) and Platyrhaphe swinhoei (H. Adams, 1866). Radula: radula is taenioglossa. Radula format is 1-2-1-2-1. The central tooth is

110 scoop-shaped, 5 cuspids, central cuspid is the largest, approximately 1/3 width of central tooth; the other cuspids gently reduce their size from inner to outer site. The inner lateral teeth are scoop-shape, 4 cuspids, the 3rd cuspid is the largest. The outer lateral teeth are 3 cuspids, mid cuspid are the largest, outer and inner side are smaller and equal size. The marginal teeth are sickle-like shaped, 3 cuspids, the central cuspid is the largest, the inner and outer cuspids are almost equal size. Distribution: Endemic to Taiwan. Lan-Yu; Liu-Dau. Habitat: live under the defoliation or on the ground.

15. Platyrhaphe minutus (H. Adams, 1866) (Fig. 1.10 G–L; 1.19 D) Records and Synonym: 1866. Cyclotus minutus H. Adams. Proc. Zool. Sci. London, p318, No. 9, PI. XXXIII, Fig. 9. 1891. Cyclotus minutus H. Adams. Schmacker, V. B. & Boettger, O. Nachrichtsblatt der Deutschen Malakozoologischen Gesellschaft, Jahrg, Vol. 23, pl92. 1905. Cyclotus minutus H. Adams. Pilsbry, H. A. & Hirase, Y. Proceedings of Academy of Natural Sciences of Philadelphia, Vol. 57, p724. 1905. Cyclotus minutus concentratus Pilsbry & Hirase. Pilsbry, H. A. & Hirase, Y. Proceedings of Academy of Natural Sciences of Philadelphia, Vol. 57, p724. 1941. Platyrhaphe minutus (H. Adams). Memoirs of the Faculty of Science and Agriculture Taihoku Imperial University, Vol. 22 (4), p80, No. 134. 1956. Platyrhaphe minutus (H. Adams). Kuroda, T. Venus, Vol. 19(2), p138. 1984. Platyrhaphe minutus (H. Adams). Chang, K.M. & al. Pei-yo. Vol. 9, p4. No. 14. 1990. Platyrhaphe minutus (H. Adams). Lai, K.Y. World of landsnail. Taiwan Museum, Taipei. p41, No. 17. 1993. Platyrhaphe minutus (H. Adams). Higo, S. & Goto, Y. A Systematic List of Molluscan Shells from the Japanese Is. And the Adjacent Area. Elle Shell Publications, Japan, p.61. No. 735. 2001. Platyrhaphe minutus (H. Adams). Lee, Y.C. & Wu, W.L. Bulletin of Malacolgy Taiwan, Vol. 25, p58-59, pl. 3, fig. 14. 2003. Platyrhaphe minutus (H. Adams). Lee, Y.C. & Chen, W.D. Natural observational illustrations 3－Land snail. Chin-Chin Publications Ltd. Taipei, p55. 2003. Platyrhaphe minutus (H. Adams). Hsieh, B.C. Landsnails of Taiwan. Council of Agriculture Executive Yuan, p69. 2004. Platyrhaphe minutus (H. Adams). Hsieh, B.C. Incredible snails, Yuan-Liou Publishing Co., Ltd., p101. 2006. Platyrhaphe minutus (H. Adams). Hsieh, B.C. & al. Landsnails of Taiwan.

111 Forestry Bureau Council of Agriculture Executive Yuan, p65. Shell: shell width 6-7mm, height 3-3.5mm. 3.75-4 whorls, which is convex. Shell is festucine to rufous in color. Apex pointed, reddish brown to purple red, succeeding whorls much depressed. Surface is sculptured with spiral cords, which are reticulated with growth lines. The body whorl close to the aperture is descending and inclined. The umbilicus is wide and perspective showing the upper whorls within. The aperture thick and gently reflex. The operculum is a multispiral type, calcareous, bone white, slight concave externally and strongly delve at nucleolus. Radula: the radula is similar to Platyrhaphe lanyuensis. There is not distinct difference within the genus Platyrhaphe of Taiwan. Distribution: Endemic to Taiwan. South Taiwan: Kaohsiung; Lung-chi, Tainan City; Nan-hua, Tainan County; Nan-huah, Tainan City; Chair-shan, Kaohsiung City; E-Luan-Bi; Sheau-liou-chyou, Ping-dung County. East Taiwan: Chung-de, Hualian County; Bei-Nan, Tai-dung County. Habitat: live under the defoliation or on the ground.

16. Platyrhaphe sunggangensis Lee & Wu, 2001 (Fig. 1.10 M–O) 2001. Platyrhaphe sunggangensis Lee & Wu. Lee, Y.C. & Wu, W.L. Bulletin of Malacolgy Taiwan, Vol. 25, p59, pl. 3, fig. 17. 2003. Platyrhaphe sunggangensis Lee & Wu. Lee, Y.C. & Chen, W.D. Natural observational illustrations 3－Land snail. Chin-Chin Publications Ltd. Taipei, p56. 2006. Platyrhaphe sunggangensis Lee & Wu. Hsieh, B.C. & al. Landsnails of Taiwan. Forestry Bureau Council of Agriculture Executive Yuan, p67. Shell: shell width 11-11.4mm, height 4-6.9mm, 3.75 convex whorls. Shell is festucine in color. Apex pointed, khaki, succeeding whorls much depressed. Surface is sculptured with spiral cords, which are reticulated with irregular axial cords and growth lines. There are distinct radial laminae near the suture and becoming a white band, which occupy 1/2 part of whorls. The body whorl close to the aperture is descending and inclined. The umbilicus is wide and perspective showing the upper whorls within. The umbilicus is about 1/2.75 of the shell diameter. The aperture thick and gently reflex. The operculum is a multispiral type, calcareous, bone white, flattish externally and strongly delve at nucleolus. Radula: I only got one living specimen, unfortunately the radula was missing. Distribution: Central Taiwan: Sung-Gang, Nan-Tou County. East Taiwan: Taidung County (Cycas taitungensis nature reserve) Habitat: live under the defoliation or on the ground.

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17. Platyrhaphe swinhoei depressus Pilsbry & Hirase, 1906 (Fig. 1.11 A–C; 1.19 E) Records and Synonym: 1905. Platyrhaphe swinhoei depressus Pilsbry & Hirase. Pilsbry, H. A. & Hirase, Y. Proceedings of Academy of Natural Sciences of Philadelphia, Vol. 57, p724. 1956. Platyrhaphe swinhoei depressus Pilsbry & Hirase. Kuroda, T. Venus, Vol. 19(2), p138. 2001. Platyrhaphe swinhoei depressus Pilsbry & Hirase. Lee, Y.C. & Wu, W.L. Bulletin of Malacolgy Taiwan, Vol. 25, p59, pl. 4, fig. 19. Shell: shell width 11-16.4mm, height 6-7.5mm, 4-4.5 whorls. Shell is similar to premier subspecies, but more depressed spire, apex purple in color. Shell is red color. The umbilicus is about 1/3.5 of the shell diameter. Radula: the radula is similar to Platyrhaphe lanyuensis. There is not distinct difference within the genus Platyrhaphe of Taiwan. Distribution: Endemic to Taiwan. North Taiwan: Iran. Central Taiwan: Dah-dong-shan, Jia-yi County. Habitat: live under the defoliation or on the ground.

18. Platyrhaphe swinhoei swinhoei (H. Adams, 1866) (Fig. 1.11 D–I; 1.19 F) Records and Synonym: 1866. Cyclotus swinhoei H. Adams. H. Adams. Proc. Zool. Sci. London, p318, No. 10 PI. XXXIII, Fig. 10. 1905. Cyclotus swinhoei H. Adams. Pilsbry, H.A. & Hirase, Y. Proceedings of Academy of Natural Sciences of Philadelphia, Vol. 57, p724. 1941. Platyrhaphe swinhoei (H. Adams). Kuroda. T. Memoirs of the Faculty of Science and Agriculture Taihoku Imperial University, Vol. 22(4), p80, No. 133. 1956. Platyrhaphe swinhoei (H. Adams). Kuroda, T. Venus, Vol. 19 (2), p138. 1984. Platyrhaphe swinhoei (H. Adams). Chang, K.M. & al. Pei-yo. Vol. 9, p4, No. 13. 1990. Platyrhaphe swinhoei (H. Adams). Lai, K.Y. World of Landsnail. Taiwan Museum, Taipei. p41. No. 16. 1993. Platyrhaphe swinhoei (H. Adams). Higo, S. & Goto, Y. A Systematic List of Molluscan Shells from the Japanese Is. And the Adjacent Area. Elle Shell Publications, Japan, p.61, No. 737. 2001. Platyrhaphe swinhoei (H. Adams). Lee, Y.C. & Wu, W.L. Bulletin of Malacolgy Taiwan, Vol. 25, p59, pl. 4, fig. 18. 2003. Platyrhaphe swinhoei (H. Adams). Lee, Y.C. & Chen, W.D. Natural observational illustrations 3－Land snail. Chin-Chin Publications Ltd. Taipei,

113 p57 2003. Platyrhaphe swinhoei (H. Adams). Hsieh, B.C. Landsnails of Taiwan. Council of Agriculture Executive Yuan, p68. 2004. Platyrhaphe swinhoei (H. Adams). Hsieh, B.C. Incredible snails, Yuan-Liou Publishing Co., Ltd., p100. 2006. Platyrhaphe swinhoei (H. Adams). Hsieh, B.C. & al. Landsnails of Taiwan. Forestry Bureau Council of Agriculture Executive Yuan, p64. Shell: shell width 7-11mm, height 4-6.5mm, 4-4.5 whorls, which is moderately convex. Shell is festucine to rufous in color. Apex pointed, reddish brown to purple red, succeeding whorls much depressed. Surface is sculptured with spiral cords, which are reticulated with irregular axial cords and growth lines. The spiral cords are faint or disappeared, it is just below the suture and is replaced with radial laminae, sometimes becoming a white band. The body whorl close to the aperture is descending and inclined. The umbilicus is wide and perspective showing the upper whorls within. The umbilicus is about 1/4.4 of the shell diameter. The aperture is thick and gentlely reflex. The operculum is a multispiral type, calcareous, bone white, slight concave externally and strongly delve at nucleolus. Radula: the radula is similar to Platyrhaphe lanyuensis. There is not distinct difference within the genus Platyrhaphe of Taiwan. Distribution: Endemic to Taiwan. North Taiwan: Taipei. Taipei County; Ji-nan Temple, Mu-Ja Taipei City; San-diau-ling, Taipei County; Da-chi-jiau, Shin-dian City; Shan-jia, Shu-lin, Taipei County; Ju-dung, Shin-ju County; Da-shi; Yi-lan. Central Taiwan: Tou-bian-keng, Tai-ping City, Taichung County. East Taiwan: Hualian. South Taiwan: Kaohsiung; Shiau-Liou-Chiou, Pingdung County. Habitat: live under the defoliation or on the ground.

Genus Cyclotus Swainson, 1840 1840. Cyclotus Swainson, W. A treatise on malacology, or the natural classification of shells and shell-fish. 186-336. Type species: Cyclotus variegatus Swainson, 1840 Diagnosis: Shell medium size, yellowish brown, with reddish brown zigzag radial stripes, usually with an interrupted reddish brown band at the peripheral whorl. Spire depressed. The body whorl close to the aperture is slightly inclined. The umbilicus is wide and perspective showing the upper whorls within. The circular aperture is duplicate peristome. The operculurn is a multispiral type, calcareous, bone white. Distribution: India, Mainland China, Taiwan, Korea, and Japan.

114 Species included: 104 species worldwide (Kobelt 1902, Goto & Poppe 1996), 5 species and subspecies in Taiwan. Key to species of Cyclotus from Taiwan 1. Shell height/shell width ≧ 0.06……………………...…………………. C. adamsi Shell height/shell width ﹤0.06…………………...………...…………..………....2 2. Shell with very extended out lip………………………….……. C. taivanus dilatus Shell without very extended out lip………………...……………..………………..3 3. Shell width 13.8–19mm………………...………...…...………C. taivanus taivanus Shell width 9.2–13.65mm, operculum very concave inside….C. taivanus diminutus

19. Cyclotus adamsi (Pilsbry & Hirase, 1906) (Fig. 1.12 A–F; 1.20 A) Records and Synonym: 1905. Cyclotus taivanus adamsi Pilsbry & Hirase. Pilsbry, H. A. & Hirase, Y. Proceedings of Academy of Natural Sciences of Philadelphia, Vol. 57, p723. 1936. Cyclotus (Procyclotus) taivanus adamsi Pilsbry. Taketani, M. Trans. Nat. Hist. Soc. Formos., Vol. 26 (154), p276, No. 17. 1956. Cyclotus taivanus adamsi Pilsbry & Hirase. Kuroda, T. Venus, Vol. 19(2), p138. 2001. Cyclotus taivanus adamsi Pilsbry & Hirase. Lee, Y.C. & Wu, W.L. Bulletin of Malacology Taiwan, Vol. 25, p62. pl. 4, fig. 22. 2003. Cyclotus taivanus adamsi Pilsbry & Hirase. Lee, Y.C. & Chen, W.D. Natural observational illustrations 3－Land snail. Chin-Chin Publications Ltd. Taipei, p62. Shell: Shell width 11.5-18.15mm, height 7.25-12.25mm, 4 whorls. The general shape and size are similar to the preceding species, but it has the highest spire, uniform yellow or tawny yellow colored. The lip is not notched near suture. The operculum is flattish outside but concaver inside, multispiral type, calcareous, bone white, strongly delve at nucleolus, with a distinct nipple inside. Radula: radula is taenioglossa. Radula format is 1-2-1-2-1. The central tooth is scoop-shaped, 5 cuspids, central cuspid is the largest, approximately 1/3 width of central tooth; the other cuspids gently reduce their size from inner to outer site. The lateral teeth are scoop-shape, 4 cuspids, the 3rd cuspid is the largest. Some individuals are 5 cuspids inner lateral teeth. The marginal teeth are sickle-like shaped, 3 cuspids, the central cuspid is the largest. Distribution: Endemic subspecies of Taiwan. North Taiwan: Taipei; Ji-shan-yan, Taipei City; Guan-yin Mountain, Taipei County; Shi-tou Mountain, Nan-juang, Shin-ju County. Habitat: live under the defoliation or on the ground.

115 20. Cyclotus taivanus dilatus Lee & Wu, 2001 (Fig. 1.12 G–L; 1.20 B) Records and Synonym: 1951. Cyclotus taivanus koshunensis (Pilsbry & Hirase, MS). Hirase, S. A handbook of illustrated shells in natural colors from Japanese Islands and their adjacent territories – revised and enlarged edition of “A collection of Japanese shells”. Bunkyokaku. Tokyo, Japan, pl. 79, fig. 4. 1956. Cyclotus taivanus koshunensis (Pilsbry & Hirase, MS). Kuroda, T. Venus, Vol. 19 (2), p138. 2001. Cyclotus taivanus dilatus Lee & Wu. Lee, Y.C. & Wu, W.L. Bulletin of Malacology Taiwan, Vol. 25, p63. pl. 4, fig. 23. 2003. Cyclotus taivanus dilatus Lee & Wu. Lee, Y.C. & Chen, W.D. Natural observational illustrations 3－Land snail. Chin-Chin Publications Ltd. Taipei, p63. Shell: shell width 16.5-20.2mm, height 8.4-10.25mm, 4 whorls. Shell color and pattern are similar to the original species, but with very extended lip. Spire depressed. The body whorl close to the aperture is slightly inclined. Surface is sculptured with irregular growth lines. The umbilicus is wide and perspective showing the upper whorls within. The circular aperture is duplicate peristome. The outer lip, which has extended reflex and evolute edge is notched near suture, the inner is thick edge, but not reflex. The parietal lip attached to the last whorl. The operculum is a multispiral type, calcareous, bone white, a little concave externally and strongly delve at nucleolus, with an indistinct nipple inside. Radula: the radula is similar to Cyclotus taivanus taivanus. There is no distinct difference between these two species. Distribution: Endemic subspecies of Taiwan. Central Taiwan: Tou-bian-keng, Tai-ping City, Taichung County; Ching-de Temple, Guo-shing, Nantou County. East Taiwan: Taroko valley, Hualian County. Habitat: live under the defoliation or on the ground.

21. Cyclotus taivanus diminutus Lee & Wu, 2001 (Fig. 1.13 A–C; 1.20 C) Records and Synonym: 1932. Cyclotus (Procyclotus) taivanus H. Adams. Kuroda. T. Bulletin of the Biogeographical Society of Japan. Vol. 3(1), p. 3, No.2. (miss identification) 1932. Cyclotus (Procyclotus) taivanus H. Adams. Kuroda, T. Venus, Vol. 3(4), p188. (miss identification) 1941. Cyclotus taivanus diminutus Kuroda & Kano. Kuroda. T. Memoirs of the Faculty of Science and Agriculture Taihoku Imperial University, Vol. 22(4), p81. No. 136-a.

116 1956. Cyclotus taivanus diminutus Kuroda & Kano. Kuroda, T. Venus, Vol. 19(2), p139. 1963. Cyclotus taivanus H. Adams (sub. sp.?). Reigle, N. J. Quarterly Journal of the Taiwan Museum, Vol. 16(1, 2), p85 1974. Cyclotus (Procyclotus) taivanus H. Adams. Lin, C.C. Bulletin of the Malacological Society of China, Vol. I, p45. No. 53. (miss identification) 1974. Cyclotus (Procyclotus) taivanus diminutus Kuroda & Kano. Lin, C.C. Bulletin of the Malacological Society of China, Vol. 1, p46, No. 54. 1984. Cyclotus taivanus diminutus Kuroda & Kano. Chang, K.M. & al. Pei-yo. Vol. 9, p4, No. 17. 1990. Cyclotus taivanus diminutus Kuroda & Kano. Lai, K.Y. World of Landsnail. Taiwan Museum, Taipei. p41, No. 14. 1993. Cyclotus (Procyclotus) taivanus diminutus Higo, S. & Goto, Y. A Systematic List of Molluscan Shells from the Japanese Is. And the Adjacent Area. Elle Shell Publications, Japan, p.62, No. 746. 2001. Cyclotus taivanus diminutus Lee & Wu. Lee, Y.C. & Wu, W.L. Bulletin of Malacology Taiwan, Vol. 25, p62. pl. 4, fig. 24. 2003. Cyclotus taivanus diminutus Lee & Wu. Lee, Y.C. & Chen, W.D. Natural observational illustrations 3－Land snail. Chin-Chin Publications Ltd. Taipei, p64. 2003. Cyclotus taivanus diminutus Kuroda & Kano. Hsieh, B.C. Landsnails of Taiwan. Council of Agriculture Executive Yuan, p61. 2006. Cyclotus taivanus diminutus Kuroda & Kano. Hsieh, B.C. & al. Landsnails of Taiwan. Forestry Bureau Council of Agriculture Executive Yuan, p55. Shell: shell width 9.2-13.65mm, height 4.5-6.6rnm, 4 whorls, spire depressed, yellowish brown, with fine reddish brown radial cascade stripes, some with an interrupted reddish brown band at the periphery of body whorl, most individuals are in faint patterns. The body whorl close to the aperture is slightly inclined. Surface is sculptured with irregular growth lines. The umbilicus is wide and perspective showing the upper whorls within. The circular aperture is single, thick peristome edge and gentlely reflex lip. The lip is not notched near suture. The parietal lip attached to the last whorl. The operculum is flattish outside but concaver inside, multispiral type, calcareous, bone white, strongly delve at nucleolus, with a distinct nipple inside. Radula: the radula is similar to Cyclotus taivanus taivanus. There is no distinct difference between these two species. Distribution: Endemic subspecies of Taiwan. East Taiwan: Lan-Yu. Habitat: live under the defoliation or on the ground.

117

22. Cyclotus taivanus taivanus H. Adams, 1870 (Fig. 1.3 D–I; 1.20 D) Records and Synonym: 1870. Cyclotus taivanus H. Adams. Adams, H. Proc. Zool. Sci. London, p378, No. 8, pl. XXVII, Fig. 11, 1la. 1891. Cyclotus taivanus H. Adams. Schmacker, V. B. & Boettger, O. Nachrichtsblatt der Deutschen Malakozoologischen Gesellschaft, Jahrg, Vol. 23, p192. 1905. Cyclotus taivanus H. Adams. Pilsbry, H. A. & Hirase, Y. Proceedings of Academy of Natural Sciences of Philadelphia, Vol. 57. p723. 1936. Cyclotus (Procyclotus) taivanus H. Adams. Taketani, M. Trans. Nat. Hist. Soc. Formos., Vol. 26(154), p275, No. 16. 1941. Cyclotus taivanus H. Adams. Kuroda. T. Memoirs of the Faculty of Science and Agriculture Taihoku Imperial University, Vol. 22(4), p80, No. 136. 1956. Cyclotus taivanus H. Adams. Kuroda, T. Venus, Vol. 19(2), p138. 1984. Cyclotus taivanus H. Adams. Chang, K.M. & al. Pei-yo. Vol. 9, p21, pl. 1, Fig. 5. 1988. Cyclotus taivanus H. Adams. Lai, K.Y. The shells. Holiday Publisher. P12, Fig. 3. 1990. Cyclotus taivanus H. Adams. Lai, K.Y. World of Landsnail. Taiwan Museum, Taipei. p41, No. 13, pl. 1, fig. 9. 1993. Cyclotus (Procyclotus) taivanus H. Adams. A Systematic List of Molluscan Shells from the Japanese Is. And the Adjacent Area. Elle Shell Publications, Japan, p.62, No. 744. 2001. Cyclotus taivanus H. Adams. Lee, Y.C. & Wu, W.L. Bulletin of Malacology Taiwan, Vol. 25, p62-63. pl. 4, fig. 21. 2003. Cyclotus taivanus H. Adams. Lee, Y.C. & Chen, W.D. Natural observational illustrations 3－Land snail. Chin-Chin Publications Ltd. Taipei, p61. 2003. Cyclotus taivanus H. Adams. Hsieh, B.C. Landsnails of Taiwan. Council of Agriculture Executive Yuan, p61. 2004. Cyclotus taivanus H. Adams. Hsieh, B.C. Incredible snails, Yuan-Liou Publishing Co., Ltd., p99. 2006. Cyclotus taivanus H. Adams. Hsieh, B.C. & al. Landsnails of Taiwan. Forestry Bureau Council of Agriculture Executive Yuan, p54. Shell: shell width 13.8-19mm, height 6.9-10.2mm, 4.3-4.75 whorls. Yellowish brown, with reddish brown zigzag radial stripes, usually with an interrupted reddish brown band at the periphery of body whorl. Spire depressed. The body whorl close to the aperture is slightly inclined. Surface is sculptured with irregular growth lines. The umbilicus is wide and perspective showing the upper whorls

118 within. The circular aperture is subduplicate peristome. The lip is reflex and is notched near suture. The parietal lip attaches to the last whorl. The operculum is multispiral type, calcareous, bone white, a little concave externally and strongly delve at nucleolus, with an indistinct nipple inside. Radula: radula is taenioglossa. Radula format is 1-2-1-2-1. The central tooth is scoop-shaped, 5 cuspids, central cuspid is the largest, approximately 1/3 width of central tooth; the other cuspids gently reduce their size from inner to outer site. The lateral teeth are scoop-shape, 4 cuspids, the 3rd cuspid is the largest. The marginal teeth are sickle-like shaped, 3 cuspids, the central cuspid is the largest. Distribution: South Taiwan: Liou-guei, Kaohsiung County; Yuan-lin, Yun-lin County; Alishan, Jia-yi County; Yan-shuei, Jia-yi County; Yu-jing, Tainan County; Chau-jou, Ping-dung County; Heng-chueng, Ping-dung County. East Taiwan: Da-wu, Taidung County. Habitat: live under the defoliation or on the ground.

Genus Cyathopoma W. & H. Blanford, 1861 1861. Cyathopoma W. & H. Blanford, Contributions to Indian Malacology, No. II. The Journal of Asiatic Society of Bengal, 30: 347–366. Type species: Cyathopoma filocinclum W. & H. Blanford, 1861 Diagnosis: Shell tiny, convex, deep suture, white to cream in color. Surface is sculptured with spiral cords. The umbilicus is widely opened. The aperture is circular, peristome simple, thin. Operculum is a multispiral type, circular, yellowish white, calcareous, concave outside. Distribution: from Indian, China, Japan to Taiwan. Species included: 42 species worldwide (Kobelt 1902, Goto & Poppe 1996), 4 species in Taiwan. Key to species of Cyathopoma from Taiwan 1. Protoconch surface sculpture with micro-pustule ……………...……...………….2 Protoconch surface smooth……………………...…………………..……………..3 2. Surface with spiral cords…………………..………………...…….. C. taiwanicum Surface without spiral cords…………………...……………...…………….. C. iota 3. The whorl is free from penultimate whorl………………...…………….. C. ogaitoi The whorl is not free from penultimate whorl…………………….………C. micron

23. Cyathopoma iota Pilsbry & Hirase, 1904 (Fig. 1.14 A–B; 1.20 E) Records and Synonym: 1904. Cyathopoma iota Pilsbry & Hirase. Pilsbry, H.A. & Hirase, Y. Proceedings of Academy of Natural Sciences of Philadelphia, Vol. 56, p 619.

119 2003. Cyclotus micron iota (Pilsbry & Hirase). Lee, Y.C. & Chen, W.D. Natural observational illustrations 3－Land snail. Chin-Chin Publications Ltd. Taipei, p59. 2006. Cyclotus micron Pilsbry. Hsieh, B.C. & al. Landsnails of Taiwan. Forestry Bureau Council of Agriculture Executive Yuan, p55. Shell: shell micro, shell width 1.3-1.9mm, height 1.4-2.0mm, 3.5-4.5 whorls, convex, deep suture, polish, white, translucent. The apex is domal, surface sculpture with micro-pustule. Spire is moderately tall. Surface is sculptured with micro-spiral lines. The umbilicus is narrowly opened. The aperture is circular, with thin edge. The operculum is a multispiral type, white, calcareous, a little concave outside. Radula: radula is taenioglossa. Radula format is 1-2-1-2-1. The central tooth is scoop-shaped, 5 equal size cuspids. There are 7–9 irregular size tiny cuspids on the convex side of the central tooth. The lateral teeth are scoop-shape, inner lateral teeth is 5-6 cuspids, the outer lateral teeth with 6–7 cuspids (variation in the same individual). The marginal teeth with 6 small cuspids on the inner side and 3 large cuspids on the outer side. Distribution: Ranges from Kumejima, Okinawa to North Taiwan. North Taiwan: Dar-Guan Mountain, Tau-Yuan County; Borling, Iran County. Habitat: live under the defoliation or on the ground.

24. Cyathopoma micron (Pilsbry, 1900) (Fig. 1.14 C–D; 1.20 F) Records and Synonym: 1900. Cyclotus (?) micron Pilsbry. Pilsbry, H.A. Nautilus, XIV(1), p12. 1905. Cyathopoma micron (Pilsbry). Pilsbry, H.A. & Hirase, Y. Proceedings of Academy of Natural Sciences of Philadelphia, Vol. 57, p 725. 1941. Cyathopoma (Nakadaella) micron (Pilsbry). Kuroda, T. Memoirs of the Faculty of Science and Agriculture Taihoku Imperial University, Vol. 22(4), p81, No. 138. 1951. Cyclotus micron Pilsbry. Hirase, S. A handbook of illustrated shells in natural colors from Japanese Islands and their adjacent territories – revised and enlarged edition of “A collection of Japanese shells”. Bunkyokaku. Tokyo, Japan. pl. 79, fig. 6; pl. 131, fig. 7. 1956. Nakadaella micron (Pilsbry). Kuroda, T. Venus, 19(2), p139. 1984. Cyathopoma (Nakadaella) micron (Pilsbry). Chang, K.M. & al. Pei-yo Vo l . 9 , p4, No.19. 1990. Nakadaella micron (Pilsbry). Lai, K.Y. World of Landsnail. Taiwan Museum, Taipei. p41, No. 15. 1993. Nakadaella micron (Pilsbry). Higo, S & Goto, Y. A Systematic List of

120 Molluscan Shells from the Japanese Is. And the Adjacent Area. Elle Shell Publications, Japan, p.62, No. 752. 1995. Nakadaella micron (Pilsbry). Azuma, M. Colored illustrations of the land snails of Japan – revised edition. Hoikusha Publishing Co., Ltd. Osaka, p72. No. 22. pl. 3, fig. 22. 2001. Cyclotus micron Pilsbry. Hirase. Lee, Y.C. & Wu, W.L. Bulletin of Malacology Taiwan, Vol. 25, p61. pl. 4, fig. 20. 2003. Cyclotus micron Pilsbry. Hirase. Lee, Y.C. & Chen, W.D. Natural observational illustrations 3－Land snail. Chin-Chin Publications Ltd. Taipei, p58. 2003. Cyclotus micron Pilsbry. Hsieh, B.C. Landsnails of Taiwan. Council of Agriculture Executive Yuan, p60. Shell: shell micro, shell width 1.6-1.8mm, height 0.75-1.2mm, 4 whorls, convex, deep suture, polish, white, translucent. Spire is depressed. Surface is sculptured with tiny growth lines. The umbilicus is widely opened, perspective showing the upper whorls. The aperture is circular, with thin edge. The operculum is a multispiral type, white, calcareous, a little concave outside. Radula: radula is taenioglossa. Radula format is 1-2-1-2-1. The central tooth is scoop-shaped, 5 equal size cuspids. There are 7–9 irregular size tiny cuspids on the convex side of the central tooth. The lateral teeth are scoop-shape, inner lateral teeth is 6 cuspids, the outer lateral teeth with 5-6 cuspids (variation in the same individual). The marginal teeth with 6-7 small cuspids on the inner side and 3 large cuspids on the outer side. The present species is similar to Cyathopoma iota, but the cuspids on the teeth were shorter and broader than the above two species. Further, the outer lateral teeth had fewer cuspids (5–6 in number). Distribution: Ranges from Japan to Taiwan. North Taiwan: Taipei. Central Taiwan: Sung-gang, Nan-Tou County. East Taiwan: Northern shore of Tszen runlet, Hualian County; South Taiwan: Chi-shan, Kaohsiung County. Habitat: live under the defoliation or on the ground.

25. Cyathopoma ogaitoi (Minato, 1988) (Fig. 1.14 E–F; 1.21 A) Records and Synonym: 1988. Nakadaella ogaitoi Minato. Minato, H. Venus, Vol. 47(3), p 155-157. 2003. Cyclotus ogaitoi (Minato). Hirase. Lee, Y.C. & Chen, W.D. Natural observational illustrations 3－Land snail. Chin-Chin Publications Ltd. Taipei, p60. Shell: shell micro, shell width 1.4mm, height 2.0mm, 3.5 whorls, convex, deep suture, polish, white, translucent. Spire is depressed. Surface is sculptured with tiny growth lines. The umbilicus is widely opened, perspective showing the upper

121 whorls. The aperture is circular, with thin edge. The operculum is a multispiral type, white, calcareous, a little concave outside. The general shape of present species is similar to Cyathopoma micron but the whorl is free from penultimate whorl. Radula: the radula is similar to Cyathopoma micron. There is not distinct difference between these two species. Distribution: Ranges from Hyogo-ken, Guei-Jou Province, China to South Taiwan. Habitat: live under the defoliation or on the ground.

26. Cyathopoma taiwanicum Pilsbry & Hirase, 1906 (Fig. 1.14 G–H; 1.21 B; 1.21 B) Records and Synonym: 1905. Cyathopoma taiwanicum Pilsbry & Hirase. Pilsbry, H. A. & Hirase, Y. Proceedings of Academy of Natural Sciences of Philadelphia, Vol. 57, p724. 1905. Cyathopoma taiwanicum degeneratum Pilsbry & Hirase. Pilsbry, H. A. & Hirase, Y. Proceedings of Academy of Natural Sciences of Philadelphia, Vol. 57, p724. 1941. Cyathopoma taiwanicum Pilsbry & Hirase. Kuroda, T. Memoirs of the Faculty of Science and Agriculture Taihoku Imperial University, Vol. 22(4), p81, No. 137. 1956. Cyathopoma taiwanicum Pilsbry & Hirase. Kuroda, T. Venus, Vol. 19(2), p139. 1984. Cyathopoma (Jerdonia) taiwanicum Pilsbry & Hirase. Chang, K.M. & al. Pei-yo. Vol. 9, p4, No. 18. 1990. Cyathopoma taiwanicum Pilsbry & Hirase. Lai, K. Y. World of Landsnail. Taiwan Museum, Taipei. p41, No. 19. 1993. Cyathopoma taiwanicum Pilsbry & Hirase. Higo, S. & Goto, Y. A Systematic List of Molluscan Shells from the Japanese Is. and the Adjacent Area. Elle Shell Publications, Japan, p62, No. 749. 2001. Cyathopoma taiwanicum Pilsbry & Hirase. Lee, Y.C. & Wu, W.L. Bulletin of Malacology Taiwan, Vol. 25, p61. pl. 4, fig. 25-26. 2003. Cyathopoma taiwanicum Pilsbry & Hirase. Lee, Y.C. & Chen, W.D. Natural observational illustrations 3－Land snail. Chin-Chin Publications Ltd. Taipei, p65. 2003. Cyathopoma taiwanicum Pilsbry & Hirase. Hsieh, B.C. Landsnails of Taiwan. Council of Agriculture Executive Yuan, p70. 2004. Cyathopoma taiwanicum Pilsbry & Hirase. Hsieh, B.C. Incredible snails, Yuan-Liou Publishing Co., Ltd., p102. 2006. Cyathopoma taiwanicum Pilsbry & Hirase. Hsieh, B.C. & al. Landsnails of Taiwan. Forestry Bureau Council of Agriculture Executive Yuan, p67.

122 Shell: shell width L5-2.5mm, height 1-1.5mm, 3-4 whorls, convex, deep suture, white to cream in color. There are 1.75 smooth whorls at protoconch. The apex is domal. Each teleoconch whorl is sculptured with 6 spiral cords. There are other 11 spiral cords between periphery and the umbilical wall. The umbilicus is widely opened and perspective showing the upper whorls. The aperture is circular, peristome simple, thin. Operculum is a multispiral type, circular, white, calcareous, concave outside. Radula: the radula is similar to Cyathopoma iota. There is not distinct difference between these two species. Distribution: Endemic to Taiwan. North Taiwan: Shi-ding, Taipei County; Ji-nan Temple, Mu-ja Taipei City; Da-chi-jiau, Shin-dian City; Wu-lai, Taipei County; Su-au, Yi-lan; Keelung City. East Taiwan: Northern shore of Tszen runlet, Hualian County. Habitat: live under the defoliation or on the ground.

Genus Chamalycaeus Kobelt & Molledorff, 1897 1897. Chamalycaeus Kobelt, W. & Molledorff, O. Catalog der gegenwärtig lebend bekannten Pneumonopomen. Nachrichtenblatt der Deutschen Malakozoologischen Gesellschaft, 137-152. Type species: Alycaeus andamaniae Benson, 1861 Diagnosis: Shell very small, depressed, ashen pale yellow to orange in color. Teleoconch whorls ornamented with axial riblets. Constricted near the aperture. Body whorl sharply descending and greatly inclined near the aperture. Sutural tube laying backward along the suture. Umbilicus widely opened. Aperture circular, continuous, oblique, peristome duplex, reflexed, thickened within. Operculum circular, multispiral. Distribution: Japan, Taiwan, South China, Indonesia, Java, Andaman, and Philippines. Species included: 88 species worldwide (Kobelt 1902, Goto & Poppe 1996), 2 species in Taiwan. Key to species of Chamalycaeus from Taiwan 1. Protoconch is reddish brown colored……………………….….. C. hungerfordianus Protoconch is festucine colored………………………...……………….…C. varius

27. Chamalycaeus hungerfordianus (Nevill, 1881) (Fig. 1.15 A–C; 1.21 C; 1.21 C) Records and Synonym: 1881. Alycaeus hungerfordianus Nevill. Nevill, G. Journal Asiatic Society of Bengal. Vol. L, Part II, 149-150.

123 1905 Alycaeus hungerfordianus Nevill. Pilsbry, H. A. & Hirase, Y. Proceedings of Academy of Natural Sciences of Philadelphia, Vol. 57, p729. 1941. Chamalycaeus hungerfordianus (Nevill). Kuroda, T. Memoirs of the Faculty of Science and Agriculture Taihoku Imperial University, Vol. 22(4), p81, No. 142. 1956. Chamalycaeus hungerfordianus (Nevill). Kuroda, T. Venus, Vol. 19(2), p140. 1984. Chamalycaeus hungerfordianus (Nevill). Chang, K.M. & al. Pei-yo. Vol. 9, p4, No. 23. 1990. Chamalycaeus hungerfordianus (Nevill). Lai, K. Y. World of Landsnail. Taiwan Museum, Taipei. p41, No. 21. 1993. Chamalycaeus hungerfordianus (Nevill). Higo, S. & Goto, Y. A Systematic List of Molluscan Shells from the Japanese Is. and the Adjacent Area. Elle Shell Publications, Japan, p63, No. 763. 2001. Chamalycaeus hungerfordianus (Nevill). Lee, Y.C. Pei-yo, Vol. 27, p7. 2001. Chamalycaeus hungerfordianus (Nevill). Lee, Y.C. & Wu, W.L. Bulletin of Malacology Taiwan, Vol. 25, p67-68. pl. 5, fig. 28. 2003. Chamalycaeus hungerfordianus (Nevill). Lee, Y.C. & Chen, W.D. Natural observational illustrations 3－Land snail. Chin-Chin Publications Ltd. Taipei, p68. 2003. Chamalycaeus hungerfordianus (Nevill). Hsieh, B.C. Landsnails of Taiwan. Council of Agriculture Executive Yuan, p73. 2006. Chamalycaeus hungerfordianus (Nevill). Hsieh, B.C. & al. Landsnails of Taiwan. Forestry Bureau Council of Agriculture Executive Yuan, p71. Shell: shell very small, shell width 3.5-3.8mm, height 2mm, 3.5 whorls, depressed, orange. Protoconch 1.75 whorls, shing, domal, sculpture with 10-11 spiral cords on last 3/4 protoconch. The following whorls ornamented with regular riblets which is denser near sutural tube and absolutely form neck toward aperture. The teteleoconch spiral cords are blurred when it is mature. Constriction of neck rather weak. Body whorl sharply descending and greatly inclined near the aperture. Sutural tube, narrow, about 1mm, laying backward along the suture. Umbilicus widely opened, perforated 1/3 of shell diameter. Aperture circular, continuous, oblique, peristome duplex, reflexed, thickened within, parietal lip attached to the last whorls. Operculum circular, ceratoid, multispiral, translucently, reddish brown in color, concave externally Radula: radula is taenioglossa. Radula format is 1-2-1-2-1. The central tooth is scoop-shaped, 7 cuspids, central cuspid is the largest, the other cuspids gently reduce their size from inner to outer site, the most outer site cuspid rapid decrease its size. The lateral teeth are scoop-shape, 4 cuspids, the 3rd cuspid of inner one is the largest. The outer lateral teeth are 4 cuspids, mid two cuspids are the larger

124 than outer and inner cuspids. The marginal teeth are sickle-like shaped, 3 cuspids are almost equal size. Distribution: Endemic to Taiwan. North Taiwan: Taipei; Dan-shuei; Da-chi-jiau, Shin-dain County; Terng-long waterfall, Miau-li County; Central Taiwan: National Fonghuanggu Bird Park, .Nan-tou County Habitat: live under the defoliation or on the ground.

28. Chamalycaeus varius (Pilsbry & Hirase, 1906) (Fig. 1.15 D–I; 1.21 D; 1.21 D) Records and Synonym: 1905. Alycaeus varius Pilsbry & Hirase. Pilsbry, H. A. & Hirase, Y. Proceedings of Academy of Natural Sciences of Philadelphia, Vol. Vol. 57, p729. 1941. Chamalycaeus varius (Pilsbry & Hirase). Kuroda. T. Memoirs of the Faculty of Science and Agriculture Taihoku Imperial University, Vol. 22(4), p81. No. 143. 1956. Chamalycaeus varius (Pilsbry & Hirase). Kuroda, T. Venus, Vol. 19(2), p140. 1984. Chamalycaeus varius (Pilsbry & Hirase). Chang, K.M. & al. Pei-yo. Vol. 9, p4, No. 24. 1990. Chamalycaeus varius (Pilsbry & Hirase). Lai, K.Y. World of Landsnail. Taiwan Museum, Taipei. p41, No. 22. 1993. Chamalycaeus varius (Pilsbry & Hirase). Higo, S. & Goto, Y. A Systematic List of Molluscan Shells from the Japanese Is. and the Adjacent Area. Elle Shell Publications, Japan, p.63, No. 762. 2001. Chamalycaeus varius (Pilsbry & Hirase). Lee, Y.C. & Wu, W.L. Bulletin of Malacology Taiwan, Vol. 25, p68. pl. 5, fig. 29. 2003. Chamalycaeus varius (Pilsbry & Hirase). Lee, Y.C. & Chen, W.D. Natural observational illustrations 3－Land snail. Chin-Chin Publications Ltd. Taipei, p69. 2003. Chamalycaeus varius (Pilsbry & Hirase). Hsieh, B.C. Landsnails of Taiwan. Council of Agriculture Executive Yuan, p73. 2004. Chamalycaeus varius (Pilsbry & Hirase). Hsieh, B.C. Incredible snails, Yuan-Liou Publishing Co., Ltd., p104. 2006. Chamalycaeus varius (Pilsbry & Hirase). Hsieh, B.C. & al. Landsnails of Taiwan. Forestry Bureau Council of Agriculture Executive Yuan, p70. Shell: shell very small, shell width 4.5mm, height 2.8mm, 4 whorls, depressed, pale yellowish. Protoconch 2 whorls, festucine, sharply pointed, sculpture with several spiral cords on last part of protoconch. The following whorls ornamented with regular riblets which is denser near sutural tube and obsoletely from neck toward aperture. The teleoconch spiral cords are blurred when it is mature. Constriction of neck rather weak. Body whorl sharply descending and greatly inclined near the

125 aperture. Sutural tube, narrow, about 1mm, laying backward along the suture. Umbilicus widely opened, perforated 1/3 of shell diameter. Aperture circular, continuous, oblique, peristome duplex, well expanded, thickened within, the columellar margin wider, parietal lip attached to the last whorls. Operculum circular, ceratoid, multispiral, translucently, reddish brown in color, concave externally. Radula: the radula is similar to Chamalycaeus hungerfordianus. There is not distinct difference between these two species. Distribution: Endemic to Taiwan. North Taiwan: Taipei; Nei-hu, Taipei City, Yin-He-Dung, Shin-dian City. Habitat: live under the defoliation or on the ground.

Genus Dioryx Benson, 1859 1859. Dioryx Benson, W.H. A sectional distribution of the genus Alycaeus Gray, with characters of 6 new species and of other Cyclostomidae, collected at Darjiling by W.T, Blanford, Esq. Ann. Mag. Nat. Hist. London, 3(3): 176-184 Type species: Alycaeus amphora W.H. Benson 1856 Diagnosis: Shell conical, fragile, white to dull reddish brown, convex, gradually increasing diameter from apex to 1/2 body whorl and rapidly increasing in diameter than contracted near the aperture. Surface reticulated with irregular growth lines, sculpture dense, regular riblets near sutural tube. Sutural tube, narrow, long, laying backward along the suture. Umbilicus narrowly opened. Aperture circular, peristome reflex, well expended. Operculum, circular, multispiral type, ceratoid, translucent yellow. Distribution: Indian, China, Burma, Vietnam and Taiwan. Species included: 16 species worldwide (Kobelt 1902, Goto & Poppe 1996), 1 species in Taiwan.

29. Dioryx swinhoei (H. Adams, 1866) (Fig. 1.16 A–P; 1.21 E; 1.21 E) Records and Synonym: 1866. Alycaeus (Dioryx) swinhoei H. Adams. H. Adams. Proc. Zool. Sci. London, p318, No. 11, pl. XXXIII, fig. 11. 1891. Alycaeus (Dioryx) swinhoei H. Adams. Schmacker, V. B. & Boettger, O. Nachrichtsblatt der Deutschen Malakozoologischen Gesellschaft, Jahrg, Vol. 23, p189. 1905. Alycaeus (Dioryx) swinhoei H. Adams. Pilsbry, H. A. & Hirase, Y. Proceedings of Academy of Natural Sciences of Philadelphia, Vol. 57, p729. 1941. Dioryx swinhoei (H. Adams). Kuroda. T. Memoirs of the Faculty of Science and

126 Agriculture Taihoku Imperial University, Vol. 22(4), p81, No. 141. 1951. Dioryx swinhoei (H. Adams). Hirase, S. A handbook of illustrated shells in natural colors from Japanese Islands and their adjacent territories. - revised and enlarged edition of "A collection of Japanese shells". Bunkyokaku. Tokyo, Japan, pl. 78, fig. 10. 1956. Dioryx swinhoei (H. Adams). Kuroda, T. Venus, 19 (2): 132-147. 1984. Dioryx swinhoei (H. Adams). Chang, K. M. & al. Pei-yo. Vol. 9, p4, No. 22. 1990. Dioryx swinhoei (H. Adams). Lai, K.Y. World of Landsnail. Taiwan Museum, Taipei. p41, No. 20. 1993. Dioryx swinhoei (H, Adams). Higo, S. & Goto, Y. A Systematic List of Molluscan Shells from the Japanese Is. And the Adjacent Area. Elle Shell Publications, Japan, p63, No. 761. 2001. Dioryx swinhoei (H. Adams). Lee, Y.C. & Wu, W.L. Bulletin of Malacology Taiwan, Vol. 25, p66-67. pl. 4, fig. 27a–f. 2003. Dioryx swinhoei (H. Adams). Lee, Y.C. & Chen, W.D. Natural observational illustrations 3－Land snail. Chin-Chin Publications Ltd. Taipei, p66-67. 2003. Dioryx swinhoei (H, Adams). Hsieh, B.C. Landsnails of Taiwan. Council of Agriculture Executive Yuan, p72. 2004. Dioryx swinhoei (H, Adams). Hsieh, B.C. Incredible snails, Yuan-Liou Publishing Co., Ltd., p103. 2006. Dioryx swinhoei (H, Adams). Hsieh, B.C. & al. Landsnails of Taiwan. Forestry Bureau Council of Agriculture Executive Yuan, p69. Shell: shell width 5-6mm, height 5.5-7.1mm, conical, fragile, color variable, translucently white to dull reddish brown. 3 1/3-3 3/4 whorls, convex, gradually increasing in diameter from apex to 1/2 body whorl and rapidly increasing in diameter than contracted near the aperture. Surface reticulated with irregular growth lines and indistinct spiral threads, sculpture dense, regular riblets near sutural tube. Sutural tube, narrow, long, laying backward along the suture. Umbilicus narrowly opened. Aperture circular, peristome reflex, well expended. Operculum circular, multispiral type, ceratoid, translucent yellow, concave externally, with a nipple inside. Radula: radula is taenioglossa. Radula format is 1-2-1-2-1. The central tooth is scoop-shaped, 7-8 cuspids, central cuspid is the largest, approximately 1/4 width of central tooth; the other cuspids are smaller in size. The lateral teeth are scoop-shape, 5 cuspids, the middle cuspid is the largest. The marginal teeth are sickle-like shaped, 3 cuspids of the tip, of these three the outer cuspid is the largest. A huge cuspids, on the lower site of the marginal teeth below 3 cuspids. Distribution: Endemic to Taiwan. North Taiwan: Wu-Lai, Taipei County;

127 Lu-jiau-keng runlet, Yang-Ming Mountain; Yin-he-dung, Shin-dian City. East Taiwan: Taroko valley, Hualian County; Northern shore of Tszen runlet, Hualian County; Bei-nan, Tai-dung County. Habitat: animals always attach to the leaf of taro and calla.

128

Fig. 1.1 Ventral, apex and basal view of Leptopoma shells (A-C) Leptopoma nitidum Sowerby, 1843 from Taroko Vally 24.159194N, 121.621361E; (D-L) Leptopoma tigris Lee & Wu, 2001, D-F: Lan-Yu 22.080389N, 121.506250E, G-L: Liu-Dau 22.659278N, 121.504444E.

129

Fig. 1.2 Ventral, apex and basal view of Japonia and Pilosphaera shells (A-C) Japonia formosana Pilsbry & Hirase, 1906 from Miau-li County 24.582528N, 120.875000E ; (D-F) Japonia lanyuensis Lee & Wu, 2001 from Lan-Yu 22.028222N, 121.579667E; (G-I) Holotype of Japonia boonkioensis n. sp. from Fen-chi-hu in Jia-i County 23.491972N, 120.699833E; (J-L) Pilosphaera zebra Pilsbry & Hirase, 1906 Wu-Lai, Taipei 24.849194N, 121.569861E. 130

Fig. 1.3 Ventral, apex and basal view of Ptychopoma wilsoni (Pfeiffer, 1865) shells A-C: Cha-jyue, San-shya, Taipei County 24.871780 N, 121.405655E, D-F: Sin-Xu 24.686917N, 121.228778E.

131

Fig 1.4 Ventral, apex and basal view of Cyclophorus formosaensis Nevill, 1881 shells. A-F: Charn-guang Temple, Taroko valley 24.168722N, 121.609111E, G-I: Shyong-kong, San-shya, Taipei County 24.868222N, 121.410111E, J-L: Chorng-der, Hualian County 24.189278 N, 121.660889E.

132

Fig. 1.5 Ventral, apex and basal view of Cyclophorus friesianus Moellendorff, 1883 shells, A-C: Shan-ping, Kaohsiung County 22.965917N, 120.683944E, D-F: Provincial Highway No.159A, 36k, Jia-yi County 23.469750N, 120.659250E, G-I: Guan-tzy-liing, Tainan County 23.339306N, 120.503111E, J-L: San-dih-men, Ping-dung County 22.807361N, 120.648250E.

133

Fig. 1.6 Ventral, apex and basal view of Cyclophorus latus (Kuroda, 1941) shells, A-C: County highway No. 136, 39.5K, Taichung County 24.099361N, 120.790389E, D-F Wu-Lai, Taipei County 24.847028N, 121.539278E, G-I: Dar-Guan Mountain, Tauyuan County 24.709250N, 121.431833E, J-L: Gu-guan, Taichung County 24.199278N, 120.999417E.

134

Fig. 1.7 Ventral, apex and basal view of Cyclophorus latus (Kuroda, 1941) shells, A-C: Bai-luh, Her-ping township, Tichung County 24.175333N, 120.909861E, D-F: Mei-feng, Nan-tou County 24.088139N, 121.171556E, G-I: Bai-liing, Iran County 24.525722N, 121.516083E, J-L: An-ping-keng, Iran County 24.792778N, 121.655889E.

135

Fig. 1.8 Ventral, apex and basal view of Cyclophorus moellendorffi Schmacker & Boettger, 1891 shells, A-C: Mu-dan township, Ping-dung County 22.112139N, 120.761472E, D-F: Provincial Highway No.23, 13.5k, Taidung County 23.074834N, 121.302564E, G-I: Taidung County 22.504278N, 120.958111E, J-L: Chair-shan, Kaohsiung City 22.650694N, 120.259000E.

136

Fig. 1.9 Ventral, apex and basal view of Cyclophorus cf. turgidus (Pfeiffer, 1851) shells, A-C: Jang-hu, Jia-yi County 23.611944N, 120.641250E, D-F: Provincial Highway No.149, Yun-lin County 23.589639N, 120.569306E, G-I: County Highway No.129, 20.5k, Jia-yi County 23.342500N, 120.691861E.

137

Fig. 1.10 Ventral, apex and basal view of Platyrhaphe shells, (A-F) Platyrhaphe lanyuensis Lee & Wu, 2001 Lan-Yu 22.028222N, 121.579667E, A-C: the surface mud removed, D-F: surface mud keeped. (G-L) Platyrhaphe minutus (H. Adams, 1866) G-I: Chair-shan, Kaohsiung City 22.650694N, 120.259000E, J-L: County highway No. 175, 3k, 23.328917N, 120.499750E. (M-O) Platyrhaphe sunggangensis Lee & Wu, 2001 Sung-gang, Nan-Tou County 24.073098N, 121.168753E

138

Fig. 1.11 Ventral, apex and basal view of Platyrhaphe shells, (A-C) Platyrhaphe swinhoei depressus Pilsbry & Hirase, 1906 Dah-dong-shan, Jia-yi County 23.498778N, 120.714500E; (D-I) Platyrhaphe swinhoei (H. Adams, 1866) D-F: Taroko Vally 24.168722N, 121.609111E, G-I: Jang-hu, Jia-i County 23.611944N, 120.64125E (J-K) Platyrhaphe hirasei (Pilsbry, 1901) Buh-luoh-wan, Taroko Vally 24.174003N, 121.575797E

139

Fig. 1.12 Ventral, apex and basal view of Cyclotus shells, (A-F) Cyclotus adamsi (Pilsbry & Hirase, 1906), (G-L) Cyclotus taivanus dilatus Lee & Wu, 2001. A-C: Fwu-shan Taipei County 24.795750N, 121.492944E, D-F: Su-au, Iran County 24.521194N, 121.824889E; G-I: Charn-guang Temple Taroko Vally 24.159250N, 121.606111E, J-L: Chorng-der, Hualian County 24.189278N, 121.660889E. 140

Fig. 1.13 Ventral, apex and basal view of Cyclotus shells, (A-C) Cyclotus taivanus diminutus Lee & Wu, 2001, (D-I) Cyclotus taivanus taivanus H. Adams, 1870. A-C: Lan-Yu, 22.028222N, 121.579667E, D-F: Liou-guei, Kaohsiung County 22.965917N, 120.683944E, G-I: Provincial Highway No.20, 79k, Bau-Lai, Kaohsiung County 23.108639N, 120.699722E.

141

Fig. 1.14 ESEM photograph of Cyathopoma shells. A-B: Cyathopoma iota Pilsbry & Hirase, 1904 Bai-liing, Iran County 24.525722N, 121.516083E, C-D: Cyathopoma micron (Pilsbry, 1900) li-shyng, Nan-tou County 24.067833N, 121.159722E. E-F: Cyathopoma ogaitoi (Minato, 1988) Lei-gong Shan, Guei-Jou Province 26.404N, 108.214E, G-H: Cyathopoma taiwanicum Pilsbry & Hirase, 1906 Da-chi-jiau, Shin-dian City 24.957972N, 121.571833E. 142

Fig. 1.15 Ventral, apex and basal view of Chamalycaeus shells, (A-C) Chamalycaeus hungerfordianus (Nevill, 1881) Da-chi-jiau, Shin-dian City 24.957972N, 121.571833E, (D-I) Chamalycaeus varius (Pilsbry & Hirase, 1906) Nei-gou, Nei-hu, Taipei City 25.088639N, 121.628444E.

143

Fig. 1.16 Ventral lateral, apex and basal view of Dioryx swinhoei (H. Adams, 1866) shells, A-D: San-diau-liing, Taipei County 25.062972N, 121.806222E, E-P: Ren-tzer, Iran County 24.545611N, 121.507278E.

144

Fig. 1.17 Radula of Cyclophoridae. A: Leptopoma nitidum (Sowerby, 1843) from Kaan-ding, Ping-dung County (21.965917N, 120.814500E) ; B: Leptopoma tigris Lee & Wu, 2001 from Liu-dau, from Taidung County (22.659278 N, 121.504444E); C: Japonia formosana Pilsbry & Hirase, 1906 from County Highway No.126, 26k, Miau-li County (24.582528N, 120.875E); D: Japonia lanyuensis Lee & Wu, 2001 from Lan-Yu (22.028222 N, 121.579667E); E: Japonia boonkioensis n. sp. from Fen-chi-hu in Jia-i County (23.491972N, 120.699833E); F: Pilosphaera zebra (Pilsbry & Hirase, 1906) from Nei-Hu, Taipei City (25.088639N, 121.628444E).

145

Fig. 1.18 Radula of Cyclophoridae. A: Ptychopoma wilsoni (Pfeiffer, 1865) from frog stone, Miau-li County (24.686917N, 121.228778N); B: Cyclophorus formosaensis Nevill, 1881 from Harn-chi, Iran County (24.583417N, 121.691167E); C: Cyclophorus friesianus Moellendorff, 1883 from Guan-tzy-liing, Tainan County (23.339306N, 120.503111E); D: Cyclophorus latus (Kuroda, 1941) from Ren-tzer, Iran County (24.545611N, 121.507278E); E: Cyclophorus latus (Kuroda, 1941) from Mei-feng, Nan-tou County (24.088139N, 121.171556E); F: Cyclophorus moellendorffi Schmacker & Boettger, 1891 from Chair-shan, Kaohsiung City (22.650694N, 120.259000E).

146

Fig. 1.19 Radula of Cyclophoridae. A: Cyclophorus cf. turgidus (Pfeiffer, 1851) from Provincial Highway No. 162A, 32.5k, Yun-lin County (23.560944N, 120.655583E); B: Platyrhaphe lanyuensis Lee & Wu, 2001 from Liu-Dau (22.669667N, 121.506444E); C: Platyrhaphe lanyuensis Lee & Wu, 2001 from Lan-Yu (22.028222N, 121.579667E); D: Platyrhaphe minutus (H. Adams, 1866) from Sheau-liou-chyou, Ping-dung County (22.336417N, 120.360639E); E: Platyrhaphe swinhoei depressus Pilsbry & Hirase, 1906 from Dah-dong-shan in Jia-i County (23.498778N, 120.7145E); F: Platyrhaphe s. swinhoei (H. Adams, 1866) from San-diau-ling, Taipei County (25.0625N, 121.809694E).

147

Fig. 1.20 A: Cyclotus adamsi (Pilsbry & Hirase, 1906) from Kai-cheng Temple, Iran County (24.813139N, 121.710583E); B: Cyclotus taivanus dilatus Lee & Wu, 2001 from Chorng-der, Hualian County (24.189278N, 121.660889E); C: Cyclotus t. diminutus Lee & Wu, 2001 from Yongsing, Lanyu Island, Taidung County (22.028222N, 121.579667E); D: Cyclotus t. taivanus H. Adams, 1870 from Bau-Lai, Kaohsiung County (23.108639N, 120.699722E); E: Cyathopoma iota Pilsbry & Hirase, 1904 from Bai-liing, Iran County (24.525722N, 121.516083E); F: Cyathopoma micron (Pilsbry, 1900) from li-shyng mountain road, Nan-tou County (24.067833N, 121.159722E)

148

Fig. 1.21 A: Cyathopoma ogaitoi (Minato, 1988) from Lei Gong Shan, Guei-Jou Province (26.404N, 108.214E); B: Cyathopoma taiwanicum Pilsbry & Hirase, 1906 from Da-chi-jiau, Shin-dian City (24.957972N, 121.571833E); C: Chamalycaeus hungerfordianus (Nevill, 1881) from Da-chi-jiau, Shin-dain County (24.958417N, 121.574306E); D: Chamalycaeus varius (Pilsbry & Hirase, 1906) from Nei-hu, Taipei City (25.088639N, 121.628444E); E: Dioryx swinhoei (H. Adams, 1866) from Ren-tzer, Iran County (24.545611N, 121.507278E).

149

150 Chapter 6

Summary and conclusion

The Cyclophorids fauna of Taiwan Before the present investigation, there were 9 genera and 29 cyclophorid species (including 3 unidentified species) in Taiwan reported by me (Lee & Wu 2001). In 2003, Cyathopoma iota and C. ogaitoi were reported as new records of Taiwan by me (Lee & Cheng 2003). The above reports were based on morphology and some viewpoints were immature. In the phylogenetic study of Cyclophoridae I chanced to find a new species of Japonia and will be report in Zootaxa magazine (Lee, Lue & Wu 2008). Base on morphologic and molecular data, the taxonomic status changed and previous miss identified species were corrected in chapter 1. In summary, there are 10 genera and 29 cyclophorid species in Taiwan.

The phylogeny of East Asia Cyclophoridae I have undertaken the first molecular phylogenetic analysis of the Cyclophoridae. Sequence of COI and 16S rRNA were examined in 32 species of 10 genera of cyclophorid encompassing the entire currently recognized major subfamily. It allows an independent test of the present classification based on morphologic characters. At the level of genus, my molecular phylogeny closely supports the current taxonomy, but at subfamily level does not although the bootstrap support is low. In the other hand, Cyathopoma micron were recognized as Cyclotus micron based on its biconcave operculum morphology (Pilsbry & Hirase 1904). Besides, C. iota was considered a closer relative to C. micron than to C. taiwanicum (Pilsbry & Hirase 1904, Higo & Goto 1993, Lee & Chen 2003). My molecular and radula data indicate that C. iota is closer to C. taiwanicum than to C. micron. In summary C. micron, C. iota, C. ogaitoi, and C. taiwanicum should be placed in Cyathopoma rather than Cyclotus. Both COI and 16S rRNA gene trees indicate Japonia and Ptychopoma are sister group of Cyclophorus and Cyclotus, respectively. The former two will be used as the out group of Cyclophorus and Cyclotus in their phylogenetic studies.

Taiwan Cyclophorus speciation model vs. C. taivanus ssp. speciation model

151 Cyclophorus and Cyclotus widely occur in Taiwan and share similar ecological niche. Both Taiwan Cyclophorus and C. taivanus ssp. span two morphologic units. In Taiwan Cyclophorus, the keel and round shell occurs in northern and southern Taiwan, respectively. In contrast, tall spire and flat spire previous C. taivanus ssp. also occurs in north and south Taiwan, respectively. The two morphologic units in different two species groups both have north form and south form. Is that a coincidence? Alternatively, are there different selective pressures on cyclophorid snails in north and south Taiwan? My result from Cyclophorus PLS analysis showed that differences between populations in some shell traits co-varied significantly with long term climatic conditions and altitude. I found climatically warmer and stable temperature tend to have keeled shell. The warm and stable climate tends to have luxuriant vegetation. The keeled shell would be at an advantage when roam over the ground in the luxuriant vegetation. Previous C. taivanus ssp. also appears a climatic gradient responsible for the distribution pattern of species (Fig. 4.12). Along this cline, C. adamsi occupies only the sites with the moist winter and arid summer season. This type of climate seems to exclude C. taivanus group (C. t. dilatus, C. t. diminutus, C. t. peraffinis, and C. t. taivanus). The actual level of water stress at a particular site depends strongly on the local microclimate, which may account for the observed intermingled pattern in the contact zone. Even though, the sampling population size is few and limited, there is probably a cline in umbilicus proportion between populations near contact zone (Fig. 4.14). Likewise, there are more or less clines in the other eight shell traits between populations near contact zone (Fig. 4.15). The above showed a highly probability of adaptation of different climate. However, the speciation model of Taiwan Cyclophorus is different from C. taivanus ssp. The molecular and morphology data indicate that there are two distinctive forms of Cyclophorus in southwest Taiwan, one with flat shell and sculpture with spiral cords on shell surface, one with tall shell and without spiral cords. The molecular data show strong geographic structure (Fig. 3.8–3.12) and current gene flow between close populations (Fig. 3.7). Taiwan Cyclophorus probably is a case of ring speciation. Both two distinctive southwest Taiwan forms are keeled shell. It may due to the adaptation of long term climatic conditions described in above section. The molecular data of previous C. taivanus ssp. show two highly divergent haplotype clades, indicated the presence of two independently evolving lineages. The sequence divergence between two clades was almost as high as between other Cyclophoridae species. Besides, the gene flow between two clades is absent. Therefore the two clades should be considered as separated species. For the environmental analysis, temperature may be a limiting factor of the distribution of C. adamsi and C. taivanus

152 group. It appears that a climatic gradient is responsible for the distribution pattern of species (Fig. 4.12). The actual level of water stress at a particular site depends strongly on the local microclimate, which may account for the observed intermingled pattern in the contact zone. Event the sampling population is few and limited; there is probably a cline in umbilicus proportion between populations near contact zone (Fig. 4.14). Likewise, there are more or less clines in the other eight shell traits between populations near contact zone (Fig. 4.15). Even though premating or postmating reproductive isolation may have evolved as a byproduct of ecological divergence. The ecological divergence appears to be sufficient to prevent immediate contact and therefore acts as an effective barrier to mating. The ecological divergence probably appears rule of speciation in C. taivanus ssp. case. The speciation process is not complete among C. t. dilatus, C. t. diminutus, C. t. peraffinis, and C. t. taivanus, and the adaptation of climatic pressure continuing be a rule of speciation process. It appears a chance to understand that even how similar two taxonomic groups is, occupied similar niche, undergo the same geological history, with morphological adaptation to the same long term climates, they may have different speciation model. The speciation cause of live-form is complex; even a tiny difference will lead to distinct speciation result.

153

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162 Acknowledgments

This study was support by a grant from the “Digital Archives of Malacofauna from Taiwan” project of Research Center for Biodiversity, Academia Sinica. Thanks are due to Dr. Hai-Tau Sht, Dr. Wei Liang of Hainan Normal University, Ming-Hui Lin and Mr. Zan-Chen Hwang for their help to collect some materials from China. The author also wishes to appreciate Mr. Chen-Lung Tung and Ms. Chih-Hui Wang for their help to collect some species from Taiwan and Mr. Ming-Hui Lin for providing living Leptopoma tigris. I also thank Mr. Tai-Lang Lin of Institute of Cellular and Organismic Biology for SEM study and all members of Malacology Laboratory for molecular study.

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