台灣產滑蜥屬（Scincella）與蜓蜥屬（Sphenomorphus）的分子與形態

國立臺灣師範大學生命科學系碩士論文

台灣產滑蜥屬（ Scincella）與蜓蜥屬（Sphenomorphus）的分子與形態鑑定 Taxonomy of Scincella and Sphenomorphus (Squamata: Scincidae) of Taiwan based on molecular data and morphological evidence

研究生：汪仁傑

Jen-Chieh Wang

指導教授：林思民博士

Dr. Si-Min Lin

中華民國 101 年 8 月

Table of contents

中文摘要 ------1

Abstract ------2

Introduction ------4

Materials and Methods ------8

Results ------14

Discussion ------19

Conclusion ------24

References ------25

Table ------28

Figures ------36

Appendix ------48

摘要

台灣與鄰近地區滑蜥屬物種的分類關係複雜且尚未明確，而根據

尾部腹面顏色可以將台灣滑蜥（Scincella formosensis）分為紅色與

黃色兩種色型，因此以形態及遺傳資料檢驗台灣滑蜥種間與種內之分

類關係並給予適當處理有其必要。另一方面，長久以來將瞼窗做為判

別滑蜥屬與蜓蜥屬的重要特徵，然而近年來的研究顯示瞼窗能否有效

區分這兩個屬仍有待檢驗。此外，台灣南部中海拔發現了兩群形態介

於滑蜥與蜓蜥的小型石龍子，這些族群的分類地位需要確認釐清。本

研究自台灣及鄰近地區取得滑蜥屬與蜓蜥屬包含外群共 15 個分類群，

309 個樣本，以粒線體基因組的 Cytochrome b、核基因組的 Cmos

與 Bach1 等三個基因片段重建親緣關係樹，同時利用外部形態的特

徵探討台灣及鄰近地區滑蜥屬與蜓蜥屬物種的分類關係。結果顯示

1.黃色型的台灣滑蜥為典型的台灣滑蜥，而紅色型則為寧波滑蜥的台

灣族群；2. 台灣滑蜥與先島滑蜥尚處於種化的早期階段，應該將先

島滑蜥併入台灣滑蜥；3.台灣中海拔的兩個族群與台灣蜓蜥關係最接

近，但形態和遺傳均存在具有鑑別力的特徵，應該是兩個尚未描述的

新種；4. 以瞼窗構造做為滑蜥與蜓蜥屬的鑑別特徵會導致這兩個屬

非單系群，台灣蜓蜥及中海拔的兩個新種應該改置於滑蜥屬。

關鍵字：形態、核基因、粒線體基因、滑蜥屬、蜓蜥屬、瞼窗 1

Abstract

The species diversity and taxonomic status of Scincella and small-sized Sphenomorphus in Taiwan and neighboring regions is a long and lasting controversy. According to the color of tails, Sc. formosensis could be separated into red-tailed and yellow-tailed morphs. On the other hand, two populations of ground skinks recently discovered at mid-elevation of Taiwan represent intermediate morphological characters between Scincella and Sphnomorphus. The species or genetic diversity of such a species complex in Taiwan and adjacent regions remains to be further studied, while the validity of the transparent disk as the diagnosis between the two genera should be re-evaluated. In this study, phylogeny of this species group was constructed with a total of 309 samples comprising 15 taxa by using mitochondria DNA Cytochrome b, nuclear Cmos and nuclear Bach1 sequences. Morphological characters were also applied to revise the taxonomic relationship between Scincella and Sphenomorphus of Taiwan and neighboring areas. My results indicated that: 1. the yellow-tailed morph represents a typical Sc. formosensis, while the red-tailed morph should be Sc. modesta; 2. with insufficient diagnosis between the two, Sc. boettgeri is suggested to be a synonym of Sc. formosensis; 3. the two mid-elevation taxa closely related to Sp. taiwanensis should be treated as new species; and 4. with a paraphyletic relationship between Scincella and Sphenomorphus, we suggest not treating transparent disk as a diagnosis between

2 the two genera, while Sp. taiwanensis species group were suggested to be transferred to Scincella, i.e., Sc. taiwanensis.

Keywords: mitochondrial DNA, morphological traits, nuclear gene, Scincella, Sphenomorphus, transparent window

Introduction

The skink genus Scincella (Mittleman, 1950), also known as the “ground skinks”, is a member of Lygosominae (Scincidae). At present, a total of 35 species of Scincella have been reported, including 27 species from Asia and other species from America (Greer, 1974; Nguyen, 2010a; Uetz P. 2008).

The phylogenetic relationships among Scincella and other genera remain unsolved because of their shared morphological characters (Nguyen et al., 2010a, b; Nguyen et al., 2011). Traditionally, the main diagnostic character of this genus is the presence of a transparent or an opaque window on lower eyelid (Boulenger, 1887; Smith, 1935; Taylor, 1963; Greer, 1974; Ouboter, 1986). However, this feature is also found in several other genera of Scincidae: Asymblepharus, Leptoseps, Lipinia, Paralipinia, and Vietnascincus (Darevsky and Orlov, 1994, 1997; Greer, 1997; Eremchenko, 2002; Shea and Greer, 2002; Greer and Shea, 2003). In contrast, some of the members within Scincella (e.g., Sc. cheerei) may lack this feature (Linkem et al., 2011). The most confusing problem is the interrelationship between Scincella and Sphenomorphus, while the latter might be the largest genus in Scincidae comprising several non-monophyletic groups (Honda et al., 2003; Grismer, 2007, 2008; Uetz, 2008; Nguyen et al., 2011). Several species have transferred from Sphenomorphus to Scincella or vice versa because of their similar morphological or

4 genetic characteristic, such as Sc. reevesii (Mittleman, 1952), Sc. rufocaudata (Nguyen et al., 2010a), Sc. devorator (Nguyen et al., 2011), Sc. assatus, Sc. cherriei, and Sc. incerta (Linkem et al., 2011). Obviously, validity and definition of Scincella respect to Sphenomorphus needs a revision. The species diversity and taxonomic status of Scincella and small-sized Sphenomorphus in Taiwan and southern Ryukyus is a long and lasting controversy. The first focal species in this study is Scincella formosensis (Van Denburgh, 1912), a small lygosominid skink endemic to northern and western Taiwan with an altitudinal distribution under 1000 meters. Scincella formosensis is closely related to Sc. boettgeri (Van Denburgh, 1912) endemic to southern Ryukyus and both species had a complicated taxonomic history since first described by Van Denburgh (1912). These taxa were first described as discriminate subspecies of Leiolopisma laterale (Say, 1823) (=Sc. lateralis) from North America, with diagnostic characters including scales around the middle and along the mid-dorsal line of body, relative positions of prefrontals, and the degree of distinctness in the lower border of the dorso-lateral dark stripe. However, Schmidt (1927a, b) revised these East Asian populations and treated them as two separate species. Later, Nakamura and Uéno (1963) considered Sc. boettgeri and Sc. formosensis are both subspecies of Lygosoma reevesii (Gray, 1838) (=Sc. reevesii). In contrast, Greer (1974) compared external and osteological characters and agreed with Schmidt’s (1927a, b)

5 view to treat them as valid species. Ouboter (1986) examined a few (n=22) samples from continent East Asia, and considered all the populations of this range as Sc. modesta (Günther, 1864), a species described from China. However, his conclusion remained problematic because specimens from Taiwan and the Ryukyus were not included in his evaluation. Finally, Chen et al. (2001) examined a large amount of specimens from Taiwan and the Ryukyus and treated them as separate taxa. However, Sc. reevesii and Sc. modesta were still not included in their evaluation. Conclusively, the complicated relationship between these Scincella skinks has never been studied with a complete sampling. Besides for the problems occurred between Sc. fomosensis and its congeners, biodiversity of this species itself might be underestimated. According to the color of bellies and tails, Sc. formosensis could be further separated into two morphs: the yellow-tailed morph, and the red-tailed morph (Fig. 1). Based on our preliminary observation, the yellow-tailed morph is wildly distributed in western Taiwan, while the red-tailed morph occurs only in northern Taiwan. The two morphs distribute sympatrically in some locations such as Wulai at New Taipei City and Fuxing at Taoyuan County. The taxonomic status of Taiwanese Scincella needs a revision both at the inter-specific and intra-specific levels. The second problematic taxon is Sphenomorphus taiwanensis (Chen & Lue, 1987), another attenuate ground skink endemic to the high mountain regions in Taiwan. This species was attributed to

6 genus Sphenomorphus because of the lack of transparent window in moveable lower eyelid (Chen & Lue, 1987). This species is also distinctly from Sc. formosensis for their differentiation in habitats: Sc. formosensis lives in low elevation hardwood forests usually under 1000 m, while Sp. taiwanensis occurs only in alpine grasslands, alpine tundra, and edge of montane coniferous forests with an altitude higher than 1800 m. Such a clear cut diagnosis did not meet any problem in the past several decades until two taxa of ground skinks recently discovered at mid-elevation (1500-1700m) of southern Taiwan (Fig. 2). These lizards represent intermediate morphological, altitudinal, and habitat characters between Sc. formosensis and Sp. taiwanensis. Obviously, the species or genetic diversity of such a species complex in Taiwan and adjacent regions remains to be further studied.

In this study, I aim to accomplish the following targets: 1. To evaluate the validity of Scincella respect to Sphenomorphus both in the viewpoint of phylogeny and morphology;

2. To solve the long and lasting questions about the species and

genetic diversity of Scincella skinks in Taiwan;

3. To recover the underestimated diversity of ground skinks in mid

elevations of Taiwan.

Materials and Methods

Sample collection A total of 290 small-sized Lygosominae specimens were obtained in this study, including: 202 Sc. formosensis from 26 localities, comprising 146 yellow-tailed and 56 red-tailed morphs respectively; 32 Sc. boettgeri from 4 Southern Ryukyu Islands; 15 Sc. reevesii from Hongkong and Guangxi; 15 Sc. modesta from Hongkong; 17 Sp. taiwanensis from 5 localities; 6 Sphenomorphus sp1. collected from Dahanshan, Pingtung County; and 3 Sphenomorphus sp2. collected from Tengzhi, Kaohsiung County. To comfirm weather Scincella and Sphenomorphus are reciprocally monophyly, 7 outgroup taxon 19 individuals were sampled from Lygosominae including: 3 Sc. lateralis; 1 Sc. cherriei; 3 Sc. rufocaudatus; 1 Sp. simus, 7 Sp. indicus, 3 Sp. incognitus, and 1 Eutropis longicaudata. Sc. lateralis is the type species of Scincella; Sc. cherriei and Sc. rufocaudatus had been placed in Sphenomorphus before, while Sc. cherriei may lack the window on lower eyelid. Although the type species of Sphenomorphus, Sp. melanopogon, was not obtained in this study, the morphologically similar Sp. indicus was included (Linkem et al., 2011). Sequences of the same mitochondrial fragments from Sc. lateralis, Sc. cherriei, and Sp. simus were downloaded from GenBank. Sample localities and voucher numbers of these specimens were shown in Fig. 3 and Table 1.

Molecular techniques Total genomic DNA was isolated from muscle tissue by using the Qiagen DNeasy Blood & Tissue Kit (Qiagen Inc., 2009). DNA was suspended in 1X TE buffer and stored at -20°C. The total genomic DNA was used in subsequent polymerase chain reactions (PCR) as template to amplify one mitochondrial (cytochrome b; cytb) and two nuclear genes (Bach1 and Cmos). In order to amplify a large variety of different taxa, specific combination of primers was applied for a certain species as listed in Appendix 1. The PCR mixtures were prepared in total volumes of 20 μM, consisting of of 1X Green GoTaq Flexi Buffer (Promega), 2.5 mM MgCl2, 0.4 μM each primer, 0.2 mM each dNTP, 0.5 U GoTaq Flexi DNA Polymerase (Promega), and 0.1-0.5 ng template DNA. PCR amplification procedure was set to initial denaturation at 94 °C for 3 minutes, followed by 35 cycles at 94 °C denaturation for 30 seconds, annealing for 40 seconds (temperature see Appendix 1), 72 °C extension for 2 minutes, and final extension at 72 °C for 5 minutes by using a iCycler Thermal Cycler (Bio-Rad). The annealing temperature of several taxa was slightly adjusted to ensure the PCR products in their best condition. The same primers used for PCR were used for the sequencing reactions carried out on an ABI 3730 automated DNA sequencer by Genomics BioSci & Tech Corp. (Taipei, Taiwan). The sequences were determined in both directions, and the original signals were proofread using SEQUENCHER software version 4.7 (Gene Codes

Corporation). The sequences obtained were compared to those of other Scincids to ensure the accuracy of the PCR ampliﬁcations.

Phylogenetic analyses of mitochondrial dataset Individual sequence dataset was first transformed to a haplotype dataset by using DnaSP 5.10 (Rozas et al., 2003). The best-fit model of sequence evolution was assessed by using Akaike information criterion (AIC) as implemented in jModelTest 3.6 (Posada and Crandall, 1998). Result of this analysis in cytochrome b sequences (1143 bp) suggested TrN+I+G (Tamura-Nei’s model with a proportion of invariable sites and a gamma shape distribution) as the best-fit substitution model for this dataset, with a gamma value (G) = 1.1310, and a proportion of invariable sites (I) = 0.4680. For phylogenetic reconstruction, maximum likelihood (ML), neighbor-joining (NJ), and Bayesian inference (BI) analyses were used. Maximum likelihood (ML) and neighbor-joining (NJ) analyses of the data set were done in PhyML (Guindon et al., 2010) and MEGA 5 (Tamura et al., 2011), respectively, the Bayesian inference analysis was performed using MrBayes 3.1.2 (Huelsenbeck and Ronquist, 2001). Non-parametric bootstrapping (both 1000 pseudoreplicates in NJ and ML) was performed to obtain robustness of node support for the resulting trees (Felsenstein, 1985). Four Markov chains were used and each Markov chain was started from a random starting tree and ran for 107 generations,

10 trees were chosen every 100 generations, and the initial 25% generations were discarded as burn-in. The remaining data were used to estimate the strict consensus tree and the Bayesian posterior probabilities (BPPs). Finally, the values of statistical supports of ML bootstraps, MP bootstraps, and BPPs were labeled on corresponding nodes.

Phylogenetic analyses of nuclear dataset Nuclear Bach1 (939 bp) and Cmos (372 bp) genes were combined to form a concatenated dataset in tree construction. For a better presentation of their phylogenetic relationship in species level, only a proportion of yellow-tailed Scincella, red-tailed Scincella, Sc. modesta and Sc. boettgeri were remained in the final analysis to prevent from the redundancy of too many similar sequences. Since homologous fragment of several non-focal species is not available from GenBank, they were not included in this analysis (e.g., Sp. simus, Sc. cherrei, and Sc. lateralis). The best-fit model of this dataset was TPM1uf+G (Kimura 3-parameter model with a gamma shape distribution), with a gamma value (G) = 0.4860. Phylogenetic analyses followed those of the mitochondrial dataset: a best tree constructed by using ML algorithm, with ML bootstraps, NJ bootstraps, and Bayesian posterior probability to obtain the nodal supports. Owing to their lower divergence and polymorphism, the interrelationship among some closely related species could not be

11 represented in a phylogenetic tree. In these cases, we generated haplotype network using phylogenetic algorithms with migration and used proper models of sequence evolution (Salzburger et al., 2011). For the two diploid locus, heterozygous sites were found when a secondary peak was at least half height of the primary peak (the 50% threshold). When more than one heterozygous site occurred within an individual, haplotype reconstruction was performed by using PHASE 2.1 (Stephens and Scheet, 2005; Stephens et al., 2001) implemented in DnaSP v5 (Librado and Rozas, 2009). Every run was set up with MCMC iterations to 100,000 and thinned every 1,000 intervals. In order to reflect the actual proportion of haplotypes in each species, each individual was included in the network by contributing a pair of phased alleles. Phylogenetic reconstructions were estimated using Maximum likelihood approach. The generated tree were used to estimate each haplotype network in Haploviewer (Salzburger et al., 2011).

Morphological Analysis In order to confirm whether morphological characters could be applied to distinguish among these related closely taxa of Scincella and Sphenomorphus, a total of 224 specimens were carefully evaluated, comprising 125 yellow-tailed Scincella, 53 red-tailed Scincella, 23 Sc. boettgeri, 8 Sc. modesta, 8 Sc. reevesii, 8 Sp. taiwanensis, 5 Sphenomorphus sp.1 from Dahanshan, and 2 Sphenomorphus sp.2 from Tengzhi (Table 1).

Ten meristic morphological characters were used in discriminant analysis (DA), included the numbers of: nuchals (N), supralabials (SL), infralabials (IL), dorsal scales (DS, counted from parietals to the just posterior margin of hindlimbs), gular and ventral scales (GVS, counted from mental to preanals), contact point number of frontal and frontonasals (F), midbody scale rows (MBSR, counted around midbody), subdigital scales of finger IV and toe IV (F4S and T4S, respectively; counted only for specimens having undamaged tips of finger IV and toe IV), and the scale rows covered by dorsal-lateral stripe (SRB), a critical trait used to distinguish between Sc. formosensis and Sc.boettgeri (Van Denburgh, 1912; Chen et al., 2001) (Fig. 4). Discriminant analyses were applied by using JMP 7.0 (SAS Institute Inc., 2007). Finally, another two qualitative traits were evaluated to serve as diagnosis among closely related taxa, including the existence of transparent window in lower eyelid (TW) used to distinguish between Sphenomorphus and Scincella; and the color of belly tail (CT) used to distinguish between the two color morphs of Scincella in Taiwan.

Results

Phylogeny of Cytochrome b The 298 small-sized Lygosominae obtained in this study yielded to a total of 124 cytochrome b haplotypes as shown in Table 1. After combined with the outgroup taxa, the resultant dataset comprised 133 OTUs with 1143 bp in length, including 577 (50.5%) variable and 530 (46.4%) parsimony informative sites respectively. Molecular phylogeny of cytochrome b gene reconstructed by ML, NJ, and Bayesian approaches represented almost identical tree topologies. Based on the consistency among these criterion and the high statistical supports, we accepted the ML tree as the best topology representing the relationship among our samples (Fig. 5). The first remarkable result revealed that both Sphenomorphus and Scincella are not monophyletic based on their current definition. Sphenomorphus simus is clustered with Sc. cherrei, while Sp. taiwanensis (A) and its sister taxa (B and C) are nested within other Scincella species on the tree. This result indicated that the interrelationship and definition between these two genera should be further discussed. Secondly, phylogeny relationship confirmed the distinctness of Sc. lateralis and Sc. reevesii (D). The primary taxonomic treatments, which treated populations in Ryukyu and Taiwan as synonym of Sc. lateralis (Van Denburgh, 1912) or Sc. reevesii (Nakamura & Uéno, 1963), could be easily excluded. All the Scincella individuals collected in Taiwan could be precisely allocated into two clades respectively representing the

14 yellow-tailed morph (H) and the red-tailed morph (F). No coloration or genetic admixture was observed between the two clades according to the current sampling shame. It is remarkable that the two color morphs of Scincella in Taiwan are not belonging to the same clade. The yellow-tailed morph is grouped with Sc. boettgeri (G), while the red-tailed morph is grouped with Sc. modesta (E). Clustering of the red-tailed Scincella and Sc. modesta is strictly supported by bootstraps and Bayesian posterior probability, and the two clades have a divergence of 0.0923 (Table 2). Clustering of the yellow-tailed Scincella and Sc. boettgeri is also highly supported, yet the differentiation between the two is not prominent (Table 3). Specimens from type locality of Sc. formosensis (Guanziling, Taiwan) and their neighboring regions represented exclusively the yellow-tailed morph, and were all attributed into the yellow-tailed clade (H) on the phylogenetic tree. The last remarkable result is the phylogenetic position the two Taiwanese mid-elevation populations (B and C). These two populations formed a monophyletic group with a distinct divergence from typical Sp. taiwanensis (A). The divergence between Sp. taiwanensis and Tengzhi or Dahanshan populations are 0.1648 and 0.1540, respectively (Table 2). The two populations per se have a divergence of 0.1458. Combination of the three taxa is referred as the Sp. taiwanensis species group in the following context.

Phylogeny and haplotype genealogy of Bach1 and Cmos

The sequences of nuclear DNA have less variable sites and much lower divergence compared to mitochondrial sequences. As in the mitochondrial dataset, phylogeny constructed from the concatenated sequences of Bach1 and Cmos genes represented almost identical tree topology among ML, NJ, and Bayesian approaches (Fig. 6). Ignoring those non-focal species for which sequences were not available from GenBank, there are three differences between nuclear and mitochondrial phylogeny, including: (1) Sc. rufocaudtus and Sc. reevesii (D) formed a weakly supported clade in mitochondrial phylogeny, yet this clade does not exist in the nuclear phylogeny; (2) the Sp. taiwanensis species group (the A-B-C clade) are more closely related to the yellow-tailed Scincella and Sc. boettgeri clade (the G-H clade) in this tree; and (3) closely related species, including members within A-B-C clade, E-F clade, and G-H clade, are not so well resolved in nuclear phylogeny. Despite of these differences, the conclusive results in mitochondrial phylogeny are still well supported by nuclear phylogeny: (1) Spenomorphus and Scincella are not reciprocal monophyly; (2) the yellow-tailed and red-tailed Scincella formosensis are distinctly separated and are closely related to Sc. boettgeri and Sc. modesta, respectively; and (3) the two mid-elevation populations occurring in Taiwan are distinct from a typical Sp. taiwanensis. In order to confirm the relationship among these closely related taxa, haplotype networks were separately constructed for Scincella

(Sc. modesta, Sc. boettgeri, yellow-tailed and red-tailed Scincella) and the Sp. taiwanensis species groups (Figs. 7 and 8). In order to reflect the actual proportion of haplotypes in each species, each individual was included in the network by contributing a pair of phased alleles. Networks of Scincella from Bach1 and Cmos (Fig. 7) represented a congruent separation between the E-F clade and the G-H clade by owing quite different haplotypes. The red-tailed Scincella and Sc. modesta do not share any common haplotype in the Bach1 network, supporting that these two clades have a long-term differentiation history both in mitochondrial and partial of the nuclear genome. In contrast, the yellow-tailed Scincella and Sc. boettgeri were found to share a most dominant haplotype in both genes, indicating that these two taxa have not yet reached reciprocal monophyly in terms of nuclear loci. A high genetic differentiation is also observed in the Sp. taiwanensis group. In the Bach1 network, the three taxa represented highly differentiated haplotypes (in terms of a nuclear gene) with no sharing. On the other hand, although a shared haplotype was found in the Cmos network, the differences in the frequency of this haplotype along with some other private ones indicated that this gene has also reached a certain level of genetic differentiation.

Morphological analysis Discriminant analyses were applied to distinguish among those

17 genetically and morphologically closely related species. The results were shown in Figs. 9, 10, 11. The classification success rate (%) for each group is presented in Table 2. The three members belonging to Sp. taiwanensis species group have a best classification rate of 100% or each taxon (Table 4C and Fig. 11), indicating a good resolution in mitochondrial, nuclear, and morphological characters. Differentiation between the yellow-tailed Scincella and Sc. boettgeri could reach 94.4% (the former) and 100% (the latter). This situation indicated that these two taxa could be finely distinguished by using morphological character (Table 4B and Fig. 10), but not well supported by mitochondrial and nuclear markers. In contrast, genetic differentiation between Sc. modesta and the red-tail Scincella is prominent, but these two clades are not so easy to be distinguished merely by morphology (Table 4A and Fig. 9).

Discussion

The underestimated diversity of Scincella in Taiwan Scincella species in Taiwan and the Ryukyus have experienced several different taxonomic revisions. They have been grouped into Sc. lateralis (Van Denburgh, 1912), Sc. reevesii (Nakamura and Uéno, 1963), and Sc. modesta (Ouboter, 1986) in different periods. Phylogeny of these taxa represented a conclusive result that did not support the first two treatments. The most remarkable result of the phylogeny indicated the coexistence of two Scincella species in Taiwan. Since all of the topotypes (from Guanziling, N=11) of Sc. formosensis are grouped into the yellow-tailed morph in morphological and genetic respects, the yellow-tailed morph should denote for a typical Sc. formosensis. On the other hand, the red-tailed morph, with a definite differentiation from the yellow-tailed morph, should not belong to the same species. This morph is closely related to Sc. modesta with a high statistic support. However, genetic differentiation between the two is still prominent: an 9.23% p-distance for mitochondrial sequences, and has reached reciprocal monophyly for one of the two nuclear loci which we have evaluated. However, these two taxa are difficult to be distinguished merely based on morphology. The 9 meristic morphological characters evaluated are highly overlapping except for the number of F4S and T4S (Table 5): Sc. modesta represents values of these characters higher than that of the red-tailed morph. However, Sc. modesta is wildly distributed

19 in China with a type locality from Ningbo, Zhejiang (Gunther, 1864), samples of Sc. modesta used in this study from Hongkong are far from the type location. In the future, a comparison among samples from southeast China, such as Guangdong, Fujian and Zhejiang provinces, is needed for a better understanding on the genetic and morphological differentiation of such a widely distributed species. With such a minor morphologic differentiation, we suggest to attribute the red-tailed morph as a unique population of Sc. modesta in Taiwan.

Taxonomic dilemma of Sc. formosensis and Sc. boettgeri The yellow-tailed morph denoting a typical Sc. formosensis is closely related to Sc. boettgeri as suggested by most literature. However, this monophyly could be further divided into 11 clades (Fig. 12) with a between-group distance ranging from 4.72% to 9.72% (Table 3). It is also worth to note that the divergence among Taiwanese populations has exceeded that between Taiwan and Ryukyu populations. For example, clade 4 and 5 distribute in northern Taiwan more closely related to Sc. boettgeri and are clustered to the latter in the Bayesian tree. A majority of individuals from these two species have a shared haplotype in both of the two nuclear genes, indicating that genetic differentiation between the two occurred in a comparatively shorter time span. Most of the meristic morphological characters evaluated in this study represented overlapping values between the two taxa (Table

5). However, there are still several diagnostic characters. The number of DS on Sc.boettgeri is more than Sc. formosensis, which fits the original description of Van Denburgh (1912). According to his description, the frontal is in contact with the frontonasal in most Sc. formosensis (14 of 19 individuals evaluated), while the prefrontals separate the frontial from the frontonasal in most Sc. boettgeri (35 of 37 individuals evaluated). Our study obtained a similar trend: the frontal is in contact with the frontonasal in 86 of 125 Sc. formosensis, and the prefrontals separate the frontial from the frontonasal in 14 of 23 Sc. boettgeri. Van Denburgh (1912) also mentioned that Sc. formosensis do not have very definite lower border on dark lateral band, while Sc. boettgeri has broader dark lateral band and with less definite lower border. In our study, the number of SRB on Sc. boettgeri is indeed more than Sc. formosensis in most individuals, indicating a much more clear lateral dark band of the former. However, some Sc. formosensis from northern Taiwan also exhibit the character of a dark lateral band. Interestingly, these individuals are those who have a closer genetic relationship to Sc. boettgeri. Owing to the imperfect resolution between the two taxa both in molecular and morphological markers, the differentiation between Sc. formosensis and Sc. boettgeri has not yet reached the threshold of a so called “good” phylogenetic species. Under the consideration of phylogenetic species concept (Futuyma, 2009), we consider to combine the two taxa as the senior synonym, Sc.

21 formosensis. However, owing to a medium degree of genetic and morphological differentiation, the southern Ryukyu population could remain its endemism and treated as an island subspecies, Sc. formosensis boettgeri.

Sphenomorphus taiwanensis species group in Taiwan The two populations from mid-elevation (1500-1700m) area of south Taiwan are grouped with Sp. taiwanensis on mitochondrial and nuclear genes. However, their genetic and morphological differentiation are large. The discriminating rate of morphological analysis is 100% for each one. The Dahanshan population could be distinguished by its unique dark lateral band with a series of tiny spots, which is never seen in any other neighboring species. The Tengzhi population is characterized by an appearance extremely similar to Sc. formosensis, but they are actually not belonging to the same clade. These two taxa are also different from a typical Sphenomorphus or Scincella by having partially developed transparent disk. Tengzhi and Dahanshan populations could only be found at mid-elevation (1500-1700m) area of southern Taiwan. These feature indicated the underestimated species diversity of small Lygosominae in Taiwan. With extremely narrow distribution and small population size, the maintaining of these unique taxa would be a critical challenge in the future.

Definition of Scincella needs to berevised In our result, molecular phylogeny on both mitochondria DNA and nuclear DNA indicate Scincella and Sphenmorphus is paraphyly, although those nodes on basal branch had low support, Sp. indicus and Sp. incognitus at the base of the tree, Sp. sinus group with Sc. cherrei, while Sp. taiwanensis form a monophyly with Sc. formosensis, Sc. boettgeri, and Sc. modesta (Fig. 5). The transparent window on lower eyelid was once treated as an important diagnostic character of Scincells, but some species such as Sc. cheerei may lack of this feature (Linkem et al., 2011).

Therefore, the effectiveness to distinguish between Sphenomorphus and Scincella only by transparent window is doubtful. Furthermore, partially developed transparent disc is observed in some of the taxa with the broad Scincella clade, such as the Tengzgi and Dahangshan populations. Conclusively, we tend to retain the validity of Scincella, but transfer some taxa from Sphenomorphus to Scincella to maintain the monophyly of this genus. Under this consideration, Sphenomorphus taiwanensis should be replaced as Scincella taiwanensis, while the two undescribed species should also grouped into Scincella. The synopomorph of the revised Scincella will need to be revised in the future.

Conclusions

1. The lowland Scincella in Taiwan previously treated as Sc. formosensis is comprising two separate taxa suggested by morphological and genetic evidences. The yellow-tailed morph represents a typical Sc. formosensis, while the red-tailed morph is similar to Sc. modesta. 2. Genetic differentiation was observed between the red-tailed morph and Sc. modesta, but lack of strong diagnostic morphological characters. Before comparing with topotype from Ningbo, Zhejiang, we tend to treat the former as a unique population of Sc. modesta in Taiwan. 3. Owing to of the imperfect genetic and morphological differentiation, the southern Ryukyu Sc. boettgeri should be a synonym of Sc. formosensis. Considering its uniqueness, we suggest to retain it as an island subspecies, i.e., Sc. formosensis boettgeri. 4. With prominent divergence both in morphological and molecular evidences, the two mid-elevation taxa from Dahanshan and Tengzhi should be treated as new species. 5. With a paraphyletic relationship between Scincella and Sphenomorphus, as well as the contiguous development of the transparent disk on lower eyelid, we suggest not treating transparent disk as a diagnosis among the two genera. We also suggest Sp. taiwanensis to be transferred to Scincella, i.e., Sc. taiwanensis.

References

Boulenger GA. 1887. Catalogue of the Lizards in the British Museum (Natural History). Vol. III. Trustees, London. Chen SH, Lue KY. 1987. A new species of skink, Sphenomorphus taiwanensis from Taiwan (Sauria: Scincidae). Bulletin of the Institute of Zoology, Academia Sinica 26: 115-121. Chen SL, Ota H, Hikida T. 2001. Geographic variation in the two smooth skinks, Scincella boettgeri and S. formosensis (Squamata: Scincidae), in the subtropical East Asian islands. Zoological Science 18: 115-130. Darevsky IS, Orlov NL. 1994. Vietnascincus rugosus, a new genus and species of the Dasia-like arboreal skinks (Sauria, Scincidae) from Vietnam. Russian Journal of Herpetology 1: 37-41. Darevsky IS, Orlov NL. 1997. A new genus and species of scincid lizards from Vietnam: the first Asiatic skink with double rows of basal subdigital pads. Russian Journal of Herpetology 31: 323-326. Eremchenko VK. 2002. Scincidae,. In: Schleich HH and Kästle W, eds. Amphibians and Reptiles of Nepal: A. R. G. Gantner Verlag. 723-768. Felsenstein J. 1985. Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39: 783-791. Greer AE, Shea G. 2003. Secondary temporal scale overlap pattern: a character of possible broad systematic importance in Sphenomorphine skinks. Journal of Herpetology 37: 545-549. Greer AE. 1974. The generic relationships of the scincid lizard genus Leiolopisma and its relatives. Australian Journal of Zoology Supplementary Series 31: 1-67. Greer AE. 1997. Leptoseps: a new genus of scincid lizards from Southeast Asia. Journal of Herpetology 31: 393-398. Grismer LL. 2007. A new species of small Montane forest floor skink (genus Sphenomorphus Fitzinger 1843) from southern Peninsular Malaysia. Herpetologica 63: 544-551. Grismer LL. 2008. A new species of insular skink (genus Sphenomorphus Fitzinger 1843) from the Langkawi Archipelago, Kedah, West Malaysia with the first report of the herpetofauna of Pulau Singa Besar and an updated checklist of the herpetofauna of Pulau Langkawi. Zootaxa 1691: 53-66. Guindon S, Dufayard JF, Lefort V, Anisimova M, Hordijk W, Gascuel O. 2010. New algorithms and methods to estimate maximum-likelihood

phylogenies: assessing the performance of PhyML 3.0. Systematic Biology 59: 307-321. Günther A. 1864. The Reptiles of British India. Ray Society: London Honda M, Ota H, Köhler G, Ineich I, Chirio L, Chen SL, Hikida T. 2003. Phylogeny of the lizard subfamily Lygosominae (Reptilia: Scincidae), with special reference to the original of the New World taxa. Genes and Genetic Systems 78: 71-80. Huelsenbeck JP, Ronquist F. 2001. MRBAYES: Bayesian inference of phylogenetic trees. Bioinformatics 17: 754-755. Librado P, Rozas J. 2009. DnaSP v5: a software for comprehensive analysis of DNA polymorphism data. Bioinformatics 25: 1451-1452. Linkem CW, Diesmos AC, Brown RM. 2011. Molecular systematics of the Philippine forest skinks (Squamata: Scincidae: Sphenomorphus): testing morphological hypotheses of interspecific relationships. Zoological Journal of the Linnean Society 163: 1217-1243. Mittleman MB. 1952. A generic synopsis of the lizards of the subfamily Lygosominae. Smithsonian Institution: Washington. Nakamura KU, S. I. Uéno 1963. Japanese Reptiles and Amphibians in Colour Hoikusha, Osala (in Japanese) Nguyen TQ, Ananjeva NB, Orlov NL, Rybaltovsky E, Böhme W. 2010. A New Species of the Genus Scincella Mittlemann, 1950 (Squamata: Scincidae) from Vietnam. Russian Journal of Herpetology 17: 269-274. Nguyen TQ, Nguyen SV, Böhme W, Ziegler T. 2010. A new species of Scincella (Squamata: Scincidae) from Vietnam. Folia Zoologica 59: 115-121. Nguyen TQ, Schmitz A, Nguyen TT, Orlov NL, Böhme W, Ziegler T. 2011. Review of the genus Sphenomorphus Fitzinger, 1843 (Squamata: Sauria: Scincidae) in Vietnam, with description of a new species from northern Vietnam and southern China and the first record of Sphenomorphus mimicus Taylor, 1962 from Vietnam. Journal of Herpetology 45: 145-154. Ouboter PE. 1986. A revision of the genus Scincella (Reptilia: Sauria: Scincidae) of Asia, with some notes on its evolution. Zoologische Verhandelingen 229: 1-66. Posada D, Crandall KA. 1998. Modeltest: testing the model of DNA substitution. Bioinformatics 14: 817-818. Rozas J, Sanchez-DelBarrio JC, Messeguer X, Rozas R. 2003. DnaSP, DNA polymorphism analyses by the coalescent and other methods. Bioinformatics 19: 2496-2497.

Saint KM, Austin CC, Donnellan SC, Hutchinson MN. 1998. C-mos,a nuclear marker useful for squamate phylogenetic analysis. Molecular Phylogenetics and Evolution 10: 259-263. Salzburger W, Ewing GB, von Haeseler A. 2011. The performance of phylogenetic algorithms in estimating haplotype genealogies with migration. Molecular Ecology 20: 1952-1963. Schmidt KP. 1927a. The reptiles of Hainan. Bulletin of the American Museum of Natural History 54: 395-465. Schmidt KP. 1927b. Notes on Chinese Reptiles. The reptiles of Hainan 54: 467-551. Shea GM, Greer AE. 2002. From Sphenomorphus to Lipinia: generic reassignment of two poorly known New Guinea skinks. Journal of Herpetology 36: 148-156. Smith MA. 1935. The Fauna of British India, including Ceylon and Burma, Reptilia and Amphibia. Vol. 2 Sauria, Taylor and Francis: London. Stephens M, Scheet P. 2005. Accounting for decay of linkage disequilibrium in haplotype inference and missing-data imputation. The American Journal of Human Genetics 76: 449-462. Stephens M, Smith NJ, Donnelly P. 2001. A new statistical method for haplotype reconstruction from population data. The American Journal of Human Genetics 68: 978-989. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. 2011. MEGA5: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Molecular Biology and Evolution 28: 2731-2739. Taylor EH. 1963. The Lizards of Thailand. The University of Kansas Science Bulletin 44: 687-1077. Townsend TM, Alegre RE, Kelley ST, Wiens JJ, Reeder TW. 2008. Rapid development of multiple nuclear loci for phylogenetic analysis using genomic resources: An example from squamate reptiles. Molecular Phylogenetics and Evolution 47: 129-142 Uetz P. 2008. The Reptile Database. Available at: http://www.reptile-database.org/ (accessed May 23 2006). Van Denburgh J. 1912. Concerning certain species of reptiles and amphibians from China, Japan, the Loo Choo Islands, and Formosa. Proceedings of the California Academy of Sciences 3: 187-258.

Table 1. Species, sample localities, sample size, and GenBank Accession number of the specimens used in this study.

Species ID Collection locality Sample size Haplotype of cytochrome b Haplotype of Bach1 Haplotype of Cmos Eutropis longicaudata Eutropis Sandimen Township, Pingtung, Taiwan 1 Hap_115 - - - - longicaudata

Sp. incognitus IG Neipu Township, Pingtung, Taiwan 3 Hap_112/114 - - - -

Sp. indicus IDBH Fuxing Township, Taoyuan, Taiwan 3 Hap_130/131 - - - -

IDDL Douliu City, Yunlin, Taiwan 2 Hap_132/133 - - - -

IDCN Guangxi, China 2 Hap_111/113 - - - -

Sc. cherrei Sc. cherrei NCBI 1 Hap_120 - - - -

Sp. simus Sp. simus NCBI 1 Hap_122 - - - -

Sc. rufocaudatus RU Con Son island, Vietnam 3 Hap_123/124/125 - - - -

Sc. reevesii (D) DT Hongkong, China 14 Hap_116/118/126/127/128/129 - - - -

KS Guangxi, China 1 Hap_116 - - - -

Sc. lateralis Sc. lateralis NCBI 3 Hap_117/119/121 - - - -

Sp. sp.1 Dahanshan Dahanshan Chunri Township, Pingtung, Taiwan 6 Hap_72/107/108 Sp_1/4 Sp_1/2/3/4/5 (B)

Sp. sp.2 Tengzhi (C) Tengzhi Taoyuan Dist., Kaohsiung, Taiwan 3 Hap_109 Sp_7/9/10/11 Sp_1

Sp. taiwanesis (A) HH Xiulin Township, Hualien, Taiwan 7 Hap_64/65/66/69/71 Sp_5/6/8 Sp_1

AL Alishan Township, Chiayi, Taiwan 4 Hap_62/67/68 Sp_3/12 Sp_1

Table 1. (Continued)

Code&Species ID Locality Sample size Haplotype of cytochrome b Haplotype of Bach1 Haplotype of Cmos

Sp. taiwanesis (A) HY Haiduan Township, Taitung, Taiwan 2 Hap_63 Sp_2/16/17 Sp_1/6

YK Haiduan Township, Taitung, Taiwan 3 Hap_63/110 Sp_3/12/13 Sp_1/6/7

YL Yuli Township, Hualien, Taiwan 1 Hap_70 Sp_2 Sp_1

Sc. modesta (E) Hap_99/100/101/102/103/104/1 Sc_1/2/4/5/6/7/9/27/32/ BF Hongkong, China 11 Sc_3/11/15/16/17/18/19/20/21 05 34

DM Hongkong, China 4 Hap_106 Sc_6/7/8/9/29 Sc_3/20/22/23

Sc. formosensis (F) KL Zhongzheng Dist., Keelung, Taiwan 17 Hap_73/79 Sc_3/12/26/31/33 Sc_3 (red-tailed morph) MJ Wenshan Dist., Taipei, Taiwan 2 Hap_76/77 Sc_26/31 Sc_3

SD Xindian Dist., Taipei, Taiwan 4 Hap_74/75/78 Sc_12/13/14/15/25 Sc_3

DS Daxi Township, Taoyuan, Taiwan 2 Hap_93/96 - - Sc_3

LT Longtan Township, Taoyuan, Taiwan 6 Hap_91/92 Sc_3/12/15/21 Sc_3

BL Fuxing Township, Taoyuan, Taiwan 15 Hap_90/97/98/101/103 Sc_3/5/15/ Sc_3

WLR Wulai Dist., Taipei, Taiwan 7 Hap_82/94/95 Sc_3/12/13/22/29 Sc_3

YM Shilin Dist., Taipei, Taiwan 3 Hap_80/81 Sc_26 Sc_3

Table 1. (Continued)

Code&Species ID Locality Sample size Haplotype of cytochrome b Haplotype of Bach1 Haplotype of Cmos

Sc. boettgeri (G) YO Yonaguni island, Japan 1 Hap_53 Sc_10 Sc_2

IR Iriomote island, Japan 9 Sc_10/11/24 Sc_2

IS Ishigaki island, Japan 10 Hap_39/40/41/42/43/45/46/47 Sc_30/48/49 Sc_1/2

MY Miyako island, Japan 12 Hap_44/49/50/51//54/55/56 Sc_36/37/44/45 Sc_2/10/12

Sc. formosensis JS Jinshan Dist., Taipei, Taiwan 18 Hap_4/83/84/85/86 Sc_10/47/50 Sc_1/2/4 (yellow-tailed morph) WL Wulai Dist., Taipei, Taiwan 2 Hap_89 Sc_10 Sc_2/4 (H) DA Nan’ao Township, Yilan, Taiwan 2 Hap_88 Sc_3/12/26/31/33 Sc_3

BH Fuxing Township, Taoyuan, Taiwan 1 Hap_87 Sc_26/31 Sc_3

BG Changhua City, Changhua, Taiwan 16 Hap_11/12/59/60/61 Sc_10/16/17 Sc_2

NT Yuchi Township, Nantou, Taiwan 2 Hap_13 Sc_42/43 Sc_2

BS Baoshan Township, Hsinchu, Taiwan 11 Hap_52/57 Sc_10/20 Sc_2/8

ZC Zaoqiao Township, Miaoli, Taiwan 1 Hap_58 - - - -

Table 1. (Continued)

Code&Species ID Locality Sample size Haplotype of cytochrome b Haplotype of Bach1 Haplotype of Cmos JL Zhuolan Township, Miaoli, Taiwan 11 Hap_31/34/35/52 Sc_41 Sc_2/5/9

BHS Heping Dist., Taichung, Taiwan 1 Hap_30 - - - -

TZ Tanzi Dist., Taichung, Taiwan 11 Hap_7/30 Sc_39/40/41 Sc_2/13

Sc. formosensis CS Gushan Dist., Kaohsiung, Taiwan 11 Hap_15/19/24/25 Sc_10 Sc_2 (yellow-tailed morph) DP Dapu Township, Chiayi, Taiwan 11 Hap_8/14/16/17 Sc_10 Sc_1/6 (H) CY East Dist., Chiayi, Taiwan 11 Hap_1/2/18 Sc_10/19 Sc_1/2

JU Zhushan Township, Nantou, Taiwan 10 Hap_20/21/22/26 Sc_10/34/46 - -

GK Gukeng Township, Yunlin, Taiwan 1 Hap_23 - - - -

DL Douliu City, Yunlin, Taiwan 15 Hap_20/26/27/28/29 Sc_10/18 Sc_2/14

GZ Baihe Dist., Tainan, Taiwan 11 Hap_3/4/5/6 Sc_10/19/23 Sc_2/7

Table 2. Pairwise p-distance of cytochrome b among ingroup taxon.

Clade A B C D E F G H A B 0.1540 C 0.1648 0.1458 D 0.2474 0.2602 0.2598 E 0.2071 0.1989 0.1983 0.2584 F 0.1927 0.1849 0.1946 0.2525 0.0923 G 0.1733 0.1762 0.1787 0.2360 0.1695 0.1709 H 0.1848 0.1878 0.1865 0.2457 0.1804 0.1707 0.0954 Abbreviations: A: Sp. taiwanensis; B: Sphenomorphus sp1. Dahanshan; C: Sphenomorphus sp2. Tengzhi; D: Sc. reevesii; E: Sc. modesta; F: red-tailed morph Sc. formosensis; G: Sc. boettgeri; H: yellow-tailed morph Sc. formosensis.

Table 3. Pair wise p-distance of cytochrome b among the 11 clades defined from Sc. boettgeri and yellow-tailed Sc. formosensis. Definition of the clades refers to Fig. 6.

Clade 1 2 3 4 5 6 7 8 9 10 11 1 2 0.0472 3 0.0810 0.0822 4 0.0864 0.0905 0.0965 5 0.0816 0.0848 0.0849 0.0863 6 0.0884 0.0952 0.0944 0.0889 0.0857 7 0.0867 0.0938 0.0986 0.0855 0.0884 0.0643 8 0.0815 0.0873 0.0884 0.0858 0.0818 0.0689 0.0679 9 0.0877 0.0945 0.0972 0.0927 0.0955 0.0754 0.0770 0.0710 10 0.0876 0.0964 0.0950 0.0955 0.0876 0.0764 0.0746 0.0673 0.0731 11 0.0863 0.0891 0.0893 0.0945 0.0804 0.0723 0.0738 0.0751 0.0761 0.0714

Table 4. Results of the discriminant analysis (DA) of 10 meristic morphological characters of the 7 closely related skinks. The classification success rate (%) for each group is presented. (A) Sc. modesta and red-tailed morph Sc. formosensis; (B) Sc. boettgeri and yellow-tailed morph Sc. formosensis; (C) Sphenomorphus taiwanensis, Sphenomorphus sp.1 Dahanshan and Sphenomorphus sp.2 Tengzhi.

(A) Red-tailed Scincella Sc. modesta % Red-tailed Scincella 46 7 86.8 Sc. modesta 1 7 87.5

(B) Yellow-tailed Scincella Sc. boettgeri % Yellow-tailed Scincella 118 7 94.4 Sc. boettgeri 0 23 100

(C) Sp. taiwanensis Sp. sp.1 Dahanshan Sp. sp.2 Tengzhi % Sp. taiwanensis 8 0 0 100 Sp. sp.1 Dahanshan 0 5 0 100 Sp. sp.2 Tengzhi 0 0 2 100

Table 5. The 10 meristic morphological characters and 2 qualitative traits of the 7 closely related skinks.

Taxa N SL IL F N DS MBSR GVS F4S T4S SRB TW CT

Sc. formosensis 125 6-7 5-7 0-2 1-4 49-64 26-30 57-73 9-13 13-20 0.5-3.5 a yellow yellow-tailed morph

Sc.boettgeri 23 7 6 0-2 2-4 55-64 26-30 58-74 9-12 15-17 2.5-4 a yellow

Sc. formosensis 53 7 6 2 2-4 46-61 26-30 54-67 8-13 11-15 - - a red red-tailed morph

Sc. modesta 8 7 6 2 2-5 46-56 26-30 57-68 10-12 13-17 - - a red

Sp. taiwanensis 8 7 6-7 1 2-5 55-63 26-31 66-74 9-13 14-17 - - b - -

Sp. sp.1 (DHS) 5 6 5-6 1 3-6 47-54 24-26 59-63 8-9 11-12 - - c - -

Sp. sp.2 (TZ) 3 6-7 5-6 1 3-4 47-52 22-24 64-67 10 13 - - c - -

Abbreviations: N: samples size; SL: supralabials; IL: infralabials; F: contact point number of frontal and frontonasals; N: nuchals; DS: dorsal scales;

MBSR: midbody scale rows; GVS: gular and ventral scales; F4S: subdigital scales of finger IV; T4S: subdigital scales of toe IV; SRB: the scale rows covered by dorsal-lateral stripe; TW: transparent window in lower eyelid; CT: color of belly tail. a: transparent disk exists, lower eyelid covered with a single enlarged and transparent scale; b: transparent disk lack, lower eyelid covered with tiny opaque scales; c: transparent disk partially developed, lower eyelid covered with tiny transparent scales.

A B

C D

Fig. 1. The two color morphs of Scincella formosensis: (A) dorsal view and (B) ventral view of the yellow-tailed morph; (C) dorsal view and (D) ventral view of the red-tailed morph.

A B 涂昭安攝 Fig. 2. The ground skinks discovered at mid-elevation (1500-1700m) of southern Taiwan: (A) Sphenomorphus sp1. Dahanshan from Dahanshan, Pingtung and (B) Sphenomorphus sp2. Tengzhi from Tengzhi, Kaohsiung.

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Hap_115 Eutropis longicaudata 100/100/100 Hap_114 Hap_112 Sp. incognitus Hap_133 Hap_132 Hap_131 Sp. indicus 100/99/100 Hap_130 Hap_113 78/82/56 Hap_111 11/43/- - Hap_122 Sp. simus Hap_120 Sc. cherrei Hap_125 Hap_123 78/82/54 Hap_124 Sc. rufocaudatus 100/100/100 Hap_129 Hap_127 36/- -/44 Hap_126 D Hap_128 Sc. reevesii 100/100/100 Hap_118 44/58/41 Hap_116 Hap_121 Hap_119 100/100/100 Hap_117 Sc. lateralis Hap_110 Hap_63 Hap_70 Hap_69 16/- -/- - Hap_68 Hap_67 Sp. taiwanensis 100/100/100 Hap_62 A Hap_71 Hap_66 96/98/100 Hap_65 Hap_64 Hap_109 Sphenomorphus sp2. Tengzhi Hap_108 C 100/100/100 Hap_72 Sphenomorphus sp1. Dahanshan 23/- -/- - 82/79/92 Hap_107 B Hap_106 100/100/100 Hap_105 Hap_104 Sc. modesta Hap_99 E Hap_102 Hap_100 Hap_95 100/100/100 Hap_82 Hap_94 Hap_97 Hap_101 Hap_98 Hap_90 89/96/100 Hap_103 100/100/100 Hap_96 Hap_92 Hap_91 F Sc. formosensis (red-tailed morph) Hap_93 Hap_81 Hap_80 Hap_79 Hap_73 Hap_78 Hap_74 Hap_77 Hap_76 Hap_75 Hap_56 Hap_51 Hap_50 70/78/96 Hap_55 Hap_54 Hap_49 Hap_44 57/- -/47 Hap_48 Hap_38 Hap_37 Sc. boettgeri Hap_36 G 99/99/100 Hap_47 Hap_41 Hap_42 Hap_43 46/57/91 Hap_45 Hap_46 100/100/100 Hap_40 Hap_39 Hap_53 Hap_88 Hap_87 57/62/63 Hap_89 Hap_85 Hap_84 80/100/100 76/79/98 Hap_83 Hap_86 Hap_58 Hap_57 Hap_52 Hap_61 47/78/59 Hap_60 Hap_59 40/40/- - Hap_11 Hap_12 Hap_13 ML: Maximum Likelihood Hap_25 Hap_24 Hap_19 100/100/100 Hap_15 NJ: Neighbor Joining Hap_17 90/88/100 Hap_14 Hap_16 BI: Bayesian Inference 70/- -/- - Hap_35 H Sc. formosensis (yellow-tailed morph) Hap_31 Hap_34 100/100/100 Hap_22 ML/NJ/BI Hap_21 Hap_23 Hap_20 Hap_26 Hap_29 40/15/63 Hap_27 Hap_28 Hap_18 Hap_2 100/100/100 Hap_1 Hap_8 Hap_5 Hap_4 Hap_3 32/30/- - Hap_6 Hap_33 Hap_32 Hap_9 100/100/100 Hap_10 Hap_7 Hap_30

0.2 Fig. 5. Phylogenetic tree of mitochondrial DNA cytochrome b gene constructed by maximum likelihood. The numbers beside each node denote statistic supports by 1000 maximum likelihood bootstraps, 1000 neighbor joining bootstraps, and Bayesian post probability, respectively.

IG02 IG03 Sp. incognitus IG01 100/99/100 91CN01 91CN02 100/99/100 81IDBH3 Sp. indicus 82IDDL1 70/92/58 81IDBH1 81IDBH2 61DT12 61DT13 61DT01 100/99/100 61DT02 D Sc. reevesii 61DT03 61KS01 61DT05 61DT04 VN03 100/99/100 VN02 Sc. rufocaudatus VN01 22MJ01 100/99/100 26YM01 65/63/79 21KL01 24BL01 F Sc. formosensis (red-tailed morph) 23LT01 99/91/100 25WLR1 52DM02 52DM01 51BF10 65/63/85 100/96/99 52DM03 E Sc. modesta 51BF11 ML: Maximum Likelihood 52DM04 32HH01 NJ:NeighborJoining 32HH03 35YK02 BI: Bayesian Inference 35YK01 66/64/93 36YL01 ML/NJ/BI 34HY02 A 34HY01 Sp. taiwanensis 35YK03 100/99/100 32HH02 33AL04 100/99/100 33AL02 33AL03 41TJ02 85/96/100 41TJ03 C Sphenomorphus sp2. Tengzhi 41TJ01 42DHT1 DH03 66/79/99 42DH01 B Sphenomorphus sp1. Dahanshan DH04 DH02 42DH05 17MY01 87/81/100 16IS01 16IS02 G Sc. boettgeri 15IR01 17MY02 01BS01 15IR02 02JL01 04BG02 18YN01 03TZ02 95/98/100 05NT01 05NT02 06DL02 07CY06 08GZ01 H Sc. formosensis (yellow-tailed morph) 09DP01 10CS07 11JS02 11JS04 12WL01 14BH01 13DA01 0.005

Fig. 6. Phylogenetic tree of nuclear Bach1 and Cmos genes constructed by maximum likelihood. The numbers beside each node denote statistic supports by 1000 maximum likelihood bootstraps, 1000 neighbor joining bootstraps, and Bayesian post probability, respectively.

Sc. formosensis (red-tailed morph) Sc. modesta Sc. boettgeri

Sc. formosensis (yellow-tailed morph)

(A)

(B)

Fig. 7. Haplotype network of the four closely related Scincella taxa generated by (A) nuclear Bach1, and (B) nuclear Cmos sequences.

Sp. taiwanensis Sphenomorphus sp1. Dahanshan Sphenomorphus sp2. Tengzhi

(A)

(B)

Fig. 8. Haplotype network of Sphenomorphus taiwanensis species group generated by (A) nuclear Bach1, and (B) nuclear Cmos sequences.

21 8 9 10 11 12 13 14 15 Fig. 9. Discriminant analysis (DA) of red-tailed morph Scincella (n=53, red squares) and Sc. modesta (n=8, pink squares) by using 9 morphological traits.

-6

-7

-8

-9

-10

-11

-12 -15 -14 -13 -12 -11 -10 -9 -8 -7

Fig. 10. Discriminant analysis (DA) of yellow-tailed morph Scincella (n=125, gold squares) and Sc. boettgeri (n=23, green squares) by 10 morphological traits.

2 1 0 -1 -2 -3 -4 -5 -6 -7 -8 40 45 50 55 60

Fig. 11. Discriminant analysis (DA) of Sphenomorphus taiwanensis (n=8, purple squares), Sphenomorphus sp1. Dahanshan (n=5, yellow squares), and Sphenomorphus sp2. Tengzhi (n=2, deep blue squares) by 9 morphological traits.

Hap_56 Hap_51 Hap_50 Hap_55 100/98/100 Hap_54 1 Hap_49 57/- -/47 Hap_44 Hap_48 100/100/100 Hap_38 Hap_37 Sc. boettgeri 99/99/100 Hap_36 Hap_47 Hap_41 Hap_42 Hap_43 46/57/91 2 100/100/100 Hap_45 Hap_46 Hap_40 3 Hap_39 Hap_53 Hap_88 100/100/100 Hap_87 57/62/63 Hap_89 4 80/100/100 Hap_85 76/79/98 Hap_84 5 100/100/100 Hap_83 Hap_86 Hap_58 100/100/100 Hap_57 Hap_52 47/78/59 6 Hap_61 Hap_60 40/40/- - Hap_59 7 Hap_11 Hap_12 Hap_13 Hap_25 Hap_24 8 Hap_19 100/100/100 Hap_15 Hap_17 Hap_14 90/88/100 70/- -/- - Hap_16 Sc. formosensis 9 Hap_35 100/100/100 Hap_31 (yellow-tailed morph) Hap_34 Hap_22 Hap_21 Hap_23 Hap_20 Hap_26 Hap_29 40/15/63 Hap_27 Hap_28 10 Hap_18 Hap_2 100/100/100 Hap_1 Hap_8 Hap_5 Hap_4 Hap_3 32/30/- - Hap_6 11 Hap_33 100/100/100 Hap_32 ML: Maximum Likelihood Hap_9 NJ: Neighbor Joining Hap_10 Hap_7 BI: Bayesian Inference Hap_30 ML/NJ/BI 0.02 Fig. 12. Haplotype genealogy of yellow-tailed Sc. formosensis and Sc. boettgeri of mitochondrial DNA cytochrome b gene constructed by maximum likelihood. Numbers beside each node denote statistic supports by 1000 maximum likelihood bootstraps, 1000 neighbor joining bootstraps, and Bayesian post probability, respectively.

Appendix 1. Primer sequences for the mitochondrial DNA cytochrome b, nuclear gene Cmos and Bach1 used in this study.

Locus Primer Sequence(5’-3’) Tm(℃) Cited source

SciCB-L AACCAAGACCTGTGAYAYGAA Cytochrome b 56 This study SciCB-H2 CGTTARGGTCCCGACTTTGG

Bach1-F3 GGGGATACATGACATTGAGG Bach1 60 Modified by Townsend et al., 2008 Bach1-R3 GTTCTGGCTGTTGTTCTGCT

G73 GCGGTAAAGCAGGTGAAGAAA Cmos 56 Saint et al., 1998 G74 TGAGCATCCAAAGTCTCCAATC

台灣產滑蜥屬（Scincella）與蜓蜥屬 （Sphenomorphus）的分子與形態

台灣產滑蜥屬（Scincella）與蜓蜥屬（Sphenomorphus）的分子與形態