國立臺灣師範大學生命科學系碩士論文

颯弄蝶屬之分子親緣與系統研究 Molecular phylogeny and systematics of the genus Satarupa Moore, 1865 (: Hesperiidae: Pyrginae)

研究生: 莊懷淳

Huai-chun Chuang

指導教授: 徐堉峰 博士

Yu-Feng Hsu

千葉秀幸 博士

Hideyuki Chiba

中華民國 102 年 7 月

致謝

首先要感謝我的父母，經過多番的波折與衝突答應讓我長久以來的心

願能夠一邊實驗一邊做田野調查。並且非常的感謝我的論文指導教

授，徐堉峰教授，給予我如此自由的環境讓我實驗。並且也要感謝實

驗室的各位夥伴，讓整個實驗室充滿了活潑的環境讓原本枯燥的研究

過程增添豐富的色彩，猶如雨天過後的七彩彩虹。感謝實驗室的學長

姐們帶領我野外調查並且獲得台灣難以獲得的樣本。並且也要感謝提

供我國外樣本的千葉秀幸博士，讓我獲得許多困難獲取的樣本，以及

提供我很多論文寫作的相關意見。當然一定要感謝提供如此高規格的

實驗環境的李壽先老師，並且在很多思考上給予很大的啟發，並且教

導我在很多表達上的細節與邏輯。感謝林思民老師在口試與報告的時

候給予我許多中肯的意見。也要感謝共同陪我奮鬥的同學們，一起在

實驗室熬夜趕 ppt 等口頭報告。最後最後一定要感謝這兩年來一起共

同奮鬥的顏嘉瑩同學，在這兩年一起度過了非常多的挫折與試煉，如

果沒有他我這兩年的色彩會黯淡許多。要感謝的人太多，寥寥字詞無

法述說滿滿的感謝，就如陳之藩所言：「要感謝的人太多，不如謝天

吧！」

Contents

中文摘要…………………………...……………………..……1

Abstract…………………………………………………..……2

Introduction………………………………….………….….…4

Materials and methods…………………………………….…8

Results……………………………………………………..….12

Discussion……………………………………………….……14

References...………………………………………………..…18

Tables………………………………………………….……...25

Figures………………………………………………….…….29

Appendix……………………………….……………………37

中文摘要

親緣關係為生物研究的基礎，弄蝶親緣關係已有高階親緣關係發表，

在屬級的親緣關係尚有不足之處。颯弄蝶屬屬特徵為前翅 2A 至 R3

脈之間有透明的斑紋與後翅具有大片的白塊。因颯弄蝶種之間的形態

太過於相似，許多發表尚有辨識錯誤的情形。Evans 在 1949 整理為 7

種，Okano (1987) 和 Chiba (1989) 則認為颯弄蝶屬為 valentini, zulla, gopala, nymphalis, splendens, monbeigi 和 formosibia。以翅形與幼蟲型

態 S. formosibia 與 S. monbeigi 應屬於同一類群，但是前者外生殖器卻

並不典型。Shirôzu 將 S. majasra 處理為 gopala 的亞種，但 Tsukiyama

處理為 nymphalis 的亞種。颯弄蝶屬近緣屬皆為熱帶分布，颯弄蝶卻

分布自爪哇至溫帶的西伯利亞。建構颯弄蝶屬親緣關係可以確立 formosibia 及 majasra 的分類地位並且了解颯弄蝶屬的溫帶分布是由

熱帶分布至溫帶或原本分布在溫帶。本研究使用 COI, COII 與 Ef1α

基因，以最大簡約法、最大概似法與貝式推斷進行親緣關係分析。結

果顯示颯弄蝶屬為一個單系群，並且各個種為單系群，可以反映現在

的分類是沒有問題。並且更能確定 formosibia 與 monbeigi 為同一群。

從樹型可知 majasra 不屬於 gopala 或是 nymphalis 的亞種，建議提升

為一個種，且颯弄蝶屬是由熱帶分布至溫帶並且適應溫帶的環境。

關鍵字：分布、分子親緣關係、颯弄蝶屬

1 Abstract The phylogenetic relationship is the foundation of biological researches. The family level of relationship of Hesperiidae had been reported, but the phylogeny of genus levels still insufficient. The genus Satarupa is recognized by the transparent spots between veins 2A to R3 on the forewing and large white area in hind wing. Because of the difficulty in identification between the Satarupa , there is many mis- identification in the publication about Satarupa. Evans recognized 7 species in 1949 and Okano (1987) and Chiba (1989) established genus Satarupa includes 7 species. The species currently identified in the genus is S. valentini, S. zulla, S. gopala, S. nymphalis, S. splendens, S. monbeigi, and S. formosibia. Even more, the gentilia of S. formosibia is unusual as the other species divided with the larvae characters and wing pattern; S. majasra treated as subspecies of gopala by Shirôzu in 1953, but Tsukiyama recognized the characters more similarity with nymphalis. Because the relatives genera of S. distribute in tropical regions, but the genus S. distributes from temperate region to tropical region. Construct the phylogenetic relationship of S. species to realize that what the ancestral region of Satarupa and clarify the states of S. formosibia and majasra. Samplings form the Asia countries, and use the PCR to amplify the target genes, two from mitochondrion: COI and II; one form nuclear, Ef1α. Use the sequence to maximum parsimony, maximum likelihood and Bayesian inference analysis to construct the phylogenetic tree. The results show that the genus Satarupa is monophyletic group and also the each species. The tree topology suggests that S. formosibia belongs to the group with monbeigi, and S. majasra belongs neither gopala nor

2 nymphalis, and it should rise as a species. The temperate distribution suggests that the adaption from the tropical region to the temperate regions.

Keyword: distribution, molecular phylogeny, Satarupa

3 Introduction: The classification of organisms is an important issue to understand the biodiversity on the earth (Wilson 2000), and it is the most fundamental of biological studies when using organisms to advance researches. Taxonomists have used similarities, observed behavioral and morphological characters without considering their phylogenetic relationships of the taxa (de Queiroz and Gauthier, 1990). However, those practices resulted in a lot of misclassifications when re-examine after a couple years using more characters (Hebert et al., 2004). Using characters to construct phylogenetic trees illustrate most of evolutionary explanations other than only using functional or adapted characters. It helps scientists to understand the system of biological diversity and the evolutionary information. Hesperiidae is the large family in Lepidoptera, it is known by stockier bodies, hooked antennae and darting flight habits. Because of the wing pattern variation, rarely know in larvae stage and ecological habits, the classification need to redefine. The genus Satarupa Moore, 1865 consists of a few relatively large skippers; it is characterized by black wings with some transparent spots between veins 2A to R3 on the forewing and large white area on the hind- wing. The Upper corner of the F cell protruding, vein Cu2 close to the base of F cell, and the posterior edge of hidewing is longer than anterior edge; Male external genitalia tegument casque, uncus short and thick, gnathos round shape (Chou 1998). All known larvae utilize Rutaceae as host plants (Uchida 1995; Hsu 2002; Harada et al., 2012). Evans (1949) recognized 7 species: S. valentini Oberthür, 1921, S. zulla Tytler, 1915, S. gopala Moore, 1865, S. nymphalis (Speyer, 1879), S.

4 splendens Tytler, 1915, S. monbeigi Oberthür, 1921, and S. majasra Fruhstorfer, 1909. This classification had been followed until Shirôzu (1960) pointed out that there were two species distributed in Taiwan, namely S. majasra and S. formosibia with the letter synonymized with majasra by Evans (1949). In addition, Shirôzu (1960) followed Evans (1956) and treated S. majasra as a subspecies of S. gopala. Because of this treatment, there are so many misidentifications and confusion found among current publications especially those from China (Io 1990; Li and Chu 1992; Gu and Chen 1997; Chou 1998; Wang 1998; Chou et al. 1994; Huang et al. 2001; Xue et. al. 2009) and shows on the appendix 1. Okano (1982) and Okano and Okano (1984) described two additional species from Emeishan, Sichuan, both of are the variation of monbeigi and which were subsequently synonymized with monbeigi by Okano himself (Okano 1987; Chiba and Tsukiyama 1989), and shown in Figure 1. Based on the adult morphological characters, the members of the genus can be classified into four lineages: valentini-group, gopala-group majasra/ zulla/nymphaslis- group, and formosibia/ splendens/ monbeigi-group (Chiba, in prep.). Morphological characters of larvae also suggest that this grouping would be valid. Larval stages of five species have been known (Hsu 2002; Harada et al., 2012). Uchida (1995) illustrated larvae of formosibia and majasra. The former has yellow and white spots on the lateral sides, while the latter has white lateral band. Igarashi and Fukuda (2000) illustrated and described four species of Satarupa larvae. However, the larva of majasra from Taiwan (illustrated as gopala) is that of formosibia and the male photographs of nymphalis from Sichuan have characters of monbeigi. The larva of gopala has white

5 lateral band as majasra and white spots on the dorsal middle line; S. valentini larvae has lateral band as majasra and four lines of white spots on the dorsal middle line (Harada et al, 2012). The gentilia of S. formosibia is unusual within the genus. The valva is smaller and the projection is shorter than other species except S. valentini. The taxonomic of S. formosibia needs to confirmation. Besides, Tsukiyama(1995) claimed that S. majasra should be treated as the subspecies of S. nymphalis rather than gopala, or as the endemic species of Taiwan. The taxonomic states of two Taiwanese species taxa S. formosibia and S. majasra needs classified. Historical biogeography reflects the spatial and temporal pattern of taxa; these days the biogeographic researches combine the phylogenetic data to understand the events of vicariance, dispersal and even the mode of speciation. Many taxon-oriented phylogenetic studies address the biogeographical implication of their results, and find the geological connections among the areas. (Murphy et al., 2001) The genus of American katydid, Neoconocephalus, distributes form the North America to the Central America, the life histories and others characters were confuse the scientists for a couple years. Using phylogeny and match the geographical distribution that shows the life history has changed multiple times independently. (Snyder et al., 2009). The genus Satarupa is distributed form south western Siberia, China, Malaysia, and to Sunda Islands. The distributions of related genera of Satarupa shown by Warren 2009 are tropical distributed, mainly in , Indo-China peninsula, and Indonesia. In the tribe Tagiadini, only two genera, and Satarupa, are distributed from

6 tropical to temperate regions. Furthermore, most of the species in the genus Satarupa are distributed from Siberia to Southern China, while S. gopala is distributed mainly in the tropics, from Indo-China peninsula to Java. The life cycle of Satarupa species except for gopala is one generation per year, and the larvae have to expose to a period of cold temperature which reflects the temperate life trait (Snyder et al., 2009) even distributes in the tropical region. But the outgroup species, with the multivoltine and without diapause, the life cycle of tropical life trait with tropical distribution. According to the description above, the aims of the study are 1) clarify the systematic states of Satarupa taxa with doubtful placement, and 2) to infer the evolution of behavior and phenological features formed in members of Satarupa.

7 Materials and Methods: Specimens 7 ingroup species (27 individuals) were used in this research; specimens were collected in Taiwan, China, Malaysia and and identified by using Evans key (1949) with the help of Dr. Hsu and Dr. Chiba (see Table 1.). The legs (or part of the tissue) were stored in 99.5% ethanol at -80oC for molecular work. Out-groups related to Satarupa were selected based on the current phylogeny of the group (Warren 2008 & 2009), and shown in table 2.

Molecular analysis DNA extraction Legs were taken from the tissues store in the tube at -20oC and contain 99.5% ethanol. The genomic DNA was purified from legs using the Qiagen PUREGENE kit (Gentra Systems, Minnesota, USA) and the working condition is: Take a legs, or a part of leg segment into the 1.5 ml microtube, and wait for the ethanol evaporate. Add 100 μl cell lysis buffer, 10 μl 0.1 M dithiothreitol (DTT) solution and 10 μl 0.5 mg/ml proteinase K solution, and grind with grinding rods. Then dry bath with 65 oC for 1.5 h. After dry bath, adding 40 μl protein precipitation solution, frozen at -20 oC for 30 minutes. The samples were centrifuged at 14000rpm for 15minutes, and take supernatant into another 1.5 ml microtube. The samples centrifuged at 14000 rpm for 15minutes again for removing impurities, and then add 150 μl 100 % isopropanol for DNA precipitation. After mixing well, centrifuge at 14000 rpm for 10 minutes and discard the supernatant. Add 400 μl 70 % (v/v) ethanol into sample for dissolve

8 excess ions and centrifuges at 14000 rpm for 5 minutes, then removing supernatant and dry at room temperature about 4 to 12 hrs. Finally, add

50 μl d2H2O for dissolve DNA for one overnight. DNAs were stored at -20oC for further PCR reaction.

Gene selection, amplification and sequencing Mitochondrial COI, COII and nuclear Ef-1α region are widely used in the phylogenetic researches of Lepidoptera, and are consider to be sufficient to infer genus-level phylogeny in some cases (Canfield et al. 2008, Hundsdoerfer et al. 2009), the COII gene also had been identified as the rapid evolving unit in the mitochondria (Schmidt et al, 2005), thus these regions were selected in this study. To amplify the region describe above, using PCR for amplification. The primers used in PCR amplification from the previous studies and list at table 3. (Caterino and Sperling, 1999; Kandul et al., 2004 and Wu, 2010). Condition of one sample for PCR amplification describe following:

22.5 μl or 23.5 μl d2H2O, 1 μl dNTP, 0.6 μl for a pair of primer, 3 μl 10X PCR reaction buffer, 0.3 μl MDTaq DNA polymerase (MDBio, Taipei, Taiwan), and 1 or 2 μl sample DNA (>10 ng/μl DNA). Total reaction volume is 30 μl. PCR program using following protocol: initial denaturation temperature 95oC for 2 minutes, and then 35 cycles of: 1) Denaturation at 95oC for 30 seconds, 2) Annealing at 50 oC - 55 oC for 30 seconds, and 3) Extension at 72 oC for 30 – 60 seconds, annealing and extension time depend on primer used. After 35 cycles as describe above, final extension at 72 oC for 7 minutes. The PCR product was electrophoresed in 0.8 - 1.0 % agarose gel to check the length of

9 amplification, and sequenced by commercial company (Genomics, Taipei, Taiwan). All sequences were checked using sequencher 4.7.1 (GeneCode, Boston, USA), and primer regions and tRNA-Leu parts between COI and COII genes by checking with the reference sequence (Hao et al., 2012) were deleted. After cleaning the sequences with bad signal, dataset were aligned using the Muscle algorithm (Edgar 2004) in MEGA 5.0 (Tamura et al. 2011) with default settings, aligned sequences were checked by eye with translated to amino acid alignment for further phylogenetic analysis.

Phylogenetic analysis To estimate the inter-specific and intra-specific evolutionary distance of ingroup species, the p-distance of the COI + COII dataset was calculated using MEGA 5.0 with the nucleotide substitution type include transitions and transversions in p-distance model. I used jModeltest (Posada, 2008) to test the appropriate nucleotide substitution model selected on the basis of corrected Akaike information criterion (AICc), the best model for the ML and BI analysis. All sequence data, using TNT 1.1 (Goloboff et al. 2008) for Maximum parsimony (MP) analysis with sectorial search, ratchet, drift and tree fusing with initial search level set to 100 (the heaviest search), and with the bootstrap test in 1000 replications; MrBayes 3.2 (Ronquist et al., 2012) for Bayesian inference (BI) analysis with appropriate nucleotide substitution model and three time independently Markov chain Monte Carlo (MCMC) method analysis in 1,000,000 generations at final average standard deviation of split frequencies below 0.01; used the PHYML 3.0 (Guindon et al. 2010) for

10 Maximum likelihood (ML) with the same as BI nucleotide substitution model, substitution rate categories in number 4,and use the gamma shape parameter analyzed by jModeltest0.0.1. The trees improved method with nearest neighbor interchange (NNI) method, and with bootstrap method test in 1000 replications. In the results and discussion sections, bootstrap support values lower than 70 is referred as “weakly supported”, values ranging from 71-90 are referred as “moderately supported” and values higher than 90 are referred as “strongly supported”; Bayesian posterior probability change as the percentage way to indicate the value, the value lower than 80 are referred as “weakly supported”, values ranging from 81-95 are referred as “moderately supported” and values ranging higher than 95 are referred as “strongly supported”. (Soltis and Soltis, 2003)

11 Result Dataset characters The sequence data were obtained from 27 individuals. Total of 2927 base pairs (bps) were used for phylogenetic analysis, which 2141 bps were from mitochondrial COI + COII region and 786 bps from nuclear Ef1α region. 27 samples were used in the COI + COII sequencing, 21 form the ingroups and 6 from the outgroups. 22 samples were used in the Ef1α sequencing, 15 from ingroups and 6 from outgroups. The COI + COII p-distance of each species are shown in table 4, and the inter-species p-distance in table 5. The intra-species p-distance is lower than 0.6%, inter-species p-distance is between 3.8% to 7.1%, and the p-distance between the S. gopala and S. majasra is 0.054 over the 0.003. (Hebert et al., 2003)

Phylogenetic relationships Using the jModeltest 0.1.1 analysis for the appropriate model of BI and ML analysis in COI + COII sequence is general time reversible (GTR) with site variation rates gamma-distributed; Ef1α sequence is sym- metrical model (SYM) with site variation rates gamma-distributed. The phylogenetic tree by using mitochondrial genes, COI and COII, and BI, MP, ML analysis show the same topology in which the genus Satarupa is a strongly supported monophyletic group, and currently recognized species are also strongly supported as monophyletic with clear relationships between species. Compare to the grouping by morpho- logical characters, the mitochondrial gene result shows that the grouping by Chiba (in prep.) is valid except S. majasra, it’s separated with S. zulla /

12 nymphalis. The S. formosibia/ monbeigi group is a monophyletic group, S. zulla / S. nymphalis group is a monophyletic group, and S. gopala and S. majasra form a paraphyletic group independently. The relationship of S. gopala is closer to the clade of S. zulla / S. nymphalis, and S. majasra refers as a sister clade with the clade of S. gopala + zulla + nymphalis. The intra-species bootstrap values were strongly supported in each species, except the S. nymphalis, is weakly supported and the each sub- species: S. nymphalis khamensis and S. nymphalis nymphalis. The phylogenetic trees by using nuclear Ef1α and analysis by MP, ML and BI are strongly support the monophyly of the genus Satarupa. The S. monbeigi and S. nymphalis as a monophyletic group in the BI tree, and others species are in the same clade. The ML trees show that the S. nymphalis is a monophyletic group, and others are in the same clade with S. nymphalis. The bootstrap values within specie nodes are very low, that the tree cannot tell any information about relationship of the species. The nuclear tree cannot tell the intra-species relationship. In outgroup, the samples in this study do not cluster in a monophyletic group in mitochondrial trees of three analysis methods, the T. japetus is the sister clade of Miminiades / Daimio / the other Tagiades species, but the bootstrap value of Miminiades and Tagiades is very low. The Daimio is nested within Tagiades species, but the result not strongly supported. In the nuclear trees, the Daimio is nested with in the Tagiades species, with the moderately supported in BI analysis and weakly supported in ML, MP analysis.

13 Discussion The gene tree can’t reflect specie tree, and there are many articles for discussing this problem (Barraclough, 2001; Nichols, 2001; Corl, 2013). However, using mitochondrial genes for phylogenetic analysis offers proven robust in inferring species limits or corroborating morphological hypotheses in many studies (Moore, 1997; Miura et al., 2000; Penz et al., 2012) Samples of S. formosibia + S. monbeigi form a monophyletic group, even though the gentilia of S. formosibia is unusual. The molecular evidence shows that the morphological grouping is valid. S. formosibia only distributes in Taiwan and regard as the endemic species of Taiwan, compare to S. majasra, another Taiwan species, because these two species are often found in the field in the same place at the same time, it not the vicariance event that separated by the barriers and divides into different characters, maybe the population dispersal multiple times from the China. If majasra is considered a Taiwanese subspecies of gopala, samples of S. gopala form a paraphyletic assemblage. The relationship showed in phylogeny tree that S. majasra, S. nymphalis and S. gopala form clades independently. It supports neither the treatment of subspecies of gopala by Shirôzu nor the treatment of subspecies of nymphalis by Tsukiyama. Besides, Tsukiyama referred if the S. majasra not belong to the subspecies of nymphalis that could treat as a species of Satarupa (Tsukiyama, 1995). The subspecies means that the same species with geographical variation or units of variation within species that had been identified by morphological difference (Mayr, 1942; Braby et al., 2012). Even though

14 the subspecies would not be a monophyletic group because it may be formed by different population from different places on the continents, but it’s be easier formed a monophyly in the island because the physical impediment (Phillimore and Owens 2006). The S. majasra forms a monophyletic group. The definition of species in phylogenetic species concept is the descendants and ancestor includes monophyletic group (Donoghue, 1985). The contemporary species concepts share the common view that the species are separately evolving from the metapopulation lineages (de Queiroz, 2007) and species would form a paraphyletic (Omland et al., 2000) or polyphyletic group when the speciation occur (Barraclough and Nee, 2001), and the example in common raven that with few phenotypic differentiation but the mitochondrial DNA evidence shows that the population with the paraphyletic groups and suggests that the peripheral isolate speciation occurs. The S. gopala majasra only distributes in Taiwan and separates from the population of Indochina and Indonesia and main land China. Furthermore, the p-distance between Satarupa species that range from 3.8% to 7.1%, and the p-distance between the S. gopala and S. gopala majasra is 5.4% over the 3%, offer another evidence to suggest that should treat as a species (Avise, 2000). The states of S. gopala majasra elevates to the species rank need to use more data like morphological, ecological and population based data for support. Another need to concern that is the sampling of the S. gopala individuals, that lack of the specimens from the other country of Indochina like , Cambodia, and Sumatera island of Indonesia. Because of wide distribution, need more locations of S. gopala for compare the genetic distance and morphological characters to support

15 the elevation. The relative genera of Satarupa distribute at tropical region, like genus Tagiades, Gerosis, and Coladenia, but the genus Satarupa distributes from tropical to temperate region. In this study treat Taiwan as the tropical region to discuss the biogeographical issues. The tree shows two major clades, clade S. monbeigi/ formosibia and clade S. gopala / zulla / nymphalis, with the same pattern of distribution, one clade in tropical region, and one clade in temperate region. May be the pattern will be 1) Satarupa invaded to the temperate region multiple times and adapted the environment. 2) Satarupa invaded to the temperate region one single time and recolonized back to the tropical regions. Base on the life history, all S. species with one generation per year, need low temperature trigger in the early larvae stage and not use the pupa stage to get through the winter, which replies the temperate life history. That suggests it is invaded to the temperate region one single time and adapted, than recolonized back to the tropical regions. The tree shows that maybe multiple times of the recolonize event. Otherwise, the total samples were 33 Satarupa individuals, but in this research only used 27 samples for analysis, the 5 samples without any DNA extractions. The reason of no extractions maybe the specimen collected in 1990s, and the storage condition not good for the DNA. Besides, found some of specimens with few glue on the abdomen and legs that have more difficulty to extract DNA. The lack of S. splendens and S. valentini, the specimens from other collector is too old to extract DNA. The nuclear gene Ef1α tree can’t show the relationship between

16 species, the nuclear gene for phylogenetic analysis usually use in the high level phylogeny or the long term evolutionary problems (Kojima, 1988; Danforth and Ji, 2001). The nuclear gene with slower evolving rate than the mitochondrial genes, even though that Reed and Sperling (1999) found that EF-1a could resolve relationships among species groups within the same genus, but not always. This result seems that it’s not appropriate for the genus Satarupa. At last, the outgroup species of the Daimio is nested within other Tagiades species. The state of Daimio is chaos in tribe Tagiadini, the distribution from the temperate regions to tropical region; maybe it can treat as the species of Tagiades that adapted to the temperate regions, but still have to use more Tagiades and Daimio species and morphological, ecological and molecular evidence to advance discuss of the state of Daimio.

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24 Table 1. Samples used in this study

Lot. Species Locality Nation

Sa001t S. formosibia Xinyi Town, Nantou Taiwan Sa002t S. formosibia Fusing Town, Taoyuan Taiwan Sa004t S. formosibia Fusing Town, Taoyuan Taiwan Sa006v S. gopala Tam Dao Vietnam Sa007v S. gopala Province Dok Nong, Dak Lac Vietnam Sa008m S. gopala Tapah Hills, Perak Malaysia Sa009m S. gopala Tapah Hills, Perak Malaysia Sa029c S. gopala Hainan China Sa024t S. majasra Fusing Town, Taoyuan Taiwan Sa028t S. majasra Northern Taiwan Taiwan Sa013c S. monbeigi Mt. Kang Jia, Sichuan China Sa015c S. monbeigi Tianquan County, Sichuan China Sa019c S. monbeigi Menghai, Yunnan China Sa025c S. monbeigi Mt. Changbai, Jilin China Sa027c S. monbeigi Wuxi Town, Jiangsu China Sa010c S. nymphalis khamensis Baiyunyuan, Hangzhou, Zhejiang China Sa014c S. nymphalis khamensis Li County, Sichuan China Sa016c S. nymphalis khamensis Yingjing County, Sichuan China Sa017c S. nymphalis khamensis Yingjing County, Sichuan China Sa018c S. nymphalis khamensis Yingjing County, Sichuan China Sa020c S. nymphalis khamensis Yingjing County, Sichuan China Sa021c S. nymphalis khamensis Yingjing County, Sichuan China Sa022c S. nymphalis khamensis Mt. Jiangjin Simian, Sichuan China Sa026c S. nymphalis nymphalis Nanzamu Town, Liaoning China Sa031r S. nymphalis nymphalis Anisimovka Partizansk, Primo. Russia Sa005b S. splendens Tainai, Kachin Sa033c S. valentini Nanling, Guangdong China Sa003c S. zulla Motou Aniqiao, Xi Zhang China

25 Sa030c S. zulla Motou Country, Xi Zhang China Sa032c S. zulla Motou Country, Xi Zhang China

Table 2. outgroup

Lot. Species Locality Nation op003t niitakana Jingmei District, Taipei Taiwan op008pe Miminiades nurscia Huanuco, Tingo Maria Peru op009ph Mooreana princeps Mt.Apo, Mindanao op010m Tapah Hills, Perak Malaysia op002m Seseria affinis Tapah Hills, Perak Malaysia op013t Seseria formosana Academia Sinica, Taipei, Taiwan op005t Tagiades cohaerens Chunri, Pingtung Taiwan op006t Tagiades cohaerens Majia, Pingtung Taiwan op007gh Tagiades flesus Hoehoe Ghana op001m Tagiades japetus Ulu Piah, Perak Malaysia op011ph Tagiades japetus Silago, Leyte island Philippines op012ph Tagiades japetus Samar, Samar island Philippines op004t Tagiades trebellius Orchid Island Taiwan

26 Table 3. Primers used in this study.

Primers 5'→3'

COI&COII cox-J-1460 TACAA TTTAT CGCCT AAACT TCAGC C Zcox-J-1530 CAACA AATCA TAAAG ATATT GG H1co1-1700R AGTCA ATTTC CRAAT CCTCC MiBocox-J-1700 AATAC TATTG TTACA GCTCA TGC H1co1-1880F TCAAG AAGAA TTGTA GAAAA TGG MiBocox-N-2010 AGTTG TAATA AAATT AATWG CTCCT A Skcox-J-2040 CTCTA CCAGT ATTAG CTGGA GC Skcox-J-2100 TTTTG ATCCT GCAGG AGGAG G cox-N-2191 CCCGG TAAAA TTAAA ATATA AACTT C Chcox-J-2200 ACCAG GATTT GGTAT AATTT CCCA Chcox-N-2360 GAGCT CATAC AATAA ATCCT AAT H1co1-2360R GAGCT CATAC AATAA ATCCT A MiBocox-J-2360 CTTTT GGATC TTTAG GAATA ATT C1-J-2500 CAAGA AAGAG GAAAA AAAGA AAC C1-J-2550 ATTTA CWGTA GGWAT AGATA TTGA H1c1-2600F2 TTGAT ATCCT TTATT TACAG G Gallc1-N-2750 CCTGC TAATC CTAAA AAATG TTG Gallc1-N-2770 GTCGA GGTAT TCCTG CTAAT CCTA Jpcox-N-2770 GATAA TCTGA ATAAC GACGA GG Jpcox-2840 TTTTG NTATC ATTCA ATAGA TGA Efcox-J-3000 ATATG TAATG GATTT AAACC CC Micox-N-3080 TTTGA CCTTC TAATA AAAAT CG C2-3138-F2 AGAGT TTCAC CTTTA ATAGA ACA cox-J-3138 AGAGC CTCTC CTTTA ATAGA ACA H1c2-3300R1 TTGTT CTTCT AATAA AAATC G cox-N-3389 TCATA ACTTC AATAT CATTG cox--3408 CAATG ATATT GAAGT TATGA cox-N-3782 GAGAC CATTA CTTGC TTTCA GTCAT CT

27 Ef-1a & wingless Efla-E15f CGGAC ACGTC GACTC CGG Efla-24F GACAC GTCGA CTCCG GCAAG TC Efla-51.9F CARGA CGTAT ACAAA ATCGG Efla-266F CACAG AGATT TCATC AAGAA CA Efla-516F CATCA AGAAG ATCGG TTACA ACC Efla-548R AACAT GTTGT CTCCG TGCCA Efla-839R AGAGC CTGCT GGTGC ATCTC Efla-843R TCYTG GAGAG CYTCG TGGTG CAT Efla-969F GACTC CAAGA ACAAC CCACC CA Efla-R-1048re AACCG TTTGA GATTT GACCA GGG Efla-Ef1242R ACRGT YTGTC TCATG TCACG Efla-J-inter1a AAATA TGCCT GGGTA TTGGA CAAAC T Efla-J-inter3a TCTGG CTGGC ACGGA GACAA CATG Efla-N-inter3b TGTTG TCTCC GTGCC AGCCA GA

28 Table 4. The p-distance of each species

species 1 2 3 4 5 6 1 Satarupa formosibia 2 Satarupa monbeigi 0.052 3 Satarup gopala 0.059 0.066 4 Satarupa majasra 0.067 0.071 0.054 5 Satarupa zulla 0.066 0.074 0.038 0.055 6 Satarupa nymphalis khamensis 0.066 0.070 0.041 0.057 0.041 7 Satarupa nymphalis nymphalis 0.061 0.067 0.040 0.060 0.042 0.005

Table 5. The intra-species p-distance species distance Satarupa formosibia 0.0013 Satarupa monbeigi 0.0007 Satarupa zulla 0.0048 Satarup gopala 0.0037 Satarupa majasra 0.0005 Satarupa nymphalis khamensis 0.0053 Satarupa nymphalis nymphalis ---

29 Figure 1. The previous study of genus Satarupa

30 Figure 2. The phylogenetic tree of genus Satarupa and outgroups by using mitochondrial genes and BI analysis.

31 Figure 3. The phylogenetic tree of genus Satarupa and outgroups by using mitochondrial genes and ML analysis.

32 Figure 4. The phylogenetic tree of genus Satarupa and outgroups by using mitochondrial genes and MP analysis.

33 Figure 5. The phylogenetic tree of genus Satarupa and outgroups by using nuclear gene and BI analysis.

34 Figure 6. The phylogenetic tree of genus Satarupa and outgroups by using nuclear gene and ML analysis.

35 Figure 7. The phylogenetic tree of genus Satarupa and outgroups by using nuclear gene and MP analysis.

36 Appendix 1. Synonymic list of Satarupa species Satarupa valentini Oberthür, 1921 Satarupa valentini Oberthür, 1921, Etud. Lép Comp. 18 (1): 75. ; Evans, 1949, Cat. Hesp. Eur. Asia Aus.: 121; Bridges, 1994, Cat. Hesp. World: VIII. 234. Type locality: Ta Tsien Lou, Sichuan

Satarupa zulla Tytler, 1915 Satarupa zulla Tytler, 1915, J. Brmbay Nat. Hist. Soc. 24 (1): 148. ; Evans, 1949, Cat. Hesp. Eur. Asia Aus.: 121; Bridges, 1994, Cat. Hesp World: VIII. 246. Type locality: Naga Hills, India

Satarupa zulla zulla Tytler, 1915 Satarupa zulla Tytler, 1915, J. Bombay Nat. Hist. Soc. 24 (1): 148 Satarupa zulla zulla, Evans, 1949, Cat. Hesp. Eur. Asua Aus.: 121 Type locality: Naga Hills, India

Satarupa zulla ouvrardi Oberthür, 1921 Satarupa ouvrardi Oberthür, 1921, Etud. Lep. Comp. 18 (1): 74. Satarupa zulla ouvrardi Evans, 1949, Car. Hesp. Eur. Asia Aus.: 121. Type locality: Tse Kou, Yunnan

Satarupa gopala Moore, 1865 Satarupa gopala Moore, 1865, Proc. Zool. Soc. Lond. 1865 (3): 780; Evans, 1949, Cat. Hesp. Eur. Asia Aus.: 121; Bridges, 1994, Cat. Hesp.

37 World: VIII. 92; Chou, 1994, Mon. Rhop. Sin.: 712; Wang et al., 1998, Ins. Faun. Henan Butt.: 203. Type locality: Sikkim Satarupa tonkiniana Fruhstorfer, 1909, Ent. Zeit. 23: 139. Type locality:Tonkuin Satarupa hainana Evans, 1932, Ident. Indian Butt. (2nd ed.): 331. Type locality: Hainan

Satarupa gopala gopala Moore, 1865 Satarupa gopala Moore, 1865, Proc. Zool. Soc. Lond. 1865 (3): 780. Satarupa gopala gopala, Eliot, 1992, Butt. Malay Pen. (4th ed.): 342; Bridges, 1994, Cat. Hesp. World: IX. 62; Gu & Chen, 1997, Butt. Hainan Type locality: Sikkim

Satarupa gopola majasra Fruhstorfer, 1909 Satarupa gopala majasra Fruhstorfer, 1909, Ent. Zeit. 23 (31): 139; Shirôzu, 1960, Butt. Formasa Col.: 385. Type locality: Formosa

Sataurpa nymphalis Speyer, 1879 Tagiades nymphalis Speyer, 1879, Stett. Ent. Ztg. 40: 348 Satarupa nymphalis, Staudinger, 1887, In: Romanoff, Mém. Lep. 3: 153; Leech, 1892, Butt.Chin. Jap. Cor.: 562; Evans, 1949, Cat. Hesp. Eur. Asia Aus.: 122; Bridges, 1994, Cat.Hesp. World: VIII. 152; Chou, 1994, Mon. Rhop. Sin.: 712; Tuzov et al., 1997, Guide Butt. Russia 1:

38 106; Wang, 1999, Mon. Butt. N. E. China: 273. Type locality: Amur

Satarupa nymphalis nymphalis (Speyer, 1829) Tagiades nymphailis Speyer, 1829, Stett. Ent. Ztg. 40: 384. Type locality: Amur Satarupa nymphalis sugitanii Matsumura, 1929, Ins. Matsum. 3: 106; Wang, 1999, Mon. Butt. N. E. China: 274. Type locality: Korea Satarupa nymphalis nymphalis, Evans, 1949, Cat. Hesp. Eur. Asia Aus.: 122; Chou, 1994, Mon. Rhop. Sin.: 712; Wang, 1999, Mon. Butt. N. E. China: 273. Staudinger, 1887, In: Romanoff, Mém. Lep. 3: 153; Leech, 1892, Butt. Chin. Jap. Cor.: 562; Evans, 1949, Cat. Hesp. Eur. Asia Aus.: 122; Bridges, 1994, Cat. Hesp. World: VIII. 152; Chou, 1994, Mon. Rhop. Sin.: 712; Tuzov et al., 1997, Guide Butt. Russia 1: 106; Wang, 1999, Mon. Butt. N. E. China: 273.

Satrupa nymphalis khamensis Alphéraky, 1897 Satrupa khamensis Alphéraky, 1897, In Romanoff, Mem. Lep. 9: 115. Type locality: Kham Satarupa oberthueri Evans, 1932, Ident. India Butt. (2nd ed.): 330. Type locality: Ta Tsien Lou, Sichuan Satarupa intermedia Evans, 1932, Ident. India Butt. (2nd ed.): 330. Type locality: Ta Tsien Lou, Sichuan Satarupa nymphalis khamensis, Evans, 1949, Cat. Hesp. Eur. Asia Aus.: 122; Lee & Zhu, 1992, Atl. Chin. Butt.: 149-150; Bridges,

39 1994, Cat. Hesp. World: VIII. 115; Chou, 1994, Mon. Rhop. Sin.: 712.

Satarupa splendens Tytler, 1915 Satarupa splendens Tytler, 1915, J. Bombay Nat. Hist. Soc. 24 (1): 148; Evans, 1949, Cat. Hesp. Eur. Asia Aus.: 122; Huang, 2003, Neue Ent. Nachr. 55: 68 (note) Type locality: Naga Hills

Satarupa monbeigi Oberthür, 1921 Satarupa monbeigi Oberthür, 1921, Etud. Eèp. Comp. 18 (1): 76. ; Evans, 1949, Cat. Hesp. Eur. Asia Aus.: 123; Chiba, 1988, J. Res, Lepid. 27 (2): 138; Bridges, 1994, Cat. Hesp World: VIII. 145; Chou, 1994, Mon. Rhop. Sin.: 712. Type locality: Siao Lou, Sichuan Satarupa omeia M. Okano, 1982, Artes Liberales 31: 91. Synonymized by Okano, 1987. Type locality: Mt. Omei, Sichuan Satarupa lii M. Okano & T. Okano, 1984, Tokurana (6/7): 125. Synonymized by Okano, 1987. Type locality: Omeishan, Sichuan

Satarupa formosana Matsumura, 1910 Satarupa formosana Matsumura, 1910, Ent. Zeit. 23 (4): 181. Satarupa formosibia Strand, 1927, Arch. Naturgesch. (A) 91 (11): 282.; Shirôzu, 1960, Butt. Formosa Col.: 386; Chou, 1994, Mon. Rhop. Sin.:

40 712. Satarupa formosicola Matsumura, 1929, Ill. Comm. Ins. Japan 1: 34. Type locality: Formosa

41