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 (Lepidoptera: 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 species, 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 Indonesia Sunda Islands. The distributions of related genera of Satarupa shown by Warren 2009 are tropical distributed, mainly in India, Indo-China peninsula, and Indonesia. In the tribe Tagiadini, only two genera, Daimio 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