國立臺灣師範大學生命科學系碩士論文 蕉弄蝶屬之分子親緣關係探討 與屬內兩種入侵農業害蟲之來源檢測 Molecular phylogeny of the genus Erionota (Lepidoptera: Hesperiidae), with inferring the origins of two notorious banana pest species 研究生: 顏嘉瑩 Chia-Ying Yen 指導教授: 徐堉峰博士、千葉秀幸博士 Yu-Feng Hsu, Hideyuki Chiba 中華民國 102 年 6 月 致謝 回首在師大的兩年碩士求學歷程，我受到諸位師長、同學、朋 友與家人的協助與鼓勵，實在有著說不盡的感謝話語。 首先，我想感謝我的指導教授徐堉峰教授的費心指導。扎實地 授予豐富的知識、不吝嗇地分享自身經驗、耐心地指導每一個研究 的小細節、適時地協助我渡過研究和寫作的瓶頸，並且帶領著研究 室一同創造歡樂、充滿笑聲的研究環境。每天最期待的事情之一， 就是和老師及實驗室夥伴們中午一起吃午飯 (特別是在大太陽天才 有的牛肉麵)，然後天南地北的大聊特聊，分享生活中的各種瑣事。 此外，老師對於研究與探求新知的熱情與熱忱更是我們後輩的好榜 樣。在這兩年間，老師還給了我充分的機會去學習更多的學術技能， 更鼓勵我勇敢譜出對於未來的願景，讓我在學術上與生活上都留下 了許多深刻且精彩的美好回憶與成長，能當您的學生真的是一件很 幸運而且很幸福的事情。接著，我想感謝林思民教授的慷慨指導， 願意接納我參與貴實驗室的 meeting 一同學習。老師風趣親切的個 性和對學生們亦師亦友的相處之道，總是能給予學生最大的溫暖和 關懷，在這短短兩年中更是從老師身上學到了許多研究、生活等做 人處事的智慧。也感謝師大和東海大學教授們的教導與勉勵，帶領 著我學習做研究的許多工夫或針對我的論文提出許多寶貴的建議。 我想特別感謝卓逸民教授，您嚴謹的指導與要求學術研究時的態度 和精準度，使我能在迥異的學習環境與截然不同的領域中方能時常 警惕自己，並站穩腳步繼續向前邁進。您永遠是我學術的燈塔，即 使霧再濃，夜再黑，甚至迷失了方位，您總是都能為我指引方向。 在碩士班的日子裡，研究室是我最重要的地方，在這裡所結識 的夥伴都是這一路幫助我成長的重要人物。感謝實驗室學長姐們在 我匍匐學步階段的細心指導，感謝夥伴和學弟妹的幫忙與鼓勵。感 謝草魚研究室的全體夥伴，除了大方的出借電腦以及在分析軟體上 的指導之外，仍給予我許多溫暖歡樂的回憶。感謝李壽先研究室學 長姐們提供舒適的實驗環境與設備，並總是不吝的給予親切的叮嚀 和照應。感謝師大研究所一同打拼的好同學好朋友們，特別是我的 實驗室夥伴兼戰友的莊懷淳同學這一路上的互相扶持，感謝你們陪 伴我度過每一個五味雜陳的日子，一起苦中作樂，一起加油打氣， 一起互相砥礪。能夠認識你們，是我碩士生涯中最重要的收穫。 另外，我要感謝台灣香蕉研究所大方地提供台灣早期香蕉耕種 以及害蟲防治等相關資料。也要感謝印度國家生物科學中心的 Dr. Krushnamegh Kunte 和日本千葉博士所提供的珍貴外國樣本。並且感 謝所有曾經提供樣本、指導分析以及給予論文指導和建議的人們， 亦感謝一路上曾經給予鼓勵或關心的朋友們，謝謝你們。 最後，我想感謝我最親愛的家人們和我最重要的人－信賓，謝 謝你們一直相信我並支持我的決定，讓我可以無後顧之憂、全心全 意地專注在自己最喜歡的事物上。並且總是在我難過時陪伴我鼓勵 我，給予我最強大的力量，使我又有勇氣面對一切挑戰，沒有你們 我無法走到這步。 往這個目標邁進，我從未後悔過，雖然在這條道路上有著始料 未及的困難與磨難，甚至曾經覺得舉步維艱，無法再往前走，但對 於研究滿滿的熱情使我燃燒不盡，並成為支持著我完成夢想的動力。 而在最後的最後，我想感謝自己，感謝自己堅持努力不懈直到最後。 也期許自己，碩士班的畢業不是結束，而是代表自己具備了接受下 一個階段挑戰的勇氣。 Contents 中文摘要………………………...………………..……..……1 Abstract……………………………………………….....……3 Introduction……………………………….…………....….…5 Materials and methods…………………………………..…19 Results……………………………………….….………..….27 Discussion……………………………………..………..……33 References...……………………………………...…...…..…44 Tables……………………………………………….……......58 Figures……………………………………………….…...….67 中文摘要 香蕉弄蝶(Erionota torus)與尖翅香蕉弄蝶(E. thrax)皆隸屬於鱗翅 目(Lepidoptera)弄蝶科(Hesperiidae)蕉弄蝶屬(Erionota)，為入侵許多國 家的外來種農葉害蟲，因其幼蟲除了取食芭蕉科植物(Musaceae)葉片 之外，亦具有捲旋葉面製作蟲巢的習性，使植物行光合作用之面積大 量減少，阻礙成長，嚴重危害香蕉產業，造成了嚴重的經濟損失。其 中香蕉弄蝶於 1986 年首次在台灣屏東縣九如鄉發現，至 90 年代早期 即已遍布全台。除了危害栽培種蕉類植物之外，本種幼蟲亦可取食原 生種臺灣芭蕉(Musa formosana)。然而，香蕉弄蝶與尖翅香蕉弄蝶的飛 行能力並不足以飛躍長距離之海洋屏障，且通常活動範圍不會離寄主 植物太遠，因此許多研究皆推測其入侵與人類活動息息相關。本研究 在第一部分分別追溯兩種蕉弄蝶之族群來源與入侵途徑並探討台灣香 蕉弄蝶之來源為單一產地還是多個產地。此外，蕉弄蝶屬中只有香蕉 弄蝶與尖翅香蕉弄蝶的食草為芭蕉科，其於六種蕉弄蝶成員與鄰近之 姐妹屬則是利用棕梠科或薑科。本研究在第二部分為了解香蕉弄蝶屬 成員之親緣關係以探討此屬的食草利用格局，則以分子證據粒線體 DNA 的 COI 和 COII 基因與核 DNA 的 Ef-1α 基因，利用最大簡約法、 最大概概似法及貝氏推論法進行親緣關係樹的建立。 第一部分，本研究採集 28 隻香蕉弄蝶樣本於 8 個國家，共 15 個 1 採集樣點，進行粒線體 COI 與 COII 的序列分析，得有 10 個基因型。 其中台灣本島的族群與沖繩的石垣島和與那國島以及中國福建省的族 群擁有相同的基因型，由於香蕉弄蝶入侵沖繩的時間較台灣晚，因此 此結果支持台灣的香蕉弄蝶是來自於中國福建之單一入侵產地。在尖 翅香蕉弄蝶部分，本研究共採集 11 隻樣本於 9 個採集樣點，共來自於 6 個國家，進行粒線體序列分析後，得有 6 個基因型。研究結果發現， 尖翅香蕉在亞洲地區的族群基因相似度與地理距離遠近具有相同模式。 第二部分，為了建構香蕉弄蝶屬的成員之親緣關係以及食性演化，本 研究採用粒線體與核序列進行分析(COI+COII: 2209 bp; Ef-1α: 1200 bp)。研究結果顯示，香蕉弄蝶屬為單系群，且屬內六種物種皆為單系 群。此外，香蕉弄蝶與尖翅香蕉弄蝶亦為一個單系群，顯示以芭蕉科 為食草的利用為單一寄主轉移事件。 關鍵字：蕉弄蝶屬、寄主轉移、親緣關係、入侵種 2 Abstract Banana skippers, which comprises two species, Erionota torus and E. thrax, belong to family Hesperiidae, and have been introduced into several counties. Their caterpillars feed on family Musaceae and construct leaf rolls to make shelters, which can cause the reduction of the banana's leaf area, the decline of the photosynthesis rate of plants, and the reduction of plant growth and fruit yields. E. torus was discovered as a new banana pest in Taiwan since 1986. And rapidly spread all over the island in the early 1990s. Their caterpillars not only damage cultivated bananas but also feed on endemic Taiwanese bananas. The flying ability of the banana skippers does not allow them to fly across the distant ocean barrier, and they usually do not leave the host plant area too long. Thus, it seems very likely that the banana skippers in Taiwan and other countries was introduced by human activities. In the first part, we employing the standard phylogeographic methodologies to test the possible geographic sources of the population of the two banana skippers introduced in various countries of the world. Moreover, the other members in this genus and the sister group of the genus Erionota mainly utilize Arecaceae and Zingiberaceae as their host plant, while only the family Musaceae are utilized by E. torus and E. thrax as their host plant. In the second part, we sequences utilized molecular evidence, mitochondrial COI+COII and nuclear Ef-1α, to reconstruct the phylogenetic relationship of the genus Erionota and map the pattern of hostplant use onto this inferred tree. We obtain 28 specimens from 15 localities in 8 counties, and distinguished 10 haplotypes. The population in Taiwan shares the same 3 haplotype with the specimens from Okinawa, Japan (Yonaguni Is and Ishigaki Is) and Fujian Province, China. Thus, the invasion source to Taiwan is very likely from Fujian Province, China. Our result showed a clear pattern that the genetic differentiation among the populations in Asia reflected the geographic distance between the collecting localities. The results highly supported that the genus Erionota is a monophyletic group. The six species we obtained in this research are also representing six monophyletic groups. Moreover, the banana skippers, E.thrax and E.torus, are a monophyletic group which represents a single host shift from Arecaceae or Zingiberaceae to Musaceae. Keywords: Erionota, host shift, invasive species, phylogeny 4 Introduction Invasive species: a serious problem worldwide Many human activities, such as those related to agriculture, aquaculture, recreation, and transportation, result in both the deliberate and accidental dispersal of species across natural barriers. Although unintentionally transported species may die out en route or soon after arriving at a new habitat, other exotic species may survive and colonize the new territory. The threat and significance of this problem is difficult to evaluate. However, evidence suggests that it is widespread and serious (Carlton 2000). Invasive species can facilitate the diffusion of diseases, which may have serious consequences for human or animal health (Stewart 1991, van Riper 1991, Atkinson et al. 1995, Beadell et al. 2006, Strayer et al. 2006). Moreover, the negative effects of predation (Vitousek et al. 1996, Wardle et al. 2001, Phillips et al. 2003, Simon and Townsend 2003, Baxter et al. 2004, Croll et al. 2005, Anderson et al. 2006, Lovett et al. 2006) and competition for resources (Evans 2004, Jones et al. 2008, Beckmann and Shine 2009) can cause the loss of indigenous biodiversity and threaten native ecosystem (Vitousek et al. 1996, Wardle et al. 2001, Phillips et al. 2003, Simon and Townsend 2003, Baxter et al. 2004, Croll et al. 2005, Anderson et al. 2006, Lovett et al. 2006). Invasive species may have devastating economic effects as well (Vitousek et al. 1996). For example, costs resulting from environmental damage and the implementation of measures to control harmful invasive species in the United States have been estimated at nearly $120 billion per year (Pimentel et al. 2005). Although the anthropogenic dispersal of plants is harmful, it may also 5 be neutral or profitable. Nearly all human societies have depended on the deliberate relocation of plants (Mark and Lonsdale 2001) and establishing plants beyond their native ranges has been essential to the development of agriculture (Hodge and Erlanson 1956). Bananas: one of the world's most important crops Bananas and plantains are one of the most widely consumed foods in the world (Rossmann et al. 2012). They belong to the largest and most economically important genus, Musa, in the family Musaceae. Species belonging to Musaceae are characterized by an above-ground pseudo stem (false stem), large, flexible, and waterproof leaves, and hanging clusters of elongated, edible fruits. According to the latest estimates (2010) by the Food and Agriculture Organization of the United Nations (FAO), land devoted to banana farming covers approximately 5,014.06 thousand hectares globally with total production of 102,028.17 thousand tons. The origin and domestication history of bananas is extremely complex (Kennedy 2008). However, archaeological and palaeoenvironmental evidence suggests that bananas were first cultivated in Papua New Guinea and can be traced back to around 8,000 BCE (Bowdery 1999, Denham et al. 2003, Lentfer 2003). Banana plants are primarily cultivated for their fruits, and to a lesser extent, to make fiber, wine, and as ornamental plants. As a nutritional, low-price, high-productivity versatile crop, bananas have spread widely across the tropical and subtropical regions, with a substantial trade history beginning at the end of the nineteenth century. Improvements in cooling and refrigerated shipping have enabled bananas to be transported over long 6 distances from the tropics to world markets and across over 150 countries. The banana was introduced to Taiwan from Fujian, China, approximately 200 years ago. Following the Japanese occupation of Taiwan, farmers focused on improving banana varieties, transforming bananas into a superior agricultural fruit, and establishing Taiwan’s banana industry (Ho 1975). In the 1960s, Taiwan’s banana cultivation area measured over 40,000 hectares, and Taiwan was among the major producers of bananas in the world. The current banana cultivation area in Taiwan is 10,664 hectares, with the total production output equaling approximately 260 tons (Agriculture and Food Agency,