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 (: Hesperiidae), with inferring the origins of two notorious banana pest

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()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()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( 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 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 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, Council of Agriculture, Taiwan, 2009).

Banana : one of the most notorious pests of bananas.

Although bananas are grown and traded widely across the world, they are plagued various pests. A wide range of antimicrobial pests were attracted, such as the banana weevil (Cosmopolites sordidus) (Abera et al. 1999, Gold et al. 2001, Gold et al. 2004), the banana aphid (Pentalonia nigronervosa) (Stechmann et al. 1996, Robson et al. 2007) or the burrowing nematode (Radopholus similis) (Mendoza and Sikora 2009). Banana skippers, which comprise two species, Erionota torus Evan, 1941 and E. thrax (Linnaeus, 1767), are one of the most notorious pests of bananas. They belong to order Lepidoptera, family Hesperiidae, subfamily Hesperiinae, and are the well-known pests in Southeast Asia. Adult banana skippers possess large dark brown wings with large yellow hyaline spots on the forewing. The hind wings are unmarked and paler with some obscure dark streaks on the underside. They resemble the species of the

7 genus , but have no violet band on the underside of the hind wing. Their body and legs are generally concolorous with wings. They both exhibit little sexual dimorphism. Banana skippers have red eyes, which is believed relating to their phototactic behavior, and is also why they are called palm redeyes. Because of their crepuscular habit, they are only active in the early morning and before dusk. Both species E. thrax and E. torus can be observed between sea level and 2,000 m in secondary forests, semi-cleared lowlands, and in and around villages, towns, and cities where bananas grow. The genitalia of E. torus and E. thrax are quite different. However, it is hard to identify them from their wing pattern characters and appearance of caterpillars. One of the common diagnostic features is: the forewing termen of E. thrax is almost straight, as compared to the more convex curve of E. torus. However, the identification based on this characteristic is not accurate enough. Banana skippers prefer laying their eggs singly or in clusters on the lamina of banana plants. The eggs usually hatch within 5–8 days and the young larvae begin feeding and constructing leaf rolls on the edges of the leaves to make shelters to hide during the daytime and come out to feed at night. As the larvae grow, the rolls also increase in size and the white waxy powders cover their entire body. The wax powder is believed to be a by-product of metabolism (Waterhouse et al. 1998) and it is highly concentrated on older larvae and prepupae. While banana trees need more leaves for fruit production, defoliation up to 20% will not cause a significant loss in fruit weight (Bauer et al. 2003). Banana skippers does

8 not directly damage to the banana fruit, but their feeding and rolling cause the significant reduction of the banana's leaf area, which will decrease the photosynthesis rate of plants, cause defoliation, reduce plant growth, delays fruit ripening in bunches, and fruit yields. Only three caterpillars per leaf may cause the leaf defoliation (Queensland Horticulture Institute 2000).

The damages by banana skippers

E. thrax, were first observed in north-western Papua New Guinea (PNG) in 1983 (Sands and Sands 1991, Waterhouse et al. 1998), which is one of the banana exporting countries in the world, the estimates of banana annual production was about to 700,000 tonnes (FAO 2002) in a very conservative estimate. Over the next six years, E. thrax spread across the mainland and to the New Guinea islands, and became a major pest of bananas. Banana production decreased about 30% as a result of the banana skipper, and affects approximately 700,000 banana growers, 28,000 banana purchasers, and around one million of banana consumers were affected (Waterhouse et al. 1998, Bauer et al. 2003). The damage was evaluated by the Australian Centre for International Agricultural Research (ACIAR) project, and the value of the damage prevented by biological control was estimated at $201.6 million (Raitzer and Lindner 2005). Banana skippers also plagued banana crops on several cities and counties in their native areas and non-native areas such as American (Guam, ), Australia, Mauritius, China (Fujian, Guangdong, Guangxi, Yunnan, Hainan), the Philippines, Vietnam, Japan (Okinawa), Indonesia (Java), , Myanmar, Thailand, and Taiwan. The total

9 economic impact caused by the banana skippers is difficult to be estimated. Moreover, in Taiwan, E. torus not only harm the exotic and breed-improvement banana cultivars, but also feed on the native species, Musa formosana (Hsu 1999). E. thrax and E. torus are both listed in the Index of economically important Lepidoptera in 1994. This index contains records of about 6000 species of harmful and beneficial Lepidoptera over the past 80 years (Zhang 1994).

Biological control of the banana skipper

Due to their shelter-building behavior, using the classical insecticides may not be effective in controlling banana skipper larvae (Waterhouse and Norris 1989, Waterhouse et al. 1998). Therefore, biological control agents, such as parasitoids (Mau et al. 1980, Sands et al. 1993), seem to be the effective management means. For example, E. thrax was discovered in Hawaii in 1973 (Davis and Kawamura 1975) and was soon became a serious pest. An eradication program was initiated by the Entomology Branch of the Hawaii Department of Agriculture. Three species of parasitic Hymenoptera, Ooencyrtus erionotae (Encyrtidae), Apanteles erionotae (Braconidae), and Scenocharops sp. (Ichneumonidae) was introduced from Malaysia in 1973 (Funasaki et al. 1988). The establishment of the egg parasite, O. erionotae, and the larval parasite, A. erionotae, greatly reduced the density of the pest (Mau et al. 1980). In Taiwan, larvae were manually removed and types of chemical pesticides were used for control, but the effort to eliminate larvae or to increase the mortality of E. torus (Tsai et al. 1990) were failed. Later, a parasitoid, A. erionotae, was introduced from Hawaii, bred and released in several counties and successfully reduced the

10 disaster caused by E. torus.

Aim

We believe that banana skippers are prominent models for ecological and evolutionary studies of agricultural pests. Finding the source and the pathways of an introduced or invaded species is useful not only for predicting the adaptive range of the species but also to anchor studies to decipher the history of introduction of the species and the pattern of human transport, investigating the sources of biological control agents, and facilitating quarantine efforts. With studies conducted on the phylogenetic relationship and the elucidation of the classified status, evolutionary history such as the evolution of the use of a host plant could be inferred. Such studies are likely to answer questions as to how and when the pest species switch their host plant usage and also to be able to ascertain the turning point where the are induced to become a pest species. Even though banana skippers have became notorious pests, previous studies on this species were focused only on their ecological characters (Okolle et al. 2006b) or their biological control by parasitoids (Nakao and Funasaki 1976, Mau et al. 1980, Okolle et al. 2006a, 2006b, Okolle et al. 2009). No explicit phylogenetic hypotheses have been proposed for the genus Erionota, and their possible invasion pathways are still unknown. In this study, we are going to use the standard phylogeographic method to trace the source of the widely and rapidly invaded pest, Erionota torus and E. thrax (part I). Then, we use the molecular evidence, cytochrome oxidase subunit I (COI) and cytochrome oxidase subunit II (COII) from the mitochondrial genome and elongation factor-1α (Ef-1α)

11 from the nuclear genome, to reconstruct the phylogenetic relationship of the genus Erionota (part II).

12

Part I Tracing the source of banana skippers

Although the type locality of E. torus is in Sikkim, India, the British Museum’s collections holds records that confirm it was also found in Myanmar, Malaysia and China (Evans 1949). However, the native area of E. torus is still unknown. Today, E. torus has been introduced into Okinawa (Teruya et al. 1973), Taiwan (Chu and Chou 1988) and the Philippines (de Jong and Treadaway 1993). The type locality of E. thrax is in Java, Indonesia, and it was also found in Malaya and the Philippines. Though several researchers propose the native area of the banana skipper to be Southeast Asia (Ahmad and Balasubramaniam 1975), no scientific evidence supports this hypothesis. As of today, E. thrax has been introduced into South Pacific islands Papua New Guinea in 1983 (Sands and Sands 1991, Sands et al. 1993) such as New Britain, Duke of York and New Ireland islands, and possibly Bougainville (Waterhouse et al. 1998). E. thrax has also spread across the ocean to Guam in 1956, Hawaii in 1973 (Mau et al. 1980) and Mauritius (Monty 1970). E. torus was discovered in Jiouru, Pingtung County in southern Taiwan in September 1986 (Chiang and Hwang 1991). It was discovered for the first time as a new banana pest in Taiwan, and it rapidly spread to the low lands and the areas at a low elevation all over the island in the early 1990s (Chiang 1988). The banana skipper larvae not only damage cultivated banana but also feed on endemic Taiwanese banana, Musa formosana (Hsu 1999). Although both torus and thrax are rapid flyers, the flying ability of

13 the skippers does not allow them to fly across the distant ocean barrier. Moreover, the adult skippers usually stay near the host plants and usually stop and rest in dark shade. Based on all these circumstances, it seems very likely that the banana skipper in Taiwan and other countries was introduced by human activities. Although there are some discussions as to where and how the banana skipper came from, no scientific evidence has been presented. The possible geographic source of the population of banana skipper introduced in Taiwan is unsolved. Identification of the source and the route taken by the banana skippers is important not only for biological control but also as a prominent model to study invasion biology and for ecological and evolutionary studies. In this study, we collected the specimens of E. torus and E. thrax from several countries, used mitochondrial COI and COII sequences and employed standard phylogeographic methodologies to test the possible geographic sources of the population of the banana skippers introduced in various countries of the world.

14

Part II Molecular phylogeny of the genus Erionota

Lepidoptera are one of the largest groups of insects, comprising of more than 16,000 species (Hasan 1994). The family Hesperiidae, commonly known as skippers, includes approximately 4,000 species (Bridges 1994), currently classified into 567 genera (Warren et al. 2008). Despite the enormous taxonomic diversity and compared to the understanding of all other families, the current information on skippers is still very limited (Wahlberg et al. 2005, Warren et al. 2008); most of the studies were forced on on the higher-level taxa (Atkins 2005, Warren 2006, Warren et al. 2008, Warren et al. 2009) or the phylogenetic position of skippers within the order Lepidotera (Scott 1985, Scott 1986, de Jong et al. 1996, Wahlberg et al. 2005). The phylogeny and taxonomy within the genus remain rare. Erionota is a genus of skippers in the family Hesperiidae, subfamily Hesperiinae, and the members in this genus are medium to large sized swift flying skippers. Their bodies are thick and solid and the length of their forewing is at least 30 mm. The forewings are dark brown and have hyaline yellow spots while the hind wings are spotless. The cells of the hind wings are relatively short and less than half the length of the forewings. The second segment of the labial palps is robust while the third segment is small and short. The tip of the antennae is hammer-like and the base is white. The members of this genus are mainly distributed in the Oriental region. The larvae are feed on Arecaceae, Musaceae, and Zingiberaceae, and the adult mainly inhabit broad-leaved forest. They have powerful and strong flights, and are obviously crepuscular and

15 nocturnal in habit. These butterflies not only visit flowers for nectar but also sip the liquid from decaying matter. Two members of this genus, E. torus and E. thrax, are the world known pests on banana plants all over the world, while other members are relatively rare. The genus Erionota was established by Mabille (1878), who recognized three species, (Linnaeus, 1767), E. hypaepa (Moore, [1858]), and E. irava (Moore, [1858]). The type species of Erionota is E. thrax, which was redirected from Papilio thrax Linnaeus, 1767 designated by Watson (1893). The taxonomy of Erionota, revised by Evans in 1941, extended the genus to includ seven species: E. torus Evans 1941, E. thrax (Linnaeus, 1767), E. acroleucus (Wood-Mason & de Nicéville, 1881), E. tribus Evans 1941, E. grandis (Leech, 1890), E. sybirita (Hewitson, 1876), and E. harmachis (Hewitson, 1878). Current researchers have largely followed this classification with only a few modifications and two species are added. One of the corrections of the classification is E. harmachis; the breeding of the larvae of this species proved an evidence that they are essentially the female of Orange Palmer (Unkana mytheca, Hespreiidae) (Igarashi and Fukuda 1997). The other correction is that E. acroleucus and E. hiraca (Moore, 1881) were considered conspecific by de Jong and Treadaway (1993). In addition, de Jong and Treadaway (1993) demonstrated that although these two species were published in the same year (1881), E. hiraca was published in a few months earlier than E. acroleucus, which would highlight E. hiraca as an valid name for this species. E. hislopi Evans 1956 and a newly described species, E. surprisa (de Jong and Treadaway 1993), were also added in this

16 genus. In the current study, we used the following eight species: E. thorus Evans 1941, E. thrax (Linnaeus, 1767), E. hiraca (Moore, 1881), E. grandis (Leech, 1890), E. hislopi Evans 1956, E. tribus Evans 1941, E. sybirita (Hewitson, 1876), and E. surprisa de Jong & Treadaway 1993, as a brief examination for species identification. The taxonomic system and phylogenetic relationships for the genus Erionota has yet to be established. Previous studies only used morphological characters to classify the species. However, the morphological characters in this genus are unsuitable for species identification and may not provide sufficient information to clarify the phylogeny of Erionota. In this study, we examined the relationship of species that belong to the genus Erionota by the molecular evidence. In phylogenetic studies of insects, DNA sequences from a multitude of gene regions, such as mitochondrial (COI, COII, ND1, and ND5) and nuclear (Ef-1α and wg), protein-coding or ribosomal RNA (16S), have been used to find regions that provide informative data to resolving phylogenies at various levels (Brower and DeSalle 1998, Caterino et al. 2000, Vila and Björklund 2004, Mallarino et al. 2005, Hundsdoerfer et al. 2009). Most protein coding mitochondrial genes show rapid increases in distance for recent divergences, but not for higher level relationships (Hebert et al. 2004). Previous study also suggested that the nuclear genes 16S and Ef-1α performed best in genus- and tribal level analyses, while EF-1α and wg can perform particularly well at higher taxonomic levels, such as the taxonomic rank of subfamily (Nazari et al., 2007). As Wahlberg and Wheat (2008) indicated, phylogeny will become more stable

17 and robust as the number of genes is increased. Thus we choose DNA sequences from three gene regions on the genome: cytochrome oxidase subunit I (COI) and cytochrome oxidase subunit II (COII) from the mitochondrial genome and elongation factor-1α (Ef-1α) from the nuclear genome to reconstruct the phylogenetic relationship of the Erionota species.

18

Materials and methods

Part I

Sample collection

To obtain the pattern of haplotype distribution, two pest species, E. torus and E. thrax, were sampled: Samples of E. torus were collecteded in Taiwan, Japan (Okinawa, Ishigaki, Yonaguni, and Yoron), P. R. China, Malaysia, Laos, Indonesia and Vietnam. E. thrax were obtained from

Malaysia, the Philippines, India, Laos, P. R. China and Indonesia. Totally 28 individuals of E. torus and 11 individuals of E. thrax were used in this study. All specimens used in this study were obtained by fieldwork or donations from other collectors and muscle tissues were preserved in

99.5% ethanol under -80℃. Details of sampling of specimens are given in

Table 1.

Molecular technique

DNA extraction

Genomic DNA was extracted from the legs or the thoracic muscle tissue of adult skippers using the Qiagen Purgene DNA Isolation Kit (Gentra Systems, Minneapolis, Minnesota, USA), following the extraction protocol of manufacturer. The tissues were taken out from 99.5% ethanol, cut into pieces and put into the 1.5 mL microcentrifuge tube for preparation. After ethanol was evaporated, the disposable polypropylene pestles were used to grinding the tissues with 100 μL cell lysis solutions. Then 10 µL of Proteinase K solution (0.5 mg/mL) and 10mM dithiothreitol

19

(DTT, 0.1M) were added before the tissues were incubated for 1.5 hours at 65 °C with gentle shaking. At this step, great agitation may disrupt the DNA fragments. After quickly spinned the tubes to get solution off inside of the cap, 40µL protein precipitation solution we added and the samples were frozen at -20℃ for 30 min. The samples were centrifuged twice at

14,000 rpm for 15 min to separate cellular debris and the supernatant was mixed with an equal volume of isopropanol for DNA precipitation, centrifuged at 14000 rpm for 10 min and the supernatant was discarded this time. The last step was to added 500μL 70℃ ice ethanol to wash the tube and to centrifuge at 14000 rpm for 5 min. After the ethanol was discarded, the tubes were air dried at room temperature for 3-8 hours. All extractions were eluted into 50µL of double-distilled H2O and stored in -20℃.

Gene selection, amplification and sequencing

To amplify the sequences of the mitochondrial COI and COII region. 28 pairs of primers were obtained from Caterino and Sperling (Caterino and Sperling 1999), Kandul et al. (Kandul et al. 2004), Lu et al. (Lu et al. 2009) and Wu et al. (Wu et al. 2010). 2 pairs of primers were designed in this research. All the primers are all listed in Table 2. Polymerase chain reactions (PCRs) were carried out in 30μL of solution containing 1μL of template, 1μL of 10μM dNTP, 1.5μL of 25mM

MgCl2, 3μL of 10X PCR reaction buffer, 0.4 or 0.6μL of each 10μM primers, 0.3μL of Power Taq (Genomics, Taipei, Taiwan) and double-distilled H2O filled to 30μL. Thermal cycle parameters for COI and

COII were as follows: initial denaturation at 95℃ for 4 min, followed by

20

35-45 cycles consisting of the denaturation at 95℃ for 30s, annealing at

48-55℃ (depending on which primers were used) for 30s, and extension at

72℃for 25-60s. Final step was the extension at 72℃ for 7 min and stop at

4℃. Amplified PCR products were subjected to horizontal gel electrophoresis on 1% agarose gel containing 0.75 μg/mL ethidium bromide (EtBr) and photographed under UV transillumination to ensure that the lengths of DNA fragments were correctly amplified. DNA was sequenced by commercial company (Genomics, Taipei, Taiwan). DNA was sequenced by commercial company (Genomics, Taipei, Taiwan). All sequences were checked by sequencher 4.7.1 (GeneCode, Boston, USA). After deleted the primer regions and tRNA-Leu gene, sequences were aligned manually using BioEdit v7.1.3 (Hall 2011), and the dataset was aligned using the Muscle algorithm (Edgar 2004) in MEGA 5.0 (Tamura et al. 2011) with default settings, Then the aligned sequences were translated to amino acid sequence to check alignment and the existence of stop codon for further phylogenetic analysis. Specimens were considered to have the same haplotype if ambiguous sites could make their sequences identical. The number of nucleotide differences between populations (Nei 1972, 1973) was calculated using ARLEQUIN v. 3.0 (Schneider et al. 2000).

Phylogenetic Analysis

For the Bayesian analysis, the calculations were run in MrBayes 3.2.1 (Ronquist and Huelsenbeck 2003). The best evolution model was selected based on AICc by jModelTest 2.1.1.(Darriba et al. 2012). Markov chain

21

Monte Carlo (MCMC) from a random starting tree was initiated in the Bayesian inference and run twice for 10 million generations to confirm that the final average standard deviation of split frequencies was below 0.01. Five chains (one cold chain and four hot chains) were run simultaneously and sampled every 1,000 generations. After discarding the first 1,000 trees (burn-in), a consensus phylogeny was made with the remaining trees. Posterior probabilities for each node were inferred from this consensus tree.

Genetic diversity and haplotype network

Standard genetic diversity indices such as the total number of distinct haplotypes, overall haplotype diversity (h; Nei 1987) and nucleotide diversity (π; Tajima 1983, Nei 1987) were calculated in DNASP v5

(Librado P 2009) to assess within population genetic variation. Tajima's D (Tajima 1989) and Fu's Fs (Fu 1997) test were used to check for deviations from neutrality, to make sure that the variations inter-populations were affected not by natural selection but only by randomly genetic drift. To illustrate the relationship among haplotypes, the minimum spanning network was constructed by the software TCS 1.21 based on the principle of parsimony (Clement et al. 2000).

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Part II

Sample collection

6 ingroup species (21 individuals) were used in this research, including E. torus, E. thrax, E. hiraca, E. grandis, E. hislopi, and E. surprisa. Fresh specimens of two extremely rare species, E. tribus and E. sybirita, were unable to acquire in this study. 6 species (6 individuals) from the genus Gangara, , Acerbas, Zela were chosen as outgroup according to previous study by Warren et al. (Warren et al. 2009). All outgroup species are known to utilize palms (the host plant of E. grandis and E. hiraca) as their larval host plants. Bananas and bamboos are also recorded as the host plants of H. irava. After species identification, the muscle tissues were preserved in

99.5% ethanol under -80℃ until further use for DNA isolation. The species sampled and their collection localities are listed in Table 1.

Molecular technique

DNA extraction

The steps of the DNA extraction process was the same as in the Part I.

Gene selection, amplification and sequencing

To amplify the entire mtDNA COI and COII gene region (COI-1508 base pairs; COII-549 base pairs), 2 pairs of primers were synthesized in this research, and 28 pairs of primers were obtained from previous researches (Caterino and Sperling 1999, Kandul et al. 2004, Lu et al. 2009, Wu et al. 2010). 5 pairs of primers from Wu et al. (2010) were used to

23 amplify the nuclear Ef-1α gene region (EF-1α-1240 bp). All the primers used in this research are all listed in Table 2. PCR amplification reaction was performed as the same procedure as in Part I and DNA were also sequenced by commercial company (Genomics, Taipei, Taiwan). All sequences were checked using sequencher 4.7.1 (GeneCode, Boston, USA), primer regions and tRNA-Leu gene were deleted. Sequences were aligned manually using BioEdit v7.1.3 (Hall 2011) and the dataset were aligned using the Muscle algorithm (Edgar 2004) in MEGA 5.0 (Tamura et al. 2011) with default settings. Then aligned sequences were translated to amino acid sequence to check alignment and the existence of stop codon for further phylogenetic analysis.

Phylogenetic analysis

The p-distance of the COI + COII dataset was calculated using MEGA 5.0 (Tamura et al. 2011), to estimate the inter-specific and intra-specific genetic distance of ingroup species. To determine the relative fit of candidate models of nucleotide evolution for gene and genomic compartment, three datasets used in this research, namely COI + COII, EF-1a and COI + COII + EF-1a, were analyzed separately in jModelTest 2.1.1 (Posada 2008, Darriba et al. 2012). The best models of nucleotide evolution were selected on the basis of corrected Akaike information criterion (AICc). As Burnham and Anderson (2002) strongly recommend using AICc, rather than Akaike information criterion (AIC), if the sample size is small. In order to choose a proper functional outgroup, 6 outgroup species from the genus Gangara, Hidari,

24

Acerbas and Zela were selected in preliminary test using Bayesian inference. We analysed three datasets, mtDNA COI + COII gene, nuclear EF-1a gene, and the combined data set of mitochondrial and nuclear sequences. The Maximum parsimony analyses (MP) were constructed using PAUP* 4.0.b10 (Swofford 2003) and the Maximum likelihood analyses (ML) were conducted using PHYML version 3.0 (Guindon and Gascuel 2003, Guindon et al. 2005). Bayesian analyses were implemented in MrBayes 3.2.1 (Ronquist and Huelsenbeck 2003). MP analyses were executed utilizing heuristic search with 5,000 random taxon additions and the tree bisection/reconnection (TBR) branch swapping method. Gaps were treated as missing. For combined dataset, the COI, COII and EF-1a gene region were defined as three character groups. Clade support values were measured by using the resampling-based bootstrap approach (Felsenstein 1985) performing 1,000 pseudo-replicates and 100 independent replicates. The ML trees were generated using the best fit model chosen by the model test. The model and parameters were indicated by jModelTest 2.1.1 (Darriba et al. 2012), based on the corrected Akaike information criterion (AICc). The confidence values of the ML tree were evaluated by the bootstrap test with 1,000 iterations. BI analyses were run using Markov chain Monte Carlo (MCMC) and stared twice for 10 million generations each, to confirm that the final average standard deviation of split frequencies was below 0.01. Trees were sampling every 1000 generations and four simultaneous chains (one cold

25 and three hot) were used in each run. The parameters and the model of evolution were unlinked across character partitions. We discarded the first one thousand sampled trees as burn-in for those were not within the stationary distribution of log-likelihoods. Trees and posterior probabilities were summarized using the 50%-majority rule consensus method.

26

Results

Part I

Erionota torus

We obtained sequence data (Total: 2,209 base pairs) corresponding to the mitochondrial COI+COII gene (COI: 1,518 bp; COII: 691 bp) from 28 individuals of E. torus. All sequences can be translated into amino acids. The neutral tests, Tajima’s D test (average value of D= -0.063; P value= 0. 85, NS) and Fu's Fs value (average value of D= -0.057; P value= 0.71, NS), all showed that the accumulated mutations of the dataset were not seriously affected by positive selection, and was suitable for analyses. The E. torus specimens were collected from 8 counties (Table 1), 15 localities, and 10 haplotypes were recognized from the samples (Figure 1). The haplotype network showed that 13 specimens from main Taiwan island, 1 specimen from China (Fujian Province) and 3 specimens from Japan (Yonaguni Is and Ishigaki Is, Okinawa) are both possessed haplotype A. There are three hyplotypes connected to haplotype A, which are haplotype B, haplotype C and haplotype D. A specimen from Taiwan, Kinmen is haplotype B, with two steps mutation to haplotype A. Haplotype D (2 specimen from Laos and Guangxi, China) and haplotype C (a specimen from Hainan, China) are also connected together by a single step mutation with haplotype A. Haplotype E contain 2 specimens from Yunnan Province and with two steps mutation to haplotype C and haplotype D. The other one specimens from Okinawa, Japan (Miyagi Is) possessed haplotype G, which is five steps mutation to haplotype A (which

27 contain the other 3 specimens from Okinawa). A specimen from Vietnam possessed haplotype F. Haplotype H (a specimen from the Philippines), haplotype I (1 specimen from Indonesia) and haplotype J (1 specimen from Malaysia) exhibit a long branch branch connection with haplotype A (haplotype H: 6 steps, haplotype I: 8steps, haplotype J: 8steps). This relationship among haplotypes supports that the E. torus population in Taiwan is close to the population in Fujian Province, China.

Erionota thrax

We also obtained 11 individuals of E. thrax with sequenced a total of 2,209 base pairs from mitochondrial COI+COII gene (COI: 1,518 bp; COII: 691 bp). The neutral tests, Tajima’s D test (average value of D= -0.045; P value= 0.78, NS) and Fu's Fs value (Fs average value of D= -0.034; P value= 0.72, NS) was negative, but non-significant. Both result showed that the accumulated mutations of the E. thrax dataset were affected by randomly genetic drift and did not seriously affected by positive selection. This result also suggested that the dataset was suitable for population analyses without bias from positive selection. The E. thrax specimens were collected from 6 counties, 9 localities (Table 1), and distinguished 6 haplotypes (Figure 2). The topology of the Bayesian tree shows that the geographic regions, Malaysia (three specimens), Indonesia (two specimens) and India (one specimen), are both monophyletic groups and are divided into three haplotype (Malaysia: haplotype D, Indonesia: haplotype B, India: haplotype F). Two specimens from China and Laos were found to have the same haplotype (haplotype E). Three specimens from the Philippines were found in different

28 haplotypes, that is, two specimens from Palawan have the same haplotype (haplotype C) and one specimen from Mindanao has its own haplotype (haplotype A). The haplotype network based on 6 haplotypes indicates that haplotype A and haplotype B are both with four steps mutation to haplotype C. While haplotype C is connected to haplotype D (with one step mutation), and haplotype D is connected to haplotype E and F (both with two steps mutation).

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Part II

Dataset characters

28 specimens were used in the COI+COII sequencing representing 6 ingroup species (22 specimens) and 6 outgroup species (6 specimens). 26 specimens were used in the analysis of the Ef-1a region, including 5 ingroup species (20 specimens) and 6 outgroup species (6 specimens). We sequenced a total of 3,409 base pairs (bp) in this study, of which 2,209 bp fragment for the mitochondrial COI+COII region (COI: 1,518 bp; COII: 691 bp) and 1,200 bp from nuclear Ef-1a region. There were no indels in the alignment of the protein-coding genes and the entire 67 bp of the intervening leucine tRNA was deleted. Substitution versus divergence pattern of COI+COII and Ef-1a dataset showed no sign of saturation for both 1st+2nd codon and 3rd codon, the phylogenetic analysis thus used all site for analysis. The COI+COII p-distance of ingroup species are showed in Table 4 and Table 5. Most inter-species p-value is higher than 0.030. The distance between E. hiraca to E. torus (0.029), E. thrax (0.026) and E. grandis (0.027) are little lower than 0.030. And the distance between E. thrax and E. torus (0.027) is also little lower than 0.030. Although the distance between the some species are lower than 0.030, they are still very close to

0.030 and may still offer a good discrimination .The intra-species distance of E. torus and E. thrax are much higher (more than 0.0400), while E. hiraca and E. surprisa are comparatively lower (0.0068 and 0.0039). There are no data on intraspecific distance in E. hislopi and E. grandis because they were represented only by one specimen in the analysis.

30

One specimen representing E. hislopi did not amplify for the entire mitochondrial fragment, and are only represented by 1291 bp of the COI gene. The other two specimens representing E. grandis and E. hiraca did not amplify for the nuclear Ef-1a region and were removed from the phylogenetic analysis of the nuclear gene. These specimens were obtained as dried material and have been stored for years. The muscle tissues may be eaten by the bacteria and fungi or presumably due to DNA degradation. mtDNA

The best fit model chosen to describe the evolution of the mtDNA sequences was the GTR+G model. Nucleotide frequencies were A= 0.3198, C= 0.1277, G= 0.1231, T= 0.4295, the proportion of invariant sites = 0, and the gamma shape parameter= 0.1450. The detail parameter estimates of sequence evolution for the genus Erionota used in this study are listed in Table 6. Results of the mitochondrial COI+COII gene in the MP analysis (Figure 3), ML analysis (Figure 4) and Bayesian analysis (Figure 5) were both strongly supported the monophyly of the genus Erionota (MP= 100

％, ML=99％, Bayesian pp=1.00). Both MP tree, ML tree and BI tree were well resolved under the evolution model with virtually all species-level relationships well supported. Six ingroup species, E. torus, E. thrax, E. hiraca, E. grandis, E. hislopi, and E. surprisa, are both monophyletic groups. And the systematic position of the species in the genus Erionota was considered stable. As the tree topology of the MP, ML and BI analysis show the same: E. hislopi+ (E.surprisa+ (E. hiraca+ (E. grandis+ (E.

31 thrax+E. torus)))). In the mitochondrial trees, our data provide no evidence of the closest sistergroup of the genus Erionota. Thus, the genera of Gangara, Hidari, Acerbas, Zela were regarded as a co-outgroup. nDNA

Model test identified the GTR+I+G model as the best fit model to the Ef-1a sequences. Nucleotide frequencies were A= 0.2719, C= 0.2536, G= 0.2379, T= 0.2366, the proportion of invariant sites = 0.4760, and the gamma shape parameter = 0.2990. The detail parameter estimates of sequence evolution for Erionota used in this study are listed in Table 6. The MP (Figure 6), ML (Figure 7) and BI (Figure 8) analysis strongly support that the genus Erionota is a monophyletic group (MP=100％,

ML=100％, Bayesian pp=1.00). And each species are both monophyletic groups. The tree topology of the genus Erionota produced by ML and BI methods both showed the same relationships of the species: E. hislopi+ (E.surprisa+ (E. hiraca+ (E. thrax+E. torus))). The tree topology of the MP tree cannot strongly clarify the relationship between the species E.thrax, E. torus, E. hiraca and E. surprise, which showed: E. hislopi+ (E.surprisa+ (E. hiraca+ E. thrax+ E. torus) Although the result strongly suggested Erionota as a monophyletic group, the outgroup did not reflect the closest sistergroup of the genus Erionota. Thus, the genera of Gangara, Hidari, Acerbas, Zela were regarded as a co-outgroup.

32

Discussion

Part I

The banana skipper, E. torus, is introduced into Japan and Taiwan. In addition, the other banana skipper, E. thrax, is found in Papua New Guinea, Guam, Hawaii and Mauritius (Monty 1970). The long distance dispersal of banana skippers is very likely caused by human activities base on the following four observations. First, the flying ability of the skippers is not good enough to fly across the distant ocean barrier. Second, due to their crepuscular habit, they only active in the early morning and before dusk, and regardless to their activity, feeding time or resting time, they usually do not leave from their host plant too far. The third observation is that the localities where the banana skippers have been found are usually near by the harbor or airport. And the fourth observation is that the freshly-emerged E. torus adult is able to survive more than a week under 4℃ without any food (Personal observation), it seems that the banana skippers may likely pass through the harsh transport processes. However, the source and the pathways of the banana skippers have received little attention.

In Japan, the banana skipper, E. torus was first found in December 1972 at several banana plantations in the Naha City, which is on the west coast of southern Okinawa island. While in 1971, there was a collector, Miyagi, had already captured few E. torus in Okinawa island. At that time, they regard E. torus as the vagrant butterfly, and speculated that the E. torus was introduced by the typhoon from China. However, there are many

33 research subsequently indicated that the invasive of E. torus is highly related with the Vietnam War (Taruya et al. 1973). The U.S. military occupied the Okinawa islands from the beginning of the Pacific War in 1945 until 1972 (nearly the end of the Vietnam War). During this period, numerous invasion species had found in Okinawa island such as sweet potato weevil (Euscepes postfaciatus), banana weevil (Odoiporus longicollis), vegetable weevil (Listroderes obliquus), brinjal fruit and shoot borer (Leucinodes orbonalis) and potato tuber moth (Phthorimaea operculella). Because the military planes and the warships do not need the quarantine procedures, it is more likely that the “hitchhikers” get the chance to establish in the new territory. Same situation in E. torus. Naha City, the place where E. torus was first discovered, was adjacent to the U.S. military base. It was 1972 when the Vietnam War had been continued. The U.S. army often went back and forth their colonies in South east Asia, where the banana skippers are distributed. It is the reason why many researchers suspected that E. torus was introduced from Vietnam by the U.S. military. The result of the haplotype network shows that the specimens from China (Fujian Province), Taiwan and Japan (Yonaguni Is and Ishigaki Is, Okinawa) share the same haplotype (haplotype A), while a specimen from the Miyagi island, Uruma City, Okinawa has a different haplotype (haplotype G), which is five steps mutation to haplotype A. The Yonaguni island is Japan's westernmost island which is located 125 km from Taiwan and 127 km from the Ishigaki island. E. torus was first discovered in Yonaguni island in 2002 (Fukasawa 2002), which was later

34 than the record in Taiwan (1986). Thus, the E. torus population in Yonaguni island was very likely came from Taiwan or China by wind or ocean current. The Kuroshio current is a north-flowing ocean current, which flows in the north-western Pacific Ocean from Taiwan island northwards to Japan. Previous study suggested that the Kuroshio current may be an important carrying or promoting vector for the species to establish their territory from Taiwan to Japan. For example, a stick (Megacrania tsudai), which is a protected species in Taiwan, had successfully established populations in Yonaguni island and Ishigaki island. Ushirokita (1998) suggested that the introduction of the Megacrania tsudai was carried by the Kuroshio current. The typhoons occurred every year may also have been a possible invasive method for E. torus. With three possible invasive methods for E. torus (natural dispersal, Kuroshio current and typhoons) and the result of the haplotype network, we suggest that the E. torus population in Yonaguni and Ishigaki island was very likely introduced from Taiwan than from China. In this study, we obtain a specimen (Tw-S-04) which was collected from Pingtung County (where E. torus was first discovered in Taiwan) in 1987 (E. torus was first found in 1986 in Taiwan). The result shows that this specimen (Tw-S-04) shares the same haplotype with other specimens which were also collected from the main Taiwan island during 2012 and 2013. The haplotypes from A to G shows a star-like network, which indicates a rapid population expansion. Comparing to the discovery

35 records of E. torus in these counties, the invasion source to Taiwan and Japan may came from China, Laos or Vietnam in a short time scale. Which also show that E. torus is a successful invasive species. An invasive species may need several conditions for successful, for example their host plant need to be available in the non-native area, they do not have any or few natural enemies or predators, and they have to adapt to the temperature or environmental conditions in non- native area or happened find a suitable place. Studies of the phylogeographic structure are helpful to understand the potential of the rapid evolution of these species and provide novel insights into the colonization dynamics and the invasive history. Our result of the phylogeographic structure of E. torus provides a brief survey basically of East Asia. In the future, adding more specimens from the South-east Asia may help to obtain an overall result of the phylogeographic relationship for the entire Asia E. torus populations. The haplotype network of E. thrax completely matches with the distance of the geographic location. Although the sample size in this study is not enough to answer the origin of E. thrax, it still provides a clear pattern of the relationship between the populations in sampled counties. The specimens from China and Laos have the same haplotype (haplotype E), and their geographic distribution give a good explanation. Haplotype F, which contains a specimen from India, is connected with haplotype E with a single step mutation. Haplotype D which contains all of the specimens from Malaysia is in the middle of haplotype C, haplotype E and haplotype F. This pattern also corresponds with the geographic distance between the collecting localities. Two specimens from Palawan, the Philippines, have

36 the same haplotype (haplotype C), while the other specimen from

Mindanao, the Philippines has a different haplotype (haplotype A). And to our surprise, the result of the haplotype network shows that haplotype A is closer to haplotype B (two specimens from Indonesia) than haplotype C

(two specimens also from the Philippines). This result indicated that the genetic relationship of the population in Mindanao, the Philippines is closer to the population in Indonesia.

Contributions and Future Work

This study, to our knowledge, provides the first phylogeographic study of the banana skippers. Although the sampling of E. thrax and E. torus populations in the global banana cultivation in this study were not enough to draw sweeping conclusions about the pattern of introductions, our study still provides a preliminary estimate of the number of introductions. Moreover, our study may as a foundation for more in-depth study. With more additional data incorporating with our study, it will be more thoroughly to resolve the relationships between native and introduced. Finally, a better understanding of the relationship between the source and the pathways of an introduced or invading species may help to moving on the other issues or shed light on new directions for research, such as, the invasion history of the species with the pattern of human transport, the reasons helps them successfully established the population in the non-native area, or the development of biological control.

37

Part II

The mitochondrial and nuclear trees both showed the strongly support of the monophyly of the genus Erionota. While the relationship of outgroup were remained unresolved. It was failed to answer which genus (Gangara, Hidari, Acerbas and Zela) is closest to Erionota. Evans (Evans 1949) placed the genus Erionota in the Erionota subgroup of the Plastingia group, which also included Prada and Tiacellia. Maruyama (1991) merged Erionota and Unkana subgroups of Evans into Erionota group. As de Jong (2006) pointed out, these subdivisions may be unsatisfactory because diagnostic characters are poor, and possible relationships with genera from other continents have not been considered. Thus, we did not choose the outgroup members from the Erionota subgroup or Erionota group. In this study, instead, we chose the outgroup members based on Warren’s molecular research (Warren et al. 2009). Although they did not put the genus Erionota in their phylogenetic tree, they still put the genus in the morphological discussion. Base on the descriptions, we used Gangara, Hidari, Acerbas and Zela to determine sister-group relationships in this study. However, in this study, both mitochondrial and nuclear region did not resolve the sistergroup relationship of the genus Erionota. Thus, more genera such as Unkana and Lotongus are the candidates for the fruther research in the future. A species, Koruthaialos rubecula rubecula, was added in outgroup for the phylogenetic analysis at the beginning of this study, because they are known to utilize the Musaceae. However, the preliminary result showed that the relationship between the genus Erionota and Koruthaialos

38 was too far. Thus, the species, Koruthaialos rubecula rubecula, was then deleted in the following analysis. Cluster the taxa that are far related may easily cause the long branch attraction. That is, the far-relationship taxon may by chance exhibit the similar characteristics as the ingroup, while the phylogenetic analysis does not down-weight these apparent similarities and will group these taxa together as a clade, which will fail to group the ingroup with their "true" sister taxa.

Phylogenetic relationships within the genus Erionota

Previous studies suggested that nuclear gene, Ef-1α, performed best at higher taxonomic levels. In this study, the result of the Ef-1α dataset in the MP analyses are failed to resolve the relationship, E. hiraca and E. surprise. But the results of Ef-1α dataset in the ML and Bayesian analyses perform well to clarify the relationship at the species level of the genus Erionota. Within the genus, the MP, ML and Bayesian analyses resulted in a very similar tree topology: each species is well defined monophyletic groups. And it also showed very high Bayesian posterior probability, Maximum parsimony and Maximum likelihood bootstrap support for the most relationships. Banana skippers, E.thrax and E. torus, are a monophyletic groups. Their relationship with other species is E. hislopi+ (E.surprisa+ (E. hiraca+ (E. grandis+ (E. thrax+E. torus)))). The morphological characters of the banana skippers, E. thrax and E. torus, are very similar, but their genitalias are quite different. The previous classification divided the banana skippers into two species based on the genitalia. Our result shows that the genetic distance between E. thrax and

39

E. torus are 2.7％. According to the research by Hebert et al. (2003), the genetic distance of the COI sequences between Lepidoptera species is approximately 3％. Thus, although it is hard to identify the banana skippers from their morphological characters, based on the genitalia and molecular evidence the banana skippers may clearly and reliably divided into two species, E. thrax and E. torus. The tree topology shows that the species E. grandis is the closest species to the banana skippers, while the morphological characters also reflect the same pattern. The wing pattern of the E. grandis is similar to the banana skippers, with three spots on the forewing and the hind wings are unmarked. The obvious difference between them is that the color of the spots (E. grandis have white spots and banana skippers have yellow spots). In our prediction, E. hiraca and E.surprisa are very likely to group as a clade because of the similarity of their adult morphology. The size of E. hiraca and E.surprisa are comparatively small to E. grandis and banana skippers. The forewings of the E. hiraca are dark brown with yellow-hyaline spots as well. But the elongated cell spot at the end cell attached to the spot next it at its inner half and attached to the spot in space three at its outer half, the species E.surprisa is more or less like E. hiraca. E. hiraca are also called the white tipped skipper as its common name, because of the distinctive whitened apical patch on their forewing above. However, the results show that E. hiraca is closer to E. grandis, E. thrax and E. torus, and these four species are a monophyletic group. And species E.surprisa is than a monophyletic group with E. hiraca, E. grandis, E. thrax and E. torus. The species E. hislopi has the most distant relationship

40 to the other species. Due to the sampling problem, other two rare species, E. tribus and E. sybirita are failed to obtain in this study. The relationships among these two species to the other Erionota species are remained ambiguous. More specimens are needed to obtain a more comprehensive result.

The hostplant evolution of the genus Erionota

Our results strongly support that banana skippers are a monophyletic group base on the evidence from the mitochondrial (MP=72％, ML=78％

BI pp=0.92) and nuclear gene regions (MP=78％, ML=74％ BI pp=0.98).

In the genus Erionota, E. thrax and E. torus are the only species known to utilize the banana plant (Musaceae). Corbet and Pendledury (1978) listed the Musaceae, Cocos nucifera (cocont palm, Arecaceae) and Saccharum officinarum (sugar cane, ) as E. thrax hostplants. Seitz (1927) also recorded other monocotyledons such as Raphis and (Arecaceae). E.torus had also sporadically recorded on the Arecaceae or found the eggs layed in clusters on lamina of the Alpinia zerumbet (Zingiberaceae) (Personal observation). However, nearly no records of E. thrax feeding on Poaceae since 1978. Moreover, our experiments showed that E. thrax and E. torus feeding on the Arecaceae had low survival rates. And there were no E. torus survived by feeding on Zingiberaceae. Thus, we regard Musaceae as banana skippers’ main hostplant. We take Arecaceae as the occasionally records and do not consider Poaceae and Zingiberaceae as the hostplants of the banana skippers.

41

Two species, E. grandis and E. hiraca are known to utilize the palms (Palmae) as their hostplant. The life history or the hostplant uses of the other species in this genus are remaining unknown. However, knowing that banana skippers are a monophyletic group still provides evidence that the hostplant use of the banana skippers had occurred a single host shift, which was shifted from the palms to the bananas.

Over 80％ of the phytophagous Lepidoptera are monophagous or oligophagous (Bernays and Chapman, 1994). And how do the phytophagous insects evolve to utilize a novel hostplant had numerous factors and had being discussed by far. For example, some butterflies may shift their hostplant to prevent inter-specific competition (Shapiro and Carde 1970). While the other study suggests that if the plant had massively grow in the insect’s territory, the insect will more likely to meet the plant and thus increase the chances for the inset to utilize the new plant (Strong et al. 1984). However, the relationships between the larval and adult butterflies and their hostplants are complicated because of the butterfly larvae adaptation to plant secondary metabolites. Bananas and plantains are the fourth most important food crop in the world, ranked after rice, wheat and maize. They are grown in at least 107 countries (FAO 2005), especially in the tropical and subtropical regions. Banana is a major food source and an important income source for the southeast Asian counties. As a tropical plant, banana perfectly grows under warm conditions in the southeast Asia, which is also the distribution range of the genus Erionota. Based on this circumstance, the ancestor of banana skippers may have had more opportunity to shift their host plant from the

42 palms to the bananas. As Merz (1959) stressed, many butterflies lay their eggs on their host plants with great precision. But on the other hand, numerous "mislays" have often been recorded (Remington, 1952; Dethier, 1959). In these cases, those larvae on the wrong hostplants may either leave to find their appropriate plant or die. There are still very few chances that the wrong plant became their new host plant. The phylogenetic relationship between the palms (Arecaceae) and bananas (Musaceae) is far. The family Arecaceae belongs to monocotyledon order Arecales, while the family Musaceae belongs to Zingiberales. However, the leaves of the bananas are palm-like, which may confuse the banana skippers when they were laying the eggs. However, the possible mechanisms of the host plant shift should need more investigation. Whether the host plant shift from the palms to the banana of the banana skippers was cause by the species competition or the adaptation of the secondary metabolites are unable to answer at the present stage. More host plant usage of the other members in the genus Erionota and the relate genera need to be known to clarify the evolution of host plant usage in the genus Erionota.

43

References

Abera, A. M. K., C. S. Gold, and S. Kyamanywa. 1999. Timing and distribution of attack by the banana weevil (Coleoptera:Curculionidae) in East African highland banana (Musa Spp.). The Florida Entomologist 82:61-64. Ahmad, Y. and A. Balasubramaniam. 1975. Major crop pests in Peninsular Malaysia. Ministry of Agriculture and Rural Development. Anderson, C. B., C. R. Griffith, A. D. Rosemond, R. Rozzi, and O. Dollenz. 2006. The effects of invasive north american beavers on riparian plant communities in Cape Horn, Chile. Do exotic beavers engineer differently in sub-antarctic ecosystems? Biological conservation 128:467-474. Atkins, A. F. 2005. Skipper butterflies: their origins? A preliminary discussion on the higher systematics and a proposed phylogeny of the Hesperiidae. The Bulletin of the Amateur Entomologists' Society 33:24–34. Atkinson, C. T., K. L. Woods, R. J. Dusek, L. S. Sileo, and W. M. Iko. 1995. Wildlife disease and conservation in Hawaii: Pathogenicity of avian malaria (Plasmodium relictum) in experimentally infected Iiwi (Vestiaria coccinea). Parasitology 111:S59-S69. Bauer, M., D. Pearce, and D. Vincent. 2003. Saving a staple crop: Impact of biological control of the banana skipper on poverty reduction in Papua New Guinea. Pages Impact Assessment Series, No. 22. Australian Centre for International Agricultural Research, Canberra, Australia.

44

Baxter, C. V., K. D. Fausch, M. Murakami, and P. L. Chapman. 2004. Fish invasion restructures stream and forest food webs by interrupting reciprocal prey subsidies. Ecology 85:2656-2663. Beadell, J. S., F. Ishtiaq, R. Covas, M. Melo, B. H. Warren, C. T. Atkinson, S. Bensch, G. R. Graves, Y. V. Jhala, M. A. Peirce, A. R. Rahmani, D. M. Fonseca, and R. C. Fleischer. 2006. Global phylogeographic limits of Hawaii's avian malaria. Proceedings. Biological sciences / The Royal Society 273:2935-2944. Beckmann, C. and R. Shine. 2009. Impact of invasive cane toads on Australian birds. Conservation biology 23:1544-1549. Bowdery, D. 1999. Phytoliths from tropical sediments: Reports from Southeast Asia and Papua New Guinea. Bulletin of the Indo-Pacific Prehistory Association (Melaka Papers, Volume 2) 18:159-168. Bridges, C. A. 1994. Catalogue of the family-group, genus-group, and species-group names of the Hesperiidae (Lepidoptera) of the world. Published by Author, Urbana, Illinois. Brower, A. and R. DeSalle. 1998. Patterns of mitochondrial versus nuclear DNA sequence divergence among nymphalid butterflies: the utility of wingless as a source of characters for phylogenetic inference. Insect Molecular Biology 7:73-82. Burnham, K. P. and D. R. Anderson. 2002. Model Selection and Multi-Model Inference: A Practical Information-Theoretic Approach. 2nd edition. Pages 323- 325. Springer Science, New York, USA. Carlton, J. T. 2000. Global change and biological invasions in the oceans. Invasive in a changing world. Pages 31-54. H. A. Mooney and R. J.

45

Hobbs, editors. Island Press, Washington, DC. Caterino, M., S. Cho, and F. Sperling. 2000. The current state of insect molecular systematics: A thriving tower of Babel. Annual Review of Entomology 45:1-54. Caterino, M. S. and F. A. H. Sperling. 1999. Papilio phylogeny based on mitochondrial cytochrome oxidase I and II genes. Molecular Phylogenetics and Evolution 11:122-137. Chiang, H. S. 1988. Bionomics and control of banana skipper, Erionota torus Evans, in Taiwan. Chinese Journal of Entomology, Special Publish 2:167-174. Chiang, H. S. and M. T. Hwang. 1991. The banana skipper, Erionota torus Evans (Hesperiidae: Lepidoptera): Establishment, distribution and extent of damage in Taiwan. Tropical Pest Management 37:207-210. Chu, Y. I. and L. Chou. 1988. The banana skipper, a new banana pest in Taiwan. Taiwan Agriculture 24:47-51. Clement, M., D. Posada, and K. A. Crandall. 2000. TCS: a computer program to estimate gene genealogies. Molecular Ecology 9:1657-1659. Corbet, A. S. and H. M. Pendlebury. 1978. The butterflies of the Malay Peninsula. 3rd edition, revised by J. N. Eliot. Malayan Nature Society, Kuala Lumpur. Croll, D. A., J. L. Maron, J. A. Estes, E. M. Danner, and G. V. Byrd. 2005. Introduced predators transform subarctic islands from grassland to tundra. Science 307:1959-1961. Darriba, D., G. L. Taboada, R. Doallo, and D. Posada. 2012. jModelTest 2:

46

more models, new heuristics and parallel computing. Nature Methods 9:772. Davis, C. J. and K. Kawamura. 1975. Notes and exhibitions. Proceedings of the Hawaiian Entomological Society 22(1):21. de Jong, R. 2006. Notes on Salanoemia Eliot, 1978, and Isma Distant, 1886 (Lepidoptera: Hesperiidae), mainly from Java and Sumatra. Tijdschrift voor Entomologie 149:15-20. de Jong, R. and C. G. Treadaway. 1993. The Hesperiidae (Lepidoptera) of the Philippines Zoologische Verhandelingen 288:1-125. de Jong, R., R. Vane-Wright, and P. R. Ackery. 1996. The higher classification of butterflies (Lepidoptera): problems and prospects. Insect Systematics and Evolution 27:65-101. Denham, T. P., S. G. Haberle, C. Lentfer, R. Fullagar, J. Field, M. Therin, N. Porch, and B. Winsborough. 2003. Origins of agriculture at Kuk swamp in the highlands of New Guinea. Science 301:189-193. Edgar, R. C. 2004. MUSCLE: a multiple sequence alignment method with reduced time and space complexity. BMC Bioinformatics 5:113. Evans, E. W. 2004. Habitat displacement of North American ladybirds by an introduced species. Ecology 85:637-647. Evans, W. H. 1949. A catalogue of the Hesperiidae from Europe, Asia and Australia in the British Museum (Natural history). Printed by order of the Trustees of the British Museum, London,. Felsenstein, J. 1985. Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39:783-791. Fu, Y. X. 1997. Statistical tests of neutrality of mutations against

47

population growth, hitchhiking and background selection. Genetics 147:915-925.

Fukasawa, K. 2002. The record of the banana skipper in Yonaguni island.

The butterfly research 192:24. (In Japanese) Funasaki, G. Y., P. Y. Lai, L. M. Nakahara, J. W. Beardsley, and A. K. Ota. 1988. A review of biological control introductions in Hawaii: 1980 to 1985. Proceedings of the Hawaiian Entomological Society 28:105-160. Gold, C., J. Pena, and E. Karamura. 2001. Biology and integrated pest management for the banana weevil, Cosmopolites sordidus (Germar) (Coleoptera: Curculionidae). Integrated Pest Management Reviews 6:79-155. Gold, C. S., G. H. Kagezi, G. Night, and P. E. Ragama. 2004. The effects of banana weevil, Cosmopolites sordidus, damage on highland banana growth, yield and stand duration in Uganda. Annals of Applied Biology 145:263-269. Guindon, S. and O. Gascuel. 2003. A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Systematic Biology 52:696-704. Guindon, S., F. Lethiec, P. Duroux, and O. Gascuel. 2005. PHYML Online-a web server for fast maximum likelihood-based phylogenetic inference. Nucleic acids research 33:W557-559. Hall, T. A. 2011. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Biosciences 41:95-98.

48

Hasan, S. A. 1994. Butterflies of Islamabad and the Murree hills. Asian Study Group, Islamabad. Hebert, P. D. N., A. Cywinska, S. L. Ball, and J. R. deWaard. 2003. Biological identifications through DNA barcodes. Proceedings of the Royal Society of London 270:313-322. Hebert, P. D. N., E. H. Penton, J. M. Burns, D. H. Janzen, and W. Hallwachs. 2004. Ten species in one: DNA barcoding reveals cryptic species in the neotropical skipper butterfly, Astraptes fulgerator. Proceedings of the National Academy of Sciences of the United States of America 101:14812-14817. Ho, C. R. 1975. The expansion and changing location of banana production in Taiwan. Journal of National Taiwan Normal University 20:98-128. Hodge, W. H. and C. O. Erlanson. 1956. Federal plant introduction-A review. Economic Botany 10:299-334. Hsu, Y. F. 1999. Butterflies of Taiwan. Volume 1. Fonghuanggu Bird and Ecology Park. Hundsdoerfer, A., H. Meier, J. Rheinheimer, and M. Wink. 2009. Towards the phylogeny of the Curculionoidea (Coleoptera): Reconstructions from mitochondrial and nuclear ribosomal DNA sequences. Zoologischer Anzeiger 248:9-31. Igarashi, S. and H. Fukuda. 1997. The life histories of Asian butterflies. Volume 1. Tokai University Pres, Tokyo, Japan. Jones, H. P., B. R. Tershy, E. S. Zavaleta, D. A. Croll, B. S. Keitt, M. E. Finkelstein, and G. R. Howald. 2008. Severity of the effects of

49

invasive ats on seabirds: A global review. Conservation Biology 22:16-26. Kandul, N., V. Lukhtanov, A. Dantchenko, J. Coleman, C. Sekercioglu, D. Haig, and N. Pierce. 2004. Phylogeny of Agrodiaetus Hübner 1822 (Lepidoptera: Lycaenidae) Inferred from mtDNA sequences of COI and COII and Nuclear Sequences of EF1-α: Karyotype diversification and species radiation. Systematic Biology 53:278-298. Kennedy, J. 2008. Pacific bananas: Complex origins, multiple dispersals? Asian Perspectives 47 (1):75-94. Lentfer, C. J. 2003. Tracing antiquity of banana cultivation in Papua New Guinea: Report on collection of modern reference material from Papua New Guinea in 2002. Unpublished report prepared for the Pacific Biological Foundation, Sydney, NSW, Australia. Librado P, R. J. 2009. DnaSP v5: a software for comprehensive analysis of DNA polymorphism data. Bioinformatics. 25:1451-1452. Lovett, G. M., C. D. Canham, M. A. Arthur, K. C. Weathers, and R. D. Fitzhugh. 2006. Forest ecosystem responses to exotic pests and pathogens in Eastern North America. BioScience 56:395-405. Lu, C. C., L. W. Wu, G. F. Jiang, H. L. Deng, L. H. Wang, P. S. Yang, and Y. F. Hsu. 2009. Systematic status of Agehana elwesi f. cavaleriei based on morphological and molecular evidence. Zoological Studies 48:270-279. Mallarino, R., E. Bermingham, K. R. Willmott, A. Whinnett, and C. D. Jiggins. 2005. Molecular systematics of the butterfly genus Ithomia

50

(Lepidoptera: Ithomiinae): a composite phylogenetic hypothesis based on seven genes. Molecular Phylogenetics and Evolution 34:625-644. Mark, R. N. and W. M. Lonsdale. 2001. Humans as global plant dispersers: getting more than we bargained for. BioScience 51(2):95-102. Maruyama, K. 1991. Butterflies of Borneo 2(2), Hesperiidae. Tobishima Corporation, Tokyo. Mau, R. F. L., K. Murai, B. Kumashiro, and K. Teramoto. 1980. Biological control of the banana skipper, thrax (Linnaeus) (Lepidoptera; Hesperiidae) in Hawaii. Proceedings of the Hawaiian Entomological Society 23(2):231-237. Mendoza, A. R. and R. A. Sikora. 2009. Biological control of Radopholus similis in banana by combined application of the mutualistic endophyte Fusarium oxysporum strain 162, the egg pathogen Paecilomyces lilacinus strain 251 and the antagonistic bacteria Bacillus firmus. Biocontrol 54:263-272. Monty, J. 1970. Notes on a new insect pest in Mauritius: the banana leaf roller Erionota thrax L. (Lepidoptera, Hesperiidae). Revue agricole et sucrière de l'île Maurice 49:107-109. Nakao, H. K. and G. Y. Funasaki. 1976. Introductions for biological control in Hawaii, 1974. Proceedings of the Hawaiian Entomological Society 22(2):329-331. Nei, M. 1972. Genetic distance between populations. The American Naturalist 106:283-292. Nei, M. 1973. Analysis of gene diversity in subdivided populations.

51

Proceedings of the National Academy of Sciences of the United States of America 70:3321-3323. Nei, M. 1987. Molecular evolutionary genetics. Columbia University Press, New York. Okolle, J. N., A. H. Ahmad, and M. Mashhor. 2009. Infestation and parasitism of banana skipper (Erionota thrax) (Lepidoptera: Hesperiidae) in relation to banana leaf age, and surface and distance from field edge. The Asia and Australasian Journal of Plant Sciences and Biotechnology 3(1):61-65. Okolle, J. N., M. Mansor, and A. H. Ahmad. 2006a. Spatial distribution of banana skipper (Erionota thrax L.) (Lepidoptera: Hesperiidae) and its parasitoids in a Cavendish banana plantation, Penang, Malaysia. Insect Science 13:381-389. Okolle, J. N., M. Mansor, and A. H. Ahmad. 2006b. Seasonal abundance of the banana skipper, Erionota thrax (Lepidoptera: Hesperiidae) and its parasitoids in a commercial plantation and a subsistence farm in Penang, Malaysia. International Journal of Tropical Insect Science 26(3):197-206. Phillips, B. L., G. P. Brown, and R. Shine. 2003. Assessing the potential impact of cane toads on Australian snakes. Conservation Biology 17(6):1738-1747. Pimentel, D., R. Zuniga, and D. Morrison. 2005. Update on the environmental and economic costs associated with alien-invasive species in the United States. Ecological Economics 52(3):273-288. Posada, D. 2008. jModelTest: phylogenetic model averaging. Molecular

52

biology and evolution 25(7):1253-1256. Queensland Horticulture Institute. 2000. Banana quarantine threats: Banana skipper. Department of Primary Industries. Raitzer, D. A. and R. Lindner. 2005. Review of the returns to ACIAR's bilateral R&D Investments. Impact Assessment Series (IAS) from Australian Centre for International Agricultural Research. Robson, J. D., M. G. Wright, and R. P. P. Almeida. 2007. Biology of Pentalonia nigronervosa (Hemiptera, Aphididae) on banana using different rearing methods. Environmental Entomology 36:46-52. Ronquist, F. and J. Huelsenbeck. 2003. MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19:1572-1574. Rossmann, B., H. Müller, K. Smalla, S. Mpiira, J. B. Tumuhairwe, C. Staver, and G. Berg. 2012. Banana-associated microbial communities in Uganda are highly diverse but dominated by Enterobacteriaceae. Applied and Environmental Microbiology 78(14):4933-4941. Sands, D. P. A., P. Bakker, and F. M. Dori. 1993. Cotesia erionotae (Wilkinson) (Hymenoptera: Braconidae), for biological control of banana skipper, Erionota thrax (L.) (Lepidoptera: Hesperiidae) in Papua New Guinea. Micronesica 4:99-105. Sands, D. P. A. and M. C. Sands. 1991. Banana skipper, Erionota thrax (L.) (Lepidoptera: Hesperiidae) in Papua New Guinea: a new pest in the South Pacific region. Micronesica 3:93-98. Schneider, S., D. Roessli, and L. Excoffier. 2000. A software for population genetics data analysis. Version 2.000. Genetics and Biometry

53

Laboratory, Department of Anthropology, University of Geneva, Switzerland. Scott, J. A. 1985. The phylogeny of the butterflies (Papilionoidea and Hesperioidea). Journal of Research on the Lepidoptera 23:241-281. Scott, J. A. 1986. On the monophyly of the macrolepidoptera, including a reassessment of their relationship to Cossoidea and Castnioidea, and a reassignment of mimallonidae to pyraloidea. he Journal of Research on the Lepidoptera 25(1):30-38. Seitz, A. 1927. Grypocera. Subfamily: Hesperiidae, Skippers. Gross-Schmetterlinge der Erde. Die Indo-Australische Tagfalter. Vol. 9. Alfred Kernen, Stuttgart. Shapiro, A. M. and R. T. Carde. 1970. Habitat selection and competition among Sibling species of Satyrid butterflies. Evolution 24(1):48-54. Simon, K. S. and C. R. Townsend. 2003. Impacts of freshwater invaders at different levels of ecological organisation, with emphasis on salmonids and ecosystem consequences. Freshwater Biology 48:982-994. Stechmann, D. H., W. Völkl, and P. Starý. 1996. Ant-attendance as a critical factor in the biological control of the banana aphid Pentalonia nigronervosa Coq. (Hom. Aphididae) in Oceania. Journal of Applied Entomology 120:119-123. Stewart, J. E. 1991. Introductions as factors in diseases of fish and aquatic invertebrates. Canadian Journal of Fisheries and Aquatic Sciences 48(1):110-117. Strayer, D. L., V. T. Eviner, J. M. Jeschke, and M. L. Pace. 2006.

54

Understanding the long-term effects of species invasions. Trends in ecology & evolution (Personal edition) 21:645-651. Strong, D. R., J. H. Lawton, and R. Southwood. 1984. Insects on plants: Community patterns and mechanisms. Harvard University Press. Swofford, D. L. 2003. PAUP*: Phylogenetic analysis using parsimony (*and other methods), version 4. Sunderland, MA: Sinauer Associates. Tadashi, T.1973. The invasive species discovered on Okinawa, banana skipper. Okinawa Prefectural Agricultural Experiment Station. (In japanese) Tajima, F. 1983. Evolutionary relationship of DNA sequences in Finite populations. Genetics 105 (2) 437-460. Tajima, F. 1989. Statistical method for testing the neutral mutation hypothesis by DNA polymorphism. Genetics 123 (3):585-595. Tamura, K., D. Peterson, N. Peterson, G. Stecher, M. Nei, and S. Kumar. 2011. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Molecular biology and evolution 28:2731-2739. Teruya, T., Y. Araki, and M. Osada. 1973. Banana skipper, a new banana pest. Shokubutsu Boeki 27(5):191-193. Tsai, Y. P., M. T. Hwang, J. M. Tsai, and H. S. Chang. 1990. Bionomics and biological control of banana skipper, Erionota torus Evans, in Taiwan. Chinese Journal of Entomology 10:419-426. van Riper, C. I. 1991. The impact of introduced vectors and avian malaria on insular passeriform bird populations in Hawaii. Bulletin of the

55

Society for Vector Ecology 16:59-83. Vila, M. and M. Björklund. 2004. The utility of the neglected mitochondrial control region for evolutionary studies in Lepidoptera (Insecta). Journal of Molecular Evolution 58:280-290. Vitousek, P., C. D'Antonio, L. Loope, and R. Westbrooks. 1996. Biological invasions as global environmental change. American Scientist 84(5):468-478. Wahlberg, N., M. F. Braby, A. V. Brower, R. de Jong, M. M. Lee, S. Nylin, N. E. Pierce, F. A. Sperling, R. Vila, A. D. Warren, and E. Zakharov. 2005. Synergistic effects of combining morphological and molecular data in resolving the phylogeny of butterflies and skippers. Proceedings. Biological sciences / The Royal Society 272:1577-1586. Wahlberg, N. and C. W. Wheat. 2008. Genomic outposts serve the phylogenomic pioneers: designing novel nuclear markers for genomic DNA extractions of Lepidoptera. Systematic Biology 57:231-242. Wardle, D. A., G. M. Barker, G. W. Yeates, K. I. Bonner, and A. Ghani. 2001. Introduced browsing mammals in New Zealand natural forests: Aboveground and belowground consequences. Ecological monographs 71:587-614. Warren, A. D. 2006. The higher classification of the Hesperiidae. PhD thesis. Oregon State Univerity. Warren, A. D., J. R. Ogawa, and A. V. Z. Brower. 2008. Phylogenetic relationships of subfamilies and circumscription of tribes in the

56

family Hesperiidae (Lepidoptera : Hesperioidea). Cladistics 24:642-676. Warren, A. D., J. R. Ogawa, and A. V. Z. Brower. 2009. Revised classification of the family Hesperiidae (Lepidoptera: Hesperioidea) based on combined molecular and morphological data. Systematic Entomology 34(3):467-523. Waterhouse, D., D. Birribi, and V. David. 1998. Economic benefits to Papua New Guinea and Australia from biological control of banana skipper (Erionota thrax). CSIRO Division of Entomology, Australia. Waterhouse, D. F. and K. R. Norris. 1989. Biological Control - Pacific prospects: supplement 1 Erionota thrax. Pages 88-99. ACIAR Monograph. Wu, L. W., D. C. Lees, S. H. Yen, C. C. Lu, and Y. F. Hsu. 2010. The complete mitochondrial genome of the endangered swallowtail, Agehana maraho (Lepidoptera: Papilionidae): evaluating sequence variability and suitable markers for conservation genetic studies. Entomological News 121:267-280. Zhang, B. C. 1994. Index of economically important Lepidoptera. CAB

International, Wallingford, UK.

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Table 1.

Collection data for samples of the genus Erionota and outgroup from Taiwan and other countries. Additional collecting details are available from the author. The samples used in part I, tracing the source of banana skippers (○). The samples used in part II, Molecular phylogeny of the genus Erionota (◎).

Species Lot number Locality Status

Ingroup

E. torus Tw-N-01 Taipei, Taiwan ○◎

E. torus Tw-N-02 New Taipei City, Taiwan ○

E. torus Tw-N-03 Keelung City, Taiwan ○

E. torus Tw-N-04 Hsinchu County, Taiwan ○

E. torus Tw-C-01 Taichung City, Taiwan ○

E. torus Tw-C-02 Nantou County, Taiwan ○

E. torus Tw-C-03 Yunlin County, Taiwan ○

E. torus Tw-S-01 Tainan City, Taiwan ○

E. torus Tw-S-02 Kaohsiung City, Taiwan ○

E. torus Tw-S-03 Pingtung County, Taiwan ○

E. torus Tw-S-04 Pingtung County, Taiwan (1987) ○

58

Table 1. continued

Species Lot number Locality Status

Ingroup

E. torus Tw-E-01 Yilan County, Taiwan ○

E. torus Tw-E-02 Hsinchu County, Taiwan ○

E. torus Tw-O-01 Kinmen, Taiwan ○

E. torus Ph-Mia-03 Mindanao, Philippines ○

E. torus Ma-Pe-06 Tapah Hills, Perak, Malaysia ○

E. torus Ch-Yn-01 Yunnan Province, China ○◎

E. torus Ch-Yn-03 Yunnan Province, China ○

E. torus Ch-Gx-01 Guilin City, Guangxi Province, China ○

E. torus Ch-Fu-01 Quanzhou City, Fujian Province, China ○

E. torus Ch-Ha-01 Hainan Is., China ○

E. torus Ja-Ok-01 Yonaguni Is., Okinawa Prefecture, Japan ○

E. torus Ja-Ok-02 Yonaguni Is., Okinawa Prefecture, Japan ○

E. torus Ja-Ok-03 Miyagi Is., Okinawa Prefecture, Japan ○◎

E. torus Ja-Ok-04 Ishigaki Is., Okinawa Prefecture, Japan ○

E. torus Vi-Ta-01 Tam Dao, Vietnam ○◎

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Table 1. continued

Species Lot number Locality Status

Ingroup

E. torus In-Ba-02 Bali Is., Indonesia ○

E. torus La-Vi-01 Vientiane, Laos ○◎

E. thrax Ph-Pa-01 Roxas, Palawan, Philippines ○◎

E. thrax Ph-Pa-02 Roxas, Palawan, Philippines ○

E. thrax Ph-Pa-03 Langogan, Palawan, Philippines ○

E. thrax Ph-Mia-01 Mindanao, Philippines ○

E. thrax In-Ba-01 Bali island Is., Indonesia ○◎

E. thrax In-Su-01 Peleng Is., Sulawesi Tengah, Philippines ○

E. thrax In-AB173 India ○◎

E. thrax Ma-Pe-03 Chenderiang, Perak, Malaysia ○◎

E. thrax Ma-Pe-04 Chenderiang, Perak, Malaysia ○

E. thrax Ma-Bo-02 Borneo, Malaysia ○

E. thrax Ch-Yn-02 Yunnan Province, China ○◎

E. thrax Ch-Gx-02 Guangxi Province, China ○

E. thrax La-Vi-02 Vientiane, Laos ○◎

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Table 1. continued

Species Lot number Locality Status

Ingroup

E. grandis Ch-Gd-01 Ruyuan County, Guangdong Province, ◎ China

E. grandis Ch-Gz-01 Guizhou City, Guizhou Province, China ◎

E. hiraca Ph-Mio-02 Mindoro Island, Philippines ◎

E. hiraca Ph-Ca-01 Catanduanes Is., Philippines ◎

E. hiraca Si-Bu-01 Bukit Timah, Singapore ◎

E. hiraca Ch-Yn-04 Yunnan Province, China ◎

E. hislopi Ma-Bo-03 Borneo, Malaysia ◎

E. surprisa Ph-Mia-02 Mindanao, Philippines ◎

E. surprisa Ph-Ro-01 Sibuyan Is., Romblon Province, ◎ Philippines

E. surprisa Ph-Po-01 Polillo Is., Philippines ◎

Outgroup

Acerbas anthea Ph-Sl-01 Mt. Balocaue, S. Leyte, Philippines ◎

Gangara lebadea Ma-Pe-07 Tapah Hills, Perak, Malaysia ◎

Gangara thysis Ph-Ne-01 Negros Is., Philippines ◎

Gangara Ch-Ha-02 Hainan Is., China ◎

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Table 1. continued

Species Lot Locality Status number

Outgroup

Hidari irava Ma-Pe-08 Bujang Melaka forest, Perk, Malaysia ◎

Zela excellens Th-Ra-01 Ranong, Thailand ◎

Annotate:

The lot number is referring to the locality (first two letters represent the country; middle two or three letters represent the city or region) and a serial numbers

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Table 2. Primers used in this study.

Primers 5'→3' F/R

COI&COII cox-J-1460 TACAA TTTAT CGCCT AAACT TCAGC C F Zcox-J-1530 CAACA AATCA TAAAG ATATT GG F Mean-J-1750 CATTT TGACT TTTAC CCCCC TC F H1co1-1700R AGTCA ATTTC CRAAT CCTCC R MiBocox-J-1700 AATAC TATTG TTACA GCTCA TGC F Efcox-J-1750 GTTTTT GACTC CTTCC CCC F H1co1-1880F TCAAG AAGAA TTGTA GAAAA TGG F MiBocox-N-2010 AGTTG TAATA AAATT AATWG CTCCT A R Skcox-J-2040 CTCTA CCAGT ATTAG CTGGA GC F Jpcox-J-2040 CTTTA CCTGT ATTAG CAGGT GC F Skcox-J-2100 TTTTG ATCCT GCAGG AGGAG G F cox-N-2191 CCCGG TAAAA TTAAA ATATA AACTT C R Ef-2191N CTTCT GGGTG ACCAA AAAAT C R Chcox-J-2200 ACCAG GATTT GGTAT AATTT CCCA F Skcox-N-2220 GGGAA ATAAT ACCAA ATCCA GG R Bocox-N-2340 GTGGC TGAAG TGAAG TAAGC TC R Chcox-N-2360 GAGCT CATAC AATAA ATCCT AAT R H1co1-2360R GAGCT CATAC AATAA ATCCT A R MiBocox-J-2360 CTTTT GGATC TTTAG GAATA ATT F Efcox-N-2450 TTAAT TCCTG TTGGT ACTGC R C1-J-2500 CAAGA AAGAG GAAAA AAAGA AAC F

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Table 2. continued

Primers 5'→3' F/R

COI&COII Bocox-J-2550 CTGTA GGAGG ACTAA CTGGA G F C1-J-2550 ATTTA CWGTA GGWAT AGATA TTGA F H1c1-2600F2 TTGAT ATCCT TTATT TACAG G F Ef-2600J GCAGT ATTTG CTATT TTTGG AGG F Gallc1-N-2750 CCTGC TAATC CTAAA AAATG TTG R Cppcox-N-2770 TGGAT AATCA GAATA TCGTC GAGG R Gallc1-N-2770 GTCGA GGTAT TCCTG CTAAT CCTA R Jpcox-N-2770 GATAA TCTGA ATAAC GACGA GG R Jpcox-2840 TTTTG NTATC ATTCA ATAGA TGA R Efcox-J-3000 ATATG TAATG GATTT AAACC CC F MiBocox-J-3040 GGATC ATATA TATCT TTAAT TTC F Micox-N-3080 TTTGA CCTTC TAATA AAAAT CG R cox-J-3138 AGAGC CTCTC CTTTA ATAGA ACA F C2-N-3300b ATAAC AGCAG GTAGA ATAGT TCA R C2-N-3380 ATTCA TAWCT TCAAT ATCAT TG R Skcox-3300 ATAAA CGTAA AGATG GTAAA GC R cox-N-3389 TCATA ACTTC AATAT CATTG R cox-J-3408 CAATG ATATT GAAGT TATGA F cox-N-3782 GAGAC CATTA CTTGC TTTCA GTCAT CT R

BB-2329＊ CTCGT GTAGT ATATA AATGG CATCC F

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Table 2. continued

Primers 5'→3' F/R

COI&COII

BB-3083＊ TACAA CGGTA AAGAT TTTTT TCCTA R

BB-2735＊ TTGTA AAAAA GGAGT TGTAA AAAAT F

BB-3417＊ AATCT TTTCA GACTC ATAAG TATTG R

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

＊ The primer synthesized in this research.

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Table 4. Inter-species COI+COII p-distance of ingroup species

1 2 3 4 5 1 E. hiraca 2 E. torus 0.029 3 E. thrax 0.026 0.027 4 E. hislopi 0.078 0.076 0.084 5 E. grandis 0.027 0.034 0.032 0.091 6 E. surprisa 0.051 0.070 0.061 0.096 0.069

Table 5. Intra-species COI+COII p-distance of ingroup species

1 E. hiraca 0.0068 2 E. torus 0.0071 3 E. thrax 0.0082 4 E. hislopi n/c 5 E. grandis n/c 6 E. surprisa 0.0039

Table 6. Model selection for the genus sequences used in this

study.

Gene Model Substitution rates region

Base composition Tr Tv a c g t I α a-g c-t a-c a-t c-g g-t

COI+COII GTR+G 0.3198 0.1277 0.1231 0.4295 0 0.1450 25.9551 170.6267 9.3286 29.0543 6.8846 1.0000

Ef-1a GTR+I+G 0.2719 0.2536 0.2379 0.2366 0.4760 0.2990 5.0121 12.2307 0.7373 1.8453 0.6201 1.0000

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Figure 1. Haplotype network of Erionota torus. The haplotype network of E. torus reconstructed by TCS 1.21 based on mitochondrial COI+COII sequences. Each circle represents a unique haplotype and the line between haplotypes represents a single step mutation. The circle area ratio reflect the specimen number contain in this haplotype. The letters on the circle represent the ten different haplotypes in this analysis.

Figure 2. Haplotype network of Erionota thrax. The haplotype network of E. thrax reconstructed by TCS 1.21 based on mitochondrial COI+COII sequences. The letters on the circle represent the six different haplotypes in this analysis.

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Figure 3. MP analysis to the mitochondrial data set of genus Erionota. Maximum parsimony tree inferred from the COI+COII data set. Bootstrap values are showed above the branch. Names of terminal taxa include the specimen’s lot number and the species name.

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Figure 4. ML analysis to the mitochondrial data set of genus Erionota. Maximum likelihood tree inferred from the COI+COII data set. Bootstrap values are showed above the branch.

69

Figure 5. BI analysis to the mitochondrial data set of genus Erionota.

Bayesian analysis tree inferred from the COI+COII data set and subjected to MCMC analysis. The scale bar represents the number of nucleotide substitutions per site.

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Figure 6. MP analysis to the nuclear data set of genus Erionota. Maximum parsimony tree inferred from the Ef-1α data set. Bootstrap values are showed above the branch.

71

Figure 7. ML analysis to the nuclear data set of genus Erionota. Maximum likelihood tree inferred from the Ef-1α data set. Bootstrap values are showed above the branch.

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Figure 8. BI analysis to the nuclear data set of genus Erionota.

Bayesian analysis tree inferred from the Ef-1α data set and subjected to MCMC analysis. The scale bar represents the number of nucleotide substitutions per site.

73