國立臺灣師範大學生命科學系 碩士論文 絨弄蝶屬之分子親緣與分類關係探討 Molecular phylogeny and systematic clarification of the genus Hasora (Lepidoptera: Hesperiidae) 研 究 生:李政學 Cheng-Hsueh Lee 指導教授: 徐堉峰 博士 Yu-Feng Hsu 千葉 秀幸 博士 Hideyuki Chiba 中華民國一零一年一月 Contents 致謝………………………………………………………..…2 中文摘要…………………………...……………………..……3 Abstract…………………………………………………..……4 Introduction………………………………….………….….…6 Materials and methods…………………………………….10 Results……………………………………………………..….15 Discussion……………………………………………….……18 Conclusion...……………………………………………….…26 References...………………………………………………..…28 Tables………………………………………………….……...32 Figures………………………………………………….…….40 1 致謝 本論文的完成，首先要感謝徐堉峰老師與千葉秀幸老師的指導， 徐堉峰老師不僅提供了豐富的經驗與知識，更提供了開放自由的研究 環境，使我能在蝴蝶實驗室這個充滿歡樂氣息的環境中學習研究，而 千葉秀幸老師則以其深厚的弄蝶分類知識提供了許多我在研究與撰 寫論文時的建議，他所提供的大量珍貴樣本更是本論文得以完成的關 鍵。另一方面，我也要感謝口試委員林思民老師對本論文提供的諸多 建議，使本論文在寫作或分析上都能更臻完善。在分生實驗與分析方 面，我要感謝立偉學長與亭瑋學姊的指導，不僅指導我實驗技巧與分 析方法，更解決了我在研究過程中的許多疑惑與難題，此外我也要感 謝秉宏學長對實驗方法提供的建議。我還要感謝小油龍學長教導我許 多知識與提供了許多方面的建議。除了研究之外，我也要感謝羅桑、 豪哥、M 大、小虎、大師、阿南、花姊、bass、郁婷、阿珠、育綺、 發哥、家源、小熊、球球、姿伶、阿賢等實驗室夥伴提供了溫馨活潑 的實驗室氣氛。最後我要感謝我的家人，尤其是爸爸媽媽，提供了我 許多有形無形的幫助，使我能在無後顧之憂的環境完成學業。感謝宣 安在我的研究期間給了我許多精神上的鼓勵與支持。本論文得力於許 多人的協助，僅在此獻給家人、師長與朋友們。 2 中文摘要 絨弄蝶屬 (Hasora)為鱗翅目 (Lepidoptera)弄蝶科 (Hesperiidae)大 弄蝶亞科 (Coeliadinae)之中大型弄蝶，主要分布於印度至澳洲間，是 大弄蝶亞科中種數最多的類群，在目前分類共有約 30 種，可被分為 6-7 個種群。本屬傳統分群與分類主要依據成蝶翅紋與交尾器特徵， 然而本屬在部分物種的分類上經常缺乏共識，而過去以交尾器與翅紋 相似性為依據進行之分群是否能合理反映絨弄蝶之親緣也仍未釐 清。本研究以粒線體 DNA 之 COI, COII 片段與核 DNA 之 Ef-1a 片段 重建絨弄蝶屬之分子親緣關係，比較其與 de Jong 提出之形態親緣是 否相符，並用以檢測絨弄蝶屬傳統分群架構的合理性與釐清本屬的分 類問題。本研究內外群共採樣 27 種與 68 隻個體，在分群關係方面 支持 Chiba 所認定的 discolor-group, celaenus-group, vitta-group 與 thridas-group 以及 Evans 所認定的 discolor-group 與 thridas-group 為 單系群。而本研究也支持將原屬於 chromus-group 的 H. schoenherr 獨立為 schoenherr-group。 種級關係方面， H. mavis 與 H. leucospila 為同種之假說受強烈支持，而 H. vitta 則可被分為三個種級分類群。 關鍵字: 分子系統學、種群、東洋區、澳洲區 3 Abstract The skipper genus Hasora (Lepidoptera: Hesperiidae: Coeliadinae), which contains of approximately 30 species classified into 6-7 groups, is the largest genus of the subfamily Coeliadinae and is distributed throughout the Indo-Australian region. Currently recognized species groups and taxonomy of Hasora are mainly based on characteristics of the wing patterns and the male genetalia, and there have been some disagreements in taxonomical treatments. To date it has not been tested if the species groups based on morphological characters correspond to the phylogeny of Hasora. The objective of this research is to reconstruct the molecular phylogeny of Hasora using the sequences of mitochondrial COI, COII region and nuclear Ef-1a region, and to test if the morphological phylogeny proposed by de Jong is consistent with the molecular phylogeny, and if the species groups defined by Evans and Chiba are monophyletic. The relationships of some taxonomically controversial taxa are also investigated. Total of 22 ingroup species, 63 ingroup individuals and 5 outgroup genus were sampled, and the monophyly of discolor-group, celaenus-group, vitta-group and thridas-group sensu Chiba as well as discolor-group and thridas-group sensu Evans were supported respectively. The present study also suggested that H. schoenherr, a species formerly assigned to chromus-group, should be placed to a schoenherr-group proposed herein. For species-level relationships, the conspecific relationship between H. mavis and H. leucospila is strongly supported, and H. vitta may be 4 divided into three different species level taxa. key words: molecular systematics, species group, Oriental Region, Australian Region, Awl 5 Introduction Compared to the other “butterfly” families, the family Hesperiidae (skippers) is comparatively poor-understood in terms of phylogeny. The phylogenetic relationships within this family remained unresolved until recently, when Warren et al. (2008, 2009) proposed a phylogeny for higher-level taxa based on both morphological and molecular data. Nevertheless, the lower-level phylogeny of most Hesperiidae lineages still lacks information and needs to be worked out. The present study chooses Hasora Moore 1881, a speciose genus with prominent diversity of wing patterns and sexual dimorphism, as the target groups of study. The purpose of the study is aimed at testing alternative taxonomic schemes established by various researchers. Generic characters of Hasora Hasora, which containing approximately 30 species, is the largest genus of the subfamily Coeliadinae (Chiba 2009). Members of this genus are medium to large sized skippers and are swift flyers. They are active at dawn and twilight but can also be seen during the daytime (Bascombe et al. 1999). The genus is widely distributed from India to China, through South-East Asia to Australia and Fiji, and has the highest species diversity in the Philippine and Indonesian areas (Tsukiyama et al. 1997, Braby 2000, de Jong and Treadaway 2007, Chiba 2009). The generic characters of Hasora that may be useful to distinguish the genus from the other genera of Coeliadinae include the following: 1) vein 1b of the forewing acutely bisinuate near wing base, 2) antenna shorter than 1/2 length of 6 costa, and 3) female often with hyaline spots on the forewing. All known larvae feed on Fabaceae (Evans 1949, de Jong and Treadaway 2007, Chiba 2009). Taxonomic history The taxonomy of Hasora has been reviewed by various authors (Elwes and Edwards 1897, Evans 1949, Chiba 2009). One of the most important work on the taxonomic history of this genus is the catalogue written by Evans (1949), in which he recognized 27 species containing 79 subspecies based on wing patterns and male genetalia. This classification has been followed by current researchers with only a few modifications (e. g. de Jong 2007, de Jong and Treadaway 2007, Chiba 2009). Since Evans (1949), 2 species and 8 subspecies have been described as new. However, there are some inconsistencies in species-level taxonomy of Hasora: i. e, de Jong (2007) recognized 31 species and Chiba (2009) recognized 29 species containing 86 subspecies. Though the inconsistencies between these taxonomic treatments were partially due to different taxonomic philosophy, it may also indicate that morphological characters are unable to provide species delimitation for some taxa. For example, Hasora caeruleostriata de Jong 1982 was initially described as a subspecies of Hasora moestissima (Mabille 1876), namely as Hasora moestissima caeruleostriata de Jong and Treadaway 1982. Later de Jong and Treadaway (1993) raised H. caeruleostriata to species level judging from the band color of the hindwing, the presence of a white dot in space 6 of male forewing and the distribution of these two taxa. On the other hand, H. caeruleostriata was treated as a subspecies of H. moestissima by 7 Chiba (2009), and was suspected to be a synonym of Hasora moestissima unica, another name described by Evans (1934) (Chiba 2009). For another example, Hasora danda Evans 1949 was initially described as a species distinct from Hasora anura de Nicéville 1889 based on a few wing pattern differences, including: 1) the subapical dot of the forewing and the white cell dot on the ventral hindwing are absent, and 2) the dark discal line on the hindwing underside of male is not indent in M1 cell. Hsu et al. (2005) mentioned that these characters described by Evans can also be seen in H. anura and modified this taxon as a subspecies of H. anura, while H. danda is still recognized by de Jong (2007). The taxonomic problem of Hasora mavis Evans 1934 is another case. According to de Jong and Treadaway (2008), H. mavis is a rare species distributed in Malay Peninsula, Borneo and Mindanao. Evans (1934) initially described this taxon as a subspecies of Hasora borneensis Elwes and Edwards 1897, and then moved to Hasora khoda (Mabille 1876) (Evans 1949). Maruyama (1991) raised H. mavis to specific level and this treatment was followed by Eliot (1992), de Jong and Treadaway (2007) and Chiba (2009). This taxon is known only from females until recently, and the male of H. mavis was then illustrated by Kitamura (2002) and de Jong and Treadaway (2007). According to the illustration of de Jong and Treadaway (2007), male H. borneensis and male H. mavis differ by the straw-colored area of the upperside hindwing, while other information is very limited. However, judging from wing shape, wing markings, female genitalia and the distribution pattern, Chiba (2009) suspected that, H. mavis may be the female of Hasora leucospila (Mabille 1891), whose 8 female is extremely rare in collections. Evans’ (1949) catalogue arranged Hasora into 6 species groups, namely, lizetta-group, myra-group, discolor-group, chromus-group, celaenus-group and thridas-group, based on the similarity of wing patterns and male genetalia, especially uncus and valva. The lizetta-group is composed of H. mus Elwes and Edwards 1897, H. lizetta Plötz 1884, H. salanga (Plötz 1885) and H. proxissima Elwes and Edwards 1897; myra-group is composed of H. anura, H. myra (Hewitson 1867) and H. zoma Evans 1934; discolor-group is composed of H. discolor (Felder and Felder 1859), H. buina Evans 1928, H. umbrina (Mabille 1891) and H. borneensis; chromus-group is composed of H. chromus (Cramer 1782), H. taminatus (Hübner 1818), H. hurama (Butler 1870) and H. schoenherr (Latreille 1823); celaenus-group is composed of H. mixta (Mabille 1876), H. celaenus (Stoll 1782), H. badra (Moore 1857), H. quadripunctata (Mabille 1876), H. subcaelestis Rothschild 1916, H. vitta (Butler 1870), H. moestissima and H. perplexa (Mabille 1876); thridas-group is composed of H. khoda Mabille 1876, H. leucospila and H. thridas (Boisduval 1932). Chiba (2009)