Lepidoptera: Hesperiidae)

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

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。

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

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

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

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) revised Evans’ (1949) 6-species groups to 7-species groups, including mus-group, myra-group, discolor-group, chromus-group, celaenus-group, thridas-group and vitta-group. Most species groups defined by Evans remained intact in Chiba’s treatment, except H. vitta, H. moestissima and H. perplexa of the celaenus-group were moved to vitta-group in Chiba’s treatment, and the name of lizetta -group was modified to mus-group. Due to the lack of comprehensive phylogenetic study, available grouping systems remain untested, and, thus,

9 necessary to be clarified by a phylogenetic framework. Both morphological and molecular subfamily-level phylogeny supported the monophyly of Hasora (de Jong 2007, Tanikawa-Dodo et al. 2008), but only one preliminary genus-level phylogeny based on morphological characters is available (de Jong 2007). Only 3 of Chiba’s 7 species groups and 2 of Evans’ 6 species groups are monophyletic judging from the previously proposed morphological phylogeny. However, the consistency index of de Jong’s (2007) phylogeny was merely 0.431, and some nodes are polytomical. Thus the use of morphological characters may not provide sufficient information to clarify the phylogeny of Hasora.

Materials and methods

Taxon sampling

Totally 22 ingroup species (63 individuals) were selected in this research, which represented about 70% of the known species of Hasora. To test the phylogenetic relationships of the species groups proposed by Evans (1949) and Chiba (2009), the selected taxa contained at least two species from each species groups as representative, as shown in Table 1. Five outgroups related to Hasora were selected, and all specimens used are listed in Table 2, the collection localities of samples are showed in Figure 1.

Molecular technique

DNA extraction

Legs of the sampled specimens was plucked, stored in 99.5% ethanol and -80℃.The genomic DNA was extracted from legs using the Qiagen

PUREGENE kit (Gentra Systems, Minnesota, USA) following the listed protocol: The preserved legs were taken out from ethanol and put into a 1.5 mL microcentrifuge tube, after ethanol was evaporated, add 100μL cell lysis solution and ground the tissue with a pestle. After the tissue was totally grinded, add 10μL Proteinase K and 10μL DTT, incubated at 65°C for 1.5 hours or 55°C for 13 hours, then add 40μL protein precipitation solution, frozen at -20°C for 30min. The samples were then centrifuged at 14000rpm for 15minutes, discarded the protein pellet and add 100μL 100% isopropanol for DNA precipitation, centrifuged at 14000rpm for 10minutes and discarded the supernatant. Finally add 500μL 70% ethanol to wash the tube and centrifuged at 14000rpm for 5 minutes and discarded the ethanol, then dried the precipitated DNA for 3-8 hours and suspended the DNA in 30-50μL of ddH2O.

Gene selection, amplification and sequencing

Currently, mitochondrial COI, COII and nuclear Ef-1a region are widely used in the phylogenetic researches of Lepidoptera, and are consider to be sufficient to infer species-level phylogeny in some cases (Canfield et al. 2008, Hundsdoerfer et al. 2009), thus these regions were selected in this study.

To amplify the sequences of the mitochondrial COI, COII region and nuclear Ef-1a region, PCR amplification was conducted, and primers for PCR amplification were obtained from Caterino and Sperling (1999),

Kandul et al. (2004) and Wu (2010), listed in Table 3. PCR amplification reaction was performed with 30μL of reaction volume, 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 PowerTaq

(Genomics, Taipei, Taiwan) and ddH2O filled to 30μL. PCR was carried out using the following protocol: initial denaturation of 4 minutes at 95℃, followed by 35-40 cycles consisting of denaturation of 30 seconds at 95

℃, annealing of 30 seconds at 55℃-48℃ depending on the primer used, and extension of 25-60 seconds at 72℃. Final step was extension of 7 minutes at 72℃. The PCR product was electrophoresed in 1% agarose gel to check the length of amplification, and sequenced by commercial company. All sequences were checked using sequencher 4.9 (GeneCode, Boston, USA), and primer regions and tRNA-Leu gene were deleted. Whole dataset were aligned using the Muscle algorithm (Edgar 2004) in MEGA 5.0 (Tamura et al. 2011) with default settings, aligned sequences were translated to amino acid to check alignment and the existence of stop codon.

Phylogenetic analysis

To estimate the inter-specific and intra-specific genetic distance of ingroup species, the p-distance of the COI + COII dataset was calculated using MEGA 5.0.

Various approaches were used to infer the phylogenies of Hasora: Maximum parsimony (MP) was performed in TNT 1.1 (Goloboff et al.

2008); Maximum likelihood (ML) was carried out using PHYML 3.0 (Guindon et al. 2010). Based on the concern that genetic tree may be inconsistence with species tree, species tree reconstruction was performed in *BEAST (Heled and Drummond 2010) extension in BEAST 1.6.2 (Drummond and Rambaut 2007). To find the best fit substitution model for ML and Bayesian approach analysis, COI + COII and Ef-1a datasets were analyzed separately in jModelTest 0.1.1 (Posada 2008) using the corrected Akaike information criterion (AICc). To choose a proper functional outgroup, 5 outgroups were selected in preliminary test using Bayesian inference, and the result supported Badamia exclamationis as the closest taxon to Hasora, thus for the rest analysis Badamia exclamationis was selected as functional outgroup.

For the MP method, the heuristic New Technology Search was selected using its all four searching algorithms: sectorial, ratchet, drift and tree fusing with initial search level set to 100 (the heaviest search). Gaps were treated as missing, for combined dataset, the COI + COII and Ef-1a region were defined as two character groups. Clade robustness was evaluated by both Bremer support with default settings and 1000 non-parametric bootstrap replication.

For the ML method, GTR + I + Г model (General Time Reversible with invariable sites and gamma distribution) was selected following the modeltest result, and tree topology search was set to SPR (Subtree Puring and Regrafting). Non-parametric bootstrap was performed with 1000 replications to evaluate the branch support.

For species tree reconstruction, GTR + I + Г model was selected following the modeltest result, parameters of COI + COII, Ef-1a partition and three codons of each partition were unlinked and calculated separately, the molecular clock of mitochondrial gene was calculated relative to nuclear gene using strict clock. A Yule speciation process was selected as a tree prior, while other settings were left to defaults. Since the definition of “species” tree in this analysis was focus on the barriers for gene flow and does not need to be correspond to the specific species in taxonomy, subspecies was used as OTU in this study. The MCMC process was run twice for 100 million generations and sampled every 1000 generations with the first 10% trees discarded as burn-in, remaining trees were used to infer the posterior probability of tree topology and other statistical values.

In the results and discussion section, Bremer support values ranging from 1-4 are referred as “weakly supported”, values ranging from 5-8 are referred as “moderately supported” and values higher than 8 are referred as “strongly supported”; bootstrap support values ranging from 50-70 are referred as “weakly supported”, values ranging from 71-90 are referred as “moderately supported” and values higher than 90 are referred as “strongly supported”; Bayesian posterior probability values ranging from 0.6-0.8 are referred as “weakly supported”, values ranging from 0.81-0.95 are referred as “moderately supported” and values ranging higher than 0.95 are referred as “strongly supported”.

Results

Dataset characters

Alignments containing a total of 3432 base pairs (bp) were used in this study, of which 2211 bp was from mitochondrial COI + COII region and 1221 bp from nuclear Ef-1a region. 64 samples were contained in the COI + COII dataset, and 44 samples were contained in the Ef-1a dataset. No stop codon was found for translated COI + COII and Ef-1a sequences. The COI + COII p-distance of ingroup species are showed in Table 4 and Table 5. Most inter-species p-value is higher than 0.032, only the distance between H. mavis and H. leucospila is extremely low (0.0023). The intra-species distance however showed various results, ranging from 0.0000 to 0.0625, which will be discussed later.

Phylogenetic relationships

The summary of the MP, ML and species trees analysis is given in Table 6, 7, 8, and selected trees are showed in Figure 2-6. The monophyly of Hasora is strongly supported in all results, for intra-genus relationships, the tree topology is similar between COI + COII (mt) and combined (mt + N) results except for some higher level clades which are not strongly supported, thus the MP and ML trees of the two datasets are combined and showed in Figure 2 and Figure 4. The Ef-1a (N) results however lacking resolution of intra-genus relationships due to the low support values and polytomical topologies in many clades (Figure 3, 5). The genus can be divided into 5 major clades: A clade (H. discolor + H. borneensis + H. umbrina), B clade (H. zoma + H. lizetta), C clade (H.

15 mixta + H. celaenus + H. badra + H. quadripunctata), D clade (H. hurama + H. taminatus + H. chromus) and E clade (H. schoenherr + H. mavis + H. leucospila + H. thridas + H. khoda + H. vitta + H. moestissima), however the relationships between these clades are not clear, all results showed either polytomical topologies or very low support values between clades (mostly lower than “weakly supported” value).

Clade A is composed of H. umbrina, H. discolor and H. borneensis, and is strongly supported in all the results except for the mt and N result of MP method which showed moderately support values (bootstrap=88, 81 respectively). The position of this clade in Hasora is not stable, depending on the datasets and methods used, but all the results are moderately or not supported. One H. umbrina, 1 H. borneensis and 3 H. discolor individuals were used in this study, and the monophyly of H. discolor is strongly supported in all results.

Clade B is composed of H. zoma and H. lizetta, and this clade is strongly supported in species tree and mt + N results in MP and ML while moderately to not supported in the other results. The position of this clade is also not stable, all results are weakly or not supported. Only one specimen of each species was used in this research, and thus unable to obtain more species-level information about this clade.

Clade C is composed of H. mixta, H. celaenus, H. quadripunctata and H. badra, and is strongly supported in all the results except the N result of MP, which is moderately supported (bootstrap=81). This clade does not group with any other clade with support values higher than weak. The ((H.

16 mixta + H. celaenus) + H. badra + H. quadripunctata) topology is strongly supported in all the results except the N results of MP and ML. One H. quadripunctata, 5 H. badra, 2 H. celaenus and 2 H. mixta individuals were used in this research, and all the species are strongly supported monophyletic groups.

Clade D is composed of H. hurama, H. taminatus and H. chromus. This clade is strongly supported in species tree and mt + N results of the ML method, and moderately to not supported in the other results. This clade does not group with any other clades with support values higher than weak. The ((H. taminatus + H. hurama) + H. chromus) topology is strongly supported in species tree, and moderately to weakly supported in the mt + N and mt results of ML. Four H. chromus, 2 H. hurama, and 6 H. taminatus individuals were used in this research. While H. chromus and H. hurama are strongly supported monophyletic group. H. taminatus can be divided into 2 strongly supported clades, which are (H. taminatus malayana + H. taminatus attenuata) clade and H. taminatus vairacana clade. The monophyly of H. taminatus is only weakly supported in mt + N and mt results of ML, and is moderately supported as a paraphyletic group in species tree (posterior probability=0.9).

Clade E is the largest clade, this clade is strongly supported in species tree and the mt + N, and mt results of ML method, and moderately to not supported in the other results. This clade can be further divided into three sub-clades, the first clade is composed of H. schoenherr only, and is strongly supported as the sister group of the remaining species in clade E. Four samples of H. schoenherr were used in this research. The

17 monophyly of this species is strongly supported in species tree and mt, and mt + N results of MP and ML, but not supported in N results. The other 2 clades are defined as E1 (H. mavis + H. leucospila + H. thridas + H. khoda) and E2 (H. vitta + H. moestissima) clade, the (H. mavis + H. leucospila) clade in E1 clade is a strongly supported clade in species tree and mt, mt + N results of MP and ML (posterior probability=1, bootstrap=100, bremer support>61) and is strongly supported as the sister group of the (H. thridas + H. khoda) clade. The topology of the E2 clade is strongly supported as ((H. vitta + H. moestissima) + H. vitta) in species tree, mt and mt + N results, which support H. vitta as a paraphyletic group.

Three taxa are not included in the 5 major clades mentioned above, and those taxa are: H. salanga, H. proxissima and H. anura. The position of these taxa is also not clear. Three subspecies of H. anura was used in this study, the mt and mt + N results of MP and ML strongly supported the paraphyly of H. anura china.

Discussion

Phylogenetic relationships and monophyly of the species-groups of Hasora

The mitochondrial COI + COII data in this study is able to resolve most species-level and species-group level clades, while the Ef-1a dataset showed insufficient resolution in species-level relationships, consistent

18 with other genus-level study in Lepidoptera (Hundsdoerfer et al. 2009, Lin 2010). Compared with the COI + COII data sorely, combined dataset improved the support values of some clades especially in species-group level, the species tree also provide good supports to these clades. However, the topology of species tree are not consistent with combined data trees especially in some intra-species clades, and it has to be mentioned that phylogenies inferred from combined dataset in this study might be biased toward the mitochondrial tree due to the difference in sequence length (2211 bp vs. 1221 bp), which will be problematic especially in recently diverged clades, thus the species tree may provide a more robust result for intra-specific relationships. Though the inter-specific group relationships of many species-groups are still not clear, this study provided an alternative evidence to support or reject the previously defined species-group and taxonomic treatments, as discussed below: lizetta-group / mus-group

This group was recognized by Evans (1949) as lizetta-group, and its name was revised to mus-group by Chiba (2009) without changing its members. Three out of four species were sampled in this study, namely H. lizetta, H. salanga and H. proxissima. This group was not supported in de Jong (2007), and only H. lizetta and H. salanga formed a monophyletic group. The result of this study, however, showed a different topology, where H. lizetta is grouped with H. zoma (Clade B), and the positions of H. salanga and H. proxissima are not clear. Thus this clade is not supported by the molecular data. 19 myra-group

This group was recognized by both Evans (1949) and Chiba (2009). In this study H. zoma and H. anura were sampled, which represents the half members of this group. The monophyly of this group was supported in de Jong (2007), but not supported in this study, while zoma was grouped with lizetta and the position of anura is not clear (Fig. 2-6). discolor-group

This group was recognized by both Evans (1949) and Chiba (2009). In this study H. umbrina, H. discolor and H. borneensis were sampled, which contains 3/4 members of this group. The monophyly of discolor-group was not supported in the morphological phylogeny proposed by de Jong (2007) but correspond to the clade A (H. discolor + H. borneensis + H. umbrina) in this study which is strongly supported. Thus the molecular evidence strongly support discolor-group as a monophyletic group. chromus-group

This group was recognized by both Evans (1949) and Chiba (2009). All members of chromus-group were sampled in this research, which are H. chromus, H. taminatus, H. hurama and H. schoenherr. This group was not supported in de Jong (2007), where only the (H. taminatus + H. hurama) clade was supported. In this study, the ((H. taminatus + H. hurama) + H. chromus) clade is strongly supported in species tree and ML results, which corresponds to Clade D. However, H. schoenherr is not showed in clade D, but grouped with the previously defined

20 thridas-group and vitta-group with strong support value (clade E), indicating that H. schoenherr may be closer to thridas / vitta-group than chromus-group. The color-patterns of larva support this hypothesis (Igarashi and Fukuda 2000), as the larva of H. schoenherr is similar to the larvae of the members of the celaenus-group and vitta-group in the red head and yellowed body with red spots in fifth instar. Besides, this relationship is supported by morphological evidence (de Jong 2007), where schoenherr was the sister group of the (celaenus-group + thridas-group + vitta-group) clade. All these evidences strongly support that H. schoenherr should be removed from the chromus-group, and placed in a newly defined schoenherr-group that contained H. schoenherr only. Removing H. schoenherr, the chromus-group forms a monophyletic group due to the highly supported monophyletic relationship and the intact sample of the member. celaenus-group / vitta-group

In this study, H. mixta, H. celaenus, H. badra, H. quadripunctata, H. vitta and H. caeruleostriata were sampled, which represented 6/8 of the celaenus-group members defined by Evans (1949), 4/6 of the celaenus-group members and 2/3 members of the vitta-group defined by Chiba (2009). Evans’ celaenus-group was not recognized as a monophyletic group in de Jong (2007), but Chiba’s treatment of this species group was supported by de Jong’s (2007) result. Similar result can be seen in this study, where Evans’ celaenus-group is separated into clade C and clade E2, which are not the sister group with each other. Chiba’s celaenus-group in this study is a strongly supported monophyletic group

(clade C), and the monophyly of vitta-group is also strongly supported, which correspond to clade E2. Moreover, the molecular data suggests that vitta-group is closest to thridas-group but not celaenus-group. Thus judging from these evidences, Chiba’s treatment that divided celaenus-group into 2 species groups is strongly supported in this study. thridas-group

This group was recognized by both Evans (1949) and Chiba (2009), and all the members of this species group were sampled in this study, including H. leucospila, H. thridas and H. khoda. A sample of H. mavis which was suspected to be conspecific with H. leucospila by Chiba (2009) was also selected. The monophyly of this group was supported in de Jong (2007), and are also strongly supported in this study, which correspond to clade E1. Thus, due to the support of both molecular and morphological evidences and the intact samples of the members, thridas-group is strongly supported as a monophyletic group in this study.

Species-level classification

The species tree, and mt and mt + N results in this study strongly indicated that the current taxonomic states of some taxa are inadequately or weakly supported. H. vitta is one of the most obvious case, 4 out of 7 subspecies of vitta were sampled in this study, which are H. vitta vitta (Butler 1870) from Myanmar to Bali Island, H. vitta indica Evans 1932 from Hong Kong and Vietnam, H. vitta sula Evans 1932 from Sulawesi and H. vitta simillima Rothschild 1916 from Yapen Island. H. v. vitta and H. v. indica formed a strongly to weakly supported clade. However H. v.

22 sula is strongly and weakly supported as the sister group of H. moestissima in species tree and MP results, while in the ML results (H. v. sula + H. v. simillima) clade is strongly supported as the sister group of H. moestissima. These results strongly supports that H. vitta is a paraphyletic group. The p-distance between H. v. sula, H. v. simillima and the other two subspecies (ranging from 0.0375 to 0.0559) also showed much higher value than most intra-species p-distance in this genus and the widely applicable species limit for Lepidoptera (0.032) (Wiemers and Fiedler 2007). Furthermore, the p-distance between H. v. sula and H. v. simillima (0.0559) is even higher than the p-distance between H. v. sula and H. moestissima (0.0371). These results indicate that H. v. sula and H. v. simillima may be treated as two species level taxa. The monophyly of H. v. vitta and H. v. indica is not supported in this phylogenetic analysis, but can be explained by incomplete lineage sorting of the mt DNA tree, especially at subspecies level. This hypothesis is supported by the low inter-subspecies p-distance between H. v. vitta and H. v. indica.

The monophyly of H. taminatus is also weakly or not supported. Three out of 10 subspecies were sampled in this study, which are H. taminatus vairacana Fruhstorfer 1911 from Taiwan, H. taminatus malayana (Felder 1860) from Java and H. taminatus attenuata (Staudinger 1889) from Sulawesi. H. t. malayana is grouped with H. t. attenuata with moderately to high support, but the species clade is only weakly supported in some results and not supported in species tree results. H. hurama is moderately supported as the sister group of the (H. t. attenuata + H. t. malayana) clade. The p-distance between H. t. vairacana and the other two

23 subspecies is also relatively high (0.0501, 0.0464), nearly the same level as the p-distance between H. t. vairacana and H. hurama (0.0489). Thus, judging from the phylogenetic relationships and genetic distance, H. t. vairacana and (H. t. attenuata + H. t. malayana) may be divided into two species-level taxa. H. t. malayana was treated as a distinct species, H. malayana by Monastyrskii & Devyatkin (2003), but considering its relatively low divergence to H. t. attenuata (0.0348), the taxonomic status of this taxon in this study still remained in subspecies level, and more individuals and subspecies should be sampled to provide more reliable result.

H. schoenherr is another clade with very high intra-species divergence. Two out of 6 subspecies were sampled in this study, which are H. schoenherr chuza (Hewitson 1867) from China to Malaysia and H. schoenherr saida (Hewitson 1867) from Luzon. Though these two subspecies formed a strongly supported clade in species tree, mt and mt + N result, and the N result did not support H. schoenherr as a monophylic group, Chiba (2009) mentioned that the taxonomic position of H. schoenherr chuza should be reconsidered judging from the characters of the larva, and this suspicion is supported by the relatively high p-value between these two subspecies (0.0596). Such a high divergence suggests that H. schoenherr saida and H. schoenherr chuza may be treated as two species-level taxon, though more subspecies and samples should be added in further analysis to give more support.

The strongly supported relationship and extremely low p-distance (0.002) between H. mavis and H. leucospila provide a strong evidence of

24 the conspecific hypothesis proposed by Chiba (2009), and if this hypothesis is correct, the currently described male H. mavis and female H. leucospila may be the result of incorrect species identification. According to Chiba (personal communication), the male H. mavis might be H. borneensis, and the female H. leucospila might be H. khoda. However the “male” H. mavis and “female” H. leucospila are not included in the analysis, and only one individual of each taxon was sampled in this study. Some literature (e. g. Maddison and Knowles 2006, Knowles and Carstens 2007) have mentioned that limited sample size of individual and loci may be sufficient to delimit species with 1onger coalescent depth. However as the depth of species tree decreased, the accuracy of species delimitation decreased largely, thus it is unable to exclude the possibility that these two taxa are closely related sister species with very short divergence time, which the nuclear and mitochondrial gene trees are still not completely sorted. Judging from these reasons, the conspecific hypothesis between H. mavis and H. leucospila is highly supported in this study, but more individual or loci and the other sex of each taxon should be added in the analysis to give a more robust support to the conspecific hypothesis.

For inter-subspecies relationships, H. a. china is a strongly supported paraphyletic group in the mt and mt + N result: H. anura taiwana Hsu, Tsukiyama and Chiba 2005 was grouped with H. a. china from Sichuan and Guizhou, while this clade is grouped with other H. a. china from Sichuan, thus no clear gap between localities is observed. Such a paraphyletic result can be explained by incomplete lineage sorting of the

25 mt DNA tree, and is supported by the relatively low inter-subspecies p-distance (0.0241). This study also supports that H. a. danda is not a species-level taxon, since H. a. danda was within H. a. china clade with high support values in MP and ML results, and in species tree result the (H. a. taiwana + (H. a. china + H. a. danda)) topology is strongly supported. Furthermore, 1 out of 3 sampled individual is grouped with H. a. china but not the other 2 individuals, and the presence or absence of the white cell dot on the ventral hindwing are not correlated to the phylogenetic structure of H. danda, suggesting that the absence of this dot is not a useful key to identify H. danda from other subspecies, as mentioned in Hsu et al. (2005).

The conspecific relationship between H. moestissima moestissima and H. moestissima unica (=H. caeruleostriata) is also supported in this study, since no phylogenetic evidences support H. m. moestissima as a distinct clade from H. m. unica, and the p-distance between H. m. moestissima and H. m. unica was 0.0171, which is lower than most of the species limit in this genus. One H. m. moestissima and three H. m. unica from Leyte were sampled. Considering the sympatric distribution of the two taxa, H. moestissima unica (=H. caeruleostriata) is not supported to be a species-level or subspecies-level taxon but merely a form of H. moestissima.

Conclusion

In this study, based on the molecular phylogeny inferred from

26 mitochondrial COI, COII and nuclear Ef-1a region, some conclusion can be pointed out:

1. The genus Hasora is a monophyletic group, which correspond to the morphological phylogeny previously proposed by de Jong (2007).

2. Two out of six species-groups defined by Evans (1949) are monophyletic, which are discolor-group and thridas-group; the monophyly of lizetta-group, myra-group, chromus-group and celaenus-group are not supported.

3. Four out of seven species-groups defined by Chiba (2009) are monophyletic, which are discolor-group, celaenus-group, vitta-group and thridas-group; the monophyly of lizetta-group, myra-group and chromus-group are not supported.

4. H. schoenherr which has been treated as a member of chromus-group by both Evans (1949) and Chiba (2009) should be move to a newly defined species-group, namely schoenherr-group to ensure the monophyly of the chromus-group.

5. H. taminatus vairacana and (H. taminatus attenuata + H. taminatus malayana) are supported to be divided into two species-level taxa.

6. The relatively high genetic distance between H. schoenherr saida

and H. schoenherr chuza suggests these two taxa may be treated as two species-level taxa.

7. H. vitta is a paraphylic group, and the relatively high genetic distance between H. vitta sula, H. vitta simillima and the other two subspecies suggested that H. v. sula and H. v. simillima may be treated as two species level taxa.

8. The conspecific relationship between H. mavis and H. leucospila is strongly supported due to the extremely low genetic distance.

9. H. moestissima unica (= H. caeruleostriata) is not supported to be a species-level or subspecies-level taxon, but a form of H. moestissima.

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Table1. Species groups of Hasora. * sampled in this study Evans (1949) Chiba (2009) lizetta-group mus-group H. mus H. mus H. lizetta* H. lizetta*

H. salanga* H. salanga* H. proxissima* H. proxissima* myra-group myra-group H. anura* H. anura* H. myra H. myra H. wilcocksi H. zoma* H. zoma* discolor-group discolor-group H. umbrina* H. umbrina* H. buina H. buina

H. discolor* H. discolor* H. borneensis* H. borneensis* chromus-group chromus-group H. chromus* H. chromus* H. taminatus* H. taminatus*

H. hurama* H. hurama* H. schoenherr* H. schoenherr* thridas-group thridas-group H. khoda* H. khoda* H. leucospila* H. leucospila* H. mavis* H. thridas* H. thridas* celaenus-group celaenus-group H. mixta* H. mixta* H. celaenus* H. celaenus* H. badra* H. badra* H. sakit H. quadripunctata* H. quadripunctata* H. subcaelestis H. subcaelestis H. vitta* vitta-group H. moestissima* H. vitta* H. perplexa H. moestissima*

H. perplexa

Table2. Samples used in this study.

Species Lot. Locality Latitude Longitude H. anura china H776 Chongqing, CHINA 29.6∘N 106.5∘E H. anura china H972 Sichuan, CHINA 30∘N 104∘E H. anura china H983 Sichuan, CHINA 30∘N 104∘E H. anura china H1273 Chongqing, CHINA 29.6∘N 106.5∘E H. anura china H1605 Guangxi, CHINA 23∘N 108∘E H. anura china H1606 Guizhou, CHINA 23.2∘N 113.2∘E H. anura danda H1664 Sagaing, MYANMAR 22.0∘N 96.0∘E H. anura danda H1665 Sagaing, MYANMAR 22.0∘N 96.0∘E H. anura danda H1666 Sagaing, MYANMAR 22.0∘N 96.0∘E H. anura taiwana H1603 Nantou, TAIWAN 23.8∘N 120.9∘E H. anura taiwana H1604 Nantou, TAIWAN 23.8∘N 120.9∘E H. badra badra Lep104 Taipei, TAIWAN 25.1∘N 121.5∘E H. badra badra H511 Serang, Java, INDONESIA 6.4∘S 107.1∘E H. badra badra H1607 Taipei, TAIWAN 25.1∘N 121.5∘E H. badra badra H1608 Taipei, TAIWAN 25.1∘N 121.5∘E H. badra badra H1611 Nantou, TAIWAN 23.8∘N 120.9∘E H. borneensis luza H1635 Mt. Apo Mindanao, PHILIPPINES 7.0∘N 125.2∘E H. celaenus H1536 Waigeo Is., INDONESIA 0∘N 131∘E H. celaenus H1643 Baru Is., S. Maluku, INDONESIA 2.2∘N 128.5∘E H. chromus chromus Lep107 Taipei, TAIWAN 25.1∘N 121.5∘E H. chromus chromus H1614 Coimbatore, INDIA 11.0∘N 77∘E H. chromus chromus H1616 Coimbatore, INDIA 11.0∘N 77∘E H. chromus chromus H1628 Lanyu, TAIWAN 22.0∘N 121.5∘E H. discolor mastusia H1530 Irian Jaya, INDONESIA 5∘N 138∘E H. discolor mastusia H1610 BIAK Is., W. Irian, INDONESIA 1.0∘S 136.0∘E H. discolor mastusia H1621 Lae, PAPUA NEW GUINEA 6.7∘S 147.0∘E H. hurama hurama H1619 Ambon Is., S. Maluku, INDONESIA 3.6∘S 128.1∘E H. hurama hurama H1667 Polopolo, N. Choiseul Is., SOLOMON 7.0∘S 156.9∘E H. khoda minsona H1640 Mt. Canlaon, Negros, PHILIPPINES 10.4∘N 123.1∘E H. leucospila leucospila H1127 Surigao del Sur, Mindanao, PHILIPPINES 8.5∘N 123.3∘E H. lizetta H1620 Jambi, S. Sumatra, INDONESIA 1.5∘S 102.6∘E H. mavis H1125 Mt. Canlaon, Negros, PHILIPPINES 10.4∘N 123.1∘E H. mixta mixta H1634 Mindoro, PHILIPPINES 13.1∘N 121.1∘E H. mixta limata H1230 Lanyu, TAIWAN 22.0∘N 121.5∘E H. moestissima unica H1517 Leyte, PHILIPPINES 11∘N 125∘E

Table2. continued

Species Lot. Locality Latitude Longitude H. moestissima unica H1531 Leyte, PHILLIPINES 11∘N 125∘E H. moestissima unica H1535 Mt. Balocaue, S. Leyte, PHILIPPINES 10.6∘N 125.0∘E H. moestissima moestissima H1673 Mt. Balocaue, S. Leyte, PHILIPPINES 10.6∘N 125.0∘E H. proxissima proxissima H1638 Mt. Balocaue, S. Leyte, PHILIPPINES 10.6∘N 125.0∘E H. quadripunctata H1662 Mindanao, PHILIPPINES 8∘N 124∘E H. salanga H1623 Kg Bundu Tuhan, Sabah, MALAYSIA 6.0∘N 116.5∘E H. schoenherr chuza H439 Yunnan, CHINA 24∘N 101∘E H. schoenherr chuza H997 Yunnan, CHINA 24∘N 101∘E H. schoenherr chuza H1637 Ulu Piah, Perak, MALAYSIA 4.9∘N 100.8∘E H. schoenherr saida H1636 Camarines Sur, Luzon, PHILIPPINES 13.5∘N 123.3∘E H. taminatus vairacana H104 Hualien, TAIWAN 23.7∘N 121.3∘E H. taminatus vairacana H1609 Nantou, TAIWAN 23.8∘N 120.9∘E H. taminatus vairacana H1612 Nantou, TAIWAN 23.8∘N 120.9∘E H. taminatus vairacana H1613 Nantou, TAIWAN 23.8∘N 120.9∘E H. taminatus malayana H513 Serang, Java, INDONESIA 6.4∘S 107.1∘E H. taminatus attenuata H1630 Palu Palolo, C. Sulawesi, INDONESIA 0.9∘S 119.8∘E H. thridas chalybeata H1622 Sorong, Irian Jaya, INDONESIA 0.9∘S 131.2∘E H. umbrina H1668 N. Sulawesi, INDONESIA 0.7∘N 123.9∘E H. vitta indica H1510 Hong Kong, CHINA 22.4∘N 114.1∘E H. vitta vitta H1529 Myitkyina, MYANMAR 25.4∘N 97.4∘E H. vitta vitta H1532 Bali Is., INDONESIA 8.4∘S 115.1∘E H. vitta simillima H1617 Yapen Is., Irian Jaya, INDONESIA 1.7∘S 136.1∘E H. vitta indica H1631 Ha giang, VIETNAM 22.8∘N 105.0∘E H. vitta sula H1632 Palu Palolo, C. Sulawesi, INDONESIA 0.9∘S 119.8∘E H. vitta sula H1633 Camba, S. Sulawesi, INDONESIA 5.0∘S 119.8∘E H. vitta sula H1663 Palu Palolo, C. Sulawesi, INDONESIA 0.9∘S 119.8∘E H. vitta vitta H1672 Ulu Piah, Perak, MALAYSIA 4.9∘N 100.8∘E H. zoma H1618 W. Sumatra, INDONESIA 0.7∘S 100.8∘E Badamia exclamations Lep103 Taipei, TAIWAN 25.1 ∘N 121.5∘E Bibasis sena H1600 Mt. Balocaue S. Leyte, PHILIPPINES 10.6∘N 125.0∘E Burara jaina H2 Taipei, TAIWAN 25.1 ∘N 121.5∘E Choaspes benjamini H1477 Taipei, TAIWAN 25.1∘N 121.5∘E Coeliades forestan H1601 Lume, D. R. CONGO 4.7∘S 26.5∘E

Table 3. 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 H1co1-1700R AGTCA ATTTC CRAAT CCTCC R MiBocox-J-1700 AATAC TATTG TTACA GCTCA TGC 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 Skcox-J-2100 TTTTG ATCCT GCAGG AGGAG G F cox-N-2191 CCCGG TAAAA TTAAA ATATA AACTT C R Chcox-J-2200 ACCAG GATTT GGTAT AATTT CCCA F 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 C1-J-2500 CAAGA AAGAG GAAAA AAAGA AAC F C1-J-2550 ATTTA CWGTA GGWAT AGATA TTGA F H1c1-2600F2 TTGAT ATCCT TTATT TACAG G F Gallc1-N-2750 CCTGC TAATC CTAAA AAATG TTG 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 Micox-N-3080 TTTGA CCTTC TAATA AAAAT CG R C2-3138-F2 AGAGT TTCAC CTTTA ATAGA ACA F cox-J-3138 AGAGC CTCTC CTTTA ATAGA ACA F H1c2-3300R1 TTGTT CTTCT AATAA AAATC G R cox-N-3389 TCATA ACTTC AATAT CATTG R cox--3408 CAATG ATATT GAAGT TATGA F cox-N-3782 GAGAC CATTA CTTGC TTTCA GTCAT CT R

Table 3. continued Primers 5'→3' F/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

Table 4. Inter-species COI + COII p-distance of ingroup species. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 1 H. taminatus 2 H. mavis 0.070 3 H. leucospila 0.071 0.002 4 H. mixta 0.050 0.072 0.071 5 H. anura 0.045 0.078 0.077 0.044 6 H. vitta 0.041 0.054 0.054 0.048 0.046 7 H. moestissima 0.060 0.073 0.072 0.068 0.068 0.027 8 H. discolor 0.051 0.087 0.088 0.065 0.056 0.057 0.078 9 H. celaenus 0.059 0.083 0.083 0.032 0.058 0.057 0.075 0.076 10 H. badra 0.060 0.086 0.088 0.049 0.064 0.060 0.085 0.078 0.058 11 H. chromus 0.056 0.088 0.089 0.068 0.069 0.064 0.084 0.083 0.079 0.075 12 H. zoma 0.058 0.092 0.091 0.067 0.064 0.061 0.085 0.074 0.073 0.079 0.082 13 H. hurama 0.018 0.060 0.062 0.039 0.034 0.032 0.058 0.049 0.045 0.059 0.048 0.050 14 H. lizetta 0.044 0.074 0.076 0.055 0.048 0.040 0.058 0.064 0.057 0.069 0.068 0.058 0.037 15 H. thridas 0.064 0.066 0.066 0.076 0.076 0.047 0.067 0.077 0.084 0.085 0.083 0.082 0.055 0.066 16 H. salanga 0.051 0.080 0.082 0.066 0.060 0.053 0.076 0.069 0.075 0.074 0.080 0.076 0.054 0.064 0.077 17 H. borneensis 0.069 0.094 0.096 0.071 0.069 0.067 0.083 0.059 0.077 0.085 0.095 0.088 0.060 0.066 0.090 0.081 18 H. schoenherr 0.043 0.068 0.068 0.051 0.053 0.043 0.063 0.060 0.061 0.067 0.066 0.062 0.038 0.057 0.059 0.062 0.074 19 H. proxissima 0.055 0.094 0.095 0.070 0.058 0.064 0.080 0.076 0.078 0.082 0.090 0.075 0.055 0.057 0.090 0.069 0.075 0.068 20 H. khoda 0.073 0.072 0.073 0.082 0.084 0.056 0.081 0.088 0.087 0.087 0.090 0.093 0.068 0.087 0.057 0.082 0.102 0.070 0.103 21 H. quadripunctata 0.062 0.082 0.082 0.053 0.069 0.061 0.083 0.072 0.062 0.064 0.083 0.079 0.056 0.071 0.084 0.081 0.082 0.064 0.086 0.089 22 H. umbrina 0.071 0.090 0.091 0.079 0.071 0.066 0.087 0.062 0.085 0.092 0.093 0.089 0.067 0.079 0.089 0.076 0.075 0.075 0.079 0.091 0.081

Table 5. Intra-species COI + COII p-distance of ingroup species. Average Highset Lowest 1 H. taminatus 0.0295 0.0516 0.0023 2 H. mixta 0.0414 0.0414 0.0414 3 H. anura 0.0252 0.0478 0.0014 4 H. vitta 0.0414 0.0625 0.0041 5 H. moestissima 0.0114 0.0192 0.0000 6 H. discolor 0.0039 0.0054 0.0023 7 H. celaenus 0.0088 0.0088 0.0088 8 H. badra 0.0007 0.0014 0.0000 9 H. chromus 0.0021 0.0032 0.0014 10 H. hurama 0.0297 0.0297 0.0297 11 H. schoenherr 0.0315 0.0606 0.0023

Table 6. Summaries of maximum parsimony analysis. Dataset Number of Consistency Retention MP trees index (CI) index (RI) COI + COII/ Ef-1a 4 0.384 0.716 COI + COII 13 0.352 0.708 Ef-1a 15 0.736 0.793

Table 7. Summaries of maximum likelihood analysis. Dataset Log-likelihood COI + COII/ Ef-1a -18107.05683 COI + COII -14576.97200 Ef-1a -2998.41094

Table 8. Summary of *BEAST analysis. Tree Log-likelihood species tree -16518.052

Figure 1. Collection localities of sampled specimens in this study.

Figure 2. Maximum parsimony tree inferred from combined dataset. Bootstrap values of combined dataset are showed above the branch, and bootstrap values of COI + COII dataset are showed below the branch.

Figure 3. Maximum parsimony tree inferred from Ef-1a dataset. Bootstrap values are showed above the branch.

Figure 4. Maximum likelihood tree inferred from combined dataset. Values on the left indicate the bootstrap values of combined dataset, and values on the right indicate the bootstrap values of COI + COII dataset.

Figure 5. Maximum likelihood tree inferred from Ef-1a dataset. Bootstrap values are showed above the branch.

Figure 6. Species tree inferred from COI + COII and Ef-1a dataset. Values above the branch indicate the posterior probability.