ZooKeys 1055: 5ELEF (202 1) A peer-reviewed open-access journal doi: 10.3897/zookeys.1055.68640 RESEARCH ARTICLE #ZooKey S https:/ / ZOO keys. pensoft.net Launched to accelerate biodiversity research Molecular and morphological evidence reveals a new genus of the subfamily Heteropterinae (Lepidoptera, Hesperiidae) from China Yongxiang Hou!', Hideyuki Chiba’, Lijuan Zhu', Zhou Chang’, Lijun Ma‘, Siyao Huang', Min Wang', Xiaoling Fan! | Department of Entomology, College of Plant Protection, South China Agricultural University, Guangzhou, Guangdong 510642, China 2 B. P Bishop Museum, 1525 Bernice Street, Honolulu, Hawaii, 96817-0916, USA 3 State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Acad- emy of Sciences, Kunming, Yunnan 650201, China 4 Institute of Plant and Environmental Protection, Beijing Academy of Agriculture and Forestry Sciences, Beijing 100097, China Corresponding authors: Min Wang (minwang@scau.edu.cn); Xiaoling Fan (fanxiaol66@scau.edu.cn) Academic editor: Martin Wiemers | Received 14 May 2021 | Accepted 6 July 2021 | Published 5 August 2021 http:/zoobank.org/3C 1 84FA2-423E-43F5-B77A-5D06C5639245 Citation: Hou Y, Chiba H, Zhu L, Chang Z, Ma L, Huang S, Wang M, Fan X (2021) Molecular and morphological evidence reveals a new genus of the subfamily Heteropterinae (Lepidoptera, Hesperiidae) from China. ZooKeys 1055: 55-67. https://doi.org/10.3897/zookeys.1055.68640 Abstract Molecular phylogenetic analysis indicates that the genus Carterocephalus is not monophyletic. Based on combined molecular and morphological evidence, we propose a new genus, Pulchroptera Hou, Fan & Chiba, gen. nov., for Pamphila pulchra Leech, 1891. The adult, wing venation, and male genitalia of Pul- chroptera pulchra comb. nov., Carterocephalus palaemon, and related genera are illustrated. Keywords Carterocephalus, Pulchroptera, new combination Introduction In recent years, the molecular phylogeny of the family Hesperiidae has attracted the attention of an increasing number of researchers (Warren et al. 2008, 2009; Sahoo et al. 2016; Toussaint et al. 2018; Cong et al. 2019; Li et al. 2019; Liu et al. 2020). At the Copyright Yongxiang Hou et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 56 Yongxiang Hou et al. / ZooKeys 1055: 55-67 (2021) subfamily level, however, the phylogeny of the family Hesperiidae has yet to be estab- lished, and multiple new subfamilies (Zhang et al. 2019, 2020) and genera (Fan et al. 2016; Huang et al. 2016, 2019; Cong et al. 2019; Li et al. 2019) have been proposed in recent years. Although Heteropterinae, including 13 genera from Africa, was established already by Aurivillius (1925), subsequent authors did not recognise the subfamily, presumably because it was a mixture of genera and species assigned to the subfamilies Hesperiinae as well as Heteropterinae in the current taxonomy. Evans, somehow, proposed three different genus group names for taxa of these skippers, namely the Astictopterus group for the African taxa (Evans 1937), the Heteropterus group for the European and Asian taxa (Evans 1949), and the Carterocephalus group for the American taxa (Evans 1955), all of which he considered a part of the subfamily Hesperiinae. This arrangement was accepted in subsequent taxonomic works until Higgins (1975) and Scott and Wright (1990) restored the subfamily Heteropterinae. Recent molecular studies strongly sup- port the monophyly of the subfamily (Warren et al. 2008; Sahoo et al. 2016, 2017; Toussaint et al. 2018, 2020; Cong et al. 2019; Li et al. 2019; Zhang et al. 2019; Liu et al. 2020). Currently, Heteropterinae includes the following 13 genera: Heteropterus Dumeril, 1806; Carterocephalus Lederer, 1852; Butleria Kirby, 1871; Argopteron, 1893; Dalla Mabille, 1904; Leptalina Mabille, 1904; Metisella Hemming, 1934; Dardarina Evans, 1937; Hovala Evans, 1937; Piruna Evans, 1955; Freemaniana Warren, 2001; Ladda Grishin, 2019; and Willema Grishin, 2019. Moreover, the most recent studies indicate that all these genera are monophyletic (Cong et al. 2019; Toussaint et al. 2020). The genus Carterocephalus includes more than 20 species distributed in the Hol- arctic and Oriental regions. However, a cursory inspection of the male genitalia in- dicates that C. pulchra (Leech, 1891) is not a congener of the type species Papilio palaemon Pallas, 1771. Indeed, the findings of our morphological and molecular phylogenetic studies have revealed closer relationships with species in the genera Het- eropterus and Leptalina. Accordingly, we consider that Carterocephalus pulchra should be placed in a new genus. In the present study, we sought to assess the monophyly of the genus Cartero- cephalus and its relationship with other genera of Heteropterinae. On the basis of the evidence obtained, we describe a new genus. Materials and methods Morphological examination For the morphological study, we followed the methods described by Fan et al. (2010). To examine wing venation, wings were removed from the thorax and cleaned with a 1:1 mixture of bleaching liquid (Blue Moon, Guangzhou, China) and water for ap- proximately 3 to 4 min. Photographs of the wing venation and male genitalia were taken using a Keyence VHX-5000 digital microscope (Keyence, Osaka, Japan). A new genus of Heteropterinae from China a, Taxon sampling We sampled specimens from all genera listed in the subfamily Heteropterinae (Warren et al. 2008, 2009; Cong et al. 2019; Toussaint et al. 2020), including as many spe- cies as possible. We used a total of 44 specimens of 38 species in 13 genera as ingroup taxa, along with 12 species from other subfamilies (Coeliadinae, Pyrginae, Eudaminae, Euschemoninae, Barcinae, Trapezitinae, and Hesperiinae) as outgroup taxa. Among these specimens, 31 were newly sequenced in this study, with the remaining sequences being obtained from the GenBank database along with supplementary data presented by Sahoo et al. (2016) and Toussaint et al. (2020). The respective voucher specimens and additional information are listed in Suppl. material 1: Table S1. Vouchers bearing codes beginning with the abbreviation SCAU have been deposited in the collection of South China Agricultural University (SCAU), Guangzhou, China, and the specimens (JU19), (Dalla), and (SZSMETI) are retained in the private collections of J. Uehara, H. Chiba, and S. Safian, respectively. Laboratory protocols DNA was extracted from two or three legs of dried adult specimens using a TIAN- amp Genomic DNA Kit (Tiangen, Guangzhou, China) following the manufacturer's instructions. We amplified a single mitochondrial gene (658 bp of COI) and three nuclear genes (1066 bp of EF-1a, 610 bp of RPS5, and 403 bp of Wingless), for a total of 2737 bp. The primers used to amplify each gene were synthesised by Sangon Biotech (Shanghai, China) and are shown in Suppl. material 2: Table S2. DNA ampli- fication was performed in 20-uL reaction volumes containing 1 uL of template DNA, 0.8 uL of each primer (10 uM), 10 uL of 2x EasyTag PCR superMix (+dye) (Transgen, Beijing, China), and 7.4 pL of ddH,O. The amplification protocol adopted is the one described by Huang et al. (2019). Sequencing of the amplicons thus obtained was performed by Sangon Biotech (Shanghai, China) and Tsingke Biological Technology (Beijing, China), and new sequences have been deposited in GenBank (Suppl. mate- rial 1: Table S1). Phylogenetic analyses Sequences were aligned using Clustal W (Thompson et al. 1997) and edited manu- ally using MEGA 7.0 (Kumar et al. 2016). Gene data from Cong et al. (2019) were extracted from the genomic assembly in IDBA-UD (Peng et al. 2012). Partition- Finder v2.1.1 (Lanfear et al. 2012, 2016; Guindon et al. 2010) was used to select the optimal codon partitioning scheme under Akaike information criterion correc- tion (AICc) (Suppl. material 3: Table S3). We inferred the phylogenetic trees using two methods, namely maximum likelihood (ML) and Bayesian inference (BI), for which we used the partition scheme produced by PartitionFinder. ML analyses were performed using IQ-TREE (Nguyen et al. 2015) as implemented in the IQ-TREE 58 Yongxiang Hou et al. / ZooKeys 1055: 55-67 (2021) web online server (igtree.cibiv.univie.ac.at, Trifinopoulos et al. 2016), with branch support values evaluated based on 1000 replicates for ultrafast bootstrap (UFBoot) (Minh et al. 2013) and SH-aLRT (Guindon et al. 2010). BI analyses were per- formed using the CIPRES Science Gateway (https://www.phylo.org/) (Miller et al. 2010) with Markov Chain Monte Carlo (MCMC) randomisation in MrBayes using XSEDE 3.2.6 (Ronquist et al. 2012). Reversible-jump MCMC was used to facilitate sampling across the entire subduction rate model. We conducted two independent MCMC runs, with four Markov chains (5 x 10° generations) for each analysis, of which the initial 25% of samples were discarded as burn-in. Bayesian posterior prob- abilities (PP) were used to evaluate branch support, and trees were visualised using Fig Tree v1.4.0. Results and discussion Phylogenetic relationships The topological structures of the concatenated dataset inferred by ML and BI analyses were found to be generally consistent and strongly supported at most nodes (PP = 0.98, SH-aLRT = 95, UFBoot = 98) (Fig. 1). Moreover, the two analyses provided strong support for the monophyly of Heteropterinae (PP = 1, SH-aLRT = 99.9, UFBoot = 100), which excludes the genera Apostictopterus, Barca, Lepella, and Tsitana originally assigned to this subfamily, and is consistent with the findings of the most recent studies (Toussaint et al. 2018, 2020; Cong et al. 2019; Zhang et al. 2019). Within the subfamily Heteropterinae, four major clades were differentiated, with 14 well-supported monophyletic subclades, corresponding to the 13 currently recognised genera and the Carterocephalus pulchra clade. Certain results were consistent with those of previous studies (Cong et al. 2019; Toussaint et al. 2020): (1) of the 13 genera, 12 genera, excluding Carterocephalus, were monophyletic; (2) Argopteron and Butleria formed a strongly supported monophyletic group (PP = 1, SH-aLRT = 99.1, UFBoot = 100) that is sister to all other genera in Heteropterinae (PP = 1, SH-aLRT = 98.3, UFBoot = 99); (3) Carterocephalus, excluding the species C. pulchra, was sister to the clade containing Metisella, Hovala, and Willema with strong support (PP = 1, SH-aLRT = 99.5, UFBoot = 100); and (4) Piruna, Dardarina, Freemaniana, Ladda and Dalla formed a strongly supported monophyletic clade (PP = 1, SH-aLRT = 98.8, UFBoot = 99). Two findings, however, are inconsistent with those reported previously. Firstly, Piruna is sister to Dardarina (PP = 0.76, SH-aLRT = 87.2, UFBoot = 94), as opposed to sister to the four genera Dardarina, Freemaniana, Ladda, and Dalla. Based on the morphology of the male genitalia (Evans, 1955), Piruna shows a relatively close similarity to Dardarina, whereas species of Dalla show extensive variation. However, previous molecular phylogenetic studies, as well as our own, sampled only some representatives of Dalla. Accordingly, the monophyly of Dalla as well as the relationships among these five genera should be subjected to further studies. A new genus of Heteropterinae from China 59 Burara aquilina 3833ADW Erynnis afranius 52ADW Urhanus dorantes AW280 Euschemon rafflesia 83ADW * Apostictopterus fuliginosus SCAU He2001 */-I- 0.65/67.4/70 Barca bicolor SCAU Hel664 CORY T Antipodia atralba 616MCZ Dares Ochlodes sylvanoides AW50 0.65/67.2/56 = Lepella lepeletier ME13E039 0-75/58.1/91 Tsitana tulbagha ADW586 Astictopterus jama 337ADW 0.94/73.6/94' Isoteinon lamprospilus 165ADW «| Argopteron aureipennis BN000139 | . 'Argopteron puelmae SCAU He2263 jie ees ka 0.7/61.4/57 vas J Butleria bissexguttatus 629AVZB | Butleria elwesi BN005146 Butleria 192.21 Butleria flavomaculatus valdivianus NVG-16108G04 | «f Leptalina unicolor BN000462 Leptalina unicelor SCAU He2239 | Leptalina - .) Carterocephalus pulchra SCAU He2223 | * Carterocephalus pulchra SCAU He2224 Pulchroptera gen. nov. «| Heteropterus morpheus REO7G368 Heteropterus morpheus SCAU He2240 Metisella aegipan SZSMETI013 is Metisella kambove gamma SZSMETI1003 | Metisella i) Metisella metis paris SZSMETIO11 | “ Hovala pardalina BN003761 Liat [aa Hovala pardalina NVG-7767 emia 7 ~— Willema willemi BN005142 | ation : Willema willemi SESMET1008 Jer arcane «p Carterocephalus abax SCAU He2225 Carterocephalus patra SCAU He2221 4 .-- Carterocephalus avanti SCAU Hel654 _|4 Carterocephalus argyrostigma SCAU He2201 Ag Carterocephalus houangty SCAU He2260 Carterocephalus i i Carterocephalus dieckmanni SCAU He2200 O72 Ne Carterocephalus alcina SCAU He2203 0.91/74/83] —/63 Carterocephalus longimaculatus SCAU He2202 */93.3/*] .y Carterocephalus palaemon SCAU He2233 Carterocephalus silvicola SCAU He2230 «p— Piruna aea 275ADW Re sof iruna pirus NVG-6454 “""e Dardarina aspila BN004729 .t Dardarina dardaris 198ADW | Dardarina Dardarina dardaris JU19005 | Freemaniana rawlinsi BN003731 | Freemaniana ‘ Ladda ticidas ssp. JU19003 | -/84.2/96 . Ladda crithote JU19004 | Ladda Ladda eburones eburones NVG-18014F02 */89.1/96| Pasa iat agathocles JU19001 Heteropterus 0.05 alla epiphaneus Dalla7 * Dalla cocha Dalla19 Be PP/SH-aLRT/UFBoot “| — Dalla cypselus Dalla2 | Dalla «g4je][7 Dalla vinca Dalla78 +] .) Dalla mesoxantha SCAU He2276 Dalla wardi Dalla93 Figure |. Maximum likelihood phylogenetic tree of the subfamily Heteropterinae. Values at nodes rep- resent the posterior probabilities (PP) of BI analyses values, SH-aLRT values (SH-aLRT), and Ultrafast bootstrap support values (UFBoot) of the maximum likelihood analysis. * indicates that one of the values at a node exceeds the standard (PP = 0.98, SH-aLRT = 95, UFBoot = 98). When the three node values all reach the standard, only one * is displayed. — indicates that the node was not recovered in the ML or BI tree. Secondly, we found that Carterocephalus is not a monophyletic group, given that the 11 species analysed in the present study were recovered in two distinct clades, with C. pulchra clustering with Leptalina and Heteropterus with strong support (PP = 1, 60 Yongxiang Hou et al. / ZooKeys 1055: 55-67 (2021) SH-aLRT = 97.7, UFBoot = 100). The other ten species, including the type species C. palaemon, were recovered as a strongly supported monophyletic clade. Although in this study we focused on relationships among the genera of Heterop- terinae, it is worth mentioning that certain intra-generic relationships, namely, those between C. abax Oberthiir, 1886 and C. patra Evans, 1939, C. avanti (de Nicéville, 1886) and C. argyrostigma (Eversmann, 1851), C. longimaculatus Hou, Fan & Chiba, 2021 and C. alcina Evans, 1939, C. palaemon (Pallas, 1771) and C. silvicola (Meigen, 1828) are strongly supported. As described by Toussaint et al. (2020), despite the lack of strong support (PP = 0.73, SH-aLRT = 85.3, UFBoot = 69), C. houangty and C. dieckmanni were clustered in a clade comprising C. palaemon, C. silvicola, C. longi- maculatus, and C. alcina. In our previous study (Hou et al. 2021), we established that C. dieckmanni is sister to C. abax and C. patra. However, owing to an oversight, the names C\ dieckmanni and C. argyrostigma were confused, which explains the discrep- ancy compared with the results reported herein. Accordingly, to determine relation- ships more comprehensively in the genus Carterocephalus, we ideally need to undertake additional and more extensive sampling. Morphologically, although C. pulchra is similar to the type species of Carterocepha- lus with respect to wing shape and pattern (Fig. 2), the origin of vein Ron the hind- wing is located nearly midway between the termen and the base in C. pulchra, Heter- opterus, and Leptalina, whereas in other species of Carterocephalus the origin of vein R, is closer to the termen than to the base (Fig. 3). With regards to the male genitalia, the uncus in C. pulchra, Heteropterus, and Leptalina is deeply bifurcated, with arms distant from each other, whereas in the type species of Carterocephalus the uncus bifurcates with arms closely aligned (Fig. 4). These morphological similarities would accordingly appear to indicate that C. pulchra is more closely related to Heteropterus and Leptalina than to other species of Carterocephalus. Of these related genera, C. pulchra is autapo- morphous with respect to its male genitalia. Notably, the gnathos is weakly sclerotized, membranous, and rounded at the tip, the valvae are asymmetrical, and the juxta is a heart-shaped ring with a narrow and long latero-central process. In summary, we pro- pose a new genus, Pulchroptera Hou, Fan & Chiba gen. nov., for the Carterocephalus pulchra clade based on its autapomorphies and molecular evidence. Pulchroptera Hou, Fan & Chiba, gen. nov. http://zoobank.org/3C184FA2-423E-43F5-B77A-5D06C5639245 Figures 2—4 Type species. Pamphila pulchra Leech, 1891 Description. Forewing length 11-12 mm. Antennae approximately half the length of forewing; nudum 8 on apiculus, dark brown. Palpi on second segment long and erect, yellow with long black hairs; on third segment black, thick, short, and por- rect. Wing venation (Fig. 3): forewing: length of discoidal cell almost equal to 2/3 forewing length, Sc ends at 1/2 forewing length; origin of vein Re before vein Re; A new genus of Heteropterinae from China 61 Figure 2. Male adults of the two skippers. Above: Pulchroptera pulchra (Leech, 1891) comb. nov. from Kunming, Yunnan, China; below: Carterocephalus palaemon (Pallas, 1771) from Moscow, Russia. lcm Figure 3. Wing venation of four genera of Heteropterinae A Pulchroptera pulchra (Leech, 1891) comb. nov. B Carterocephalus palaemon (Pallas, 1771) © Heteropterus morpheus (Pallas, 1771) D Leptalina uni- color (Bremer & Grey, 1852). origin of vein M, in middle of veins M, and M,; veins CuA,, CuA,, and 1A+2A almost parallel to each other; origin of vein CuA, nearly midway between vein CuA, and base. Hindwing: costa longer than dorsum; length of discoidal cell almost equal to 3/5 hindwing; origin of vein Rs midway between base and termen; origin of vein M, slightly nearer M, than M,. Wing ground colour and wing patterns: upper side dark 62 Yongxiang Hou etal. / ZooKeys 1055: 55-67 (2021) B 1 mm D 1 mm Figure 4. Male genitalia of four genera of Heteropterinae A Pulchroptera pulchra (Leech, 1891) comb. nov. B Carterocephalus palaemon (Pallas, 1771) © Heteropterus morpheus (Pallas, 1771) D Leptalina uni- color (Bremer & Grey, 1852). brown with small yellow spots in central and submarginal areas; underside light brown, forewing patterns similar to upper side, hindwing with small silvery spots in spaces Rs, M,, CuA,, and CuA,, and with a silvery longitudinal central streak. Mid and hind tibiae each with pair of spurs. Male genitalia: Tegument small and narrow, constricted at middle in dorsal view; uncus deeply bifurcated, V-shaped dorsally; gnathos long and wide, longer than tegument, membranous, undivided from basal 1/3; saccus long; valvae asymmetrical, bifid, distal end of left valva more sclerotized than right valva; ae- deagus long, subzonal sheath shorter than suprazonal sheath, ratio of subzonal sheath to suprazonal sheath approximately 1:2, vesica with cornuti; juxta a heart-shaped ring with membranous extensions dorsally. Remarks. The new genus superficially resembles Carterocephalus Lederer, 1852, although it is distinguishable from the latter with regards to the following characters: hindwing undersides with silver spots, a deeply bifurcated V-shaped uncus, juxta a heart-shaped ring, and valvae asymmetrical. The new genus contains only the type species Pulchroptera pulchra (Leech, 1891) comb. nov., with the nominotypical subspecies and a further subspecies, Pulchroptera pulchra ops (Grum-Grshimailo, 1891) comb. nov. According to the description of Ev- ans (1949), in Pulchroptera pulchra pulchra comb. nov. the upper side of the hindwing has a cell spot and the submarginal markings are notably more conspicuous, whereas in Pulchroptera pulchra ops comb. nov. the upper side of the hindwing lacks a cell spot A new genus of Heteropterinae from China 63 and has conspicuous submarginal markings. Whether the subspecies status of the latter is valid is subject to further verification. Etymology. The name of the genus is taken from the specific epithet of the type spe- cies ‘pulchr-’, meaning beautiful, and ‘optera’, meaning wing. The gender is feminine. Distribution. Pulchroptera pulchra pulchra comb. nov.: China (Sichuan, Yunnan) Pulchroptera pulchra ops comb. nov.: China (Gansu, Qinghai, Xizang) Acknowledgments This work was supported by the National Natural Science Foundation of China (no. 31872264, 31471984). We are grateful to Messes Jiro Uehara (Japan) and Szabolcs Safian (UK) for providing samples and the Cave Biology Laboratory of South China Agricultural University for taking the wing venation and genitalia photographs. We would like to thank Editage (www.editage.cn) for English language editing. References Abouheif E, Wray GA (2002) Evolution of the gene network underlying wing polyphenism in ants. Science 297: 249-252. https://doi.org/10.1126/science.1071468 Aurivillius C (1925) Die afrikanischen Tagfalter. In Seitz, Gross-schmett Erde 13: 11-613. [80 pls.] Chou I (1994) Monographia Rhopalocerorum Sinensium. Henan Scientific and Technological Publishing House, Zhengzhou, 852 pp. Cong Q, Zhang J, Shen JH, Grishin NV (2019) Fifty new genera of Hesperiidae (Lepidoptera). Insecta Mundi 0731: 1-56. https://journals.flvc.org/mundi/article/view/ 117773 Evans WH (1937) A Catalogue of the African Hesperiidae indicating the classification and nomenclature adopted in the British Museum (Natural History). British Museum (Natural History), London, 212 pp. Evans WH (1949) A Catalogue of the Hesperiidae from Europe, Asia & Australia in the British Museum (Natural History). British Museum (Natural History). London, 502 pp. https:// www. biodiversitylibrary.org/page/48223970 Evans WH (1955) A Catalogue of the American Hesperiidae Indicating the Classification and Nomenclature Adopted in the British Museum (Natural History). Part IV. Hesperiinae and Megathyminae. British Museum (Natural History), London, 499 pp. Fan XL, Chiba H, Huang ZE Fei W, Wang M, Safian S (2016) Clarification of the Phyloge- netic Framework of the Tribe Baorini (Lepidoptera: Hesperiidae: Hesperiinae) Inferred from Multiple Gene Sequences. PLoS ONE 11(7): e0156861. https://doi.org/10.1371/ journal.pone.0156861 Fan XL, Chiba H, Wang M (2010) The genus Scobura Elwes & Edwards, 1897 from China, with descriptions of two new species (Lepidoptera: Hesperiidae). Zootaxa 2490(1): 1-15. https://doi.org/10.11646/zootaxa.2490.1.1 64 Yongxiang Hou et al. / ZooKeys 1055: 55-67 (2021) Folmer O, Black M, Hoeh W, Lutzet R, Vrijenhoek RC (1994) DNA primers for amplifica- tion of mitochondrial cytochrome coxidase subunit I from diverse metazoan invertebrates. Molecular marine biology and biotechnology 3(5): 294-299. Grum-Grshimailo (1891) Lepidoptera nova in Asia centrali novissime lecta et descripta. Horae societatis entomologicae rossicae 25(3—4): e460. https://archive.org/details/trudyrusska- goent2526189092russ/page/n505/mode/ 1up?view=theater Guindon S, Dufayard JF, Lefort V, Anisimova M, Hordijk W, Gascuel O (2010) New al- gorithms and methods to estimate maximum-likelihood phylogenies: assessing the per- formance of PhyML 3.0. Systematic biology 59(3): 307-321. https://doi.org/10.1093/ sysbio/syq010 Hauser CL (1993) Die inneren weiblichen Genitalorgane der Tagfalter (Rhopalocera): Ver- gleichende Morphologie und phylogenetische Interpretation (Insecta, Lepidoptera). Zo- ologische Jahrbiicher. Abteilung fiir Systematik, Okologie und Geographie der Tiere 120: 389-439. Higgins LG (1975) The Classification of European Butterflies. Collins, London, 320 pp. Hou YX, Li YE Wang M, Chiba H, Fan XL (2021) The relationships of Carterocephatus al- cina Evans, 1939 and relatives (Lepidoptera: Hesperiidae: Heteropterinae). Journal of South China Agricultural University 42(1): 101-108. http://doi.org/10.7671/j.issn.1001- 411X.202009046 Huang ZF, Chiba H, Jin J, Kizhakke AG, Wang M, Kunte K, Fan XL (2019) A multilocus phy- logenetic framework of the tribe Aeromachini (Lepidoptera: Hesperiidae: Hesperiinae), with implications for taxonomy and biogeography. Systematic Entomology 44: 163-178. https://doi.org/10.1111/syen.12322 Huang ZF, Chiba H, Wang M, Fan XL (2016) Establishment of a new genus for Abraximor- pha heringi and A. pieridoides (Lepidoptera: Hesperiidae: Pyrginae: Tagiadini). Zootaxa 4173(2): 147-154. https://doi.org/10.11646/zootaxa.4173.2.5 Kumar S, Stecher G, Tamura K (2016) MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets.Molecular Biology and Evolution 33: 1870-1874. https:// doi.org/10.1093/molbev/msw054 Lanfear R, Calcott B, Ho SY, Guindon S (2012) PartitionFinder: combined selection of parti- tioning schemes and substitution models for phylogenetic analyses. Molecular biology and evolution 29(6): 1695-1701. https://doi.org/10.1093/molbev/mss020 Lanfear R, Frandsen PB, Wright AM, Senfeld T, Calcott B (2016) PartitionFinder 2: new methods for selecting partitioned models of evolution formolecular and morphological phylogenetic analyses. Molecular biology and evolution. https://doi.org/10.1093/molbev/ msw260 Leech JH (1891) New Species of Rhopalocera from Western China. Entomologist 24: 57-61. https://archive.org/details/entomologist189195brit/page/59/mode/1up?view=theater Li WL, Cong Q, Shen JH, Zhang J, Hallwachs W, Janzen DH, Grishin NV (2019) Genomes of skipper butterflies reveal extensive convergence of wing patterns. Proceedings of the National Academy of Sciences of the United States of America 116: 6232-6237. https:// doi.org/10.1073/pnas.1821304116 A new genus of Heteropterinae from China 65 Liu GC, Chang Z, Chen L, He JW, Dong ZW, Yang J, Lu SH, Zhao RP, Wan WT, Ma GL, Li J, Zhang R, Wang W, Li XY (2020) Genome size variation in butterflies (Insecta, Lepidotera, Papilionoidea): a thorough phylogenetic comparison. Systematic Entomology 45(3): 571-582. https://doi.org/10.1111/syen.12417 Miller MA, Pfeiffer W, Schwartz T (2010) Creating the CIPRES Science Gateway for Infer- ence of Large Phylogenetic Trees. Proceedings of the Gateway Computing Environments Workshop 14: 1-8. https://doi.org/10.1109/GCE.2010.5676129 Minh BQ, Nguyen MAT, Haeseler VA (2013) Ultrafast approximation for phylogenetic boot- strap. Molecular Biology and Evolution 30: 1188-1195. https://doi.org/10.1093/molbev/ mst024 Monteriro A, Pierce NE (2001) Phylogeny of Bicyclus (Lepidoptera: Nymphalidae) inferred from COI, COIL and EF-1la gene sequences. Molecular Phylogenetics And Evolution 18(2): 264-281. https://doi.org/10.1006/mpev.2000.0872 Nguyen LI, Schmidt HA, von Haeseler A, Minh BQ (2015) IQ-TREE: A fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Molecular Biology and Evolution 32(1): 268-274. https://doi.org/10.1093/molbev/msu300 Peng Y, Leung HCM, Yiu SM, Chin FYL (2012) IDBA-UD: a de novo assembler for single- cell and metagenomic sequencing data with highly uneven depth. Bioinformatics 28(11): 1420-1428. https://doi.org/10.1093/bioinformatics/bts174 Ronquist F, Teslenko M, Mark PVD, Ayres DL, Darling A, Hohna S, Larget B, Liu L, Suchard MA, Huelsenbeck JP, Notes A (2012) MrBayes 3.2: Efficient Bayesian Phylogenetic Infer- ence and Model Choice Across a Large Model Space. Systematic Biology 61(3): 539-542. https://doi.org/10.1093/sysbio/sys029 Sahoo RK, Warren AD, Wahlberg N, Brower AV, Lukhtanov VA, Kodandaramaiah U (2016) Ten genes and two topologies: An exploration of higher relationships in skipper butterflies (Hesperiidae). Peer] 4: e2653. https://doi.org/10.7717/peerj.2653 Sahoo RK, Warren AD, Collins SC, Kodandaramaiah U (2017) Hostplant change and pale- oclimatic events explain diversification shifts in skipper butterflies (Family: Hesperiidae). BMC Evolutionary Biology 17: e174. https://doi.org/10.1186/s12862-017-1016-x Scott JA, Wright DM (1990) Butterfly phylogeny and fossils. In: Kudrna O (Ed.) Butterflies of Europe 2, Aula Verlag, Wiesbaden, 152-208. Thompson J, Gibson TJ, Plewniak F, Jeanmougin FE, Higgins DGG (1997) The CLUSTAL_X Windows Interface: Flexible Strategies for Multiple Sequence Alignment Aided by Qual- ity Analysis Tools. Nucleic Acids Research 25(24): 4876-4882. https://doi.org/10.1093/ nar/25.24.4876 Toussaint EFA, Breinholt JW, Earl C, Warren AD, Brower AVZ, Yago M, Dexter KM, Espe- land M, Pierce NE, Lohman DJ, Kawahara AY (2018) Anchored phylogenomics illumi- nates the skipper butterfly tree of life. BMC Evolutionary Biology 18(1): e101. https://doi. org/10.1186/s12862-018-1216-z Toussaint EFA, Ellis EA, Gott RJ, Warren AD, Dexter KM, Storer C, Lohman DJ, Kawahara AY (2020) Historical biogeography of Heteropterinae skippers via Beringian and post- Tethyan corridors. Zoologica Scripta 00: 1-12. https://doi.org/10.1111/zsc.12457 66 Yongxiang Hou et al. / ZooKeys 1055: 55-67 (2021) Trifinopoulos J, Nguyen LT, Haeseler AV, Minh BQ (2016) W-IQ-TREE: a fast online phylo- genetic tool for maximum likelihood analysis. Nucleic Acids Research 44. gkw256. https:// doi.org/10.1093/nar/gkw256 Wahlberg N, Wheat CW (2008) Genomic outposts serve the phylogenomic pioneers: Design- ing novel nuclear markers for genomic DNA extractions of Lepidoptera. Systemactic Biol- ogy 57(2): 231-242. https://doi.org/10.1080/10635 150802033006 Ward PS, Downie DA (2005) The ant subfamily Pseudomyrmecinae (Hymenoptera: Formi- cidae): phylogeny and evolution of big-eyed arboreal ants. Systematic Entomology 30: 310-335. https://doi.org/10.1111/j.1365-3113.2004.0028 1.x Warren AD, Ogawa JR, Brower AV (2008) Phylogenetic relationships of subfamilies and cir- cumscription of tribes in the family Hesperiidae (Lepidoptera: Hesperioidea). Cladistics 24(5): 642-676. https://doi.org/10.1111/j.1096-0031.2008.00218.x Warren AD, Ogawa JR, Brower AV (2009) Revised classification of the family Hesperiidae (Lepidoptera: Hesperioidea) based on combined molecular and morphological data. Sys- tematic Entomology 34(3): 467-523. https://doi.org/10.1111/j.1365-3113.2008.00463.x Wiemers M, Chazot N, Wheat CW, Schweiger O, Wahlberg N (2020) A complete time-cali- brated multi-gene phylogeny of the European butterflies. ZooKeys 938: 97-124. https:// doi.org/10.3897/zookeys.938.50878 Wu CS, Hsu YF (2017) Butterflies of China. The Straits Publishing and Distributing Group, Fuzhou, 2036 pp. Yuan EK Yuan XQ, Xue GX (2015) Fauna Sinica (Insecta: Lepidoptera: Hesperiidae). Science Press, Beijing, 769 pp. Zhang J, Cong Q, Shen JH, Brockmann E, Grishin NV (2019) Three new subfamilies of skip- per butterflies (Lepidoptera, Hesperiidae). ZooKeys 861: 91-105. https://doi.org/10.3897/ zookeys.861.34686 Zhang J, Lees DC, Shen JH, Cong Q, Huertas B, Martin G, Grishin NV (2020) The mi- togenome of a Malagasy butterfly Malaza fastuosus (Mabille, 1884) recovered from the holotype collected over 140 years ago adds support for a new subfamily of Hesperiidae (Lepidoptera). Genome 63: 195-202. https://doi.org/10.1139/gen-2019-0189 Supplementary material | Table S1 Author: Yongxiang Hou Data type: table Explanation note: Label information and GenBank accession numbers of the speci- mens used in this study. Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/zookeys.1055.68640.suppl1 A new genus of Heteropterinae from China 67 Supplementary material 2 Table S2 Author: Yongxiang Hou Data type: table Explanation note: Primers used in this study. Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODDbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/zookeys.1055.68640.suppl2 Supplementary material 3 Table S3 Author: Yongxiang Hou Data type: table Explanation note: The best-fit partition schemes and model calculated by Partition- Finder v2.1.1. Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODDbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/zookeys. 1055.68640.suppl3