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Home , Achalarus, Achalarus lyciades, Aguna (butterfly), Astraptes, Aurina, Cabirus

Genomes of butterflies reveal extensive convergence of wing patterns

Wenlin Lia,b, Qian ,b,1, Jinhui Shena,b, Jing Zhanga,b, Winnie Hallwachsc, Daniel H. Janzenc,2, and Nick V. Grishina,b,d,2

aDepartment of Biophysics, University of Southwestern Medical Center, Dallas, TX 75390-9050; bDepartment of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX 75390-9050; cDepartment of Biology, University of Pennsylvania, Philadelphia, PA 19104; and dHoward Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, TX 75390-9050

Contributed by Daniel H. Janzen, January 4, 2019 (sent for review December 17, 2018; reviewed by John Shuey and James Walters)

For centuries, biologists have used phenotypes to infer evolution. specimens were collected after 2012 and preserved in a DNA- For decades, a handful of gene markers have given us a glimpse friendly manner. Others were pinned in collections listed in SI of the genotype to combine with phenotypic traits. Today, we Appendix, Table S1 (also see Acknowledgments) and included can sequence entire genomes from hundreds of and gain specimens gathered over a century ago. Nevertheless, sequence yet closer scrutiny. To illustrate the power of genomics, we have reads targeting 10× coverage of the genome allowed us to assem- chosen skipper butterflies (Hesperiidae). The genomes of 250 rep- ble genomic regions by emphasizing -coding genes. We resentative species of skippers reveal rampant inconsistencies used 12,618 nuclear genes from the two reference genomes between their current classification and a genome-based phy- of Hesperiidae we have sequenced (13, 14). Aligned nuclear logeny. We use a dated genomic tree to define tribes (six new) genomic regions covered 8,100,834 ± 2,387,641 positions in each and subtribes (six new), to overhaul genera (nine new) and sub- specimen. Mitogenomes covered 10,663 ± 674 positions. genera (three new), and to display convergence in wing patterns Genomic Tree of Skippers. Weconstructedgenomictreesfromcon- that fooled researchers for decades. We find that many skippers catenated alignments of coding regions (Fig. 1) and from coding with similar appearance are distantly related, and several skippers regions with introns, and both trees had identical topologies. - with distinct morphology are close relatives. These conclusions are thermore, we constructed gene trees and combined them using strongly supported by different genomic regions and are consis- the program ASTRAL (15), which produced the tree with the tent with some morphological traits. Our work is a forerunner to same topology. The trees were dated, i.e., transformed to predict genomic biology shaping research. absolute ages for its nodes (SI Appendix, Methods). The major phy- logenetic groups in these trees agree with those defined by Warren genomics | | higher classification | | et al. (2, 3) and follow-up publications that used larger sets of species and genes (4–6). Subfamily (the awls) is sister t is the phenotype that is relevant when an interacts with its to all other Hesperiidae (Fig. 1). Australian endemic , Ienvironment. Phenotypic traits have defined the place of an organ- as suggested by several studies (2, 3, 16), is unique and forms a ism within its ecosystem and within a human-made classification system. Due to phenotypes being riddled by adaptive convergence, Significance it is challenging to decipher phylogeny, and thus deduce phyloge- netic classification from morphology. The phenotype is encoded by the genotype, which equally bears the footprint of evolu- With decreasing cost, biodiversity genomics can be done by tion. DNA sequences are prime for evolutionary studies. Sanger- small laboratories, not large centers. Here, we overview the sequencing of select gene markers revolutionized phylogenetic genomic landscape of an entire of . By sequenc- research and refined morphology-based classification. However, ing 250 representatives of skipper butterflies, we infer their frequent homoplasies are a serious obstacle to phylogeny recon- phylogeny and search for genotypic determinants of pheno- struction from small datasets. Since genomes are composed of typic traits. We find that wing patterns are frequently conver- millions of base pairs, the study of complete genotypes is expected gent. This likely mimetic convergence is diversified, resulting to resolve many outstanding questions. While large- genomic in five distinct parallel patterns. Each of the five patterns studies are still scarce, they are most enlightening (1). occurs within at least two genera as well as in more distant To exemplify a large-scale genomics project possible within a relatives diverged more than 20 Mya. This work offers an small laboratory, we use skipper butterflies (Hesperiidae). This example of how each of the nearly 6,000 families of animals family of about 4,000 species worldwide received less attention will be studied in the future.

than other butterflies. Only a handful of studies have focused Author contributions: D.H.J. and N.V.G. designed research; W.L., Q.C., J.S., J.Z., W.H., and on skipper phylogeny (2–6). Pioneering work by Warren et al. N.V.G. performed research; W.L., Q.C., J.Z., D.H.J., and N.V.G. analyzed ; and W.L., (2, 3) revealed many surprising phylogenetic relationships com- Q.C., J.S., J.Z., W.H., D.H.J., and N.V.G. wrote the paper.y pared with the morphological treatment (7–12). However, many Reviewers: J.S., The Conservancy; and J.W., Kansas University.y questions remained unanswered. In particular, the subfamily The authors declare no conflict of interest.y was not analyzed in detail. This open access article is distributed under Creative Commons Attribution-NonCommercial- We obtained and analyzed genomic shotgun reads of 250 NoDerivatives License 4.0 (CC BY-NC-ND).y species of skippers covering all Eudaminae genera. We con- Data deposition: The sequences reported in this paper have been deposited in the NCBI structed a genome-level phylogeny of Hesperiidae and found Sequence Read Archive (accession no. SRP147939) and the GenBank database (accession that, due to convergence in wing patterns, many skippers have nos. MH357724–MH357835). This work was registered with ZooBank (www.zoobank.org) as 8A8E82C4-AC8A-4B7B-8DF4-6BFF4BD48F3D, and registration numbers for 24 nomen- been placed in presumed evolutionary groups in which they clatural acts are given in Tables 1 and 2.y do not belong. We also discovered a group of close relatives 1 Present address: Institute for Protein Design and Department of Biochemistry, Univer- with disparate morphology. Furthermore, sequence data suggest sity of Washington, Seattle, WA 98195.y possible genomic determinants of morphological traits. 2 To whom correspondence may be addressed. Email: [email protected] or [email protected] Results and Discussion This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. Genomes of 250 Skippers. We selected 250 species of Hesperiidae 1073/pnas.1821304116/-/DCSupplemental.y from all subfamilies and tribes (SI Appendix, Table S1). Only 36 Published online March 15, 2019.

6232–6237 | PNAS | March 26, 2019 | vol. 116 | no. 13 www.pnas.org/cgi/doi/10.1073/pnas.1821304116 Downloaded by guest on September 26, 2021 Downloaded by guest on September 26, 2021 ie al. et Li utieta rvosyaei lefn ihihe ngen emnso h recrepnigt ifrn rbsaehglgtdi ifrn colors. different in or highlighted are different tribes See a different font. in to brown placed corresponding in Taxa numbers tree font. by the red tree of in the in Segments in are are green. names previously genera with in than New associated highlighted yellow. combinations are in font -species species highlighted blue different Hesperiidae are in Illustrated in taxa are New used tribes; genera. previously those places), only), than and several (Eudaminae in yellow, subtribe yellow (broken in shaded orange highlighted subfamilies; green red, and font groups: subtribes; red taxonomic only), major (Eudaminae delineate red lines horizontal shaded Colored yellow below. and above shown are segments 1. Fig. ieclbae eoi reo epria osrce rmncergn oigrgoso 5 pce.Tete scti af is n second and First half: in cut is tree The species. 250 of regions coding gene nuclear from constructed Hesperiidae of tree genomic Time-calibrated IAppendix SI PNAS | ac 6 2019 26, March o details. for | o.116 vol. | o 13 no. | 6233

EVOLUTION subfamily of its own, sister to all other Hesperiidae except Coeliad- their current classification, with several exceptions discussed in SI inae. The next split in the genomic tree is different from that Appendix. We used this mitogenome + barcode tree together with appearing in a number of DNA studies, but in agreement with the nuclear genome tree (Fig. 1) as the basis for our proposed clas- the morphological view. The Spreadwing skippers (mostly dicot sification of Hesperiidae (SI Appendix, Taxonomic Appendix). Dif- feeders) and the (mostly monocot feeders) are sis- ferences in barcodes and morphology suggested that 30 subspecies ters. Both Spreadwing and Grass skippers have been previously as defined by Evans (10–12) are more likely to be species, and divided into several subfamilies. Genomic data very strongly sup- such cases were analyzed in detail for wing pattern and genitalic port (bootstrap above 99%) of these subfamilies. The differences (SI Appendix, Taxonomic Appendix). two latest diverging subfamilies (Hesperiinae and ) havesplitoffabout 50Mya (Fig.1).Pyrginae,asdefined byWarren Eudaminae Tribes and Subtribes. Previous studies refrained from et al. (3), diversified from their common ancestor before that time defining tribes in the subfamily Eudaminae. We focused on this (>55 Mya). Moreover, while other subfamilies except Pyrginae subfamily and attempted to delineate tribes consistently with the (i.e., Eudaminae, Heteropterinae, and Hesperiinae) are well sep- of the tribes in Pyrginae (sensu stricto). The four Pyrginae arated from each other, Pyrginae sensu Warren have split into tribes (, Achlyodini, , and ) outlined three compact groups, within a short time and before divergence by Warren et al. (3) diverged around 42 Mya. Our genomic tree of Grass skippers into subfamilies. Thus, these three groups within agrees with Warren’s, except that it places Grais, , Morv- Warren’s Pyrginae are no less distinct than Hesperiidae subfam- ina, , Xispia, Pseudodrephalys, , , and Spi- ilies, and we treat them as such. Accordingly, we unify the sister oniades in Achlyodini; Cornuphallus in Carcharodini; in tribes Tagiadini and Celaenorrhinini in a subfamily Tagiadinae, Erynnini; and in Pyrrhopyginae. Unexpectedly, Emmelus and reinstate the subfamily Pyrrhopyginae. The latter are strik- belongs to Eudaminae. Conversely, is not Eudaminae ingly distinct in appearance from other skippers, and have been (4), but Pyrginae: Achlyodini. All Pyrginae tribes received 100% traditionally considered a subfamily. They diverged from their bootstrap support and are conspicuous groups in the subfamily. common ancestor with Pyrginae about 55 Mya, before the diver- To achieve consistent classification, we cut the dated genomic genceofGrassskippers.Thus,genomicdatasuggestthatthefamily tree around the time of Pyrginae divergence into tribes and Hesperiidae consists of nine subfamilies (Fig. 1 and SI Appendix), find four Eudaminae phylogenetic groups supported by 100% and diversification into subfamilies occurred about 50 Mya. bootstrap that we define as tribes. Two of the tribes that form best-separated groups are described in Table 1 as Oileidini and Mitogenomes and Cytochrome Oxidase I Barcodes. In addition to a Entheini. The two others are closely related sisters Eudamini and nuclear genome tree, we constructed a tree from mitogenomes. Phocidini that diverged about 40 Mya, and are given a tribal rank The resulting tree recapitulates major phylogenetic groups of due to their morphological distinction. the nuclear genome tree, but with weaker support. All of These four Eudaminae tribes correspond to groups with the subfamilies and tribes are composed of the same species similar morphology (10, 11). For example, Entheini is largely in either mitogenome or nuclear genome phylogenies. Next, the “B. Augiades group” of Evans. He defined it by the using mitogenomes of 250 species as a backbone, we increased “divergent” third segment of palpi that stems from the outer taxonomic coverage by adding 290 species with cytochrome edge of the second segment. Inconsistently, Evans included in oxidase I (COI) barcodes only (SI Appendix, Fig. S1). These this group three genera that have the central third segment: species grouped with mitogenomes of their expected relatives. and Hypocryptothrix, that do not belong to Entheini, and Most of these barcode-only species were placed according to Cabirus, that is not even in Eudaminae (Fig. 1). Interestingly,

Table 1. Description of new tribes and subtribes of Hesperiidae

New tribe or subtribe Type genus Diagnostic characters* ZooBank ID†

Entheini Grishin, new Hubner,¨ [1819] 276665.26:A192G; 85.28:C176T; 378.19:G1099C; 374.14:G1169T; keys to 303C1FD0-07CB-4919-900E-EA3D6347E5DD tribe B.3a in ref. 10, but exclude B.9

Loboclina Grishin, new Moore, 1884 208.2:G145T, T146G; 18312.8:A619C, G620A; keys to B.4 in ref. 8 or C.5, C606FC35-323D-4E55-AF5A-A86C6366BAFA subtribe C.10a, C.15.2 or C.18 in ref. 11

Cephisina Grishin, new Evans, 1952 COI.bc:A44T, C84G, T479A; genitalia and palpi as described in ref. 17 22B59811-F174-4FDF-A9D2-799897F4D44E subtribe (pp. 182–183) for Cephise are diagnostic

Telemiadina Grishin, Hubner,¨ [1819] 536.149:G1488C; 997.8:G514T; 860.7:A748G; 3001.3:C1773T; keys to 4AE0E59C-8B92-4C84-8651-E7A1C45C93C1 new subtribe B.2, C.3, C.7a (exclude C.7.6b), E.6a or E.9 in refs. 10 and 11

Oileidini Grishin, Oileides Hubner,¨ [1825] 1139.19:T562A; 851.8:C423A, G443A; 11945.11:G391A; 65.4:C330A; tuft CF9C3D29-523A-4D17-B140-9A69CFA98731 new tribe of scales by anal fold from the base of hindwing, either above or below

Typhedanina Grishin, Butler, 1870 1341.12:T25841C; 489.5:G307T; 3446.8:T2308A, C2309G, A2500C; tuft of B4D56F93-67F9-476F-B69C-133D98BFBD58 new subtribe scales by anal fold from the base of hindwing above, but not below

Netrocorynini Grishin, C. & R. Felder, [1867] 2284.30:399A; 904.14:T439G; 275215.7:C925G; 998.8:G308A; DE61F048-02CF-4F8E-9392-D18A4618BABD new tribe 214.24:3520C; keys to B.1, C.1 or C.15 in ref. 8

Jerini Grishin, Jera Lindsey, 1925 103.23:796A; 420.27:G308A; 671.27:935C; 425.5:G1558T; keys to E.3 in AF3B5CEA-880A-4CB2-AF40-E6D87C39C040 new tribe ref. 11; forewing cell > 3/5 of costa

Pythonidina Grishin, Hubner,¨ [1819] 274.29:G397A; 3478.6:T116C; 7985.5:G916A; 925.10:G199C; in ref. 11, CB890271-5483-4B5A-A7BC-27DBC5E23DE5 new subtribe keys to E.44a, E.49.1 or, if uncus undivided, then E.37a or 40d

Clitina Grishin, Clito Evans, 1953 COI.bc:G29T, 81A, 169A, 266A, 302T, A353T, A521T; keys to E.52 or 971884E2-E5F7-46A3-B182-657729B6A778 new subtribe E.13.8 in ref. 11

Butleriini Grishin, Butleria Kirby, 1871 2627.8:A1459T; 141.4:C104A; 37338.38:G133T, G134C; keys to H.4 & 5 in D621EF81-FA65-4858-9450-E0C041598D7A new tribe ref. 12

Pericharini Grishin, Scudder, 1872 596.8:C1601G; 144.41:G201C; 83.15:G8658A,T8657G; keys to K.27a in 94B68BD2-7F83-4E58-80E1-7F5AC8C56511 new tribe ref. 12

See SI Appendix for the lists of genera included in each taxon and sequences of protein-coding regions with diagnostic characters. *Notation 272.1:A192G means position 192 in the gene 1 on the scaffold 272 is G, changed from A in the ancestor; 169A, means position 169 is A, but the ancestral state is unclear; COI.bc is the COI barcode region. †ZooBank registration URL given for each taxon should be preceded by http://www.zoobank.org/.

6234 | www.pnas.org/cgi/doi/10.1073/pnas.1821304116 Li et al. Downloaded by guest on September 26, 2021 Downloaded by guest on September 26, 2021 e eu rsbeu yespecies Type Hesperiidae of subgenera and subgenus or genera genus new New of Description 2. Table , Telemi- gonus (17). features genera: morphological three its includes in adina unique is and Mya the 35 for subtribes monotypic as is Cephisina, here subtribe genus described them, of are One that 1). reveals (Table tribe tree the genomic The within subfamily. the groupings of half than more passes Eudami- with in together found species that (not suggests pouch tree thoracic genomic a The nae). into fits that tibiae hind and removing ie al. ancestor; et the Li in A from changed T, is 79 position means A79T Notation species. these of genera former from unclear. distinguish is to state ‡ diagnostic ancestral is the combination but their C, only is † and 59 region position barcode means COI 59C the for are characters *Sequence Tekliades of sexually group” adding are after when “D. species (10) the flat many Evans is spread and Phocidini usually Our wings crepuscular, is dimorphic. hold are tornus many skippers hindwing resting, other, lobed), How- each (not R4 tribe. near veins expanded one originate forewing be R5 Their ecologically: could and and and stand skippers morphologically branch Phocidini out and monophyletic, short strongly is a each ever, by other each hindwing. the of groove sides in both the structures on found in Similar are others). scales (Oileides) (the below -like either above males, or of in 1A+2A tufts vein genera) hindwing (six by the along tribe characterized smallest Evans is the between by is morphology and This in genera Eudaminae. intermediate Pyrginae be and may Pyrginae some tribe with the and grouped (11), were tribe this convergent. are palpi skipper along peculiar unusual possess, its groove this that males places the implying tree Its Phocidini, in Our in scales Entheini. 1A+2A. hair-like vein in of hindwing not the tufts Entheini, is in group) absent Augiades the in Phareas e genus new Chirgus genus new subgenus new Asina subgenus new Caudatractus Salantoia genus e genus new Duroca genus new Burnsius Tiana genus new Lobotractus genus new Zeutus subgenus new Urbanoides genus new Spicauda genus new oBn eitainULgvnfrec ao hudb rcddby preceded be should taxon each for singular. given nominative URL the registration in ZooBank nouns as treated are names All uaiii h ags n otdvretie tencom- It tribe. diverse most and largest the is Eudamini See from separated are Phocidini and Eudamini tribes sister The in Genera 1). (Fig. Eudaminae other to sister is Oileidini Grishin, Grishin, hs ae aeatf fln clso the on scales long of tuft a have males whose Celaenorrhinus, IAppendix SI Grishin, Grishin, Grishin, Grishin, and Grishin, hc iegdfo t itrtieTelemiadina tribe sister its from diverged which Cephise, Grishin, rsi,new Grishin, Grishin, Grishin, hthsadvrettidsget(n a included was (and segment third divergent a has that Grishin, and , hc a oe rtie idigtornus, hindwing tailed or lobed has which Cephise, nowihw sink we which into Ectomis, o h it fseisicue nec taxon. each in included species of lists the for hml ramanatek Thymele uau asine Eudamus eriopis Eudamus epralimbata alcaeus Eudamus yitu communis Syricthus niger valeriana Eudamus esmeraldus Goniurus procne Goniurus epraduroca Hesperia zeutus Cecropterus ilas&Bl,14 est .. r4i e.11 ref. in 4 or F.7.3 to 1876 keys Erschoff, 1940 Bell, & Williams 1867 Hewitson, 1867 Hewitson, 1833 Boisduval, Pl 1872 Grote, Pl M 1877 Butler, Pl 1867 Hewitson, t,1882 otz, 1881 otz, 1881 otz, she,1879 oschler, ¨ ¨ ¨ ¨ Phocides, Telemiades eogt Phocidini. to belong Speculum and Phareas, Oileides oG15 ,o 0 nrf 11 ref. in 10a or 8, G.1.5, to hook-shaped not flat, harpe 10; ref. in 7T 54;ky oG12 r9i e.11 ref. in 9 or G.1.2e to keys T574A; 479T, 10 ref. in C.7.2a to keys 373A; hindwing tailed with 7 ref. in 69 rG est .36 nrf 10 ref. in C.13.6a to keys G; or T619A 8,C0,T4C 1T 5A 8T est .. acln,abedof blend a Masculine, I.1.9 to keys 583T; 553A, 412T, T145C, C90T, 68A, 7A 17,T9C 0T 0A 26,T4A eiie eie rmtetype the from derived Feminine, T346A, A256T, 208A, 206T, T197C, T127A, T70A, of blend a Feminine, 3 or D.3.2 T637A; 598A, 530T, T163A, A79T, 59C, 5,25,23,21,23ntC,T7A 45,Msuie ln of blend a Masculine, A415T, T277A, C), 263(not 241A, 223A, 205A, 85A, A592T; T556A, 355A, 0T 2A 4T 6(o ) 27,49;ky acln,hnr skipper honors Masculine, keys 479T; T277A, C), 263(not 241T, 223A, 205T, of blend a Feminine, 283C; T278A, A196G, T142C, G86A, A43T, T16C, of blend a Masculine, as T619A; C542T, 517T, A477G, 401T, T400A, 49A, to similar means Masculine, and of blend a Feminine, T, or A607C T544A, C542T, T212A, C, or A85T T49A, to keys T562A; G506A, T), 424(not T349A, 307T, 2T 6C 4C 44;ky oJ3.ain J.39.5a to keys T424C; 349C, 163C, 127T, A607T; T548C, A526T, T278A, C271A, A22T, e.1,hrebod hook-shaped broad, harpe 12, ref. 196) (p. 17 ref. in group” ”cyda the for given spiked dorsally harpe 10; ref. in C.13.13c eiai sfor as genitalia Hypocryptothrix, ihiscoesister close its with (see http://www.zoobank.org/. Phareas splpyei:one polyphyletic: is igotccharacters* Diagnostic nan Diver- Uncanny zeutus Emmelus (Phocidini) nrf 9(.27) (p. 19 ref. in Heronia, Poly- and C1 nrf 0 acln,icue aldspecies tailed includes Masculine, 10) ref. in (C.11 lcmnso pce nognr hyd o eogt.The to. belong not do they genera mis- of into number species astounding the of is Shapes. placements study and this of Patterns result Wing unexpected in Convergence Widespread as genera. most such with compared and subgenera, smaller genomic is their distinctness but the morphological decades, subgenera for of genera as new used Some been three have 2). rybes, and (Table Appendix ), described (SI are subgenera as distinct. from defined morphologically are diverged and Mya genera 18 least four at taxa These sister their Pyrginae. from transferred and from decreased 4: 1 genera to monotypic as (Fig. of 10 described number result are The 2. which a Table of in 4 As new genera, genus. Eudaminae species 50 a other delineated of we to relationships name indicate the to by dis- wish strongly we is genus because the tinct, unless genera, monotypic of number the as of such pairs distinct genera morphologically genera and traditional well-known separates it time keeps ever, The It branches. point. cut such , to the genera corresponds by as Mya supported treat 15 species we of and groups features, the morphological are by that genera defined most current well merges the nor with splits agreement neither maximize This phyloge- to classification. dated point a time cut a at We a tree (18). within netic genus existing a being ancestor as common timeframe a certain with species of cluster Subgenera. netic and Genera Eudaminae detailed as convergence mimetic misclassified in possibly largely and been widespread have It to that Eudaminae. due skippers of of group array “crown” an “C. the includes to the belong Others from 2). (Table club plus Evans antennal of arcuate group” with the Along with Genus). genera a Within gence ihnsm eea esegop fseista a be may that species of groups see we genera, some Within iepedCnegnei igPten n Shapes. and Patterns Wing in Convergence Widespread and , pce name species [Coda]tractus and cauda Urbanus n [Py]rgus and [Il]iana name species of [ramana]Tek aooitJh .Burns M. John taxonomist [Sarmie]ntoia pce name species eiie costetype the echoes Feminine, type the echoes Masculine, Nicephellus, Codatractus eiaino h name the of Derivation n [Coe]liades and PNAS and , , ts and Sala[tis] T Chi Lobo[cla] ot]and [osta] | [lean] rau proteus Urbanus ac 6 2019 26, March † and n e genus, new a and Aguna, eatmtt reduce to attempt We . DF0F3C91-F56E-4B65-B86C-385A36F9D7FD B3B7A6F6-A95C-4A2E-B9FB-80A7A8F86761 14D26B57-940C-407B-8E70-4E25203044B8 48996B74-3AB1-4DEA-9A64-B8F112E62343 20FAC3B6-F038-40A0-B182-3C7F32A40702 476FE13C-5895-4139-BB11-44F835E21565 75715B9C-46AB-40F5-B738-420DABD56B63 081564BA-DA0C-4C46-AEAB-6C00131AC8BD 3F82E9DE-A5A2-44B3-A13D-53CF8A673FAE 7B1905F1-9471-4BBF-90BF-32360783AB1E B9382699-24FB-4466-B39B-94E6B544C425 C6E5B5DF-1C74-4DBD-85C3-7285209F6F03 edfieamjrphyloge- major a define We ooln unifies Loboclina Lobocla, Zeutus | o.116 vol. ru npi.How- unsplit. group oBn ID ZooBank 1) plus (19), | IAppendix), SI o 13 no. ‡ h most The Emmelus Zeutus | Tho- 6235

EVOLUTION out genus in Fig. 2). In retrospect, assignment to a correct genus could have been possible through detailed comparison of male genitalia. In Telegonus, the dorsal side of the valva is concave in the middle and forms a mouth-like structure with two “kissing lips.” In Cecropterus, the valva is dorsally and terminally con- vex, without a “kiss.” These genitalic features agree with genomic phylogeny and reinforce our conclusions.

Uncanny Divergence Within a Genus. Before our work, each of the three genera Ectomis, Hypocryptothrix, and Heronia consisted of single species of unique appearance. No taxonomist had ever thought of them being a group. To our surprise, all phyloge- netic trees we obtained (even from COI barcodes only) revealed only a slight divergence among these three and two other gen- era, Polythrix and Chrysoplectrum, suggesting that it is best to place all these skippers in a single genus, Ectomis. Moreover, their most divergent phylogenetic was part of Polythrix, and is described here as a new subgenus Asina (Table 2). Bar- code divergence among the subgenus Ectomis is within 10% and is less than within the genus of swallowtail butterflies Pterourus (24), which some researchers consider a subgenus of . Despite limited genetic differentiation, the expanded Ectomis contains species of profound phenotypic divergence. All skip- pers in the former genus Polythrix (subsumed by Ectomis) are tailed. Others are not, although their hindwing is usually lobed Fig. 2. Convergent wing patterns in Telegonus, Cecropterus, and others. at the tornus. While most Ectomis are brown skippers with a Previously, they were placed in crossed out genera. Genitalic valvae are pale forewing band frequently divided into spots, some vary from shown for the first two genera. solid dark brown to dark metallic green with a forewing central spot, or tawny with many white spots. Some are even part of the mimetic complex with brilliant blue thorax and wing bases above genera themselves, proposed over the of classic entomo- (20). Males of some species possess tufts of hairlike scales on logical studies, mostly stood the test of genomic data: 55 genera the forewing below, while others have a double row of yellow were recognized before our work, and we revise them to be 50. spines on the hind tibiae. Male genitalia are as diverse as wing We eliminated several monotypic genera for which visual - shapes and patterns, and the phylogenetic closeness is not appar- logical differences hindered close relationships with other species, ent from genitalia. The valva varies from a simple curved plate and merged several phenotypically diverse but genotypically close without elaborations (in Ectomis cythna) to very complex with genera (SI Appendix). Apparently, phenotypic distinction (e.g., several processes (in “Heronia” labriaris). It would be of interest wing shape, such as a tailed hindwing, or wing pattern, such as a to investigate genetic mechanisms for such a rapid phenotypic pale stripe across the forewing) may be indicative of genetic dif- divergence within Ectomis. ferentiation. However, placement of a species into a genus by its Connecting Genotype to Phenotype. Telegonus chalco (Fig. 2) and dominant to human phenotypic feature is more problematic. Telegonus brevicauda are sister species that do not look alike. For example, before our work, many tailed skippers were placed T. brevicauda belongs to the “blue-wing-base” mimicry complex in the genus Urbanus based on the tail. Genomic data imply that (Figs. 1–3) and is nearly indistinguishable from its more dis- half of them do not belong there, and we transferred them to three tant relative Urbanus tucuti (Fig. 1). We compared genomes of other genera. One genus is named here (Spicauda, Table 2), while these three species to find rapidly diverging between the the others have not previously included tailed skippers. Moreover, two sisters (SI Appendix, Methods). Genes involved in the circa- we transferred some skippers without long tails from dian clock system, transcription regulation, wing morphogenesis, to Urbanus. They were formerly misclassified due to similar wing fatty acid, and vitamin metabolism (SI Appendix, Tables S4 and patterns consisting of shiny metallic-cyan wing bases and white S5) stood out. The differences in morphogenesis genes may be forewing spots. related to the hindwing tail development in T. chalco. Next, we We found misclassifications to be widespread across Eudami- looked for fixed mutations in the uniquely patterned T. chalco nae and attribute them to convergence driven by the selec- compared with the two similar species. Xanthine dehydrogenase tion for large mimicry rings (20–22). This convergence is not confined to one or two basic patterns, but is more diverse. Some patterns are common in both the Old and New World, and, in addition to skippers, include butterflies from other families and even , flies, and (20, 23). We find five different phenotypes (Fig. 2) that parallel each other in two genera (Telegonus and Cecropterus) and their more distant relatives (outgroups): (i) greenish bases of brown wings, white stripe on the forewing, and hindwing with a white tail and margins; (ii) metallic-blue wing bases, and forewing with white stripe (20); (iii) brown forewing with a yellow stripe across and apical white spots; (iv) cream-white, semitranslucent spots on the brown forewing; and (v) brown wings, and hindwing with yellow tornus. At least four of these phenotypes are not ancestral and thus are Fig. 3. Uncanny divergence within Ectomis. These skippers belonged to five convergent. Curiously, every species (10 out of 10) was placed genera listed above each image. Type species for these genera are marked by its appearance in a wrong genus before our study (crossed with red asterisks.

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National critical Department David for fruitful Wildlife Gerardo Director Schaeffer and for Dustin Program Parks specimens; Texas R. manuscript; Pelham for and the Kinch, of Jonathan Scott database N. Lisa and A. (BOLD) Hermier, Hermier, Bernard James System Bernard and Data Lamas, MacDonald, Life John of Hermier, Brock- Barcode barcode COI Ernst sharing the ( Hermier, generously and through Bernard specimens for discussions; sequences Opler Mey A. stimulating Paul Wolfram for and mann, Center); and Sciences); Biodiversity care of Texas their Academy of f ( (University Museum (Berlin Lee Reddell Vince County R. Field History); Angeles (The James Baquiran Natural (Los Rebekah Rawlins of and Xie John Maier Museum Weiping Crystal Collection), History); History); Natural (McGuire Wright, of Natural Museum Karen University Sourakov of Riley, A&M Museum Andrei G. (Texas (Carnegie and Edward Oswald Biodiversity); Warren John and and D. 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