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N.V.G. and and D.H.J., W.H., D.H.J., J.Z., J.Z., and J.S., W.H., Q.C., Q.C., J.Z., W.L., J.S., research; Q.C., W.L., performed research; N.V.G. designed N.V.G. and D.H.J. contributions: Author in listed DNA- collections S1 a in Table in pinned Appendix, preserved were and Others 2012 manner. friendly after collected were specimens ssgetdb eea tde 2 ,1) suiu n om a forms and unique is 16), 3, (2, studies several of by sets suggested endemic Australian as larger 1). 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EVOLUTION Fig. 1. Time-calibrated genomic tree of Hesperiidae constructed from nuclear gene coding regions of 250 species. The tree is cut in half: First and second segments are shown above and below. Colored horizontal lines delineate major taxonomic groups: red, subfamilies; orange (broken in several places), tribes; yellow shaded red (Eudaminae only), subtribes; and green shaded yellow (Eudaminae only), genera. New taxa are highlighted in yellow. New genera are in red font highlighted in yellow, and those used in different -species combinations than previously are in red font. Taxa placed in a different tribe or subtribe than previously are in blue font highlighted in green. Segments of the tree corresponding to different tribes are highlighted in different colors. Illustrated Hesperiidae species are associated with names in the tree by numbers in brown font. See SI Appendix for details.

2 of 6 | www.pnas.org/cgi/doi/10.1073/pnas.1821304116 Li et al. Downloaded by guest on September 29, 2021 Downloaded by guest on September 29, 2021 e tribe new Grishin, Pericharini tribe new Grishin, Butleriini subtribe new Grishin, Clitina subtribe new yhdnn Grishin, Typhedanina Grishin, Oileidini subtribe new Grishin, Telemiadina subtribe e tribe new yhndn Grishin, Pythonidina Ntto 7.:12 en oiin12i h ee1o h cfod22i ,cagdfo nteacso;19,maspsto 6 sA u h neta tt sucer O.ci h O acd region. barcode COI the is COI.bc unclear; is state ancestral the but A, al. is et 169 Li position means 169A, ancestor; the in A from changed G, is 272 scaffold the on 1 gene the in 192 † position means 272.1:A192G *Notation new Grishin, Loboclina tribe new Grishin, Entheini Hesperiidae of subtribes and subtribe or tribes tribe new New of Description to 1. according Table placed were species relatives. barcode-only expected these their cytochrome of of mitogenomes with ( Most with only species grouped barcodes 290 species (COI) increased adding we I Next, backbone, by oxidase of a phylogenies. species as coverage All genome same species taxonomic 250 the nuclear support. of of or mitogenomes weaker composed using mitogenome with are either tribes but in and of tree, subfamilies groups genome the phylogenetic major nuclear recapitulates mitogenomes. the from tree tree resulting a constructed The we tree, genome Barcodes. nuclear I Oxidase Cytochrome and Mitogenomes Mya. 50 about occurred subfamilies into and diversification 1 and (Fig. subfamilies nine of consists diver- Hesperiidae the before their Mya, 55 from genceofGrassskippers.Thus,genomicdatasuggestthatthefamily about diverged Pyrginae with They been ancestor have subfamily. common and a skippers, considered other strik- from traditionally are appearance latter in The distinct Pyrrhopyginae. ingly Tagiadinae, subfamily sister subfamily the the a reinstate unify in and we Celaenorrhinini Accordingly, and such. Tagiadini as tribes them subfam- treat Hesperiidae we than and distinct ilies, less no are Pyrginae within groups Warren’s three divergence into these before Thus, subfamilies. split and into time skippers have Grass short of Warren a within sensu groups, Pyrginae compact three other, each from sep- Pyrginae well arated are except Hesperiinae) and subfamilies Heteropterinae, other Eudaminae, (i.e., while Moreover, time that Mya). before ancestor (>55 common their from diversified (3), byWarren al. et (Fig.1).Pyrginae,asdefined Trapezitinae) 50Mya and (Hesperiinae havesplitoffabout subfamilies diverging The subfamilies. latest these two of monophyly 99%) sup- above strongly previously (bootstrap very port data been Genomic have subfamilies. several skippers into Grass divided and Spreadwing sis- are Both feeders) with monocot ters. dicot that (mostly agreement skippers (mostly from Grass in skippers the different and but Spreadwing feeders) studies, is The view. DNA tree morphological of genomic the number the a in in split appearing next The Coeliad- except inae. Hesperiidae other all to sister own, its of subfamily e tribe new new Grishin, Cephisina subtribe eiiGrishin, Jerini tribe new Grishin, Netrocorynini subtribe new oBn eitainULgvnfrec ao hudb rcddby preceded be should taxon each for given URL registration ZooBank See IAppendix SI o h it fgnr nlddi ahtxnadsqecso rti-oigrgoswt igotccharacters. diagnostic with regions protein-coding of sequences and taxon each in included genera of lists the for Perichares Butleria Typhedanus Oileides Telemiades Pythonides Lobocla Entheus Cephise Jera Netrocoryne ide,1925 Lindsey, vn,1953 Evans, or,1884 Moore, vn,1952 Evans, ib,1871 Kirby, H H be,[1819] ubner, be,[1825] ubner, ¨ ¨ cde,1872 Scudder, yegenus Type H H ulr 1870 Butler, .&R edr 16]28.039;941:49;251.:95;998.8:G308A; 275215.7:C925G; 904.14:T439G; 2284.30:399A; [1867] Felder, R. & C. be,[1819] ubner, be,[1819] ubner, ¨ ¨ .These S1). Fig. Appendix, SI http://www.zoobank.org/. nadto oa to addition In e.12 ref. 12 ref. 11 ref. in E.13.8 40d or E.37a then undivided, uncus if or, E.49.1 E.44a, to keys .,C3 .a(xld ..b,E6 rE9i es 0ad11 and 10 refs. in E.9 or E.6a C.7.6b), (exclude C.7a C.3, B.2, for 182–183) (pp. e.1;frwn cell forewing 11; ref. .ai e.1,btecueB.9 exclude but 10, ref. in B.3a fsae yaa odfo h aeo idig ihraoeo below or above either hindwing, of base the from fold anal by scales of 11 ref. in C.18 or C.15.2 C.10a, 1.432C est .,C1o .5i e.8 ref. in C.15 or C.1 B.1, to keys 214.24:3520C; below not but above, hindwing of base the from fold anal by scales 311:281;495G0T 468T38,C39,A50;tf of tuft A2500C; C2309G, 3446.8:T2308A, 489.5:G307T; 1341.12:T25841C; O.cA4,C4,T7A eiai n ap sdsrbdi e.17 ref. in described as palpi and genitalia T479A; C84G, COI.bc:A44T, 191:52;818C2A 43;1951:31;6.:30;tuft 65.4:C330A; 11945.11:G391A; G443A; 851.8:C423A, 1139.19:T562A; 678A49;114C0A 73.8G3T 14;ky oH4&5in 5 & H.4 to keys G134C; 37338.38:G133T, 141.4:C104A; 2627.8:A1459T; 3.4:18C 9.:54;807A4G 013C73;ky to keys 3001.3:C1773T; 860.7:A748G; 997.8:G514T; 536.149:G1488C; 0.:15,T4G 81.:69,G2A est . nrf rC.5, or 8 ref. in B.4 to keys G620A; 18312.8:A619C, T146G; 208.2:G145T, 7.9G9A 486T1C 955G1A 2.0G9C nrf 11, ref. in 925.10:G199C; 7985.5:G916A; 3478.6:T116C; 274.29:G397A; 7652:12;8.8C7T 7.9G09;341:16T esto keys 374.14:G1169T; 378.19:G1099C; 85.28:C176T; 276665.26:A192G; 0.376;402:38;612:3C 2.:15T est . in E.3 to keys 425.5:G1558T; 671.27:935C; 420.27:G308A; 103.23:796A; 9.:10G 4.1G0C 31:85AT67;ky oK2ain K.27a to keys 83.15:G8658A,T8657G; 144.41:G201C; 596.8:C1601G; O.cG9,8A 6A 6A 0T 33,A2T est .2or E.52 to keys A521T; A353T, 302T, 266A, 169A, 81A, COI.bc:G29T, IAppendix), SI Cephise > / fcosta of 3/5 igotccharacters* Diagnostic r diagnostic are hti o vni uaia Fg ) Interestingly, 1). (Fig. Eudaminae segment: in even third not central the is the that by have Cabirus, in that it outer included genera the Evans defined Phocides three from Inconsistently, He group segment. stems second this that Evans. the palpi of of of largely edge segment group” is third Entheini Augiades “divergent” example, “B. For 11). the (10, morphology similar rank tribal distinction. a morphological given their are to and due Mya, 40 and about form and Eudamini diverged sisters that Oileidini that related Phocidini as tribes closely are 1 the others Table two of The in Entheini. and Two described tribes. 100% are tribes as groups by into best-separated define supported we divergence groups that Pyrginae phylogenetic bootstrap of Eudaminae four time find the around tree subfamily. the in groups conspicuous 100% are received and tribes support Pyrginae bootstrap All Achlyodini. Pyrginae: but Conversely, (4), Eudaminae. to belongs and ; places oniades tree it genomic that Our except ina, Mya. 42 Warren’s, around with diverged Pyrginae agrees (3) four al. et The Warren stricto). outlined by Pyrgini) (sensu and Erynnini, Pyrginae Achlyodini, in (, tribes tribes the the with this of consistently on tribes age delineate focused to We attempted Eudaminae. and subfamily subfamily the in tribes defining Subtribes. and Tribes Eudaminae and genitalic and species, pattern be wing to Appendix, for (SI detail likely differences in more analyzed are were (10–12) cases such Evans by defined as subspecies 30 that suggested morphology and barcodes in ferences clas- Appendix, proposed our (SI for Hesperiidae of basis sification the as 1) (Fig. tree with genome together nuclear tree the barcode + mitogenome this used in We discussed Appendix. exceptions several with classification, current their hs orEdmnetie orsodt ruswith groups to correspond tribes Eudaminae four These genomic dated the cut we classification, consistent achieve To Myrinia, nAchlyodini; in and htd o eogt nhii and Entheini, to belong not do that Hypocryptothrix, Xispia, Jera nPrhpgne Unexpectedly, Pyrrhopyginae. in Pseudodrephalys, Cornuphallus aooi Appendix). Taxonomic rvossuisrfandfrom refrained studies Previous B4D56F93-67F9-476F-B69C-133D98BFBD58 22B59811-F174-4FDF-A9D2-799897F4D44E CF9C3D29-523A-4D17-B140-9A69CFA98731 D621EF81-FA65-4858-9450-E0C041598D7A 4AE0E59C-8B92-4C84-8651-E7A1C45C93C1 DE61F048-02CF-4F8E-9392-D18A4618BABD C606FC35-323D-4E55-AF5A-A86C6366BAFA CB890271-5483-4B5A-A7BC-27DBC5E23DE5 303C1FD0-07CB-4919-900E-EA3D6347E5DD AF3B5CEA-880A-4CB2-AF40-E6D87C39C040 94B68BD2-7F83-4E58-80E1-7F5AC8C56511 971884E2-E5F7-46A3-B182-657729B6A778 Cabirus .Dif- Appendix). Taxonomic nCarcharodini; in Mimia, NSLts Articles Latest PNAS Grais, oBn ID ZooBank sntEudaminae not is and , Tosta, † Emmelus Clito | Morv- f6 of 3 Spi- SI in

EVOLUTION Phareas that has a divergent third segment (and was included gence Within a Genus). Along with Lobocla, Loboclina unifies in the Augiades group) is not in Entheini. Its males possess, genera with the arcuate antennal club from the “C. Urbanus absent in Entheini, tufts of hair-like scales in the groove along group” of Evans plus Venada, Aguna, and a new genus, Zeutus the hindwing vein 1A+2A. Our tree places this unusual skipper (Table 2). Others belong to the “crown” group of Eudaminae. It in Phocidini, implying that its peculiar palpi are convergent. includes an array of skippers that have been largely misclassified Oileidini is sister to other Eudaminae (Fig. 1). Genera in due to widespread and possibly mimetic convergence as detailed this tribe were grouped with some Pyrginae genera by Evans in Widespread Convergence in Wing Patterns and Shapes. (11), and the tribe may be intermediate in morphology between Pyrginae and Eudaminae. This is the smallest tribe (six genera) Eudaminae Genera and Subgenera. We define a major phyloge- and is characterized by tufts of hair-like scales in the groove netic cluster of species with a common ancestor existing within a along the hindwing vein 1A+2A in males, either below (Oileides) certain timeframe as being a genus (18). We cut a dated phyloge- or above (the others). Similar structures in Phareas (Phocidini) netic tree at a time point to maximize agreement with the current are found on both sides of the hindwing. classification. This neither splits nor merges most genera that are The sister tribes Eudamini and Phocidini are separated from well defined by morphological features, and we treat as genera each other by a short branch and could be one tribe. How- the groups of species supported by the cut branches. The time ever, each is strongly monophyletic, and Phocidini skippers stand 15 Mya corresponds to such point. It keeps well-known genera out morphologically and ecologically: Their forewing veins R4 Aguna, Udranomia, and Urbanus proteus group unsplit. How- and R5 originate near each other, hindwing tornus is usually ever, it separates traditional and morphologically distinct pairs of expanded (not lobed), skippers hold wings spread flat when genera such as Epargyreus and Chioides. We attempt to reduce resting, many are crepuscular, and many species are sexually the number of monotypic genera, unless the genus is strongly dis- dimorphic. Our Phocidini is the “D. Celaenorrhinus group” of tinct, because we wish to indicate relationships to other species Evans (10) after adding Phocides, Phareas, and Emmelus and by the name of a genus. As a result (Fig. 1 and SI Appendix), removing Cephise, which has lobed or tailed hindwing tornus, we delineated 50 Eudaminae genera, 4 of which are described as and Celaenorrhinus, whose males have a tuft of long scales on the new in Table 2. The number of monotypic genera decreased from hind tibiae that fits into a thoracic pouch (not found in Eudami- 10 to 4: Nicephellus, Spathilepia, and Zeutus (19), plus Emmelus nae). The genomic tree suggests that Oileides is polyphyletic: one transferred from Pyrginae. These four genera diverged from species together with Aurina belong to Phocidini. their sister taxa at least 18 Mya and are morphologically distinct. Eudamini is the largest and most diverse tribe. It encom- Within some genera, we see groups of species that may be passes more than half of the subfamily. The genomic tree reveals defined as subgenera (SI Appendix), and three new subgenera groupings within the tribe that are described here as subtribes are described (Table 2). Some of the subgenera, such as Tho- (Table 1). One of them, subtribe Cephisina, is monotypic for the rybes, have been used as genera for decades, but their genomic and genus Cephise, which diverged from its sister tribe Telemiadina morphological distinctness is smaller compared with most genera. 35 Mya and is unique in its morphological features (17). Telemi- adina includes three genera: Telemiades with its close sister Poly- Widespread Convergence in Wing Patterns and Shapes. The most gonus and Ectomis, into which we sink Hypocryptothrix, Heronia, unexpected result of this study is the astounding number of mis- Polythrix, Chrysoplectrum, and Speculum (see Uncanny Diver- placements of species into genera they do not belong to. The

Table 2. Description of new genera and subgenera of Hesperiidae

New genus or subgenus Type species Diagnostic characters* Derivation of the name† ZooBank ID‡

Tekliades Grishin, new Thymele ramanatek 68A, C90T, T145C, 412T, 553A, 583T; keys to I.1.9 Masculine, a blend of 081564BA-DA0C-4C46-AEAB-6C00131AC8BD genus Boisduval, 1833 in ref. 7 [ramana]Tek and [Coe]liades

Salantoia Grishin, Eudamus eriopis 59C, A79T, T163A, 530T, 598A, T637A; D.3.2 or 3 Feminine, a blend of Sala[tis] and 3F82E9DE-A5A2-44B3-A13D-53CF8A673FAE new genus Hewitson, 1867 in ref. 10; harpe flat, not hook-shaped [Sarmie]ntoia

Spicauda Grishin, Goniurus procne 307T, T349A, 424(not T), G506A, T562A; keys to Feminine, a blend of spica and 14D26B57-940C-407B-8E70-4E25203044B8 new genus Plotz,¨ 1881 C.13.13c in ref. 10; harpe dorsally spiked cauda

Urbanoides Grishin, Goniurus esmeraldus T49A, A85T or C, T212A, C542T, T544A, A607C or T, Masculine, means similar to 20FAC3B6-F038-40A0-B182-3C7F32A40702 new subgenus Butler, 1877 T619A or G; keys to C.13.6a in ref. 10 Urbanus

Zeutus Grishin, Cecropterus zeutus A22T, C271A, T278A, A526T, T548C, A607T; Masculine, echoes the type 75715B9C-46AB-40F5-B738-420DABD56B63 new genus Moschler,¨ 1879 genitalia as for zeutus in ref. 19 (p. 27) species name

Lobotractus Grishin, Eudamus valeriana 49A, T400A, 401T, A477G, 517T, C542T, T619A; as Masculine, a blend of Lobo[cla] C6E5B5DF-1C74-4DBD-85C3-7285209F6F03 new genus Plotz,¨ 1881 given for the ”cyda group” in ref. 17 (p. 196) and [Coda]tractus

Caudatractus Grishin, Eudamus alcaeus 355A, T556A, A592T; Codatractus (C.11 in ref. 10) Masculine, includes tailed species DF0F3C91-F56E-4B65-B86C-385A36F9D7FD new subgenus Hewitson, 1867 with tailed hindwing of Codatractus

Asina Grishin, Eudamus asine T70A, T127A, T197C, 206T, 208A, A256T, T346A, Feminine, derived from the type B3B7A6F6-A95C-4A2E-B9FB-80A7A8F86761 new subgenus Hewitson, 1867 373A; keys to C.7.2a in ref. 10 species name

Tiana Grishin, Ebrietas niger T16C, A43T, G86A, T142C, A196G, T278A, 283C; Feminine, a blend of T[osta] and B9382699-24FB-4466-B39B-94E6B544C425 new genus Williams & Bell, 1940 keys to F.7.3 or 4 in ref. 11 [Il]iana

Chirgus Grishin, Hesperia limbata 85A, 205A, 223A, 241A, 263(not C), T277A, A415T, Masculine, a blend of Chi[lean] 7B1905F1-9471-4BBF-90BF-32360783AB1E new genus Erschoff, 1876 479T, T574A; keys to G.1.2e or 9 in ref. 11 and [Py]rgus

Burnsius Grishin, Syricthus communis 205T, 223A, 241T, 263(not C), T277A, 479T; keys Masculine, honors skipper 48996B74-3AB1-4DEA-9A64-B8F112E62343 new genus Grote, 1872 to G.1.5, 8, or 10a in ref. 11 taxonomist John M. Burns

Duroca Grishin, Hesperia duroca 127T, 163C, 349C, T424C; keys to J.39.5a in Feminine, echoes the type 476FE13C-5895-4139-BB11-44F835E21565 new genus Plotz,¨ 1882 ref. 12, harpe broad, hook-shaped species name

See SI Appendix for the lists of species included in each taxon. *Sequence characters are for the COI barcode region and only their combination is diagnostic to distinguish from former genera of these species. Notation A79T means position 79 is T, changed from A in the ancestor; 59C means position 59 is C, but the ancestral state is unclear. †All names are treated as nouns in the nominative singular. ‡ZooBank registration URL given for each taxon should be preceded by http://www.zoobank.org/.

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Downloaded by guest on September 29, 2021 Downloaded by guest on September 29, 2021 yisapaac nawoggnsbfr u td (crossed study our before genus placed wrong was a 10) in of appearance out its (10 by species every Curiously, convergent. tornus. yellow with hindwing forewing; and brown wings, the brown (v) on and spots spots; white semitranslucent (ii) apical cream-white, and margins; across (iv) stripe and yellow (iii) a tail (20); with stripe forewing white white brown with a on forewing genera and stripe with bases, wing white two hindwing metallic-blue wings, and in brown forewing, of other the bases different greenish each five (i) find parallel (outgroups): We that 23). 2) (Telegonus (20, beetles (Fig. in and and and, phenotypes families World, flies, other New moths, from and butterflies even Old include Some the not skippers, diverse. to both selec- is more addition in is convergence the but common patterns, This are by basic patterns (20–22). two driven or rings one convergence to mimicry confined to large them for tion attribute and nae white and bases wing metallic-cyan spots. forewing shiny of from consisting patterns tails long without to skippers some transferred Moreover, we skippers. tailed included while previously 2), not Table have others (Spicauda, the here named three is to genus them One transferred genera. we and other there, belong not do them of placed half were skippers genus tailed the many in work, problematic. our more before is its example, feature by For phenotypic genus eye a human dif- into to genetic species dominant of a a indicative of as placement be such However, may pattern, ferentiation. forewing) wing the or across hindwing, stripe tailed (e.g., pale a as distinction such phenotypic shape, Apparently, wing Appendix ). 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EVOLUTION had the largest number of such mutations (SI Appendix, in a vial with RNAlater. For pinned and dry specimens from museum col- Table S6). This enzyme is involved in pterin metabolism, and lections, DNA was extracted from a whole abdomen or a leg. We have pterin derivatives were suggested to be responsible for wing previously reported details of methods for DNA extraction, genomic library coloration in pierid butterflies (25). The T. chalco-specific muta- preparation, next-generation sequencing, and computational analysis of tions may alter interactions between the enzyme and the trans- nuclear and mitochondrial genomes (13, 27). Phylogenetic trees were constructed with the programs RAxML (28) and ASTRAL (15), and were port proteins that deliver the enzyme to different pigment gran- dated using the procedure described in SI Appendix, where further details ules (26), and thus affect the wing color in T. chalco. Some of the methods are given. Genomic data obtained in the project have of these mutations are present in other skipper species with been deposited in the SRA database (accession SRP147939), and COI bar- extensive white scaling on hindwings. codes have GenBank accessions MH357724 through MH357835. ZooBank registration for this work, published on March 15, 2019, is 8A8E82C4- Discussion: A Broad Picture. Today, and more so tomorrow, AC8A-4B7B-8DF4-6BFF4BD48F3D. genome sequencing provides an efficient and cost-effective way ACKNOWLEDGMENTS. We thank the Area de Conservacion´ Guanacaste, to gain rapid insights about biodiversity. Given a taxonomic Costa Rica (ACG) parataxonomists who found and reared many specimens group, comparative analysis of genomic sequence along with used in this study. We are grateful to Robert K. Robbins, John M. Burns, and morphology and ecology will be rich in discoveries. Even now, Brian Harris (National Museum of Natural History, Smithsonian Institution); this can be done within a small laboratory. With the family Hes- Blanca Huertas, David Lees, and Geoff Martin (Natural History Museum, London); David Grimaldi and Courtney Richenbacher (American Museum periidae, we have shown how such a project can be accomplished of Natural History); Andrew D. Warren and Andrei Sourakov (McGuire and have reported some of the results that can be expected. Center for Lepidoptera and Biodiversity); Edward G. Riley, Karen Wright, Not only is this family interesting as a diverse group of but- and John Oswald (Texas A&M University Collection), John Rawlins terflies, it also emphasizes several generalities. First, while the (Carnegie Museum of Natural History); Weiping Xie (Los Angeles County Museum of Natural History); Crystal Maier and Rebekah Baquiran (The Field reference genomes required freshly collected specimens, the Museum of Natural History); Vince Lee (California Academy of Sciences); bulk of the project was done with old museum samples. Even James R. Reddell (University of Texas Biodiversity Center); Wolfram Mey specimens collected a century ago yielded usable genomic data. (Berlin Museum fur¨ Naturkunde) for granting access to the collections under Second, we provided a genome-based phylogeny of the family their care and for stimulating discussions; Bernard Hermier, Ernst Brock- mann, and Paul A. Opler for specimens and generously sharing COI barcode and reclassified it taxonomically. We found that many species sequences through the Barcode of Life Data System (BOLD) database were classified differently than previously thought. Other fami- (www.boldsystems.org/); Jim P. Brock, William R. Dempwolf, Bernard lies may display the same problem when examined genomically. Hermier, John MacDonald, and James A. Scott for specimens; Gerardo Third, we encountered many examples of phenotypic conver- Lamas, Bernard Hermier, and Jonathan Pelham for fruitful discussions; Bernard Hermier, Lisa N. Kinch, and R. Dustin Schaeffer for critical reading gence and divergence, and mined genomic data for the links of the manuscript; Texas Parks and Wildlife Department (Natural Resources between genotype and phenotype. With the ever-decreasing cost Program Director David H. Riskind) for Research Permit 08-02Rev; and US of sequencing, we expect that, soon, any phylogenetic project will National Park Service for the research permits: Big Bend (Raymond Skiles) start from sequencing of genomes. for BIBE-2004-SCI-0011 and Yellowstone (Erik Oberg and Annie Carlson) for YELL-2017-SCI-7076. We acknowledge the Texas Advanced Computing Cen- ter at The University of Texas at Austin for providing high-performance Materials and Methods computing resources. The study was supported, in part, by National Insti- For freshly collected specimens, DNA was extracted from tissue of a speci- tutes of Health Grants GM094575 and GM127390 (to N.V.G.) and the Welch men (minus wings and genitalia that were kept in an envelope) field-stored Foundation Grant I-1505 (to N.V.G.).

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