SI Appendix to "Genomes of skipper butterflies reveal extensive convergence of wing patterns" by Wenlin Li, Qian Cong, Jinhui Shen, Jing Zhang, Winnie Hallwachs, Daniel H. Janzen and Nick V. Grishin Published in the Proceedings of the National Academy of Sciences of the United States of America (Online ISSN 1091-6490) on March 15, 2019 as part of the article "Genomes of skipper butterflies reveal extensive convergence of wing patterns" and archived together with it. ZooBank registration for this work is 8A8E82C4-AC8A-4B7B-8DF4-6BFF4BD48F3D. Table of Contents Taxonomic Appendix .................................................................................................................................... 3 T1. Taxonomic Abstract ................................................................................................................................ 3 T2. Subfamilies, tribes and subtribes in the family Hesperiidae ................................................................... 6 T3. New tribes and subtribes described in Table 1 ....................................................................................... 8 T4. New genera and subgenera described in Table 2 ................................................................................... 9 T5. Systematic list of Eudaminae proposed in this work ............................................................................ 10 T6. Systematic list of Eudaminae prior to this publication ......................................................................... 28 T7. New World Pyrgus, Heliopetes and Heliopyrgus reclassified ................................................................ 46 T8. Justification for changes to Hesperiidae genera ................................................................................... 48 T9. Justifications for the change of status from subspecies to species ...................................................... 49 T10. Taxonomic discussion ......................................................................................................................... 53 Modified (vertical and uncut) Fig. 1 from the main text .......................................................................... 56 Expanded legend to Fig. 1 from the main text (and shown above, modified) ......................................... 57 Methods ...................................................................................................................................................... 58 M1. Sample collection and genomic DNA extraction ................................................................................. 58 M2. Sequencing library preparation protocol ............................................................................................ 58 M2.1. Paired-end library preparation protocol ...................................................................................... 58 M2.2. Preparation for sequencing on the Illumina Hiseq X ten platform .............................................. 60 M3. Nuclear and mitochondrial protein-coding gene assembly ................................................................ 61 M3.1. Processing of sequencing reads ................................................................................................... 61 M3.2. Preparation for protein-coding gene assembly ............................................................................ 61 M3.3. Protein-coding sequence assembly for phylogenetic study from sequencing reads ................... 62 M3.4. Coding sequence assembly for speciation hotspot studies.......................................................... 62 M3.5. Assembling mitochondrial protein-coding genes ......................................................................... 63 M3.6. Special procedure to handle extremely poor samples ................................................................. 63 1 w ww.pnas.org/cgi/doi/10.1073/pnas.1821304116 M4. High quality COI barcode assembly strategy ....................................................................................... 64 M4.1. Preparing database of barcodes ................................................................................................... 64 M4.2. Selecting barcode baits ................................................................................................................. 64 M4.3. Checking consistency and removing contamination .................................................................... 65 M4.4. Reference-based assembly ........................................................................................................... 65 M4.5. De novo assembly ......................................................................................................................... 65 M4.6. Barcode validation ........................................................................................................................ 66 M5. Phylogenetic analysis ........................................................................................................................... 66 M5.1. Construction of nuclear and mitochondrial genome alignments................................................. 66 M5.2. Construction of phylogenetic trees .............................................................................................. 67 M5.3. Detection of diagnostic nucleotide characters ............................................................................. 68 M6. Identification of genes and SNPs that correlate with morphological differences between mimicry complexes ................................................................................................................................................... 69 M6.1. Detection of genomic islands for speciation ................................................................................ 69 M6.2. Detection of fixed mutations in mimicry complexes .................................................................... 70 M7. References ........................................................................................................................................... 70 Table S1. Data for 250 sequenced Hesperiidae specimens ...................................................................... 72 Table S2. Data for Hesperiidae specimens with COI barcodes ................................................................. 75 Table S3. Mitochondrial genomes downloaded from NCBI ...................................................................... 77 Table S4. Proteins that are of low intra-species but high inter-species divergence between Telegonus brevicauda and Telegonus chalco .............................................................................................................. 77 Table S5. Enriched GO terms associated with proteins in Table S4 ......................................................... 88 Table S6. Genes and mutations that may be related to the phenotype of “extensive white scaling on hindwings” .................................................................................................................................................. 90 Table S7. Abbreviations of collections we used specimens from ............................................................. 90 Figure S1. Phylogenetic tree of Hesperiidae based on coding regions from mitogenome...................... 91 Legend to Figure S1: Phylogenetic tree of Hesperiidae based on coding regions from mitogenome .... 92 SI: Diagnostic nucleotide characters mapped to the reference genome of Cecropterus lyciades .......... 93 2 Taxonomic Appendix (pages 3 to 55) This section should be viewed together with Tables 1 and 2 in the main text, and is illustrated with Figure 1 in the main text and Figure S1 in the SI Appendix (pp. 91-92 below) T1. Taxonomic abstract On the basis of genome-scale phylogenetic analysis augmented with the analysis of COI barcodes and morphology, we revised higher classification of the family Hesperiidae Latreille, 1809 (Lepidoptera). As a result, the family is partitioned into 9 subfamilies, and 23 tribes and 20 subtribes are delineated. New status (a subfamily) is given to Tagiadinae Mabille, 1878 (includes Celaenorrhinini Swinhoe, 1912) and Pyrrhopyginae Mabille, 1877 is reinstated as a subfamily. The subfamily Eudaminae Mabille, 1877 is reclassified into 50 genera and a comprehensive taxonomic list is provided at the subspecies level. The status of a tribe is given to: Phocidini Tutt, 1906, Astictopterini Swinhoe, 1912, Erionotini Distant, 1886, and Megathymini Comstock & Comstock, 1895. Six tribes are described as new: Entheini Grishin, trib. n., Oileidini Grishin, trib. n., Netrocorynini Grishin, trib. n., Jerini Grishin, trib. n., Butleriini Grishin, trib. n., and Pericharini Grishin, trib. n. New status (a subtribe) is given to: Thymelicina Tutt, 1905, Calpodina Clark, 1948, Carystina Mabille 1878, Anthoptina A. Warren, 2009, and Moncina A. Warren, 2008. Six subtribes are described as new: Loboclina Grishin, subtr. n., Cephisina Grishin, subtr. n., Telemiadina Grishin, subtr. n., Typhedanina Grishin, subtr. n., Pythonidina Grishin, subtr. n., and Clitina Grishin, subtr. n. The following genera are transferred: Emmelus O. Mielke & Casagrande, 2016 to Phocidini; Jera Lindsey, 1925 to Pyrrhopyginae; Cornuphallus Austin, 1997 to Carcharodini Verity, 1940; Cabirus Hübner, [1819], Grais Godman & Salvin, 1894, Tosta Evans, 1953, Morvina Evans, 1953, Myrinia Evans, 1953, Xispia Lindsey, 1925, Pseudodrephalys Burns,