Alignement and phylogenetic tree of 106 Lepidoptera

We used both published and de novo sequences from one mitochondrial gene and seven nuclear genes, representing a total length of 7433 bp to infer a molecular phylogeny for 106 lepidopteran species. Müllerian mimicry is a positive interspecific interaction, whereby co-occurring defended prey species share a common aposematic signal. In Lepidoptera, aposematic species typically harbour conspicuous opaque wing colour patterns with convergent optical properties among co-mimetic species. Surprisingly, some aposematic mimetic species have partially transparent wings, raising the questions of whether optical properties of transparent patches are also convergent, and of how transparency is achieved. Here we conducted a comparative study of wing optics, micro and nanostructures in neotropical mimetic clearwing Lepidoptera, using spectrophotometry and microscopy imaging. We show that transparency, as perceived by predators, is convergent among co-mimics. Underlying micro- and nanostructures are also convergent despite a large structural diversity. We reveal that while transparency is primarily produced by microstructure modifications, nanostructures largely influence light transmission, maybe enabling additional fine-tuning in transmission properties. This study shows that transparency might not only enable camouflage but can also be part of aposematic signals. Methods We used 106 Lepidoptera species listed at the end of this section. We used published sequences from eight gene regions to infer a molecular phylogeny: the mitochondrial cytochrome oxidase c subunit 1 (COI) gene and the nuclear genes carbamyl-phosphate synthase II (CAD), malate dehydrogenase (MDH), elongation factor 1 alpha (EF-1a), tektin (TKT), ribosomal protein S5 (RpS5), isocitrate dehydrogenase (IDH) and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), which represent a total length of 7433 bp. To improve phylogeny topology, we added 35 species representing 8 additional families to the dataset. When no sequence was available for a particular species on Genbank, we sequenced de novo the COI, CAD and MDH genes of that species. We have missing data for some species, but we had at least the COI sequence for each species considered. For de novo sequencing, DNA was extracted from butterfly legs with a DNeasy® Blood & Tissue Kit (QIAGEN laboratory) and targeted genes were amplified with PCR conditions adapted from Wahlberg and Wheat (2008). COI, CAD and MDH were amplified in two pieces with the primers described in Wahlberg and Wheat (2008). PCR were performed in a volume of 25 µL with 2 to 4 µL of genomic DNA, 1 µL of each primer at a concentration of 100 pmol/µL, 1 µL of nucleotides at a concentration of 2 mM, 2.5 µL of DreamTaq buffer, 0.125 µL of DreamTaq polymerase. The elongation phase was reduced to 70 seconds. For CAD and MDH, the annealing temperature was reduced to 50°C for most specimens. Eurofins Genomics sequenced the PCR products with Sanger method. Sequences were aligned with CodonCodeAligner (version 3.7.1.1, CodonCode Corporation, http://www.codoncode.com/) and concatenated with PhyUtility (version 2.2, Smith and Dunn 2008). The dataset was then partitioned by gene and codon positions and the best models of substitution were selected over all models implemented in BEAST, using the ‘greedy’ algorithm and linked rates implemented in Partition Finder 1.0.1 (Lanfear et al. 2012). We performed a Bayesian inference of the phylogeny using BEAST 1.8.3 (Baele et al., n.d.) on the Cipres server (Miller et al., 2010). We constrained some clades to be monophyletic (notably Ithomiini, Danainae, Nymphalidae, Riodinidae, Pieridae, Papilionidae, Erebidae, Notodontidae, Geometridae, Noctuoidae, Papilionoidae) and we calibrated the crown age and divergence time of some groups, following Kawahara et al. (2019). Four independent analyses were run for 50 million generations, with one Monte Carlo Markov chain each and a sampling frequency of one out of 50 000 generations (resulting in 1000 posterior trees). After checking for convergence of the two best analyses, the posterior distributions of these two runs were combined (using logCombiner 1.8.2, Drummond and Rambaut 2007), with a burnin of 10%. The maximum clade credibility (MCC) tree with median node ages was computed using TreeAnnotator 1.8.2. Species not represented in our dataset were then pruned from the tree. The MCC tree was used for subsequent phylogenetic analyses. List of species : Genus species ssp Tip label in MCCtree Bombyx mori Bombyx_mori Cyclotorna sp. Cyclotorna_sp Drepana curvatula Drepana_curvulata Epicopeia hainesii Epicopeia_hainesii Calodesma albiapex Calodesma_albiapex Dysschema leucophaea Dysschema_leucophaea Dysschema sp. ME15_88_DYSSSP1 Episcea extravagans Episcea_extravagans Euchlaenidia transcisa Euchlaenidia_transcisa Hyalurga egeon ME16_67_HYALEGE Hyalurga fenestrata Hyalurga_fenestrata Hyalurga grandis TR17_17_HYALGRA Hypocrita confluens Hypocrita_confluens Hypocrita strigifera ME16_58_HYPOSTRI Hypocrita strigifera ME16_44_HYPOSTR Hypocrita strigifera ME16_64_HYPOSTR Notophyson tiresias ME16_63_NOTOTIR Notophyson tiresias TR17_9_NOTOTIRE Notophyson tiresias TR17_8_NOTOTIR Notophyson tiresias TR17_4_NOTOTIRE Pseudophaloe troetschi Pseudophaloe_troetschi Sthenognatha gentilis Sthenognatha_gentilis Metastatia pyrrhorhoea Metastatia_pyrrhorhoea ME16_16_METASP2 TR17_13_CHTEN2 11_996_CHTEN1 Arctiinae1 TR17_7_MOTH1 Arctiinae2 11_1065_MOTH2 Arctiinae3 ME16_15_METASP1 ME16_91_MOTH3 Inurois fumosa Inurois_fumosa Biston panterinaria Biston_panterinaria Plutodes costatus Plutodes_costatus Geo1 11_1002_GEO1 Geo2 ME16_100_GE02 Hagnagora mortipax Hagnagora_mortipax Operophtera brumata Operophtera_brumata Geo9 TR17_16_GEO9 Geo12 ME16_105_GEO1 Geo3 11_1879_GEO3 ME16_87_GEO1 11_1001_GEO2 Tolype velleda Tolype_velleda Phalera bucephala Phalera_bucephala Noto1 ME16_92_GEO4 Noto2 TR17_1_GEO11 11_994_GEO5 11_1064_GEO6 TR17_15_GEO4 Vila azeca LS11_2414_ERES1 Vila emilia ME16_56_ERESCLI Lycorea ilione Lycorea_ilione Callithomia lenea zelie Callithomia_lenea_zelie Godyris hewitsoni Godyris_hewitsoni Godyris panthyale panthyale Godyris_panthyale_panthyale Heterosais nephele Heterosais_nephele_nephele Hyalenna paradoxa praestigiosa Hyalenna_paradoxa_praestigiosa Hypomenitis enigma pseudortygia Hypomenitis_enigma Hypomenitis lydia Hypomenitis_lydia Hypomenitis oneidodes Hypomenitis_oneidodes Hypomenitis ortygia ortygia Hypomenitis_ortygia_ortygia Hypomenitis theudelinda zalmunna Hypomenitis_theudilinda_zalmunna Ithomia agnosia agnosia Ithomia_agnosia_agnosia Ithomia amarilla Ithomia_amarilla Ithomia avella epona Ithomia_avella Mcclungia cymo Mcclungia_cymo Megoleria orestilla orestilla Megoleria_orestilla Methona curvifascia Methona_curvifascia Napeogenes harbona Napeogenes_harbona Napeogenes inachia Napeogenes_inachia Napeogenes larilla Napeogenes_larilla Napeogenes sylphis corena Napeogenes_sylphis_corena Oleria athalina banjana Oleria_athalina_banjana Oleria onega Oleria_onega Oleria sexmaculata sexmaculata Oleria_sexmaculata_sexmaculata Ollantaya olerioides Oleria_olerioides Pseudoscada florula aureola Pseudoscada_florula_aureola Pseudoscada timna utilla Pseudoscada_timna_utilla Pteronymia oneida oneida Pteronymia_oneida_oneida Veladyris pardalis Veladyris_pardalis Eresia nauplius plagiata Eresia_nauplius_plagiata Nymp1 ME16_46_NYMP1 Papilio glaucus Papilio_glaucus Parides hahneli Parides_iphidamas Dismorphia teresa TR17_18_DISMSP1 Dismorphia theucharila leucone TR17_14_DISMTHEU Dismorphia theucharila orange tip TR17_6_DISMTHEU Dismorphia theucharila yolanda LS11_2240_DISTH Dismorphia zathoe Dismorphia_zathoe Moschoneura pinthous TR17_3_MOSCPINT Moschoneura pinthous Moschoneura_pinthous_GB Patia orise Patia_orise Itaballia demophile Itaballia_demophile Itaballia pandosia ME16_62_ITAPAND Itaballia pandosia Itaballia_pandosia Perrhybris ? Pier1 MECN_140_PIER1 Pyralis farinalis Pyralis_farinalis Ithomiola callixena Ithomiola_callixena Ithomiola floralis orange tip 11-1116_ITHOFLO Ithomiola floralis white-band 11_1127_ITHOFLO Stalachtis euterpe TR17_5_STALEUT Stalachtis euterpe Stalachtis_euterpe_GB Riodin2 TR17_12_GEO7 Riodin1 MECN_133_BUT1 Riodin3 TR17_11_GEO8 Aglia tau Aglia_tau