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Alignement and phylogenetic tree of 106 Lepidoptera


Pinna, Charline; Piron-Prunier, Florence; Elias, Marianne (2021), Alignement and phylogenetic tree of 106 Lepidoptera, Dryad, Dataset,


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.


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, CodonCode Corporation, 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
  Arctiinae1   TR17_7_MOTH1
  Arctiinae2   11_1065_MOTH2
  Arctiinae3   ME16_15_METASP1
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
Tolype velleda   Tolype_velleda
Phalera bucephala   Phalera_bucephala
  Noto1   ME16_92_GEO4
  Noto2   TR17_1_GEO11
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


Human Frontier Science Program, Award: RGP0014/2016

Agence Nationale de la Recherche, Award: ANR-16-CE02-0012