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 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 :