bioRxiv preprint doi: https://doi.org/10.1101/844175; this version posted November 16, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

A complete time-calibrated multi-gene phylogeny of the European butterflies

Martin Wiemers1,2*, Nicolas Chazot3,4,5, Christopher W. Wheat6, Oliver Schweiger2, Niklas Wahlberg3

1Senckenberg Deutsches Entomologisches Institut, Eberswalder Straße 90, 15374 Müncheberg, Germany

2UFZ – Helmholtz Centre for Environmental Research, Department of Community Ecology, Theodor- Lieser-Str. 4, 06120 Halle, Germany

3Department of Biology, Lund University, 22362 Lund, Sweden

4Department of Biological and Environmental Sciences, University of Gothenburg, Box 461, 405 30 Gothenburg, Sweden.

5Gothenburg Global Biodiversity Centre, Box 461, 405 30 Gothenburg, Sweden.

6Department of Zoology, Stockholm University, 10691 Stockholm, Sweden

*corresponding author: e-mail: [email protected]

Abstract

With the aim of supporting ecological analyses in butterflies, the third most species-rich superfamily of Lepidoptera, this paper presents the first time-calibrated phylogeny of all 496 extant butterfly species in Europe, including 18 very localized endemics for which no public DNA sequences had been available previously. It is based on a concatenated alignment of the mitochondrial gene COI and up to 11 nuclear gene fragments, using Bayesian inference of phylogeny. To avoid analytical biases that could result from our region-focus sampling, our European tree was grafted upon a global genus- level backbone butterfly phylogeny for analyses. In addition to a consensus tree, we provide the posterior distribution of trees and the fully-concatenated alignment for future analyses.

Keywords

Phylogeny; time tree; European Butterflies;

Background & Summary

The incorporation of phylogenetic information in ecological theory and research has led to significant advancements by facilitating the connection of large-scale and long-term macro-evolutionary processes with ecological processes in the analysis of species interactions with their abiotic and biotic environments1,2. Phylogenies are increasingly used across diverse areas of macroecological research3, such as studies on large-scale diversity patterns4, disentangling historical and contemporary processes5, latitudinal diversity gradients6 or improving species area relationships7. Phylogenetic information has also improved studies on assembly rules of local communities8-10, including spatiotemporal community dynamics11 and multi-spatial and -temporal context- dependencies12. Additionally, phylogenetic information has provided insights into the mechanisms and consequences of biological invasions13-16. They also contribute to assessments of ecosystem functioning and service provisioning17,18, though phylogenetic relationships cannot simply be taken as a one-to-one proxy for ecosystem functioning19,20. However, they are of great value for studies of species traits and niche characteristics by quantifying the amount of phylogenetic conservatism21 and bioRxiv preprint doi: https://doi.org/10.1101/844175; this version posted November 16, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

ensuring statistical independence22 in multi-species studies. Using an ever increasing toolkit of phylogenetic metrics23,24, and a growing body of phylogenetic insights, the afore mentioned advances across diverse research fields document how integrating evolutionary and ecological information can enhance assessments of future impacts of global change on biodiversity25-27 and consequently inform conservation efforts (but see also 20,28).

Although the amount of molecular data has increased exponentially during the last decades, most available phylogenetic studies are either restricted to a selected subset of species, higher taxa, or to small geographic areas. Complete and dated species-level phylogenetic hypotheses for species-rich taxa of larger regions are usually restricted to vascular plants29 or vertebrates, such as global birds30 or European tetrapods31, or the analyses are based on molecular data from a small subset of species (e.g. 5% in ants6). Surprisingly, comparable phylogenetic hypotheses are rare for insects, which comprise the majority of multicellular life on earth32, have enormous impacts on ecosystem functioning, provide a multitude of ecosystem services33, and have long been used as biodiversity indicators34.

Here, we present the first comprehensive time-calibrated molecular phylogeny of all 496 extant European butterfly species (Lepidoptera: Papilionoidea), based on one mitochondrial and up to eleven nuclear genes, and the most recent systematic list of European butterflies35. European butterflies are well-studied, ranging from population level analyses36 to large-scale impacts of global change37, with good knowledge on species traits and environmental niche characteristics38,39, population trends40,41 and large-scale distributions42,43 and are thus well placed for studies in the emerging field of ecophylogenetics1.

Compared to other groups of insects, the phylogenetic relationships of butterflies are reasonably well-known, with robust backbone molecular phylogenies at the subfamily44-46 and genus-level47. In addition, molecular phylogenies also exist for most butterfly families48-58 as well as major subgroups59-65 and comprehensive COI data on species level are available from DNA barcoding studies66-71. Some ecological studies on butterflies have already incorporated phylogenetic information, e.g. on the impact of climate change on abundance trends72,73, the sensitivity of butterflies to invasive species13,74 or the ecological determinants of butterfly vulnerability75. However, the phylogenetic hypotheses used in these studies had incomplete taxon coverage (but see 76) and were not made available for reuse by other researchers. To fill these gaps in the literature, and to facilitate the growing field of ecophylogenetics, here we present the first complete and time- calibrated species-level phylogeny of a speciose higher invertebrate taxon above the family level for an entire continent. Importantly, we provide this continent-wide fully resolved phylogeny in standard analysis formats for further advancements in theoretical and applied ecology.

Methods

Taxonomic, spatial and temporal coverage

We analyse a dataset comprising all extant European species of butterflies (Papilionoidea), including the families Papilionidae, Hesperiidae, Pieridae, Lycaenidae, Riodinidae and Nymphalidae. We base our species concepts, as well as the area defined as Europe, on the latest checklist of European butterflies35.

Acquisition of sequence data bioRxiv preprint doi: https://doi.org/10.1101/844175; this version posted November 16, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

The data were mainly collated from published sources and downloaded from NCBI GenBank (Table S1). One mitochondrial gene, cytochrome c oxidase subunit I (COI, 1464 bp), was available for all species in the data matrix, in particular the 5' half of the gene (658 bp, also known as the DNA barcode). Eleven nuclear genes were included when available: elongation factor-1α (EF-1α, 1240 bp), ribosomal protein S5 (RpS5, 617 bp), ribosomal protein S2 (RpS2, 411 bp), carbamoylphosphate synthase domain protein (CAD, 850 bp), cytosolic malate dehydrogenase (MDH, 733 bp), glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 691 bp), isocitrate dehydrogenase (IDH, 711 bp), wingless (412 bp), Arginine kinase (ArgK, 596 bp) and Dopa Decarboxylase (DDC, 373 bp) and histone 3 (H3, 329 bp). H3 has been sequenced almost exclusively from the family Lycaenidae, while the other gene regions have been sampled widely also in the other butterfly families. For each gene, the longest available sequence was used. However, in the case of several available sequences of similar length, those of European origin were preferentially used. Sequences were aligned manually to maintain protein reading frame, and were curated and managed using VoSeq77.

In several cases, new sequences were generated for this study. For these specimens, protocols followed Wahlberg and Wheat 78 or Wiemers and Fiedler 66. These include several species that did not have any available published sequences, many of which are island endemics (Table 1). The new sequences have been submitted to GenBank (accessions xxxx-xxxx).

Table 1. Newly sequenced species for which no published sequences had previously been available

Taxon Origin Coenonympha orientalis Greece Glaucopsyche paphos Cyprus Gonepteryx maderensis Portugal: Madeira Hipparchia azorina Portugal: Azores Hipparchia bacchus Spain: Canary Islands Hipparchia cretica Greece: Crete Hipparchia gomera Spain: Canary Islands Hipparchia maderensis Portugal: Madeira Hipparchia mersina Greece: Samos Hipparchia miguelensis Portugal: Madeira Hipparchia sbordonii Italy: Pontine Islands Hipparchia tamadabae Spain: Canary Islands Hipparchia tilosi Spain: Canary Islands Hipparchia wyssii Spain: Canary Islands Lycaena bleusei Spain Pieris balcana North Macedonia Pieris wollastoni Portugal: Madeira Thymelicus christi Spain: Canary Islands

Almost all genera are represented by multiple genes, except Borbo, Gegenes, Laeosopis, Callophrys and Cyclyrius (the latter recently synonymized with Leptotes79) which are represented only by the COI gene. Species represented by only the DNA barcode tend to be closely related to species with more genes sequenced (Table S1), minimizing the potential bias these samples could have in our analyses.

Phylogenetic tree reconstruction: bioRxiv preprint doi: https://doi.org/10.1101/844175; this version posted November 16, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

A biogeographically restricted tree of a given taxon is inherently very asymmetrically sampled. To avoid potentially strong biases when estimating topology and divergence times we chose to build upon the recent genus-level tree of butterflies47, which provides a well-supported time-calibrated backbone and corresponds well with a recent phylogenomic analysis of Lepidoptera80. This backbone tree contains 994 taxa, each taxon representing a genus across all Papilionoidea. The tree was time- calibrated using a set of 14 fossil calibration points, which provided minimum ages and 10 calibration points based on ages of host plant clades taken from the literature, which provided maximum ages. Importantly, Chazot, et al. 47 tested the robustness of their results to a wide range of alternative assumptions made in the time-calibration analysis, and showed that the estimated times of divergences were robust.

Analysis overview

In order to produce our time-calibrated tree of European butterflies, we identified the position of the European lineages and designed a grafting procedure accordingly. We split the European butterflies that needed to be added to the tree into 12 subclades. For each of these subclades we combined the DNA sequences of the taxa already included in the backbone to the DNA sequences of the European taxa to assemble an aligned molecular matrix. After identifying the best partitioning scheme, we performed a tree reconstruction without time-calibration (only estimating relative branch lengths). The subclade trees were then rescaled using the ages estimated in the backbone and were subsequently grafted. This procedure was repeated using 1000 trees from BEAST posterior distributions of the backbone and subclade trees in order to obtain a posterior distribution of grafted trees. The details of these procedures are described below.

Backbone and subclades

The time-calibrated backbone tree provided by Chazot, et al. 47 contained about 55% of all butterfly genera, including most genera occurring in Europe. A fixed topology was obtained using RAxML81 and node ages where estimated with BEAST v.1.8.3.82. We used this fixed topology from Chazot, et al. 47 to identify at which nodes European clades should be grafted. We partitioned the analysis into 12 subclades. For each subclade, the DNA sequences of all taxa already included in the global backbone (including also non-European taxa) were combined with the DNA sequences of all the new European taxa that were added. In addition to the focal taxa, we added between two and four outgroups.

The subclades, sorted by families, were defined as follows:

Papilionidae – All Papilionidae were placed into one subclade.

Hesperiidae – We identified two main clades to graft within the Hesperiidae: Hesperiinae and Pyrginae. The Hesperiinae subclade was extended to also encompass the subfamilies Heteropterinae and Trapezitinae. The genus Muschampia, not available in the backbone, was included in the Pyrginae subclade.

Pieridae – All Pieridae were considered as a single clade.

Lycaenidae – All Lycaenidae were considered as a single clade.

Riodinidae – The only European Riodinidae species, Hamearis lucina, was already available in the backbone tree. bioRxiv preprint doi: https://doi.org/10.1101/844175; this version posted November 16, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

Nymphalidae – European Nymphalidae were divided into seven subclades. (i) A subclade for the Apaturinae. (ii) In order to add Danaus chrysippus we generated a tree of Danainae. (iii) We combined the sister clades Heliconiinae and Limenitidinae into a single subclade. (iv) Nymphalinae was treated as a single subclade. (v) A first clade of Satyrinae contained the genera Kirinia, Pararge, Lasiommata, Tatinga, Chonala and Lopinga. (vi) A second Satyrinae clade contained the genera Calisto, Euptychia, Callerebia, Proterebia, Gyrocheilus, Strabena, Ypthima, Ypthimomorpha, Stygionympha, Cassionympha, Neocoenyra, Pseudonympha, Erebia, Boerebia, Hyponephele, Cercyonis, Maniola, Aphantopus, Pyronia, Faunula, Grumia, Paralasa, Melanargia, Hipparchia, Berberia, Oeneis, Neominois, Karanasa, Brintesia, Arethusana, Satyrus, Pseudochazara and Chazara. (vii) A third Satyrinae clade was created for the genus Coenonympha. Charaxinae were not treated separately from the backbone. Charaxes jasius is the only Charaxinae occuring in Europe and Charaxes castor (which is very closely related to C. jasius83) was already included in the backbone tree from Chazot et al. 47. Hence, we used the position of Charaxes castor for Charaxes jasius.

Partitioning the dataset

For each subclade we ran Partition Finder 2 84 in order to partition the data and choose substitution models. The dataset was initially partitioned into genes and codon positions. Branch lengths were set to linked and the comparison between partitioning strategies was made using the greedy algorithm and BIC score85.

Phylogenetic reconstruction

For each subclade, the dataset was imported in BEAUTi v.1.8.386 and partitioned according to the partitioning strategy identified by Partition Finder 2. We enforced the monophyly of the clade to be grafted (i.e., excluding the outgroups). All other relationships were estimated by BEAST v.1.8.3.82. We used an uncorrelated relaxed clock with lognormal distribution. By default, we started by setting one molecular clock per partition. If convergence or good mixing could not be obtained after running BEAST we reduced the number of molecular clocks (see details for each dataset further below). We did not add any time-calibration and therefore only estimated the relative timing of divergence. We performed at least two independent runs with BEAST for each subclade. We checked for convergence and mixing of the MCMC using Tracer v.1.6.087 and in the case of full convergence of the runs, the posterior distribution of trees from different runs were combined after removing the burn-in fraction.

Grafting procedure

Subclades were grafted on the backbone as follows. One backbone was sampled from the posterior distribution of time-calibrated trees from Chazot, et al. 47. For each subclade, one subclade tree was sampled from the posterior distribution of trees, the outgroups removed and the tree was rescaled based on the crown age of the subclade extracted from the backbone tree. Finally, the rescaled subclade tree was grafted on the backbone after removing all lineages belonging to this subclade in the backbone (i.e. only keeping the stem branch). We repeated this procedure for 1000 backbone trees and 1000 subclade trees and thus we obtained a posterior distribution of 1000 grafted trees. The topology of the backbone was fixed (see 47) but the topologies of the subclades were free. Hence the posterior distribution of grafted trees includes a posterior distribution of topologies and node ages. bioRxiv preprint doi: https://doi.org/10.1101/844175; this version posted November 16, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

We describe below the details of the phylogenetic tree reconstruction for each subclade.

1- Papilionidae

Dataset – The dataset for the Papilionidae consisted of 36 taxa to which three outgroups were added: Macrosoma tipulata (Hedylidae), Achlyodes busiris (Hesperiidae), Pieris rapae (Pieridae). We concatenated 11 gene fragments (COI, CAD, EF-1α, GAPDH, ArgK, IDH, MDH, RpS2, RpS5, DDC, wingless).

Partition Finder – Partition Finder identified 12 subsets.

Subset # Substitution model Gene fragments and codon positions Subset 1 TRN+I+G ArgK_pos1, RpS2_pos1, EF-1α_pos1, RpS5_pos1 Subset 2 TRN+I+G COI-begin_pos2, RpS5_pos2, ArgK_pos2, GAPDH_pos2, CAD_pos2, MDH_pos2, IDH_pos2, COI- end_pos2 Subset 3 HKY ArgK_pos3 Subset 4 HKY+I+G CAD_pos3 Subset 5 GTR+G wingless_pos1, IDH_pos1, GAPDH_pos1, MDH_pos1, CAD_pos1 Subset 6 TRN+G COI-end_pos3, COI-begin_pos3 Subset 7 GTR+G DDC_pos2, COI-end_pos1, COI-begin_pos1 Subset 8 GTR+I+G DDC_pos3, wingless_pos3, RpS2_pos3 Subset 9 K80+G DDC_pos1, wingless_pos2 Subset 10 GTR+G EF-1α_pos3 Subset 11 TRNEF+I RpS2_pos2, EF-1α_pos2 Subset 12 GTR+G RpS5_pos3, GAPDH_pos3, IDH_pos3, MDH_pos3

BEAST analysis –In order to improve the quality of our runs we replaced the default priors for rates of substitutions by uniform prior ranging between 0 and 10 for the following cases: subset5.at, subset5.cg, subset7.cg, subset7.gt, subset12.cg, subset12.gt. We used one molecular clock per subset identified by Partition Finder and obtained good mixing and convergence. We used a Birth-Death tree prior. We performed three runs of 40 million generations, sampling trees and parameters every 4000 generations.

Grafting – For grafting, the outgroups were removed, as well as Baronia brevicornis, the first Papilionidae to diverge and endemic to Mexico, i.e. we grafted at the most recent common ancestor (mrca) of all Papilionidae but Baronia brevicornis.

2- Hesperiidae: Hesperiinae

Dataset – The dataset for the Hesperiinae consisted of 169 taxa to which two outgroups were added: Typhedanus ampyx (Hesperiidae), Mylon pelopidas (Hesperiidae). We concatenated 10 gene fragments (COI, CAD, EF-1α , GAPDH, ArgK, IDH, MDH, RpS2, RpS5, wingless).

Partition Finder – Partition Finder identified 17 subsets.

Subset # Substitution model Gene fragments and codon positions Subset 1 GTR+I+G COI-end_pos1, COI-begin_pos1, ArgK_pos1 bioRxiv preprint doi: https://doi.org/10.1101/844175; this version posted November 16, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

Subset 2 GTR+I+G ArgK_pos2, IDH_pos2, EF-1α_pos2 Subset 3 GTR+G ArgK_pos3 Subset 4 TRN+I+G CAD_pos3, IDH_pos3 Subset 5 GTR+I+G IDH_pos1, CAD_pos1 Subset 6 GTR+I+G CAD_pos2, MDH_pos2 Subset 7 TRN+I+G COI-begin_pos3, COI-end_pos3 Subset 8 HKY+I+G RpS5_pos2, COI-begin_pos2, GAPDH_pos2, COI- end_pos2 Subset 9 GTR+I+G EF-1α_pos3 Subset 10 GTR+I+G EF-1α_pos1, MDH_pos1 Subset 11 GTR+I+G RpS2_pos3, GAPDH_pos3, RpS5_pos3 Subset 12 GTR+I+G GAPDH_pos1, wingless_pos2 Subset 13 TRN+I+G MDH_pos3 Subset 14 JC+I RpS2_pos2 Subset 15 GTR+I+G RpS2_pos1, RpS5_pos1 Subset 16 GTR+I+G wingless_pos3 Subset 17 SYM+I+G wingless_pos1

BEAST analysis – Preliminary analyses showed problems with the subset 3 (ArgKin_pos3) and was therefore removed from the analyses. In order to improve the quality of our runs we replaced the default priors for rates of substitutions by uniform priors ranging between 0 and 10 for the following cases: subset17.cg. The substitution model for the subset 14 was also changed into HKY+I after preliminary analyses. We used one molecular clock per subset identified by Partition Finder and obtained good mixing and convergence. We used a Birth-Death tree prior. We performed two runs of 150 million generations, sampling trees and parameters every 15000 generations.

Grafting – For grafting, the outgroups were removed and the subclade grafted at the mrca of Hesperiinae.

3- Hesperiidae: Pyrginae

Dataset – The dataset for the Pyginae consisted of 77 taxa to which three outgroups were added: Typhedanus ampyx (Hesperiidae), Pyrrhopyge zenodorus (Hesperiidae) and Hasora khoda (Hesperiidae). We concatenated 10 gene fragments (COI, CAD, EF-1α , GAPDH, ArgK, IDH, MDH, RpS2, RpS5, wingless).

Partition Finder – Partition Finder identified 14 subsets.

Subset # Substitution model Gene fragments and codon positions Subset 1 TRN+I+G EF-1α_pos1, IDH_pos1, RpS2_pos1, RpS5_pos1, ArgK_pos1 Subset 2 TRN+I+G EF-1α_pos2, GAPDH_pos2, MDH_pos2, COI- begin_pos2, COI-end_pos2, IDH_pos2, CAD_pos2, RpS5_pos2, ArgK_pos2 Subset 3 GTR+G ArgK_pos3 Subset 4 HKY+I+G CAD_pos3 Subset 5 GTR+I+G CAD_pos1, MDH_pos1, GAPDH_pos1 Subset 6 HKY+G COI-begin_pos3 bioRxiv preprint doi: https://doi.org/10.1101/844175; this version posted November 16, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

Subset 7 GTR+I+G COI-end_pos1, COI-begin_pos1 Subset 8 TRN+I+G COI-end_pos3 Subset 9 GTR+G EF-1α_pos3 Subset 10 GTR+I+G RpS2_pos3, GAPDH_pos3, RpS5_pos3 Subset 11 GTR+I+G MDH_pos3, IDH_pos3 Subset 12 JC+G wingless_pos2, RpS2_pos2 Subset 13 HKY+G wingless_pos3 Subset 14 SYM+G wingless_pos1

BEAST analysis – In order to improve the quality of our runs we replaced the default priors for rates of substitutions by uniform prior ranging between 0 and 10 for the following cases: subset7.ac, subset7.gt, subset14.cg, subset3.cg. Preliminary analyses showed problems when using a separate molecular clock for each subset identified by Partition Finder. We restricted the analysis to one molecular clock. We used a Birth-Death tree prior. We performed two runs of 100 million generations, sampling trees and parameters every 10000 generations.

Grafting – For grafting, the outgroups were removed and the subclade grafted at the mrca of Pyrginae.

4- Pieridae

Dataset – The dataset for the Pieridae consisted of 126 taxa to which three outgroups were added: Bicyclus anynana (Nymphalidae), Achylodes busiris (Hesperiidae) and Papilio glaucus (Papilionidae). We concatenated 11 gene fragments (COI, CAD, EF-1α , GAPDH, ArgK, IDH, MDH, RpS2, RpS5, DDC, wingless).

Partition Finder – Partition Finder identified 17 subsets.

Subset # Substitution model Gene fragments and codon positions Subset 1 GTR+I+G ArgK_pos1, EF-1α_pos1, GAPDH_pos1 Subset 2 TRN+I+G ArgK_pos2, DDC_pos2, CAD_pos2, IDH_pos2 Subset 3 KHY ArgK_pos3 Subset 4 HKY+I+G CAD_pos3 Subset 5 GTR+I+G CAD_pos1, RpS2_pos1, IDH_pos1, RpS5_pos1, MDH_pos1 Subset 6 HKY+G COI-begin_pos3 Subset 7 GTR+I+G COI-begin_pos1, COI-end_pos1 Subset 8 GTR+I+G MDH_pos2, COI-begin_pos2, RpS5_pos2, COI- end_pos2, EF-1α_pos2, GAPDH_pos2 Subset 9 TRN+I+G COI-end_pos3 Subset 10 K80+G DDC_pos3, RpS2_pos3 Subset 11 SYM+G DDC_pos1, wingless_pos1 Subset 12 GTR+I+G EF-1α_pos3 Subset 13 GTR+I+G RpS5_pos3, GAPDH_pos3 Subset 14 GTR+I+G IDH_pos3, MDH_pos3 Subset 15 JC+I RpS2_pos2 Subset 16 GTR+I+G wingless_pos3 Subset 17 K80+G wingless_pos2 bioRxiv preprint doi: https://doi.org/10.1101/844175; this version posted November 16, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

BEAST analysis – In order to improve the quality of our runs we replaced the default priors for rates of substitutions by uniform prior ranging between 0 and 10 for the following case: subset7.cg. The substitution model for the subset 7 was also changed into GTR+G after preliminary analyses. We used one molecular clock per subset identified by Partition Finder and obtained good mixing and convergence. We used a Birth-Death tree prior. We performed two runs of 100 million generations, sampling trees and parameters every 10000 generations.

Grafting – For grafting, the outgroups were removed and the subclade grafted at the mrca of Pieridae.

5- Lycaenidae

Dataset – The dataset for the Lycaenidae consisted of 187 taxa to which three outgroups were added: Bicyclus anynana (Nymphalidae), Pieris rapae (Pieridae) and Hamearis lucina (Riodinidae). We concatenated 12 gene fragments (COI, CAD, EF-1α, GAPDH, ArgK, IDH, MDH, RpS2, RpS5, DDC, wingless and H3).

Partition Finder – Partition Finder identified 12 subsets.

Subset # Substitution model Gene fragments and codon positions Subset 1 TRN+I+G ArgK_pos1, H3_pos1, EF-1α_pos1, RpS5_pos1, GAPDH_pos1 Subset 2 GTR+I+G RpS5_pos2, GAPDH_pos2, ArgK_pos2, DDC_pos2, MDH_pos2, COI-begin_pos2, COI-end_pos2, EF- 1α_pos2, IDH_pos2, CAD_pos2 Subset 3 GTR+G H3_pos3, ArgK_pos3, wingless_pos3 Subset 4 HKY+I+G CAD_pos3 Subset 5 GTR+G wingless_pos2, RpS2_pos1, MDH_pos1, IDH_pos1, CAD_pos1 Subset 6 GTR+G COI-begin_pos3, COI-end_pos3 Subset 7 GTR+I+G COI-begin_pos1, COI-end_pos1 Subset 8 TRNEF DDC_pos3, RpS2_pos3 Subset 9 SYM+G wingless_pos1, DDC_pos1 Subset 10 GTR+I+G EF-1α_pos3 Subset 11 GTR+G MDH_pos3, RpS5_pos3, GAPDH_pos3, IDH_pos3 Subset 12 JC+I RpS2_pos2, H3_pos2

BEAST analysis – In order to improve the quality of our runs we replaced the default priors for rates of substitutions by uniform prior ranging between 0 and 10 for the following cases: subset3.cg, subset6.ag, subset6.at, subset11.gt_subst7.cg. We used one molecular clock per subset identified by Partition Finder and obtained good mixing and convergence. We used a Birth-Death tree prior. We performed two runs of 150 million generations, sampling trees and parameters every 15000 generations.

Grafting – For grafting, the outgroups were removed and the subclade grafted at the mrca of Lycaenidae. bioRxiv preprint doi: https://doi.org/10.1101/844175; this version posted November 16, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

6- Nymphalidae: Danainae

Dataset – The dataset for the Danainae consisted of 7 taxa to which two outgroups were added: Euploea camaralzeman (Nymphalidae) and Lycorea halia (Nymphalidae). We concatenated 9 gene fragments (COI, CAD, EF-1α, GAPDH, IDH, MDH, RpS2, RpS5, wingless).

Partition Finder – Partition Finder identified 8 subsets.

Subset # Substitution model Gene fragments and codon positions Subset 1 HKY+I MDH_pos3, CAD_pos3 Subset 2 GTR+G RpS5_pos1, RpS2_pos1, GAPDH_pos1, EF-1α_pos1, wingless_pos1, MDH_pos1, CAD_pos1, IDH_pos1 Subset 3 HKY+I COI-end_pos2, COI-begin_pos2, RpS5_pos2, GAPDH_pos2, MDH_pos2, EF-1α_pos2, CAD_pos2, IDH_pos2 Subset 4 HKY+G COI-end_pos3, COI-begin_pos3 Subset 5 TRN+G COI-end_pos1, COI-begin_pos1 Subset 6 HKY+G EF-1α_pos3, RpS5_pos3 Subset 7 GTR+G GAPDH_pos3, RpS2_pos3, IDH_pos3, wingless_pos3 Subset 8 JC RpS2_pos2, wingless_pos2

BEAST analysis – We used one molecular clock per subset identified by Partition Finder and obtained good mixing and convergence. We used a Birth-Death tree prior. We performed two runs of 20 million generations, sampling trees and parameters every 2000 generations.

Grafting – For grafting, the outgroups were removed and the subclade grafted at the mrca of Danainae.

7- Nymphalidae: Apaturinae

Dataset – The dataset for the Apaturinae consisted of 9 taxa to which two outgroups were added: Timelaea albescens (Nymphalidae) and Biblis hyperia (Nymphalidae). We concatenated 10 gene fragments (COI, CAD, EF-1α , GAPDH, ArgK, IDH, MDH, RpS2, RpS5, wingless).

Partition Finder – Partition Finder identified 7 subsets.

Subset # Substitution model Gene fragments and codon positions Subset 1 TRN+G wingless_pos1, CAD_pos1, IDH_pos1, ArgK_pos1, RpS5_pos1, RpS2_pos1, EF-1α_pos1, MDH_pos1, GAPDH_pos1 Subset 2 GTR+I COI-begin_pos2, EF-1α_pos2, ArgK_pos2, MDH_pos2, CAD_pos2, COI-end_pos2, IDH_pos2, RpS5_pos2, GAPDH_pos2 Subset 3 GTR+G EF-1α_pos3, RpS2_pos3, RpS5_pos3, GAPDH_pos3, ArgK_pos3, wingless_pos3 Subset 4 HKY+I IDH_pos3, MDH_pos3, CAD_pos3 Subset 5 HKY+G COI-end_pos3, COI-begin_pos3 Subset 6 TRN+G COI-end_pos1, COI-begin_pos1 Subset 7 JC RpS2_pos2, wingless_pos2 bioRxiv preprint doi: https://doi.org/10.1101/844175; this version posted November 16, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

BEAST analysis – We used one molecular clock per subset identified by Partition Finder and obtained good mixing and convergence. We used a Birth-Death tree prior. We performed two runs of 20 million generations, sampling trees and parameters every 2000 generations.

Grafting – For grafting, the outgroups were removed and the subclade grafted at the mrca of Danainae.

8- Nymphalidae: Heliconiinae + Limenitidinae

Dataset – The dataset combined the sister clades Heliconiinae and Limenitidinae and consisted of 92 taxa to which three outgroups were added: Amnosia decora (Nymphalidae), Apatura iris (Nymphalidae) and Libythea celtis (Nymphalidae). We concatenated 11 gene fragments (COI, CAD, EF-1α , GAPDH, ArgK, IDH, MDH, RpS2, RpS5, DDC, wingless).

Partition Finder – Partition Finder identified 14 subsets.

Subset # Substitution model Gene fragments and codon positions Subset 1 GTR+I+G wingless_pos2, RpS2_pos1, ArgK_pos1 Subset 2 GTR+I+G DDC_pos2, IDH_pos2, GAPDH_pos2, RpS5_pos2, EF- 1α_pos2, ArgK_pos2 Subset 3 GTR+G ArgK_pos3 Subset 4 HKY+I+G MDH_pos3, CAD_pos3 Subset 5 GTR+I+G DDC_pos1, wingless_pos1, CAD_pos1, RpS5_pos1, MDH_pos1, IDH_pos1 Subset 6 GTR+I+G CAD_pos2, MDH_pos2, COI-begin_pos2, COI- end_pos2 Subset 7 GTR+I+G COI-end_pos3, COI-begin_pos3 Subset 8 GTR+I+G COI-end_pos1, COI-begin_pos1 Subset 9 GTR+I+G DDC_pos3, wingless_pos3, RpS5_pos3, RpS2_pos3 Subset 10 GTR+I+G EF-1α_pos3 Subset 11 TRN+I+G EF-1α_pos1, GAPDH_pos1 Subset 12 GTR+I+G GAPDH_pos3 Subset 13 GTR+I+G IDH_pos3 Subset 14 JC+I RpS2_pos2

BEAST analysis – Preliminary analyses showed problems with the subset 14 (RpS2_pos2) which was therefore removed from the analyses. In order to improve the quality of our runs we replaced the default priors for rates of substitutions by uniform priors ranging between 0 and 10 for the following case: subset7.cg. We used one molecular clock per subset identified by Partition Finder and obtained good mixing and convergence. We used a Birth-Death tree prior. We performed two runs of 100 million generations, sampling trees and parameters every 10000 generations.

Grafting – For grafting, the outgroups were removed and the subclade grafted at the split between Limenitidinae and Heliconiinae.

9- Nymphalidae: Nymphalinae bioRxiv preprint doi: https://doi.org/10.1101/844175; this version posted November 16, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

Dataset – The dataset of Nymphalinae consisted of 83 taxa to which two outgroups were added: Historis odius (Nymphalidae) and Pycina zamba (Nymphalidae). We concatenated 11 gene fragments (COI, CAD, EF-1α , GAPDH, ArgK, IDH, MDH, RpS2, RpS5, DDC, wingless).

Partition Finder – Partition Finder identified 12 subsets.

Subset # Substitution model Gene fragments and codon positions Subset 1 TRN+I+G wingless_pos1, ArgK_pos1, MDH_pos1, CAD_pos1, IDH_pos1, GAPDH_pos1 Subset 2 GTR+I+G DDC_pos2, IDH_pos2, ArgK_pos2, EF-1α_pos2, RpS5_pos2, MDH_pos2, CAD_pos2 Subset 3 GTR+G EF-1α_pos3, ArgK_pos3 Subset 4 HKY+I+G MDH_pos3, CAD_pos3 Subset 5 GTR+G COI-end_pos3, COI-begin_pos3 Subset 6 GTR+G COI-end_pos1, COI-begin_pos1 Subset 7 GTR+I+G COI-begin_pos2, COI-end_pos2 Subset 8 TRNEF wingless_pos3, DDC_pos3 Subset 9 SYM+G DDC_pos1, wingless_pos2 Subset 10 GTR+I+G RpS5_pos1, RpS2_pos1, EF-1α_pos1 Subset 11 GTR+G IDH_pos3, RpS2_pos3, GAPDH_pos3, RpS5_pos3 Subset 12 JC+I RpS2_pos2, GAPDH_pos2

BEAST analysis – In order to improve the quality of our runs we replaced the default priors for rates of substitutions by uniform priors ranging between 0 and 10 for the following case: subset5.cg. Preliminary analyses revealed problems when using one molecular clock per subset identified by Partition Finder. We restricted the analysis to one molecular clock for the mitochondrial gene fragments and one molecular clock for the nuclear gene fragments. We used a Birth-Death tree prior. We performed two runs of 100 million generations, sampling trees and parameters every 10000 generations.

Grafting – For grafting, the outgroups were removed and the subclade grafted at the mrca of Nymphalinae.

10- Nymphalidae: Satyrinae 1

Dataset – The first Satyrinae dataset consisted of 13 taxa, belonging to the genera Kirinia, Pararge, Lasiommata, Tatinga, Chonala and Lopinga, to which three outgroups were added: Bicyclus anynana (Nymphalidae), Acrophtalmia leuce (Nymphalidae) and Ragadia makuta (Nymphalidae). We concatenated 5 gene fragments (COI, EF-1α, GAPDH, RpS5, wingless).

Partition Finder – Partition Finder identified 6 subsets.

Subset # Substitution model Gene fragments and codon positions Subset 1 HKY+G COI-begin_pos3, COI-end_pos3 Subset 2 TRN+G COI-begin_pos1, COI-end_pos1 Subset 3 HKY+I EF-1α_pos2, RpS5_pos2, GAPDH_pos2, COI- end_pos2, COI-begin_pos2 Subset 4 GTR+G GAPDH_pos3, EF-1α_pos3, RpS5_pos3 Subset 5 TRN+G wingless_pos2, wingless_pos1, GAPDH_pos1, EF- bioRxiv preprint doi: https://doi.org/10.1101/844175; this version posted November 16, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

1α_pos1, RpS5_pos1 Subset 6 GTR wingless_pos3

BEAST analysis – We used one molecular clock per subset identified by Partition Finder and obtained good mixing and convergence. We used a Birth-Death tree prior. We performed two runs of 20 million generations, sampling trees and parameters every 2000 generations.

Grafting – For grafting, the outgroups were removed and the subclade grafted at the crown of the clade after removing the outgroups.

11- Nymphalidae: Satyrinae 2

Dataset – The second Satyrinae dataset consisted of 161 taxa, belonging to the genera Calisto, Euptychia, Callerebia, Proterebia, Gyrocheilus, Strabena, Ypthima, Ypthimomorpha, Stygionympha, Cassionympha, Neocoenyra, Pseudonympha, Erebia, Boerebia, Hyponephele, Cercyonis, Maniola, Aphantopus, Pyronia, Faunula, Grumia, Paralasa, Melanargia, Hipparchia, Berberia, Oeneis, Neominois, Karanasa, Brintesia, Arethusana, Satyrus, Pseudochazara and Chazara, to which three outgroups were added: Coenonympha pamphilus (Nymphalidae), Taygetis virgilia (Nymphalidae) and Pronophila thelebe (Nymphalidae). We concatenated 10 gene fragments (COI, CAD, EF-1α , GAPDH, ArgK, IDH, MDH, RpS2, RpS5, wingless).

Partition Finder – Partition Finder identified 11 subsets.

Subset # Substitution model Gene fragments and codon positions Subset 1 GTR+I+G ArgK_pos1, wingless_pos1, RpS2_pos1, IDH_pos1, CAD_pos1, EF-1α_pos1, GAPDH_pos1, RpS5_pos1, MDH_pos1 Subset 2 HKY+I+G MDH_pos2, ArgK_pos2, CAD_pos2, IDH_pos2, RpS5_pos2, EF-1α_pos2, GAPDH_pos2 Subset 3 GTR+G ArgK_pos3, wingless_pos3 Subset 4 HKY+G IDH_pos3, MDH_pos3, CAD_pos3 Subset 5 GTR+G COI-begin_pos3, COI-end_pos3 Subset 6 GTR+G COI-begin_pos1, COI-end_pos1 Subset 7 TRN+I+G COI-begin_pos2, COI-end_pos2 Subset 8 GTR+I+G EF-1α_pos3, RpS5_pos3 Subset 9 HKY+G GAPDH_pos3 Subset 10 TRNEF+G RpS2_pos2, wingless_pos2 Subset 11 K80+G RpS2_pos3

BEAST analysis – In order to improve the quality of our runs we replaced the default priors for rates of substitutions by uniform prior ranging between 0 and 10 for the following cases: subset5.ac, subset5.ag, subset5.at, subset5.cg, subset5.gt. We used one molecular clock per subset identified by Partition Finder and obtained good mixing and convergence. We used a Birth-Death tree prior. We performed two runs of 100 million generations, sampling trees and parameters every 10000 generations. bioRxiv preprint doi: https://doi.org/10.1101/844175; this version posted November 16, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

Grafting – For grafting, the outgroups were removed and the subclade grafted at the crown of the clade after removing the outgroups.

12- Nymphalidae: Satyrinae 3

Dataset – The third Satyrinae dataset consisted of 15 taxa all belonging to the genus Coenonympha, to which two outgroups were added: Sinonympha amoena (Nymphalidae) and Oressinoma sorata (Nymphalidae). We concatenated 9 gene fragments (COI, CAD, EF-1α , GAPDH, IDH, MDH, RpS2, RpS5, wingless).

Partition Finder – Partition Finder identified 6 subsets.

Subset # Substitution model Gene fragments and codon positions Subset 1 HKY CAD_pos3 Subset 2 TRN+G RpS2_pos2, COI-begin_pos1, COI-end_pos1, CAD_pos1, IDH_pos1 Subset 3 HKY RpS5_pos2, MDH_pos2, GAPDH_pos2, EF-1α_pos2, IDH_pos2, CAD_pos2 Subset 4 GTR+G COI-end_pos3, COI-begin_pos3 Subset 5 HKY+I COI-end_pos2, COI-begin_pos2 Subset 6 HKY+G wingless_pos3, RpS2_pos3, EF-1α_pos3 Subset 7 TRN+G wingless_pos1, RpS2_pos1, MDH_pos1, GAPDH_pos1, RpS5_pos1, EF-1α_pos1 Subset 8 HKY+G RpS5_pos3, MDH_pos3, GAPDH_pos3, IDH_pos3 Subset 9 JC wingless_pos2

BEAST analysis – We used one molecular clock per subset identified by Partition Finder and obtained good mixing and convergence. We used a Birth-Death tree prior. We performed two runs of 20 million generations, sampling trees and parameters every 2000 generations.

Grafting – For grafting, the outgroups were removed and the subclade grafted at the crown of Coenonympha.

Data Records

The analysed dataset (a concatenated alignment of the genes COI, CAD, EF-1α, GAPDH, ArgK, IDH, MDH, RpS2, RpS5, DDC, wingless, and H3) is available in FASTA format at DOI: 10.5281/zenodo.3531555. The posterior distribution of ML trees and the consensus tree are available in NEWICK format at DOI: 10.5281/zenodo.3531555.

Technical Validation

Species identities of the chosen sequences for the dataset were validated by blasting the DNA barcode sequence against the Barcode Of Life Database, which has a good representation of European butterfly species due to a number of barcoding projects implemented in different countries. In almost all cases, the sequences came from the same voucher specimen itself. In 88 cases (Supporting Information), the sequences used were from different individuals. In these cases bioRxiv preprint doi: https://doi.org/10.1101/844175; this version posted November 16, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

special care was taken to use sequences from reliable sources, preferably those with voucher photographs.

We based our time-calibration from a recent re-evaluation of the timing of divergence of higher-level Papilionoidea. We used the topology inferred by Chazot et al. 47 as a backbone in our grafting procedure. This topology was fixed in Chazot et al. 47, hence only node ages were estimated. Within each subclade we grafted however, we let BEAST estimate the topology in addition to node height. Several sections of the European butterfly tree remain poorly supported. This most likely arises from the lack of molecular information as well as recent and rapid diversification events within Polyommatus, Hipparchia, or Pseudochazara for example. We show here a synthetic tree summarizing the posterior distribution of topologies and node ages but the posterior distribution of grafted trees can be found in Supporting Information, providing a distribution of alternative topologies and node ages estimated by BEAST. We strongly advise any researcher using our phylogenetic framework to repeat the analyses on at least 100 trees randomly sampled from this posterior distribution in order to account for topology and node age uncertainty. This tree can also help identify the sections of the tree lacking molecular information and therefore points at the sections that should be targeted in the future when generating new molecular data.

Usage Notes

We have generated a robust phylogenetic hypothesis for all European species of butterflies with associated times of divergence (Fig. 1). Our purpose is to provide a complete phylogenetic framework for use by the ecological and evolutionary communities. The demand for such a phylogenetic information is high at the moment and various proxies have been used that are not ideal, starting already in 200588. We provide a posterior distribution of topologies and node ages, in order for researchers to be able to take phylogenetic and node age uncertainty into account if they so wish. The tree files are provided in standard Newick format as output from BEAST. Future studies will not necessarily be as comprehensive as the tree we provide. In such cases we recommend using tools such as the ape package89 in R90 to remove tips from the tree that are not relevant to a given study.

Acknowledgements

MW thanks Brigitte Gottsberger (University of Vienna) for assistance in the lab and the following colleagues for specimen samples or sequences: Benedicto Acosta-Fernandez (Spain), Bernard Turlin (France), Dirk Gerber (Germany), Eddie John (UK), John Coutsis (Greece), Javier García (Spain), Karen van Dorp (Netherlands), Klaus Schurian (Germany), Pedro Oromí (Spain), Peter Russell (UK), Roger Vila (Spain), Vlad Dinca (Finland), Xavier Merit (France), Zdenek Fric (Czech Republic), and Zdravko Kolev (Bulgaria). NW acknowledges funding from the Department of Biology, Lund University, the Swedish Research Council (grant number 2015-04441). NC acknowledges funding from BECC (Biodiversity and Ecosystem services in a Changing Climate). CWW acknowledges funding from the Swedish Research Council (grant number 2017-04386). The study was also supported by iDiv through the sDiv working group sECURE (https://www.idiv.de/secure).

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90 R: A language and environment for statistical computing. https://www.R-project.org/ (R Foundation for Statistical Computing, Vienna, Austria, 2018).

25.06 Parnassius_mnemosyne 17.43 Parnassius_apollo 12.02 Parnassius_phoebus 44.63 Archon_apollinus 20.23 68.87 39.11

2 8 Zerynthia_rumina 23.8 6.82 Zerynthia_cassandra 2.35 Zerynthia_polyxena 12.21 59.37 Zerynthia_cretica 2.99Zerynthia_cerisy 2.51 Zerynthia_caucasica

40.27 Iphiclides_feisthamelii 1.61 Iphiclides_podalirius 33.88 27.77 55.83 44.06 23.83 Papilio_alexanor 47.96 20.16 Papilio_hospiton 3.67 Papilio_machaon 41.7 33.17 27.55 40.97

33.42 13.85 Carcharodus_tripolinus 29.58 5.88 Carcharodus_alceae 19.97 99.44 Spialia_phlomidis 28.36 12.93 Spialia_therapne 44.92 6.35 Spialia_sertorius 3.46 Spialia_rosae 1.49 24.64 Spialia_orbifer Muschampia_cribrellum 20.86 18.01 Muschampia_tessellum 14.81 65.75 Muschampia_proto 23.16 44.14 12.92 Carcharodus_orientalis 15.14 1.7 Carcharodus_floccifera 10.55 6.01 Carcharodus_lavatherae 9.72 Carcharodus_baeticus 4.97 Carcharodus_stauderi

18.21 10.07 Erynnis_tages 26.45 4.86 Erynnis_marloyi

41.97 23.56

52.3 30.76 24.99 28.73 27.15 12.59 22.24 Pyrgus_sidae 3.8 Pyrgus_carthami 17.74 7.02 Pyrgus_malvoides 1.39 Pyrgus_malvae 6.04 Pyrgus_andromedae 3.67 Pyrgus_cacaliae 10.53 2.45 Pyrgus_centaureae Pyrgus_serratulae Pyrgus_armoricanus 8.12 Pyrgus_cinarae 108.3 6.98 3.89 Pyrgus_cirsii 1.16 Pyrgus_carlinae 6.1 Pyrgus_onopordi 5.4 Pyrgus_warrenensis 2.01Pyrgus_foulquieri 1.46 Pyrgus_alveus

45.96 29.68 50.67 43.39 44.25 4 9 33.04 Heteropterus_morpheus 47.73 28.21 23.32 27.09 44.09 21.59 Carterocephalus_silvicola 1.87 Carterocephalus_palaemon 18.93 42.25 30.25 38.91 33.91 35.233.48 19.5 34.2 31.48 33.2629.84

32.63 11.05 9.82 Borbo_borbonica 3.9 32.08 12.59 Pelopidas_thrax 10.21 10.91 Gegenes_nostrodamus 4.41 29.9 1.81 Gegenes_pumilio 10.13 8.8 24.47 29.5 25.76 16.8 23.02 12.64 28.65 Thymelicus_christi 16.48 3.06 Thymelicus_acteon 8.65 Thymelicus_hyrax 5.26 Thymelicus_sylvestris 1.77 26.46 Thymelicus_lineola 14.74 18.7 15.24 17.6 11.66 17.04 9.63 14.8111.07 25.43 13.13 7.39 11.349.09 9.59 3.39 Hesperia_comma 24.21

Leptidea_duponcheli 13.15 Leptidea_morsei 3.87 Leptidea_juvernica 43.23 2.25Leptidea_sinapis 1.3 Leptidea_reali 27.98 40.92 77.34 46.83 Gonepteryx_rhamni 29.87 2.94 36.74 Gonepteryx_cleobule 107.07 3.71 Gonepteryx_farinosa 1.67Gonepteryx_cleopatra 0.96 32.42 Gonepteryx_maderensis 11.61 72.82 27.81 18.84 Catopsilia_florella 5.53 21.21 Colias_hyale 1.41 14.13 Colias_alfacariensis 4.05 Colias_chrysotheme Colias_aurorina 2 3.25Colias_phicomone Colias_erate 2.870.61 Colias_crocea 2.67Colias_caucasica 1.39 65.2 Colias_myrmidone 2.03Colias_hecla 0.75Colias_palaeno 0.39 Colias_tyche 37.45 45.44 28.46 42.07 Colotis_evagore 28.91 54.41 33.82

50.28 24.05 43.55 Euchloe_tagis 14.7 Euchloe_charlonia 35.51 7.31 Euchloe_bazae 4.95 Euchloe_penia Anthocharis_euphenoides 18.64 5.28 Anthocharis_cardamines 2.9 Anthocharis_damone 2.17 Anthocharis_gruneri Zegris_eupheme 58.14 15.81 13.46 Zegris_pyrothoe 5.05 Euchloe_eversi 14.07 3.53 Euchloe_grancanariensis 3.04Euchloe_hesperidum 2.51 11.37 Euchloe_belemia Euchloe_ausonia 2.86 Euchloe_simplonia 6.97 Euchloe_insularis 4.84 Euchloe_crameri 48.79 31.74 54.04 31.15 39.29 32.87 44.26 35.86 30.69 17.9 28.96 Aporia_crataegi 42.96 27.79 23.1 20.97 20.84 26.46 20.09 Pontia_chloridice 24.43 15.57 Pontia_callidice 15.08 10.81 Pontia_edusa 23.73 5.43 Pontia_daplidice

Pieris_brassicae 18.25 2.11Pieris_wollastoni 0.78 Pieris_cheiranthi 13.25 Pieris_krueperi Pieris_rapae 11.4 3.68 Pieris_mannii 10.46 Pieris_ergane 4.28 Pieris_bryoniae 2.22Pieris_napi 0.94 Pieris_balcana

58.43 47.5 42.12 73.74 33.87 Hamearis_lucina 29.86

58.86

88.16 56.54

7.9 Cigaritis_acamas Lycaena_dimorpha 44.88 71.29 Lycaena_phlaeas Lycaena_bleusei 12.92 8.55 6.12 Lycaena_virgaureae 5.67 Lycaena_ottomana 11.11 4.36 Lycaena_tityrus 64.34 Lycaena_helle 8.2 Lycaena_alciphron 7.92 Lycaena_dispar Lycaena_thetis 7.19 3.98 Lycaena_thersamon 5.76 Lycaena_hippothoe 0.73 Lycaena_candens

31.25 54.91 25.02 Laeosopis_roboris 101.63 Thecla_betulae 35.56 22.53 Favonius_quercus Tomares_ballus 5.55 Tomares_nogelii 30.98 3 Tomares_callimachus

22.62 12.06 Callophrys_avis 3.33 Callophrys_rubi 0.88Callophrys_suaveola 0.6 Callophrys_chalybeitincta 16.66

9.11 Neolycaena_rhymnus 6.58 50.51 Satyrium_pruni 10.42 Satyrium_w-album Satyrium_acaciae 8.09 4.66 Satyrium_spini 6.76 Satyrium_ledereri 5.89 Satyrium_ilicis 2.75 Satyrium_esculi

18.36 27.22 Azanus_ubaldus 18.35 Azanus_jesous 46.12 9.02

18.79 Zizeeria_knysna 2.01 Zizeeria_karsandra 21.14 35.89 13.29 17.23 12.84 14.76 Tarucus_theophrastus 2.41 Tarucus_balkanicus

19.65 Cacyreus_marshalli 15.07 25.18 Lampides_boeticus 17.71 22.43 8.84 35.18 19.98 17.25 14.57 1.83 23.36 Celastrina_argiolus

13.67 Phengaris_alcon 5.12 Phengaris_arion 17.34 4.39 Phengaris_teleius 2.53 Phengaris_nausithous Scolitantides_orion 10.07 Praephilotes_anthracias 15.56 7.81

33.59 11.7 Iolana_iolas 3.39 Iolana_debilitata 10.1 Glaucopsyche_melanops 4.89 Glaucopsyche_alexis 3.52 13.49 3.02 Glaucopsyche_paphos

6.35 Turanana_taygetica 0.42 12.26 Pseudophilotes_bavius Pseudophilotes_barbagiae 8.12 1.76 Pseudophilotes_abencerragus 3.11Pseudophilotes_panoptes 1.34Pseudophilotes_baton 0.96 Pseudophilotes_vicrama 29.49 24.31 31.5 16.26 30.15 Cyclyrius_webbianus 8.12 Leptotes_pirithous 6.51 29.02 Tongeia_fischeri 9.73

27.13 Cupido_argiades 12.82 4.4 Cupido_decoloratus 0.63 Cupido_alcetas 6.58 Cupido_osiris 3.87 Cupido_minimus 23.5 1.85 Cupido_lorquinii 16.52

10.18 18.35 Luthrodes_galba Freyeria_trochylus 17.21 Plebejus_argus Plebejus_idas 14.55 6.3 1.37 1.88 Plebejus_bellieri 0.5 Plebejus_argyrognomon 12.62 Plebejidea_loewii 6.72 Eumedonia_eumedon 8.16 Kretania_eurypilus 3.3 Kretania_hesperica 4.17 Kretania_trappi 10.79 1.75Kretania_sephirus 8.72 0.47 Kretania_pylaon Cyaniris_semiargus Agriades_optilete 7.346.03 Kretania_psylorita 5.85 Agriades_orbitulus 6.57 Agriades_dardanus 0.87 Agriades_pyrenaicus 4.26 Agriades_zullichi 97.93 9.84 1.53Agriades_aquilo 0.79 Agriades_glandon Glabroculus_cyane Aricia_morronensis 8.92 4.6 Aricia_anteros 3.46 Aricia_cramera 5.82 Aricia_nicias 3.3 Aricia_artaxerxes 1.81Aricia_montensis 9.43 1.34 Aricia_agestis Neolysandra_coelestina Lysandra_bellargus 1.76Lysandra_corydonius 8.34 1.43Lysandra_coridon 0.83Lysandra_caelestissima 0.Ly7sandra_hispana 7.66 0.52 Lysandra_albicans Polyommatus_escheri Polyommatus_thersites 6.34 Polyommatus_amandus 4.9 Polyommatus_daphnis 6.11 Polyommatus_icarus 4.3 Polyommatus_eros 3.55 5.81 Polyommatus_celina 5.11 Polyommatus_nivescens 1.25Polyommatus_dorylas 0.53 Polyommatus_golgus 5.57 Polyommatus_damon Polyommatus_iphigenia 3.26Polyommatus_damocles 4.63 2.73Polyommatus_admetus 1.12Polyommatus_nephohiptamenos 0.56 3.9 Polyommatus_ripartii Polyommatus_damone Polyommatus_violetae 3.6 Polyommatus_fulgens 2.210.73Polyommatus_fabressei 0.61 Polyommatus_dolus 1.79 Polyommatus_humedasae 1.07Polyommatus_orphicus 0.82Polyommatus_aroaniensis 0.66 Polyommatus_timfristos

12.45 48.44 36.76 42.74 20.15 Neptis_rivularis 14.79 Neptis_sappho 2.81

41.75 32.71

61.23 3 7 13.8 12.46 Limenitis_populi 16.85 10.14 Limenitis_reducta 39.64 8.82 Limenitis_camilla 15.11 32.99 40.71 49.01 37.77 42.41 31.23 30.71 36.12

33.81 Issoria_eugenia 6.47 26.45 Issoria_lathonia Brenthis_hecate 13.37 3.41 Brenthis_daphne 2.2 Brenthis_ino 23.43 10.61 Argynnis_pandora 7.31 Argynnis_paphia 6.61 Argynnis_laodice 8.92 Speyeria_aglaja 6.16 Fabriciana_adippe 16.79 3.17Fabriciana_elisa 2.06 Fabriciana_niobe Boloria_eunomia 73.05 10.66 Boloria_graeca 2.59Boloria_pales 1.93Boloria_napaea 1.22Boloria_aquilonaris 1.04 12.86 Boloria_alaskensis Boloria_polaris 6.07 Boloria_tritonia Boloria_frigga 3.51 9.69 Boloria_improba 8.35 Boloria_selene 4.96 Boloria_thore 9.14 Boloria_dia Boloria_freija 5.95 8.48 Boloria_euphrosyne 7.03 Boloria_oscarus 3.61 Boloria_selenis 5.72 Boloria_titania 2.92Boloria_chariclea 1.25 Boloria_angarensis 44.58 30.6 33.81 18.54 9.33 15.93 63.15 15.13 8.03 52.95 13.99 Apatura_iris 6.37 Apatura_metis 2.14 Apatura_ilia 57.09 40.54 33.51

51.94 11.02 32.28 Araschnia_levana 47.92 24.92 36.82 22.61

29.59 Vanessa_cardui 6.97 Vanessa_virginiensis 46.16 24.87 13.06 Vanessa_vulcania 9.72 Vanessa_atalanta 23.56 Aglais_io 21.57 1 0 Aglais_urticae 1.1 Aglais_ichnusa 42.8 15.49 Polygonia_egea 6.71 Polygonia_c-album 5.51 82.58 11.45

10.06 Nymphalis_vaualbum 8.45 Nymphalis_polychloros 5.83 Nymphalis_xanthomelas 4.65 Nymphalis_antiopa

25.06 39.57 32.91 37.17 18.73 8.62 35.74 23.26 Hypolimnas_misippus 20.53 34.29

Euphydryas_desfontainii 11.46 3.34 32.66 Euphydryas_aurinia 1 0 Euphydryas_cynthia 6.43 Euphydryas_iduna 28.15 1.75Euphydryas_intermedia 0.56 Euphydryas_maturna 21.17 2523.17.85 Melitaea_didyma 24.44 Melitaea_trivia 15.31 Melitaea_arduinna 15.3111.72 Melitaea_aetherie 5.75 Melitaea_ornata 3.59 14.14 Melitaea_phoebe Melitaea_cinxia Melitaea_diamina 12.03 Melitaea_asteria 0.83 7.15 Melitaea_aurelia 3.18 Melitaea_varia 2.85 5.38 Melitaea_parthenoides Melitaea_deione 4.05 Melitaea_celadussa 2.88Melitaea_athalia 2.51 Melitaea_britomartis

21.09 Libythea_celtis 12.41 70.77 33.81 10.61 26.8 15.62 Danaus_plexippus 8.3 42.39 24.09 Danaus_chrysippus 14.37 79.5 38.43

25.29 39.34 33.98 41.34 17.76 68.7 14.51 Charaxes_jasius 39.23 48.71 64.38 Lasiommata_maera 14.18 Lasiommata_deidamia 11.04 Lasiommata_petropolitana 4.78 Lasiommata_paramegaera 2.3 54.88 Lasiommata_megera 21.75 Pararge_xiphia 10.83 Pararge_xiphioides 5.93 Pararge_aegeria 19.17 38.54 Kirinia_climene 7.93 Kirinia_roxelana 48.03 17.27 Lopinga_achine 12.33 10.69 36.68 29.47 31.21 43.08

41.28 Coenonympha_phryne 13.61 30.47 Coenonympha_oedippus Coenonympha_glycerion 17.78 46.1 37.45 Coenonympha_thyrsis 7.06 Coenonympha_pamphilus 1 3 10.26 Coenonympha_tullia 5.7 Coenonympha_amaryllis 3.05 12.28 Coenonympha_rhodopensis 35.98 Coenonympha_dorus 11.67 Coenonympha_corinna 8.85 Coenonympha_hero 5.29 Coenonympha_leander 5.04 Coenonympha_gardetta 3.11Coenonympha_arcania 1.49 Coenonympha_orientalis 44.5 34.68 36.75 32.35 33.73 31.62 16.89 Proterebia_phegea 34.83 Ypthima_asterope 17.49 22.84 30.97 11.66 23.07 Melanargia_arge 4.74 Melanargia_occitanica 3.2 Melanargia_pherusa 14.22 Melanargia_ines 13.52 Melanargia_russiae 7.12 Melanargia_larissa 3.41 37.53 Melanargia_lachesis 1.99 Melanargia_galathea Hipparchia_statilinus 2.69 Hipparchia_fatua 8.79 Hipparchia_tamadabae 6.33 Hipparchia_wyssii Hipparchia_tilosi 5.483.18 Hipparchia_bacchus 4.83 Hipparchia_fidia 16.19 3.6 Hipparchia_gomera 28.67 Hipparchia_neomiris Hipparchia_fagi 4.99 0.61 14.16 Hipparchia_syriaca 2.76 Hipparchia_autonoe 1.85 35.91 Hipparchia_hermione 7.33 Hipparchia_mersina 4.23 Hipparchia_miguelensis 2.78 Hipparchia_azorina Hipparchia_maderensis 4.71 1.47 Hipparchia_senthes Hipparchia_aristaeus 2.971.Hippa3 rchia_neapolitana 0.99Hipparchia_blachieri 0.99Hipparchia_volgensis 19.98 1.790.67 Hipparchia_semele Hipparchia_leighebi 1.25Hipparchia_pellucida 1.12Hipparchia_cretica 0.95Hipparchia_christenseni 0.58Hipparchia_cypriensis 0.45 Hipparchia_sbordonii

14.34 Oeneis_tarpeia 11.43 Oeneis_bore 2.76 9.61 Oeneis_ammon Oeneis_melissa 6.56 1.8Oeneis_jutta 1.17 Oeneis_magna 3.67 15.86 Oeneis_norna 33.15 2.58Oeneis_polixenes 1.6 Oeneis_glacialis Arethusana_arethusa 12.3 Minois_dryas 10.12 Brintesia_circe Satyrus_ferula 14.46 3.55 Satyrus_actaea 1.26 Satyrus_virbius Chazara_briseis 12.59 5.1 Chazara_persephone 3.52 Chazara_prieuri Pseudochazara_graeca 0.61 10.86 Pseudochazara_amymone 1.95 Pseudochazara_geyeri 1.03

5.32 Pseudochazara_anthelea 1.22 Pseudochazara_amalthea Pseudochazara_cingovskii 3.61 0.65Pseudochazara_orestes 0.46 Pseudochazara_tisiphone 1.23 Pseudochazara_euxina 0.94Pseudochazara_williamsi 0.79 Pseudochazara_mercurius 23.42

15.4 Hyponephele_huebneri 11.88 Hyponephele_lycaon 8.05 4.05 bioRxiv preprint doi: https://doi.org/10.1101/844175; this version posted November 16, 2019. The copyright holder for this preprint (which Hyponephele_lupina was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made 27.77 Pyronia_bathseba available under aCC-BY 4.0 International license. 13.12 Aphantopus_hyperantus 15.4 30.99 Pyronia_tithonus 11.94 Pyronia_cecilia 17.07 Maniola_cypricola 1.Maniola_halicarnassu2 s 0.92 Maniola_telmessia 2.81Maniola_jurtina 1.47 Maniola_chia 2.21 Maniola_nurag 29.54 2.12 Maniola_megala

Erebia_edda 14.42 6.66 Erebia_fasciata 4.95 Erebia_discoidalis 10.06 Erebia_rossii 7.76 Erebia_cyclopius 4.98 Erebia_embla 2.75 Erebia_disa 16.6 Erebia_epistygne Erebia_ottomana 4.99 Erebia_callias 2.53Erebia_rondoui Erebia_hispania 1.97 13.09 Erebia_nivalis 1.0.568Erebia_calcarius 0.55 Erebia_tyndarus 0.9 Erebia_arvernensis 0.61Erebia_cassioides 0.52 11.74 Erebia_neleus Erebia_claudina Erebia_christi 7.15 Erebia_pharte 6.74 Erebia_epiphron 3.71 Erebia_orientalis 10.25 Erebia_jeniseiensis Erebia_dabanensis 11.469.657.53 Erebia_meolans 2.27 Erebia_palarica 9.51 Erebia_albergana 8.13 Erebia_triarius 4.67 Erebia_polaris 0.34 Erebia_medusa Erebia_aethiops 10.76 Erebia_pandrose 9.36 2.42 Erebia_sthennyo 8.39 Erebia_flavofasciata 7.64 Erebia_gorge 9.997.28 Erebia_pluto Erebia_sudetica 0.77 Erebia_melampus 8.27 Erebia_manto 6.61 Erebia_eriphyle 10.44 6.07 Erebia_euryale 3.58 Erebia_ligea Erebia_oeme 7.92 Erebia_gorgone 3.75 Erebia_aethiopella 2.53Erebia_mnestra 1.53 9.08 Erebia_rhodopensis Erebia_scipio 6.84 Erebia_melas Erebia_pronoe 6.25 Erebia_lefebvrei 5.193.33 Erebia_zapateri 1.02 Erebia_neoridas 4.37 Erebia_montana 2.11Erebia_stiria 0.32 Erebia_styx

120 100 8 0 6 0 4 0 2 0 0 Parnassius mnemosyne 17.43 Parnassius apollo 12.02 Parnassius phoebus 44.63 Archon apollinus Zerynthia rumina 39.11 6.82 Zerynthia cassandra 12.21 Zerynthia polyxena 59.37 Zerynthia cretica Zerynthia cerisy Zerynthia caucasica Iphiclides feisthamelii Iphiclides podalirius 55.83 Papilio alexanor 20.16 Papilio hospiton Papilio machaon Carcharodus tripolinus 5.88 Carcharodus alceae 19.97 Spialia phlomidis 12.93 Spialia therapne 6.35 Spialia sertorius Spialia rosae 24.64 bioRxiv preprint doi: https://doi.org/10.1101/844175; this version posted November 16, 2019. The copyright holder forSpialia this preprint orbifer (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.Muschampia It is made cribrellu m 20.86 Muschampia tessellum available under aCC-BY 4.0 International license. 14.81 Muschampia proto 23.16 Carcharodus orientalis Carcharodus floccifera 10.55 44.14 Carcharodus lavatherae 9.72 Carcharodus baeticus 4.97 Carcharodus stauderi Erynnis tages 4.86 Erynnis marloyi Pyrgus sidae 3.8 Pyrgus carthami 41.97 7.02 Pyrgus malvoides Pyrgus malvae 6.04 Pyrgus andromedae 3.67Pyrgus cacaliae 108.3 10.53 Pyrgus centaureae Pyrgus serratulae 52.3 Pyrgus armoricanus 8.12 Pyrgus cinarae 6.98 Pyrgus cirsii Pyrgus carlinae 6.1 Pyrgus onopordi 5.4 Pyrgus warrenensis Pyrgus foulquieri Pyrgus alveus Heteropterus morpheus 28.21 Carterocephalus silvicola Carterocephalus palaemon Borbo borbonica 44.09 12.59 Pelopidas thrax 10.91 Gegenes nostrodamus 4.41 Gegenes pumilio 29.9 Thymelicus christi Thymelicus acteon 8.65 Thymelicus hyrax 5.26 Thymelicus sylvestris 26.46 Thymelicus lineola Ochlodes sylvanus 14.81 Hesperia comma Leptidea duponcheli 13.15 Leptidea morsei Leptidea juvernica Leptidea sinapis Leptidea reali Gonepteryx rhamni 107.07 Gonepteryx cleobule 3.71Gonepteryx farinosa Gonepteryx cleopatra 32.42 Gonepteryx maderensis Catopsilia florella Colias hyale 72.82 21.21 Colias alfacariensis 4.05 Colias chrysotheme Colias aurorina Colias phicomone Colias erate Colias crocea Colias caucasica Colias myrmidone Colias hecla Colias palaeno 65.2 Colias tyche Colotis evagore Euchloe tagis 14.7 Euchloe charlonia 54.41 7.31 Euchloe bazae 4.95 Euchloe penia 18.64 Anthocharis euphenoides 5.28 Anthocharis cardamines Anthocharis damone Anthocharis gruneri 15.81 Zegris eupheme 13.46 Zegris pyrothoe Euchloe eversi 101.63 14.07 58.14 Euchloe grancanariensis Euchloe hesperidum 11.37 Euchloe belemia Euchloe ausonia Euchloe simplonia 6.97 Euchloe insularis 4.84 Euchloe crameri Aporia crataegi Pontia chloridice 15.57 Pontia callidice 42.96 15.08 Pontia edusa 5.43 Pontia daplidice 23.73 Pieris brassicae Pieris wollastoni Pieris cheiranthi 13.25 Pieris krueperi 11.4 Pieris rapae Pieris mannii 10.46 Pieris ergane Pieris bryoniae Pieris napi Pieris balcana Riodinidae - Lycaenidae Nymphalidae

100 8 0 6 0 4 0 2 0 0 Time (Ma) Papilionidae Hesperiidae Pieridae Hamearis lucina Cigaritis acamas Lycaena dimorpha 44.88 12.92 Lycaena phlaeas 8.55 Lycaena bleusei Lycaena virgaureae 108.3 Lycaena ottomana 11.11 Lycaena tityrus 88.16 Lycaena helle 8.2 Lycaena alciphron Lycaena dispar Lycaena thetis Lycaena thersamon Lycaena hippothoe Lycaena candens Laeosopis roboris 107.07 Thecla betulae 54.91 22.53 35.56 Favonius quercus Tomares ballus 30.98 5.55 Tomares nogelii bioRxiv preprint doi: https://doi.org/10.1101/844175; this version posted November 16, 2019. The copyright holderTomares for this callimachu preprint (whichs was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprintCallophrys in perpetuity. avi sIt is made available under aCC-BY 4.0 International 22license.62 . 3.33Callophrys rubi Callophrys suaveola Callophrys chalybeitincta 16.66 Neolycaena rhymnus 6.58 Satyrium pruni 10.42 Satyrium w-album Satyrium acaciae 8.09 Satyrium spini 50.51 Satyrium ledereri Satyrium ilicis Satyrium esculi Azanus ubaldus 101.63 18.35 Azanus jesous Zizeeria knysna Zizeeria karsandra 21.14 Tarucus theophrastus Tarucus balkanicus 25.18 Cacyreus marshalli 15.07 Lampides boeticus 22.43 Celastrina argiolus Phengaris alcon 35.18 5.12 Phengaris arion 23.36 Phengaris teleius Phengaris nausithous 15.56 Scolitantides orion 10.07 Praephilotes anthracias 11.7 Iolana iolas Iolana debilitata 10.1 Glaucopsyche melanops Glaucopsyche alexis 13.49 33.59 Glaucopsyche paphos Turanana taygetica 12.26 Pseudophilotes bavius Pseudophilotes barbagiae 8.12 Pseudophilotes abencerragus Pseudophilotes panoptes Pseudophilotes baton Pseudophilotes vicrama Cyclyrius webbianus 8.12 Leptotes pirithous Tongeia fischeri Cupido argiades 12.82 27.13 Cupido decoloratus Cupido alcetas 6.58 Cupido osiris Cupido minimus 23.5 Cupido lorquinii Luthrodes galba 97.93 Freyeria trochylus Plebejus argus 17.21 6.3 Plebejus idas Plebejus bellieri Plebejus argyrognomon 14.55 Plebejidea loewii 6.72 Eumedonia eumedon 8.16 Kretania eurypilus Kretania hesperica Kretania trappi 10.79 Kretania sephirus 8.72 Kretania pylaon Cyaniris semiargus Agriades optilete Kretania psylorita Agriades orbitulus 6.57 Agriades dardanus Agriades pyrenaicus Agriades zullichi 9.84 Agriades aquilo Agriades glandon Glabroculus cyane Aricia morronensis 8.92 Aricia anteros Aricia cramera 5.82 Aricia nicias Aricia artaxerxes 9.43 Aricia montensis Aricia agestis Neolysandra coelestina Lysandra bellargus Lysandra corydonius 8.34 Lysandra coridon Lysandra caelestissima Lysandra hispana 7.66 Lysandra albicans Polyommatus escheri Polyommatus thersites 6.34 Polyommatus amandus Polyommatus daphnis 6.11 Polyommatus icarus Polyommatus eros Polyommatus celina 5.81 Polyommatus nivescens Polyommatus dorylas 5.57 Polyommatus golgus Polyommatus damon Polyommatus iphigenia Polyommatus damocles Polyommatus admetus Polyommatus nephohiptamenos Polyommatus ripartii 3.9 Polyommatus damone Polyommatus violetae Polyommatus fulgens Polyommatus fabressei Polyommatus dolus Polyommatus humedasae Polyommatus orphicus Polyommatus aroaniensis Polyommatus timfristos Nymphalidae

100 8 0 6 0 4 0 2 0 0 Time (Ma) bioRxiv preprint doi: https://doi.org/10.1101/844175; this version posted November 16, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

Papilionidae Hesperiidae Pieridae Riodinidae - Lycaenidae 108.3 Neptis rivularis 14.79 Neptis sappho 41.75 Limenitis populi 10.14 Limenitis reducta 8.82 Limenitis camilla 107.07 Issoria eugenia 6.47 Issoria lathonia Brenthis hecate 13.37 61.23 Brenthis daphne Brenthis ino 10.61 Argynnis pandora 7.31 Argynnis paphia 101.63 6.61 Argynnis laodice 8.92 Speyeria aglaja 6.16 Fabriciana adippe 16.79 Fabriciana elisa Fabriciana niobe Boloria eunomia 10.66 Boloria graeca Boloria pales Boloria napaea Boloria aquilonaris 12.86 Boloria alaskensis Boloria polaris 6.07 Boloria tritonia 97.93 9.69 Boloria frigga 73.05 Boloria improba 8.35 Boloria selene Boloria thore 9.14 Boloria dia 8.48 Boloria freija Boloria euphrosyne 7.03 Boloria oscarus Boloria selenis 5.72 Boloria titania Boloria chariclea Boloria angarensis Apatura iris 6.37 Apatura metis Apatura ilia Araschnia levana Vanessa cardui 6.97 Vanessa virginiensis 29.59 13.06 Vanessa vulcania 57.09 9.72 Vanessa atalanta 23.56 Aglais io 1 0 Aglais urticae Aglais ichnusa 15.49 Polygonia egea 6.71 Polygonia c-album 42.8 11.45 Nymphalis vaualbum 8.45 Nymphalis polychloros 82.58 5.83 Nymphalis xanthomelas Nymphalis antiopa Hypolimnas misippus Euphydryas desfontainii Euphydryas aurinia 34.29 1 0 Euphydryas cynthia 6.43 Euphydryas iduna Euphydryas intermedia 28.15 Euphydryas maturna Melitaea didyma Melitaea trivia 15.31 Melitaea arduinna 11.72 Melitaea aetherie 15.31 5.75 Melitaea ornata 14.14 Melitaea phoebe Melitaea cinxia Melitaea diamina 12.03 Melitaea asteria 7.15 Melitaea aurelia Melitaea varia Melitaea parthenoides 5.38 Melitaea deione Melitaea celadussa Melitaea athalia Melitaea britomartis Nymphalidae - 2

100 8 0 6 0 4 0 2 0 0 Time (Ma) Papilionidae 108.3 Hesperiidae Pieridae 107.07 Riodinidae - Lycaenidae 101.63 Nymphalidae - 1 Libythea celtis 97.93 70.77 Danaus plexippus 8.3 Danaus chrysippus 82.58 Charaxes jasius Lasiommata maera 14.18 Lasiommata deidamia 11.04 Lasiommata petropolitana 79.5 Lasiommata paramegaera 21.75 Lasiommata megera Pararge xiphia 10.83 Pararge xiphioides 5.93 Pararge aegeria 19.17 Kirinia climene 64.38 7.93 Kirinia roxelana 17.27 Lopinga achine Coenonympha phryne 13.61 Coenonympha oedippus Coenonympha glycerion 17.78 Coenonympha thyrsis 1 3 7.06 Coenonympha pamphilus 10.26 Coenonympha tullia 5.7 Coenonympha amaryllis 46.1 12.28 Coenonympha rhodopensis Coenonympha dorus 11.67 Coenonympha corinna 8.85 Coenonympha hero 5.29 Coenonympha leander Coenonympha gardetta Coenonympha arcania Coenonympha orientalis Proterebia phegea 34.83 Ypthima asterope Melanargia arge 4.74 Melanargia occitanica Melanargia pherusa 14.22 Melanargia ines 44.5 13.52 Melanargia russiae 7.12 Melanargia larissa Melanargia lachesis Melanargia galathea Hipparchia statilinus Hipparchia fatua 8.79 Hipparchia tamadabae 6.33 Hipparchia wyssii Hipparchia tilosi 5.48 Hipparchia bacchus Hipparchia fidia 16.19 Hipparchia gomera 28.67 Hipparchia neomiris 4.99 Hipparchia fagi Hipparchia syriaca Hipparchia autonoe Hipparchia hermione 7.33 Hipparchia mersina Hipparchia miguelensis 35.91 Hipparchia azorina 4.71 Hipparchia maderensis Hipparchia senthes Hipparchia aristaeus Hipparchia neapolitana bioRxiv preprint doi: https://doi.org/10.1101/844175; this version posted November 16, 2019. The copyright holder forHipparchia this preprint blachier (whichi was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.Hipparchia It volgensiis made s available under aCC-BY 4.0 International license19. .98 Hipparchia semele Hipparchia leighebi Hipparchia pellucida Hipparchia cretica Hipparchia christenseni Hipparchia cypriensis Hipparchia sbordonii Oeneis tarpeia Oeneis bore 11.43 Oeneis ammon Oeneis melissa 6.56 Oeneis jutta Oeneis magna 3.67Oeneis norna 15.86 Oeneis polixenes Oeneis glacialis Arethusana arethusa 12.3 Minois dryas 33.15 10.12 Brintesia circe Satyrus ferula 14.46 Satyrus actaea Satyrus virbius 12.59 Chazara briseis 5.1 Chazara persephone Chazara prieuri Pseudochazara graeca 10.86 Pseudochazara amymone Pseudochazara geyeri 5.32 Pseudochazara anthelea Pseudochazara amalthea Pseudochazara cingovskii Pseudochazara orestes Pseudochazara tisiphone Pseudochazara euxina Pseudochazara williamsi Pseudochazara mercurius Hyponephele huebneri 11.88 Hyponephele lycaon 8.05 Hyponephele lupina Pyronia bathseba 27.77 13.12 Aphantopus hyperantus 15.4 Pyronia tithonus 11.94 Pyronia cecilia 17.07 Maniola cypricola Maniola halicarnassus Maniola telmessia Maniola jurtina Maniola chia Maniola nurag 29.54 Maniola megala Erebia edda 6.66 Erebia fasciata Erebia discoidalis 10.06 Erebia rossii 7.76 Erebia cyclopius Erebia embla Erebia disa 16.6 Erebia epistygne Erebia ottomana 4.99 Erebia callias Erebia rondoui Erebia hispania 13.09 Erebia nivalis Erebia calcarius Erebia tyndarus Erebia arvernensis Erebia cassioides 11.74 Erebia neleus Erebia claudina Erebia christi 7.15 Erebia pharte Erebia epiphron Erebia orientalis 10.25 Erebia jeniseiensis 9.65 Erebia dabanensis Erebia meolans 11.46 Erebia palarica 9.51 Erebia albergana 8.13 Erebia triarius Erebia polaris Erebia medusa 10.76 Erebia aethiops 9.36 Erebia pandrose Erebia sthennyo 8.39 Erebia flavofasciata Erebia gorge 9.99 Erebia pluto Erebia sudetica Erebia melampus 8.27 Erebia manto Erebia eriphyle Erebia euryale 10.44 Erebia ligea Erebia oeme 7.92 Erebia gorgone Erebia aethiopella Erebia mnestra 9.08 Erebia rhodopensis Erebia scipio 6.84 Erebia melas Erebia pronoe Erebia lefebvrei Erebia zapateri Erebia neoridas Erebia montana Erebia stiria Erebia styx

100 8 0 6 0 4 0 2 0 0 Time (Ma) 1 Parnassius_mnemosyne 1 Parnassius_apollo 1 Parnassius_phoebus 1 Archon_apollinus 1 1 1

1 Zerynthia_rumina 1 1 Zerynthia_cassandra 1 Zerynthia_polyxena 1 1 Zerynthia_cretica 1 Zerynthia_cerisy 0.48 Zerynthia_caucasica

1 Iphiclides_feisthamelii 1 Iphiclides_podalirius 0.95 1 0.98 0.86 1 Papilio_alexanor 1 0.36 Papilio_hospiton 1 Papilio_machaon 1 1 1 1

1 0.66 Carcharodus_tripolinus 1 1 Carcharodus_alceae 0.99 1 Spialia_phlomidis 0.81 1 Spialia_therapne 1 1 Spialia_sertorius 1 Spialia_rosae 1 0.65 Spialia_orbifer Muschampia_cribrellum 0.06 0.18 Muschampia_tessellum 0.75 1 Muschampia_proto 0.6 0.6 0.6 Carcharodus_orientalis 0.22 0.85 Carcharodus_floccifera 0.06 0.04 Carcharodus_lavatherae 0.01 Carcharodus_baeticus 0.52 Carcharodus_stauderi

0.99 0.11 Erynnis_tages 1 0.98 Erynnis_marloyi

0.94 1

1 1 1 0.94 0.32 0.93 1 Pyrgus_sidae 0.89 Pyrgus_carthami 1 0.95 Pyrgus_malvoides 1 Pyrgus_malvae 0.09 Pyrgus_andromedae 0.99 Pyrgus_cacaliae 0.93 0.17 Pyrgus_centaureae Pyrgus_serratulae Pyrgus_armoricanus 0.87 Pyrgus_cinarae 1 0.47 0.92 Pyrgus_cirsii 0.99 Pyrgus_carlinae 0.63 Pyrgus_onopordi 0.2 Pyrgus_warrenensis 0.99Pyrgus_foulquieri 0.48 Pyrgus_alveus

1 1 1 1 1 1 1 Heteropterus_morpheus 1 1 1 0.55 1 1 Carterocephalus_silvicola 1 Carterocephalus_palaemon 1 0.52 1 1 0.75 1 0.99 1 0.97 0.5 1 0.98

0.47 0.39 0.44 Borbo_borbonica 0.28 0.71 0.98 Pelopidas_thrax 0.37 0.42 Gegenes_nostrodamus 0.32 0.98 0.1 Gegenes_pumilio 1 1 0.22 0.56 0.31 1 1 0.85 0.94 Thymelicus_christi 1 0.92 Thymelicus_acteon 0.83 Thymelicus_hyrax 0.98 Thymelicus_sylvestris 1 1 Thymelicus_lineola 1 1 1 0.8 1 0.99 1 1 1 0.97 1 1 1 0.61 1 1 Hesperia_comma 1

Leptidea_duponcheli 1 Leptidea_morsei 1 Leptidea_juvernica 1 1 Leptidea_sinapis 1 Leptidea_reali 1 1 1 1 Gonepteryx_rhamni 0.67 0.11 1 Gonepteryx_cleobule 1 1 Gonepteryx_farinosa 0.78Gonepteryx_cleopatra 0.76 0.99 Gonepteryx_maderensis 1 0.46 1 0.86 Catopsilia_florella 1 1 Colias_hyale 1 1 Colias_alfacariensis 1 Colias_chrysotheme Colias_aurorina 1 0.98 Colias_phicomone Colias_erate 0.971 Colias_crocea 0.76Colias_caucasica 0.66 1 Colias_myrmidone 0.88Colias_hecla 1Colias_palaeno 1 Colias_tyche 1 1 1 0.82 Colotis_evagore 1 0.75 1

0.71 1 1 Euchloe_tagis 0.47 Euchloe_charlonia 1 1 Euchloe_bazae 0.6 Euchloe_penia Anthocharis_euphenoides 1 1 Anthocharis_cardamines 1 Anthocharis_damone 0.83 Anthocharis_gruneri Zegris_eupheme 1 0.62 0.18 Zegris_pyrothoe 0.46 Euchloe_eversi 0.57 1 Euchloe_grancanariensis 0.2 Euchloe_hesperidum 0.67 0.54 Euchloe_belemia Euchloe_ausonia 0.93 Euchloe_simplonia 0.8 Euchloe_insularis 0.66 Euchloe_crameri 0.18 1 0.62 1 1 0.96 1 1 1 1 0.81 Aporia_crataegi 0.64 0.84 1 1 0.96 1 0.94 Pontia_chloridice 0.99 0.89 Pontia_callidice 0.42 1 Pontia_edusa 0.81 1 Pontia_daplidice

Pieris_brassicae 0.84 1 Pieris_wollastoni 1 Pieris_cheiranthi 0.71 Pieris_krueperi Pieris_rapae 0.04 1 Pieris_mannii 0.5 Pieris_ergane 1 Pieris_bryoniae 0.98Pieris_napi 0.98 Pieris_balcana

1 1 1 1 1 Hamearis_lucina 1

1

1 1

1 Cigaritis_acamas Lycaena_dimorpha 0.65 1 Lycaena_phlaeas Lycaena_bleusei 1 0.85 0.94 Lycaena_virgaureae 0.14 Lycaena_ottomana 0.55 0.12 Lycaena_tityrus 1 Lycaena_helle 0.25 Lycaena_alciphron 0.59 Lycaena_dispar Lycaena_thetis 0.59 1 Lycaena_thersamon 0.87 Lycaena_hippothoe 1 Lycaena_candens

0.46 0.71 0.2 Laeosopis_roboris 1 Thecla_betulae 1 0.94 Favonius_quercus Tomares_ballus 1 Tomares_nogelii 0.53 1 Tomares_callimachus

0.99 0.78 Callophrys_avis 1 Callophrys_rubi 1Callophrys_suaveola 0.13 Callophrys_chalybeitincta 0.92

0.42 Neolycaena_rhymnus 0.79 0.8 Satyrium_pruni 1 Satyrium_w-album Satyrium_acaciae 0.99 0.66 Satyrium_spini 0.49 Satyrium_ledereri 0.04 Satyrium_ilicis 1 Satyrium_esculi

1 1 Azanus_ubaldus 1 Azanus_jesous 1 1

0.78 Zizeeria_knysna 1 Zizeeria_karsandra 0.78 1 1 1 0.65 0.66 Tarucus_theophrastus 1 Tarucus_balkanicus

1 Cacyreus_marshalli 0.76 1 Lampides_boeticus 0.97 0.62 1 0.37 1 1 1 1 0.6 Celastrina_argiolus

1 Phengaris_alcon 1 Phengaris_arion 1 0.95 Phengaris_teleius 1 Phengaris_nausithous Scolitantides_orion 0.69 Praephilotes_anthracias 0.94 0.45

1 1 Iolana_iolas 1 Iolana_debilitata 0.87 Glaucopsyche_melanops 1 Glaucopsyche_alexis 0.99 1 0.15 Glaucopsyche_paphos

1 Turanana_taygetica 1 1 Pseudophilotes_bavius Pseudophilotes_barbagiae 1 0.99 Pseudophilotes_abencerragus 1 Pseudophilotes_panoptes 1 Pseudophilotes_baton 1 Pseudophilotes_vicrama 0.37 0.95 0.86 1 0.77 Cyclyrius_webbianus 1 Leptotes_pirithous 0.57 0.82 Tongeia_fischeri 1

0.76 Cupido_argiades 1 1 Cupido_decoloratus 1 Cupido_alcetas 1 Cupido_osiris 1 Cupido_minimus 1 0.99 Cupido_lorquinii 1

1 1 Luthrodes_galba Freyeria_trochylus 0.82 Plebejus_argus Plebejus_idas 1 1 0.37 1 Plebejus_bellieri 0.84 Plebejus_argyrognomon 1 Plebejidea_loewii 0.92 Eumedonia_eumedon 0.54 Kretania_eurypilus 0.48 Kretania_hesperica 1 Kretania_trappi 1 0.89Kretania_sephirus 1 1 Kretania_pylaon Cyaniris_semiargus Agriades_optilete 0.770.06 Kretania_psylorita 0.07 Agriades_orbitulus 0.24 Agriades_dardanus 1 Agriades_pyrenaicus 1 Agriades_zullichi 1 1 1 Agriades_aquilo 0.97 Agriades_glandon Glabroculus_cyane Aricia_morronensis 0.95 0.54 Aricia_anteros 0.06 Aricia_cramera 1 Aricia_nicias 1 Aricia_artaxerxes 0.99Aricia_montensis 0.94 0.2 Aricia_agestis Neolysandra_coelestina Lysandra_bellargus 1 Lysandra_corydonius 0.98 0.85Lysandra_coridon 1Lysandra_caelestissima 0.54Lysandra_hispana 0.95 1 Lysandra_albicans Polyommatus_escheri Polyommatus_thersites 1 Polyommatus_amandus 0.36 Polyommatus_daphnis 0.58 Polyommatus_icarus 0.85 Polyommatus_eros 0.17 0.54 Polyommatus_celina 0.38 Polyommatus_nivescens 1 Polyommatus_dorylas 0.89 Polyommatus_golgus 0.11 Polyommatus_damon Polyommatus_iphigenia 0.74 Polyommatus_damocles 1 0.89Polyommatus_admetus 1 Polyommatus_nephohiptamenos 0.94 1 Polyommatus_ripartii Polyommatus_damone Polyommatus_violetae 0.29 Polyommatus_fulgens 1 1Polyommatus_fabressei 0.3 Polyommatus_dolus 0.97 Polyommatus_humedasae 1 Polyommatus_orphicus 0.96Polyommatus_aroaniensis 0.36 Polyommatus_timfristos

1 1 1 1 1 Neptis_rivularis 1 Neptis_sappho 1

0.37 0.99

1 1 1 0.71 Limenitis_populi 1 1 Limenitis_reducta 0.91 0.89 Limenitis_camilla 1 1 1 1 1 1 1 0.98 1

0.99 Issoria_eugenia 1 Issoria_lathonia 1 Brenthis_hecate 1 1 Brenthis_daphne 1 Brenthis_ino 0.91 1 Argynnis_pandora 1 Argynnis_paphia 0.76 Argynnis_laodice 1 Speyeria_aglaja 1 Fabriciana_adippe 1 1 Fabriciana_elisa 1 Fabriciana_niobe Boloria_eunomia 1 0.46 Boloria_graeca 1 Boloria_pales 0.98Boloria_napaea 1 Boloria_aquilonaris 0.34 1 Boloria_alaskensis Boloria_polaris 1 Boloria_tritonia Boloria_frigga 1 1 Boloria_improba 0.76 Boloria_selene 1 Boloria_thore 0.5 Boloria_dia Boloria_freija 0.99 0.88 Boloria_euphrosyne 1 Boloria_oscarus 0.86 Boloria_selenis 1 Boloria_titania 1 Boloria_chariclea 1 Boloria_angarensis 1 1 1 1 1 1 1 0.4 1 1 0.64 Apatura_iris 1 Apatura_metis 1 Apatura_ilia 1 1 1

1 1 1 Araschnia_levana 1 1 1 0.94

1 Vanessa_cardui 1 Vanessa_virginiensis 1 1 1 Vanessa_vulcania 1 Vanessa_atalanta 0.71 Aglais_io 1 1 Aglais_urticae 1 Aglais_ichnusa 1 1 Polygonia_egea 1 Polygonia_c-album 0.95 1 1

0.98 Nymphalis_vaualbum 1 Nymphalis_polychloros 1 Nymphalis_xanthomelas 1 Nymphalis_antiopa

1 1 0.86 1 1 1 1 1 Hypolimnas_misippus 0.96 0.76

Euphydryas_desfontainii 1 1 1 Euphydryas_aurinia 0.72 Euphydryas_cynthia 1 Euphydryas_iduna 1 1 Euphydryas_intermedia 1 Euphydryas_maturna 1 1 1 Melitaea_didyma 0.48 Melitaea_trivia 1 Melitaea_arduinna 0.02 0.98 Melitaea_aetherie 1 Melitaea_ornata 1 0.86 Melitaea_phoebe Melitaea_cinxia Melitaea_diamina 1 Melitaea_asteria 1 1 Melitaea_aurelia 1 Melitaea_varia 0.27 1 Melitaea_parthenoides Melitaea_deione 1 Melitaea_celadussa 0.99 Melitaea_athalia 0.75 Melitaea_britomartis

1 Libythea_celtis 1 1 1 1 1 1 Danaus_plexippus 1 1 1 Danaus_chrysippus 1 1 1

1 1 1 1 1 1 1 Charaxes_jasius 1 1 1 Lasiommata_maera 1 Lasiommata_deidamia 0.99 Lasiommata_petropolitana 1 Lasiommata_paramegaera 1 1 Lasiommata_megera 1 Pararge_xiphia 1 Pararge_xiphioides 1 Pararge_aegeria 0.79 1 Kirinia_climene 1 Kirinia_roxelana 1 0.7 Lopinga_achine 1 0.92 1 1 1 1

1 Coenonympha_phryne 1 1 Coenonympha_oedippus Coenonympha_glycerion 1 1 1 Coenonympha_thyrsis 1 Coenonympha_pamphilus 1 1 Coenonympha_tullia 1 Coenonympha_amaryllis 1 0.33 Coenonympha_rhodopensis 1 Coenonympha_dorus 0.26 Coenonympha_corinna 1 Coenonympha_hero 1 Coenonympha_leander 0.06 Coenonympha_gardetta 1 Coenonympha_arcania 1 Coenonympha_orientalis 1 1 1 0.06 0.35 0.18 1 Proterebia_phegea 0.71 Ypthima_asterope 1 1 1 1 1 Melanargia_arge 1 Melanargia_occitanica 0.76 Melanargia_pherusa 1 Melanargia_ines 0.08 Melanargia_russiae 1 Melanargia_larissa 1 1 Melanargia_lachesis 1 Melanargia_galathea Hipparchia_statilinus 1 Hipparchia_fatua 1 Hipparchia_tamadabae 0.99 Hipparchia_wyssii Hipparchia_tilosi 0.420.93 Hipparchia_bacchus 0.19 Hipparchia_fidia 1 0.41 Hipparchia_gomera 1 Hipparchia_neomiris Hipparchia_fagi 1 1 1 Hipparchia_syriaca 1 Hipparchia_autonoe 0.3 1 Hipparchia_hermione 1 Hipparchia_mersina 0.3 Hipparchia_miguelensis 0.97 Hipparchia_azorina Hipparchia_maderensis 1 1 Hipparchia_senthes Hipparchia_aristaeus 1 0.04Hipparchia_neapolitana 0.43Hipparchia_blachieri 0.53Hipparchia_volgensis 1 0.980.77 Hipparchia_semele Hipparchia_leighebi 0.25Hipparchia_pellucida 0.Hipparchia_cretic4 a 0.68Hipparchia_christenseni 0.86Hipparchia_cypriensis 0.31 Hipparchia_sbordonii

0.97 Oeneis_tarpeia 1 Oeneis_bore 1 1 Oeneis_ammon Oeneis_melissa 1 1 Oeneis_jutta 0.99 Oeneis_magna 1 1 Oeneis_norna 0.96 0.94Oeneis_polixenes 0.9 Oeneis_glacialis Arethusana_arethusa 0.97 Minois_dryas 0.41 Brintesia_circe Satyrus_ferula 0.96 1 Satyrus_actaea 1 Satyrus_virbius Chazara_briseis 1 1 Chazara_persephone 0.58 Chazara_prieuri Pseudochazara_graeca 1 0.99 Pseudochazara_amymone 1 Pseudochazara_geyeri 0.99

1 Pseudochazara_anthelea 1 Pseudochazara_amalthea Pseudochazara_cingovskii 0.95 0.99Pseudochazara_orestes 0.93 Pseudochazara_tisiphone 1 Pseudochazara_euxina 0.Pseudochazara_williams3 i 0.15 Pseudochazara_mercurius 0.99

1 Hyponephele_huebneri bioRxiv preprint doi: https://doi.org/10.1101/844175; this version posted November 16, 2019. The copyright holder for this preprint (which 0.57 Hyponephele_lycaon was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 1 1 Hyponephele_lupina 0.96 Pyronia_bathseba 0.48 Aphantopus_hyperantus 0.98 0.96 Pyronia_tithonus 0.5 Pyronia_cecilia 1 Maniola_cypricola 1 Maniola_halicarnassus 0.47 Maniola_telmessia 1 Maniola_jurtina 0.37 Maniola_chia 0.69 Maniola_nurag 0.94 0.13 Maniola_megala

Erebia_edda 1 1 Erebia_fasciata 0.97 Erebia_discoidalis 1 Erebia_rossii 1 Erebia_cyclopius 1 Erebia_embla 0.94 Erebia_disa 1 Erebia_epistygne Erebia_ottomana 1 Erebia_callias 1 Erebia_rondoui Erebia_hispania 0.92 1 Erebia_nivalis 0.470.15Erebia_calcarius 0.32 Erebia_tyndarus 1 Erebia_arvernensis 0.05Erebia_cassioides 0.15 0.6 Erebia_neleus Erebia_claudina Erebia_christi 1 Erebia_pharte 0.25 Erebia_epiphron 0.99 Erebia_orientalis 0.16 Erebia_jeniseiensis Erebia_dabanensis 0.050.081 Erebia_meolans 1 Erebia_palarica 0.89 Erebia_albergana 0.96 Erebia_triarius 1 Erebia_polaris 1 Erebia_medusa Erebia_aethiops 0.54 Erebia_pandrose 0.54 1 Erebia_sthennyo 0.95 Erebia_flavofasciata 0.09 Erebia_gorge 0.23 0.12 Erebia_pluto Erebia_sudetica 1 Erebia_melampus 0.84 Erebia_manto 0.84 Erebia_eriphyle 0.29 0.61 Erebia_euryale 1 Erebia_ligea Erebia_oeme 0.48 Erebia_gorgone 1 Erebia_aethiopella 0.85Erebia_mnestra 0.95 0.46 Erebia_rhodopensis Erebia_scipio 0.92 Erebia_melas Erebia_pronoe 0.5 Erebia_lefebvrei 0.811 Erebia_zapateri 1 Erebia_neoridas 0.98 Erebia_montana 1 Erebia_stiria 1 Erebia_styx bioRxiv preprint doi: https://doi.org/10.1101/844175; this version posted November 16, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

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vicrama Pontia chloridice Lycaenidae Pseudophilotes baton Aporia crataegi Colias tyche Colias palaeno 10 genes Colias hecla Colias myrmidone Cyclyrius webbianus Colias caucasica Leptotes pirithous Colias crocea Colias erate Nymphalidae Colias phicomone Colias aurorina Cupido decoloratusTongeia fischeri Colias chrysotheme Cupido argiades Colias alfacariensis Colias hyale Catopsilia florella Gonepteryx maderensis Cupido alcetas Gonepteryx cleopatra Cupido minimus Gonepteryx farinosa Cupido osiris 5 genes Gonepteryx cleobule Cupido lorquinii Gonepteryx rhamni FreyeriaLuthrodes trochylus galba Leptidea reali Leptidea sinapis Plebejus argyrognomon Leptidea juvernica Plebejus argus Leptidea morsei Leptidea duponcheli PlebejusPlebejus bellieri idas Pyrgus alveus Eumedonia eumedon Pyrgus foulquieri Plebejidea loewii Pyrgus warrenensis Pyrgus onopordi Kretania eurypilus Pyrgus carlinae Kretania hesperica Pyrgus cirsii Pyrgus cinarae Pyrgus armoricanus KretaniaKretania sephirus trappi Pyrgus serratulae CyanirisKretania semiargus pylaon Pyrgus centaureae Pyrgus cacaliae Agriades optilete Pyrgus andromedae Kretania psylorita Pyrgus malvae Agriades orbitulus Pyrgus malvoides Agriades dardanus Pyrgus carthami Agriades pyrenaicus Pyrgus sidae Erynnis marloyi Agriades zullichi Erynnis tages Carcharodus stauderi AgriadesAgriades glandon aquilo Carcharodus baeticus Glabroculus cyane Carcharodus lavatherae Aricia morronensis Carcharodus floccifera Carcharodus orientalis Muschampia proto Aricia anteros Muschampia tessellum Aricia cramera Muschampia cribrellum AriciaAricia artaxerxes nicias Spialia orbifer Aricia montensis Spialia rosae Neolysandra coelestina Spialia sertorius Aricia agestis Spialia therapne Lysandra bellargus Spialia phlomidis Lysandra corydonius Carcharodus alceae Carcharodus tripolinus LysandraLysandra caelestissima coridon Thymelicus lineola Thymelicus sylvestris Lysandra hispana Thymelicus hyrax Lysandra albicans Thymelicus acteon Polyommatus escheri Thymelicus 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montana Erebia neoridas Neptis rivularis Erebia zapateri Neptis sappho Erebia lefebvrei Limenitis populi Erebia pronoe Limenitis reducta Erebia melas Limenitis camilla Erebia scipio Issoria eugenia Erebia rhodopensis Issoria lathonia Erebia mnestra Brenthis hecate Erebia aethiopella Brenthis daphne Erebia gorgone Brenthis ino Erebia oeme Erebia ligea Argynnis pandora Erebia euryale Argynnis paphia Erebia eriphyle Argynnis laodice Erebia manto Speyeria aglaja Erebia melampus Fabriciana adippe Erebia sudetica Fabriciana elisa Erebia pluto Fabriciana niobe Erebia gorge Boloria eunomia Erebia flavofasciata Boloria graeca Erebia sthennyo Boloria pales Erebia pandrose Erebia aethiops Boloria napaea Erebia medusa Boloria aquilonaris Erebia polaris Erebia triarius BoloriaBoloria alaskensis polaris Erebia albergana Boloria tritonia Erebia palarica Boloria frigga Erebia meolans Boloria improba Erebia dabanensis Boloria selene Erebia jeniseiensis Boloria thore Erebia orientalis Boloria dia Erebia epiphron Boloria freija Erebia pharte Erebia christi Erebia claudina BoloriaBoloria euphrosyne oscarus Erebia neleus Boloria selenis Erebia cassioides Boloria titania Erebia arvernensis Erebia tyndarus Boloria chariclea Erebia calcarius Apatura iris Erebia nivalis Boloria angarensis Erebia hispania ApaturaApatura metis ilia Erebia rondoui Erebia callias Erebia ottomana Araschnia levana Erebia epistygne Vanessa cardui Erebia disa Erebia embla Aglais io Erebia cyclopius VanessaVanessa virginiensis vulcania Erebia rossii Vanessa atalanta Erebia discoidalis Aglais urticae Erebia fasciata Aglais ichnusa Erebia edda Maniola megala Polygonia egea Maniola nurag Maniola chia Polygonia c-album Maniola jurtina Maniola telmessia Maniola halicarnassus Nymphalis vaualbum Maniola cypricola Pyronia cecilia NymphalisNymphalis polychloros antiopa Pyronia tithonus Aphantopus hyperantus Nymphalis xanthomelas Pyronia bathseba Hypolimnas misippus Hyponephele lycaon Hyponephele lupina Euphydryas aurinia 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nomma sieneO nomma

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Proterebia phegea s

Coenonympha Coenonymphapamphilus amaryllisCoenonympha corinna Melanargia larissa

Melanargia russiae Coenonympha leander Melanargia pherusa

Coenonympha arcania Melanargia lachesis Coenonympha gardetta Melanargia occitanica Melanargia galathea Coenonympha rhodopensis Coenonympha orientalis