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Migration is typically associated with risk and uncertainty at the population level, but little is known about its cost¿benefit trade-offs at the species level. Migratory insects in particular often exhibit strong demographic fluctuations due to local bottlenecks and outbreaks. Here, we use genomic data to investigate levels of heterozygosity and long-term population size dynamics in migratory insects, as an alternative to classical local and short-term approaches such as regional field monitoring. We analyse whole-genome sequences from 97 Lepidoptera species and show that individuals of migratory species have significantly higher levels of genome-wide heterozygosity, a proxy for effective population size, than do nonmigratory species. Also, we contribute whole-genome data for one of the most emblematic insect migratory species, the painted lady butterfly (Vanessa cardui), sampled across its worldwide distributional range. This species exhibits one of the highest levels of genomic heterozygosity described in Lepidoptera (2.95 ± 0.15%). Coalescent modelling (PSMC) shows historical demographic stability in V. cardui, and high effective population size estimates of 2 -20 million individuals 10,000 years ago. The study reveals that the high risks associated with migration and local environmental fluctuations do not seem to decrease overall genetic diversity and demographic stability in migratory Lepidoptera. We propose a "compensatory" demographic model for migratory r-strategist organisms in which local bottlenecks are counterbalanced by reproductive success elsewhere within their typically large distributional ranges. Our findings highlight that the boundaries of populations are substantially different for sedentary and migratory insects, and that, in the latter, local and even regional field monitoring results may not reflect whole population dynamics. Genomic diversity patterns may elucidate key aspects of an insect's migratory nature and population dynamics at large spatiotemporal scales., This work was funded by the National Geographic Society (grant WW1-300R-18) and by the grant PID2020-117739GA-I00 from MCIN/AEI/10.13039/501100011033 to G.T., by fellowship FPU19/01593 to A.G.-B., by project LINKA20399 from the CSIC i-LINK-2021 programme to G.T., N.E.P., R.V. and N.B., by grants from the Putnam Expeditionary Fund of the Museum of Comparative Zoology to G.T. and N.E.P., by projects PID2019-107078GB-I00/MCIN/AEI/10.13039/501100011033 and 2017-SGR-991 (Generalitat de Catalunya) to R.V. and G.T., by the University of Malaya (grant H50001-A-000027) to K.G.C., by the Swedish Collegium for Advanced Science (SCAS; Natural Sciences Programme, Knut and Alice Wallenberg Foundation, Postdoc funding) to D.S., by NSF grant DEB-1541560 to N.E.P., and by the Swedish Research Council FORMAS (grant 2019-00670) to N.B., G.T. and R.V., 1 INTRODUCTION2 MATERIALS AND METHODS2.1 Heterozygosity estimates2.2 Behavioural scoring2.3 Comparative analyses2.4 Vanessa cardui sampling and sequencing2.5 Vanessa cardui reference-based assemblies2.6 Vanessa cardui demographic inference3 RESULTS3.1 Heterozygosity rates in Lepidoptera3.2 Vanessa cardui demographic history4 DISCUSSION4.1 Genomic heterozygosity, effective population size and migration4.2 Short-term migratory cost but long-term demographic stability4.3 Delimiting populations in migratory insect speciesAUTHOR CONTRIBUTIONSACKNOWLEDGEMENTSCONFLICT OF INTEREST

The study of ecological interactions between plants, phytophagous insects and their natural enemies is an essential but challenging component for understanding ecosystem dynamics. Molecular methods such as DNA barcoding can help elucidate these interactions. In this study, we employed DNA barcoding to establish hostplant and parasitoid interactions with hesperiid butterflies, using a complete reference library for Hesperiidae of continental Europe and north-western Africa (53 species, 100% of those recorded) based on 2934 sequences from 38 countries. A total of 233 hostplant and parasitoid interactions are presented, some recovered by DNA barcoding larval remains or parasitoid cocoons. Combining DNA barcode results with other lines of evidence allowed 94% species-level identification for Hesperiidae, but success was lower for parasitoids, in part due to unresolved taxonomy. Potential cases of cryptic diversity, both in Hesperiidae and Microgastrinae, are discussed. We briefly analyse the resulting interaction networks. Future DNA barcoding initiatives in this region should focus attention on north-western Africa and on parasitoids, because in these cases barcode reference libraries and taxonomy are less well developed., Support for this research was provided by the Spanish National Research Council (CSIC) with a JAE-Intro fellowship for the introduction to research to ETD (reference numbers JAEINT_20_00248 and JAEINT20_EX_0638) and by projects PID2019-107078GB-I00/MCIN/AEI/10.13039/501100011033 and 2017-SGR-991 (Generalitat de Catalunya) to RV, and PID2020-117739GA-I00/MCIN/AEI/10.13039/501100011033 to GT. We thank the Rachadaphiseksomphot Fund, Graduate School, Chulalongkorn University, for the award of a Senior Postdoctoral Fellowship to DLJQ. Further support for this research was provided by the Academy of Finland (Academy Research Fellow, decision no. 328895) to VD. PDNH acknowledges support from Genome Canada through Ontario Genomics. BV has been funded by the CERCA Programme of the Generalitat de Catalunya and by the Grant RYC-22243-2017, whose PI is Josep Sardanyés. SV was supported by the Spanish Ministry of Economy and Competitiveness, grant PID2020-117822GB-I00 MINEICO/AEI/ FEDER and the European Union., INTRODUCTION MATERIAL AND METHODS RESULTS DISCUSSION CONCLUSION SUPPORTING INFORMATION ACKNOWLEDGEMENTS DATA AVAILABILITY REFERENCES Supplementary data

Butterfly migration across great distances is testimony to their impressive aeronautical skills. Wing size and shape are two key determinants of flight performance, and in butterflies, the specific configuration of forewings to hindwings including the overlapping area between them can have profound effects on overall size and shape. Here we use quantitative morphometrics to analyze the wing size, shape, and flight performance of 190 exemplar species representing a tribal-level phylogeny of Papilionoidea. We identify three morphological ‘flight systems’ that are characterized by these fore- and hindwing configurations (small [beating], medium, and large [gliding]), and our data suggest that the emergence of these systems coincided with periods of paleoclimatic upheaval. A higher proportion of migratory species have wings characteristic of gliders, and their degree of host specialization appears to be more flexible than that of counterparts with beating wings.

Dispersal and migratory movements of phytophagous insects are strongly tight to vegetation phenology. The succeeding broods of seasonally migrating species connect distant regions that differ in the timing of plant growth. Also, the overall plant production and the extent of the growing season determine the breeding capacity of the insects, and thus influence their demographic trends. The use of photosynthetic activity proxies based on remote sensing observations, such as NDVI or EVI, opens new avenues to study migratory patterns of insects, a largely understudied field. Here, we present two applications of NDVI to study the migration of the Painted Lady butterfly, Vanessa cardui, an obligate migratory species with a large migratory range encompassing the Palaearctic and the Afrotropics.We develop a spatio-temporal Species Distribution Model (SDM) to determine monthly suitable areas for reproduction across its migratory range. We use a comprehensive dataset of V. cardui breeding occurrences and time-series of climatic and vegetation growth variables, including EVI and monthly difference (EVI change). Vegetation indexes proved to be relevant variables explaining V. cardui breeding suitability, having higher importance in the Afrotropical region. Moderate EVI values showed best conditions for breeding. EVI change had a better fit with slight increases of vegetation growth, discarding sharp changes in greening. These patterns agree with the phenology of V. cardui herbaceous hostplants in the growing season, distributed in open-areas such as meadows, weeds and bushland. In the temperate zone, vegetation growth was not a limiting factor and suitability was mostly explained by variables related to temperature.At a temporal scale, we assess the role that anomalies in photosynthetic activity might have in modulating demographic trends of V. cardui. We performed a pixel-based time-series analysis of monthly NDVI values from 2000 to 2022. We observe that four demographic outbreaks of the butterfly observed in Europe are immediately preceded by anomalous vegetation growth events in suitable breeding regions in Africa and/or the Middle East, suggesting a strong association between both events. We investigate in higher detail the largest of the outbreak episodes in 2019. The resulting maps of anomalies showed high signal in regions of the Middle East from December 2018 to May 2019. The highest anomalies were detected in rocky deserts and arid and semi-arid shrublands, while sand deserts were not affected. The large extent found with exceptional greening could have functioned as massive breeding grounds for V. cardui. This hypothesis strongly matches massive citizen science data of V. cardui observations that were first observed in the Arabian Peninsula in March, and that further spread all over Europe in unprecedented numbers.Taken together, we highlight the potential of remote-sensing vegetation indices to study seasonal migratory movements of phytophagous insects. We show how NDVI can inform models about the potential shifting distributions of migratory species, and how NDVI anomalies can be used to predict potential population outbreaks. In a world where insects represent the majority of terrestrial diversity, the use of vegetation indices may become standard in the fields of insect movement ecology and population dynamics.

Migratory insects serve an important role to ecosystems and economies as they participate in the long-distance transfer of nutrients, pollen, and biomass. However, migratory insects are understudied, especially compared to birds and mammals, partially because traditional tracking techniques (e.g. mark-recapture, biologgers) are often ineffective for insects because insects are small, short-lived, and numerous. Thus, isotope geolocation has become an effective tool for studying dispersing insects. The painted lady butterfly (Vanessa cardui (L.)) is a virtually cosmopolitan species that was recently found to make regular, annual multi-generational migrations across the Sahara Desert. Previous work geolocating painted ladies with hydrogen isotopes has shown early spring migratory movements of painted ladies from sub-Saharan Africa to Mediterranean Europe and autumn movements to the sub-Sahara from Europe. However, these previous works using hydrogen isotopes were unable to offer refined estimates of natal origin due to the inherent limitations of the technique. Here, we update previous hydrogen isotope-based geographic assignment by (1) using an updated model of hydrogen isotope variations across the landscape (i.e., isoscape), (2) combining with strontium isotope-based geographic assignment, and (3) expanding the number of geolocated butterflies to include capture locations on both sides of the Sahara Desert across different years. Using this method, we spatially refine previous estimates of the natal origins of successful trans-Saharan migrants and estimate the distances that successful migrants travelled. Overall, this study demonstrates the advantages of combining hydrogen and strontium isotopes for the geographic assignment of migratory butterflies and further advances our understanding of long-distance insect migration.

Migratory insects may move in very large numbers, even surpassing migratory vertebrates in biomass. However, the extent of aerial flows of insects circulating around the planet, as well as their impact on ecosystems and biogeography, remain almost unstudied because of methodological challenges associated with tracking small, short-lived, organisms. In this presentation, I will show how a novel integrative approach allows reconstructing long-range insect movements, through a combination of tools on genetics, isotope ecology, ecological niche modelling, pollen metabarcoding, field ecology, and citizen science.I will show the latest discoveries on the migrations of the Painted Lady butterfly (Vanessa cardui). This butterfly species is the most cosmopolitan of all butterflies, and it is known by its regular trans-Saharan migrations, that entail distances of >4000 km, similar to those of some birds. First, we track a migratory outbreak of V. cardui butterflies taking place at a continental scale in Europe, the Middle East, and Africa from March 2019 to November 2019. We use DNA metabarcoding to identify plants from pollen transported by the insects. From 265 butterflies collected in 14 countries over 7 months, we molecularly identify 398 plants. We develop a novel geolocation approach based on combining probability rasters from species distribution modelling of each identified plant, and thus trace back the location of the outbreak’s origin and the origin of each of the subsequent generations. We show a strong representation of plants of Middle Eastern distribution in butterfly swarms collected in Eastern Europe in early spring. Swarms collected in Northern Europe in late spring were highly represented by plants of Mediterranean origin, and swarms collected in the summer in the Mediterranean likely originated in central and Northern Europe.Second, we report the first proven transatlantic crossing by individual insects, a journey of at least 4,200 km from West Africa to South America. This discovery was possible through gathering evidence from multiple sources, including coastal field surveys, wind trajectory modelling, phylogeography, pollen metabarcoding, and multi-isotope geolocation of natal origins. Wind trajectories were exceptionally favourable for the butterflies to disperse across the Atlantic from West Africa. Population genetic analyses clustered the butterflies collected in South America with the European-African population, ruling out the possibility that the migrants originated in America. Pollen metabarcoding showed highly represented plants endemic to the Sahelian region. Finally, a dual isotope analysis of hydrogen (δ2H) and strontium (87Sr/86Sr) combined with a spatio-temporal niche model of suitable reproductive habitat geolocated the natal origins of the migrants to regions in Mali, Morocco, or Portugal, and thus not discarding a journey also involving a trans-Saharan crossing.In summary, this work contributes new methodological avenues to advance our understanding of the dispersal and migration of insects. The findings here reported suggest that we may be underestimating long-range dispersal in insects, and highlight the importance of aerial highways connecting continents by trade winds. Overall, we will discuss the scale and potential implications that insect migratory movements represent for ecosystems and nature conservation worldwide.

Migratory insects are key players in ecosystem functioning and services, but their spatiotemporal distributions are typically poorly known. Ecological niche modeling (ENM) may be used to predict species seasonal distributions, but the resulting hypotheses should eventually be validated by field data. The painted lady butterfly (Vanessa cardui) performs multigenerational migrations between Europe and Africa and has become a model species for insect movement ecology. While the annual migration cycle of this species is well understood for Europe and northernmost Africa, it is still unknown where most individuals spend the winter. Through ENM, we previously predicted suitable breeding grounds in the subhumid regions near the tropics between November and February. In this work, we assess the suitability of these predictions through i) extensive field surveys and ii) two-year monitoring in six countries: a large-scale monitoring scheme to study butterfly migration in Africa. We document new breeding locations, year-round phenological information, and hostplant use. Field observations were nearly always predicted with high probability by the previous ENM, and monitoring demonstrated the influence of the precipitation seasonality regime on migratory phenology. Using the updated dataset, we built a refined ENM for the Palearctic-African range of V. cardui. We confirm the relevance of the Afrotropical region and document the missing natural history pieces of the longest migratory cycle described in butterflies., This work was funded by the National Geographic Society (grant WW1-300R-18); by the British Ecological Society (grant LRB16/1015); by the Research and Conservation Projects of the Fundació Barcelona Zoo; by the grant PID2020-117739GA-I00/MCIN/AEI/10.13039/501100011033 of the Spanish Ministry of Science and Innovation and the Spanish State Research Agency to G.T.; by the grant LINKA20399 from the Spanish National Research Council iLink program to G.T., C.P.B., N.E.P., and R.V.; by fellowship FPU19/01593 of the program Formación de Profesorado Universitario (FPU) to A.G.-B.; by the Turkana Basin Institute, National Geographic Society, and Whitley Fund for Nature to D.J.M.; and by grant 2018-00738 of the New Frontiers in Research Fund (Government of Canada) to G.T. and C.P.B., Significance Abstract Results Field Surveys, Larval Hostplants, and Field-Based Model Validation Monitoring Results and Population Dynamics across Regions A Refined Model for the Afrotropical Region Discussion The Afrotropical Breeding Grounds of V. cardui: Multiple Generations Shift South Toward the Tropics Diversity and Phenology of Larval Hostplants in the Afrotropics The Ecological Relevance of Delimiting Spatiotemporal Distributions in Migratory Insects Conclusion Methods December-January Field Surveys and Year-Round Monitoring Spatiotemporal Ecological Niche Modeling Data, Materials, and Software Availability Acknowledgments Supporting Information References

The oogenesis-flight syndrome reflects the temporal allocation of energy resources between dispersal / migration and reproduction and is a key concept in research on migratory behaviour in animals. In migratory butterflies, host plant abundance and quality may act as environmental cues to switch between the two states, but the mechanisms regulating this process are virtually unknown. Here, we used an experimental set-up to assess how variation in host plant abundance affected the activity of regulatory elements in the painted lady butterfly (Vanessa cardui), a model species for insect migratory behaviour studies. Chromatin immunoprecipitation (ChIP-seq) was used to evaluate histone tail modifications of H3K27ac and H3K4me3, as a proxy for regulatory activity. The results indicate that recently eclosed females that had access to host plants invested in reproduction at an earlier stage and that variation in host plant abundance triggered significant differences in regulatory element activity via histone tail acetylation. The functions of genes in the vicinity of differentially activated regions were primarily associated with metabolism, egg shell formation, female receptivity, muscle activity, pheromone binding and chromosome maintenance. Our results provide a first glimpse into the regulatory underpinnings of how females perceive the environment and allocate resources for either migration or reproduction and a starting point for more detailed understanding of the links between environmental variation, gene regulation and behaviour in butterflies.

Butterflies are a diverse and charismatic insect group that are thought to have evolved with plants and dispersed throughout the world in response to key geological events. However, these hypotheses have not been extensively tested because a comprehensive phylogenetic framework and datasets for butterfly larval hosts and global distributions are lacking. We sequenced 391 genes from nearly 2,300 butterfly species, sampled from 90 countries and 28 specimen collections, to reconstruct a new phylogenomic tree of butterflies representing 92% of all genera. Our phylogeny has strong support for nearly all nodes and demonstrates that at least 36 butterfly tribes require reclassification. Divergence time analyses imply an origin ~100 million years ago for butterflies and indicate that all but one family were present before the K/Pg extinction event. We aggregated larval host datasets and global distribution records and found that butterflies are likely to have first fed on Fabaceae and originated in what is now the Americas. Soon after the Cretaceous Thermal Maximum, butterflies crossed Beringia and diversified in the Palaeotropics. Our results also reveal that most butterfly species are specialists that feed on only one larval host plant family. However, generalist butterflies that consume two or more plant families usually feed on closely related plants., Funding came from the US National Science Foundation (NSF) GoLife ‘ButterflyNet’ collaborative grant (DEB-1541500, 1541557, 1541560) to A.Y.K., R.P.G., D.J.L. and N.E.P. Specimen collection and preservation was funded by NSF DBI-1349345, 1601369, DEB-1557007 and IOS-1920895 (A.Y.K.), NSF DEB-1120380 (D.J.L.), grants 9285-13 and WW-227R-17 from the National Geographic Society (D.J.L.), NSF DBI-1256742 (A.Y.K. and K.R.W.), NSF DEB-0639861 (K.R.W.) and NSF SES-0750480, DEB-0447244 and DEB-9615760 (N.E.P.). M.E. was supported by the Research Council of Norway (no. 204308) and the Hintelmann Scientific Award for Zoological Systematics. F.L.C. was supported by the European Research Council under the European Union’s Horizon 2020 research and innovation programme (project GAIA, no. 851188). M.P.B. was supported by the Swedish Research Council (IPG no. 2020‐06422). R.V. was supported by the Spanish Ministry of Science and Innovation grant PID2019-107078GB-I00/AEI/10.13039/501100011033. G.T. was supported by the Spanish Ministry of Science and Innovation (grants PID2020-117739GA-I00/AEI/10.13039/501100011033 and RYC2018-025335-I). V.L. was supported by the Russian Science Foundation (grant 19-14-00202) and by the Ministry of Science and Higher Education of the Russian Federation (grant 075-15-2021-1069). M.Y. was supported by MEXT KAKENHI no. 19916010 and JSPS KAKENHI grants 13010131, 23570111, 26440207, 17K07528 and 21H02215. A.B.B.M., H.P.R. and N.O.M. were supported by CNPQ grants proc 563332/2010-7 and 304273/2014-7., Main Results and discussion Methods Data availability Code availability References Acknowledgements Author information Ethics declarations Peer review Additional information Supplementary information Rights and permissions About this article

Seasonal environmental fluctuations provide formidable challenges for living organisms, especially small ectotherms such as butterflies. A common strategy to cope with harsh environments is to enter diapause, but some species avoid the unsuitable conditions by migrating. Despite a growing understanding of migration in the life cycles of some butterfly species, it is unknown how individuals register and store environmental cues to determine whether and where to migrate. Here, we explored how competition and host plant availability during larval development affect patterns of DNA methylation in the migratory painted lady (Vanessa cardui) butterfly. We identify a set of potentially functional methylome shifts associated with differences in the environment, indicating that DNA methylation is involved in the response to different conditions during larval development. By analysing the transcriptome for the same samples used for methylation profiling, we also uncovered a non-linear relationship between gene body methylation and gene expression. Our results provide a starting point for understanding the interplay between DNA methylation and gene expression in butterflies in general and how differences in environmental conditions during development can trigger unique epigenetic marks that might be important for behavioural decisions in the adult stage.

Anthropogenic activities are exposing insects to abnormal levels of toxic metals, with unknown implications for migratory insects. Simultaneously, metals and metal isotopes have become promising tools for the geolocation of migratory insects. Furthering our understanding of metal cycling in insect tissues is essential, both for the development of metals and metal isotopes as geolocation tools, and for assessing the toxicity risks of metals to insects. We conducted a diet-switching experiment on monarch butterflies (Danaus plexippus) with controlled larval and adult diets to evaluate the dietary and environmental sources of 23 metals and metalloids, strontium isotopes, and lead isotopes to insect wing tissues over a period of 8 weeks. Concentrations of Ca, Co, and Sb differed between the sexes. Ni and Zn bioaccumulated in the insect wing tissues over time, likely from the adult diet, while increases in Al, Cr, Cd, Cu, Fe, and Pb were likely from external sources (i.e., dust aerosols). Bioaccumulation of Pb in the monarch wings was confirmed by Pb isotopes to be from external anthropogenic sources, revealing the potential of Pb isotopes to become an indicator and tracer of metal pollution exposure along migratory paths. Concentrations of Ba, Cs, Mg, Na, Rb, Sr, Ti, Tl, and U appeared to be unaffected by dietary or environmental contamination and should be further developed for geolocation purposes. Strontium isotope ratios remained indicative of the larval diet, at least in males, supporting its potential as a geolocation tool. However, the difference in strontium isotope ratios between sexes, as well as the possibility of external contamination by wetting, requires further investigation. Our results demonstrate the complexity of metal cycling in insects and the need for further investigations, as well as the value of studying metals to develop new tools to quantify pollution exposure, metal toxicity and insect mobility.

Taxa are frequently labeled incertae sedis when their placement is debated at ranks above the species level, such as their subgeneric, generic, or subtribal placement. This is a pervasive problem in groups with complex systematics due to difficulties in identifying suitable synapomorphies. In this study, we propose combining DNA barcodes with a multilocus backbone phylogeny in order to assign taxa to genus or other higher-level categories. This sampling strategy generates molecular matrices containing large amounts of missing data that are not distributed randomly: barcodes are sampled for all representatives, and additional markers are sampled only for a small percentage. We investigate the effects of the degree and randomness of missing data on phylogenetic accuracy using simulations for up to 100 markers in 1000-tips trees, as well as a real case: the subtribe Polyommatina (Lepidoptera: Lycaenidae), a large group including numerous species with unresolved taxonomy. Our simulation tests show that when a strategic and representative selection of species for higher-level categories has been made for multigene sequencing (approximately one per simulated genus), the addition of this multigene backbone DNA data for as few as 5¿10% of the specimens in the total data set can produce high-quality phylogenies, comparable to those resulting from 100% multigene sampling. In contrast, trees based exclusively on barcodes performed poorly. This approach was applied to a 1365-specimen data set of Polyommatina (including ca. 80% of described species), with nearly 8% of representative species included in the multigene backbone and the remaining 92% included only by mitochondrial COI barcodes, a phylogeny was generated that highlighted potential misplacements, unrecognized major clades, and placement for incertae sedis taxa. We use this information to make systematic rearrangements within Polyommatina, and to describe two new genera. Finally, we propose a systematic workflow to assess higher-level taxonomy in hyperdiverse groups. This research identifies an additional, enhanced value of DNA barcodes for improvements in higher-level systematics using large data sets., This work was funded by projects PID2019-107078GB-I00/AEI/10.13039/501100011033 and 2017-SGR-991 (Generalitat de Catalunya) to R.V. and G.T., by the Committee for Research and Exploration of the National Geographic Society [grant WW1300R18 to G.T.], by the Putnam Expeditionary Fund of the Museum of Comparative Zoology (to all authors), by the U.S. National Science Foundation [DEB-0447244, and DEB-1541560 to N.E.P], and by the Ramón y Cajal programme of the Spanish Ministry of Science and Innovation [RYC2018-025335-I to G.T.]. Taxonomic studies and descriptions of new genera and subgenera were supported by the Russian Science Foundation [19-14-00202] to the Zoological Institute of the Russian Academy of Sciences to V.L., Materials and MethodsResultsDiscussionConclusionsSupplementary MaterialAcknowledgmentsFundingReferences

Migration is typically associated with risk and uncertainty at the population level, but little is known about its cost and benefit trade-offs at the species level. Migratory insects often exhibit strong demographic fluctuations due to local bottlenecks and outbreaks. Here, we use genomic data to investigate levels of heterozygosity and long-term population size dynamics in migratory insects, as an alternative to classical local and short-term approaches such as regional field monitoring. We analyze whole-genome sequences from 97 Lepidoptera species and show that migratory species have significantly higher levels of genome-wide heterozygosity, a proxy for effective population size, than non-migratory species. Also, we contribute whole-genome data for one of the most emblematic insect migratory species, the painted lady butterfly (Vanessa cardui), sampled across its worldwide distribution range. This species exhibits one of the highest levels of heterozygosity described in Lepidoptera (2.95% ± 0.15). Coalescent modeling (PSMC) shows historical demographic stability in V. cardui, and high effective population size estimates of 2 to 20 million individuals 10,000 years ago. The study reveals that the high risks associated with migration and local environmental fluctuations do not seem to decrease overall genetic diversity and demographic stability in migratory Lepidoptera. We propose a “compensatory” demographic model for migratory r-strategist organisms in which local bottlenecks are counterbalanced by reproductive success elsewhere within their typically large distributional ranges. Our findings highlight that the delimitations of populations are substantially different for sedentary and migratory insects, and that, in the latter, local and even regional field monitoring results may not reflect whole population dynamics. Genomic diversity patterns may elucidate key aspects of an insect’s migratory nature and population dynamics at large spatiotemporal scales.

Butterflies are a diverse and charismatic insect group that are thought to have diversified via coevolution with plants and in response to dispersals following key geological events. These hypotheses have been poorly tested at the macroevolutionary scale because a comprehensive phylogenetic framework and datasets on global distributions and larval hosts of butterflies are lacking. We sequenced 391 genes from nearly 2,000 butterfly species to construct a new, phylogenomic tree of butterflies representing 92% of all genera and aggregated global distribution records and larval host datasets. We found that butterflies likely originated in what is now the Americas, ∼100 Ma, shortly before the Cretaceous Thermal Maximum, then crossed Beringia and diversified in the Paleotropics. The ancestor of modern butterflies likely fed on Fabaceae, and most extant families were present before the K/Pg extinction. The majority of butterfly dispersals occurred from the tropics (especially the Neotropics) to temperate zones, largely supporting a “cradle” pattern of diversification. Surprisingly, host breadth changes and shifts to novel host plants had only modest impacts.

Motivation Butterflies represent a model in biology and a flagship group for invertebrate conservation. We provide four new resources for the Western Palaearctic butterflies: (1) an updated checklist comprising 552 species; (2) a curated dataset of 32,126 mitochondrial cytochrome c oxidase subunit I (COI) sequences for 532 species, including a de novo reference library for the Maghreb (Morocco, northern Algeria and Tunisia) and Macaronesia (Azores, Madeira and Canary Islands); (3) seven indexes of intraspecific genetic variation (IGV): observed and expected number of haplotypes, haplotype and nucleotide diversity, two fixation indexes and maximum p-distance; and (4) species-level maps illustrating the distribution of COI variability and haplotype networks. The updated checklist will be fundamental for any application dealing with butterfly diversity in the Western Palaearctic. The IGV indexes provide measures for genetic polymorphism and spatial structure and represent proxies for dispersal capacity. These resources will facilitate comparative studies of macrogenetics, foster integrative taxonomy and aid conservation strategies. Main types of variables contained A complete species checklist in table format, 32,126 mitochondrial DNA barcodes provided with metadata (species membership, WGS84 coordinates and sequence length) and a book in PDF format, including the IGV atlas and indexes, are provided. Spatial location and grain The checklist encompasses Europe up to the Urals in the east, north Macaronesia (the Azores, Madeira and the Canary Islands) and the Maghreb (Morocco, northern Algeria and Tunisia). COI sequences have been retained in the geographical interval of -31.3 to 67.5° of longitude and 27.5 - 71.2° of latitude. Time period and grain COI sequences originate from studies published between 1998 and 2022 and from de novo sequencing of 2541 specimens done between 2007 and 2022. Major taxa and level of measurement Butterflies (Lepidoptera: Papilionoidea), analysed from individual to species level. Software format Data and functions to manage the dataset are provided in the iodatabase R package (https://github.com/leondap/iodatabase) and in Dryad (https://doi.org/10.5061/dryad.9w0vt4bjj)., Support for this research was provided by the Academy of Finland (Academy Research Fellow, decision no. 328895) and by a Marie Curie International Outgoing Fellowship within the 7th European Community Framework Programme (project no. 625997) to V.D., by “la Caixa” Foundation (ID 100010434) to M.M. (grant LCF/BQ/DR20/11790020), and by projects CGL2010-21226/BOS and CGL2013-48277-P (Spanish Ministerio de Economía y Competitividad), CGL2016-76322 (AEI/FEDER, UE), PID2019-107078GB-I00 (MCIN/AEI/10.13039/501100011033) and 2017-SGR-991 (Generalitat de Catalunya) to R.Vi., the grant BES-2017-080641 funded by MCIN/AEI/10.13039/501100011033 and by “ESF Investing in your future” to J.C.H., the project PID2020-117739GA-I00 (MCIN/AEI/10.13039/501100011033) to G.T., and by the Direttiva Biodiversità 2019 and 2020 projects (Ministero della Transizione Ecologica) to L.D., 1 INTRODUCTION 2 METHODS 2.1 Checklists and geographical ranges 2.2 Data acquisition, curation and quality control 2.3 Indexes of genetic variation 2.4 Maps of genetic variation 2.5 Haplotype networks 2.6 Script availability 3 RESULTS AND DISCUSSION ACKNOWLEDGMENTS FUNDING INFORMATION CONFLICT OF INTEREST BIOSKETCH

One of the main consequences of globalization is the intensification of biological introductions. Because of their negative impact on environments, the early detection and monitoring of introduced species through molecular approaches is gaining increased uptake. This study assembles 2,278 DNA barcode records to examine contemporary patterns of sequence variation in mitochondrial cytochrome c oxidase I (COI) in five butterfly species introduced to the Nearctic, with a focus on Pieris rapae Linnaeus (Lepidoptera: Pieridae) and Thymelicus lineola Ochsenheimer (Lepidoptera: Hesperiidae). Parameters of genetic diversity were low (i.e., h < 0.606, ¿ < 0.0039) for Nearctic populations of all analyzed species. Those of P. rapae and T. lineola showed marked genetic differentiation from their source populations in the Palearctic. Haplotype distributions in their Nearctic populations exposed a starburst pattern with a few common haplotypes known from Palearctic, and infrequent haplotypes diverging from them at only one or two nucleotide sites. Some uncommon haplotypes were only found in the Nearctic suggesting they originated after invasion, while others also occur in the Palearctic. This study provides an example of genetic paradox of invasion, where species often rapidly expand their distribution and become dominant in the new habitat despite their depleted levels of sequence variation., This study was supported, in part, by a NSERC Discovery grant to PDNH and by an award from the Canada First Research Excellence Fund for the support of Food from Thought research program. This research was also supported by the Academy of Finland (Academy Research Fellow, decision no. 328895) to VD, by project PID2020-117739GA-I00/MCIN/AEI/10.13039/501100011033 to GT, by project PID2019-107078GB-I00/MCIN/AEI/ 10.13039/501100011033 to RV, and by projects related to the Direttiva Biodiversità del Ministero della Transizione Ecológica to LD, INTRODUCTION MATERIAL AND METHODS Sampling Genetic analysis RESULTS Sampling Genetic diversity and haplotype distribution DISCUSSION ACKNOWLEDGEMENTS FUNDING REFERENCES

Characterization of gene family expansions and crossing over is crucial for understanding how organisms adapt to the environment. Here, we develop a high-density linkage map and detailed genome annotation of the painted lady butterfly (Vanessa cardui) - a non-diapausing, highly polyphagous species famous for its long-distance migratory behavior and almost cosmopolitan distribution. Our results reveal a complex interplay between regional recombination rate variation, gene duplications and transposable element activity shaping the genome structure of the painted lady. We identify several lineage specific gene family expansions. Their functions are mainly associated with protein and fat metabolism, detoxification, and defense against infection - critical processes for the painted lady's unique life-history. Furthermore, the detailed recombination maps allow us to characterize the regional recombination landscape, data that reveal a strong effect of chromosome size on the recombination rate, a limited impact of GC-biased gene conversion and a positive association between recombination and short interspersed elements., Financial support for this project was provided by FORMAS (Research grant 2019-00670 to N.B.) and The Swedish Collegium for Advanced Science (Natural Sciences Programme, Knut and Alice Wallenberg Foundation, Postdoc funding for D.S.). R.V. was supported by the grant PID2019-107078GB-I00 funded by MCIN/AEI/10.13039/501100011033. G.T. was supported by the grant PID2020-117739GA-I00 funded by MCIN/AEI/10.13039/501100011033 and by “La Caixa” Foundation (ID 100010434) through the grant LCF/BQ/PR19/11700004., 1. Introduction 2. Results 2.1. Linkage map and genome annotation 2.2. Synteny 2.3. Gene family evolution 2.4. Patterns of recombination rate variation 2.4.1. Global and chromosome specific recombination rates 2.4.2. Intra-chromosomal variation in recombination rate and associations with genomic elements 3. Discussion 3.1. The genome of the painted lady butterfly 3.2. Sex chromosomes 3.3. Gene family analysis 3.4. Patterns of recombination rate variation 4. Conclusions 5. Methods 5.1. Linkage map 5.1.1. Sampling and DNA-extraction 5.1.2. Building the linkage map 5.2. Genome annotation and whole genome statistics 5.2.1. Genome assembly statistics 5.2.2. Gene and repeat annotation 5.3. Manual curation 5.4. Gene family evolution 5.5. Gene ontology enrichment 5.6. Recombination rate analysis 5.6.1. Chromosome level analysis 5.6.2. Window-based analysis Data access CRediT authorship contribution statement Declaration of Competing Interest Acknowledgements Appendix A. Supplementary data Data availability References

Male butterflies in the hyperdiverse tribe Eumaeini possess an unusually complex and diverse repertoire of secondary sexual characteristics involved in pheromone production and dissemination. Maintaining multiple sexually selected traits is likely to be metabolically costly, potentially resulting in trade-offs in the evolution of male signals. However, a phylogenetic framework to test hypotheses regarding the evolution and maintenance of male sexual traits in Eumaeini has been lacking. Here, we infer a comprehensive, time-calibrated phylogeny from 379 loci for 187 species representing 91% of the 87 described genera. Eumaeini is a monophyletic group that originated in the late Oligocene and underwent rapid radiation in the Neotropics. We examined specimens of 818 of the 1096 described species (75%) and found that secondary sexual traits are present in males of 91% of the surveyed species. Scent pads and scent patches on the wings and brush organs associated with the genitalia were probably present in the common ancestor of Eumaeini and are widespread throughout the tribe. Brush organs and scent pads are negatively correlated across the phylogeny, exhibiting a trade-off in which lineages with brush organs are unlikely to regain scent pads and vice versa. In contrast, scent patches seem to facilitate the evolution of scent pads, although they are readily lost once scent pads have evolved. Our results illustrate the complex interplay between natural and sexual selection in the origin and maintenance of multiple male secondary sexual characteristics and highlight the potential role of sexual selection spurring diversification in this lineage., Proceedings of the Royal Society B: Biological Sciences, 288 (1950), ISSN:0080-4649, ISSN:0950-1193, ISSN:1471-2954, ISSN:0962-8452

Extremely spezialized life history strategies are sometimes seen as evolutionary dead-ends. The Afrotropical butterfly genus Lepidochrysops exhibits such a life history. All 137 species are known or assumed to be phyto-predaceous, with caterpillars feeding on plants for the first few instars and later switching to parasitizing ants in the Formicinae, either by consuming ant brood or being fed by the ants through trophallaxis. The only other butterflies that exhibit this phyto-predaceous life history are those in the Palaearctic genus Phengaris (= Maculinea), a group of only a bout 11 species whose caterpillars parasitize ants in the Myrmicinae. We hypothesize that the move into ant nests by species of Lepidochrysops was an adaptive response to the aridification of Africa during the Miocene that facilitated subsequent radiation. Ant colonies have the advantage of providing both shelter from threats like fire and desiccation, as well as a stable food source when vegetation is scarce or unpredictable. To evaluate this hypothesis, we inferred a time-calibrated phylogeny using >400 loci for Lepidochrysops and its closest, non-parasitic relatives. We showed that this clade originated with the emerging Miombo woodlands about 22 million years ago (Mya), and spread to drier biomes as they became available. The diversity of the non-parasitic lineages decreased as aridification intensified around 10 Mya, culminating in an abrupt extinction. In contrast, the phyto-predaceous Lepidochrysops lineage radiated rapidly from around 6.5 Mya when phyto-predation likely evolved, showing the importance of this innovation for diversification and survival during the intensifying aridification of Africa.

Endemics co-occur because they evolved in situ and persist regionally or because they evolved ex situ and later dispersed to shared habitats, generating evolutionary or ecological endemicity centres, respectively. We investigate whether different endemicity centres can intertwine in the region ranging from Alps to Sicily, by studying their butterfly fauna. We gathered an extensive occurrence data set for butterflies of the study area (27,123 records, 269 species, in cells of 0.5 × 0.5 degrees of latitude-longitude). We applied molecular-based delimitation methods (GMYC model) to 26,557 cytochrome c oxidase subunit 1 (COI) sequences of Western Palearctic butterflies. We identified entities based on molecular delimitations and/or the checklist of European butterflies and objectively attributed occurrences to their most probable entity. We obtained a zoogeographic regionalisation based on the 69 endemics of the area. Using phylogenetic ANOVA we tested if endemics from different centres differ from each other and from nonendemics for key ecological traits and divergence time. Endemicity showed high incidence in the Alps and Southern Italy. The regionalisation separated the Alps from the Italian Peninsula and Sicily. The endemics of different centres showed a high turnover and differed in phylogenetic distances, phenology and distribution traits. Endemics are on average younger than nonendemics and the Peninsula-Sicily endemics also have lower variance in divergence than those from the Alps. The observed variation identifies Alpine endemics as paleoendemics, now occupying an ecological centre, and the Peninsula-Sicily ones as neoendemics, that diverged in the region since the Pleistocene. The results challenge the common view of the Alpine-Apennine area as a single “Italian refugium”., Support for this research was provided by ‘La Caixa’ Foundation (ID 100010434) to MM (grant LCF/BQ/DR20/11790020), the Ramón y Cajal program of the Spanish Ministry of Science and Innovation to GT (RYC2018-025335-I), the Academy of Finland to VD (Academy Research Fellow, decision no. 328895), by project PID2019-107078GB-I00/AEI/10.13039/501100011033 to RV and by the projects "Ricerca e conservazione sui lepidotteri diurni di sei Parchi Nazionali dell’Appennino centro-settentrionale”, “Ricerca e conservazione sugli Impollinatori dell’Arcipelago Toscano e divulgazione sui Lepidotteri del parco” and “Servizio di attuazione delle azioni per la protezione degli impollinatori e diffusione dell’entomofauna del Parco Nazionale dell'Alta Murgia” to LD.

Satyrinae butterflies occurring in the Mediterranean apparently have reduced gene flow over sea straits, and for several species, recent wide‐scale biodiversity surveys indicate the existence of divergent mitochondrial lineages. Here, we apply an integrative approach and examine the phylogeography of the genus Lasiommata in the Western Palearctic. Our research comprised molecular analyses (mitochondrial and nuclear DNA) and geometric morphometrics (wings and genitalia) for two main species groups, and a comparative GMYC analysis, based on COI, of all the tribes within Satyrinae from this region. The GMYC approach revealed a particularly fast coalescence rate in the Parargina subtribe. The Lasiommata group was divided into 12 evolutionary significant units: six clades for the L. maera species group, five for the L. megera species group, and one for L. petropolitana, with divergences of about 1%. The patterns of COI were mirrored by ITS2 in L. maera, but the two markers were generally inconsistent in L. megera. On the contrary, morphological differences were coherent with the results of COI for L. megera, but less clearly so for L. maera. L. paramegaera and L. meadewaldoi were considerably differentiated for all the analyzed markers and likely proceeded faster in the process of speciation because of geographic isolation and reduced effective population size, rendering the rest paraphyletic. Our study illustrates the continuous nature of speciation and the difficulties of delimiting species. In Lasiommata, the recognition of taxa as diverging lineages or distinct, possibly paraphyletic species, mostly depends on the criteria adopted by different species concepts., Funding for this research came from the Spanish MINECO and AEI/FEDER, UE (CGL2013‐48277‐P and CGL2016‐76322‐P to RVi), Marie Skłodowska‐Curie Train2Move to RVo (grant 609402–2020), Marie Sklodowska‐Curie IOF grant (project 625997) to VD, from Tuscan Archipelago National Park and Florence University to LD and from the project “Barcoding‐Italian‐Butterflies.”

We present a genome assembly from an individual female Vanessa atalanta (the red admiral; Arthropoda; Insecta; Lepidoptera; Nymphalidae). The genome sequence is 370 megabases in span. The majority of the assembly (99.44%) is scaffolded into 32 chromosomal pseudomolecules, with the W and Z sex chromosome assembled. Gene annotation of this assembly on Ensembl has identified 12,493 protein coding genes., Species taxonomy Background Genome sequence report Methods Sample acquisition and nucleic acid extraction Sequencing Genome assembly Gene annotation Ethical/compliance issues Data availability Author information References

Although best known for its extraordinary radiations of endemic plant species, the South African fynbos is home to a great diversity of phytophagous insects, including butterflies in the genus Chrysoritis (Lepidoptera: Lycaenidae). These butterflies are remarkably uniform morphologically; nevertheless, they comprise 43 currently accepted species and 68 currently valid taxonomic names. While many species have highly restricted, dot-like distributions, others are widespread. Here, we investigate the phylogenetic and biogeographic history underlying their diversification by analyzing molecular markers from 406 representatives of all described species throughout their respective ranges. We recover monophyletic clades for both C. chrysaor and C. thysbe species-groups, and identify a set of lineages that fall between them. The estimated age of divergence for the genus is 32 Mya, and we document significantly rapid diversification of the thysbe species-group in the Pleistocene (~2 Mya). Using ancestral geographic range reconstruction, we show that West Fynbos is the most likely region of origin for the radiation of the thysbe species-group. The colonization of this region occurred 9 Mya and appears to have been followed by a long period of relative stasis before a recent increase in diversification. Thus, the thysbe radiation does not appear to have resulted from the colonization of new biogeographic areas. Rather, the impact of species interactions (with ants and plants), the appearance of key innovations, and/or the opening of new ecological niche space in the region might explain the sudden burst of speciation that occurred in this group 2 Mya. The biogeographic model suggests two different diversification processes with few historical cross-colonisations, one in eastern South Africa for the C. chrysaor group and the other in western South Africa for the remaining taxa. Distributional range assessments and ecological niche models for each species show important niche overlap, and in a few cases, complete overlap. However, these shared traits are not explained by phylogenetic history. Chrysoritis taxa frequently fly in sympatry and gene tree reticulation appears to be widespread at the species level, suggesting that several episodes of range shifts might have led to secondary sympatries, allowing limited gene flow that challenges species delimitation efforts. In addition, the unusually high diversification rate for the thysbe clade of 1.35 [0.91–1.81] lineages per million years also suggests the possibility of taxonomic oversplitting. The phylogeny presented here provides a framework for a taxonomic revision of the genus. We highlight cases of potential synonymy both in allopatry and sympatry, and stress the importance of dedicated studies to assess potential pre- and post-zygotic barriers giving rise to species delimitations of the thysbe group., GT was supported by the MINECO programme Juan de la Cierva Incorporación (IJCI‐2016‐29083), the Marie Curie Actions FP7‐People‐2013 IOF (project 622716), “La Caixa” Foundation (LCF/BQ/PR19/11700004), and the National Geographic Society (grant #WW1‐300R‐18). ZAK was supported by an NSF Graduate Research Fellowship. Collecting expeditions were made possible by grants from the Putnam Expeditionary Fund of the Museum of Comparative Zoology (MCZ, Harvard University) to ZAK, and the research was supported by NSF DEB-0447244 and DEB-1541560 to NEP.

The small cabbage white butterfly, Pieris rapae, is a major agricultural pest of cruciferous crops and has been introduced to every continent except South America and Antarctica as a result of human activities. In an effort to reconstruct the near-global invasion history of P. rapae, we developed a citizen science project, the “Pieris Project,” and successfully amassed thousands of specimens from 32 countries worldwide. We then generated and analyzed nuclear (double-digest restriction site-associated DNA fragment procedure [ddRAD]) and mitochondrial DNA sequence data for these samples to reconstruct and compare different global invasion history scenarios. Our results bolster historical accounts of the global spread and timing of P. rapae introductions. We provide molecular evidence supporting the hypothesis that the ongoing divergence of the European and Asian subspecies of P. rapae (∼1,200 y B.P.) coincides with the diversification of brassicaceous crops and the development of human trade routes such as the Silk Route (Silk Road). The further spread of P. rapae over the last ∼160 y was facilitated by human movement and trade, resulting in an almost linear series of at least 4 founding events, with each introduced population going through a severe bottleneck and serving as the source for the next introduction. Management efforts of this agricultural pest may need to consider the current existence of multiple genetically distinct populations. Finally, the international success of the Pieris Project demonstrates the power of the public to aid scientists in collections-based research addressing important questions in invasion biology, and in ecology and evolutionary biology more broadly.

Modelling ecological niches of migratory animals requires incorporating a temporal dimension, in addition to space. Here, we introduce an approach to model multigenerational migratory insects using time-partitioned environmental variables (by months and years) and time- and behaviour-partitioned records (breeding records to model reproductive habitat). We apply this methodology to modelling the Palearctic-African migratory cycle of the Painted Lady butterfly (Vanessa cardui), based on data encompassing 36 years (646 breeding sites from 30 countries). Each breeding record is linked to a particular time (month and year), and the associated values of the bioclimatic variables are used for an ensemble modelling strategy, to finally obtain monthly projections. The results show obligated movements, mostly latitudinal, for the species' successive generations across the overall range, and only scattered locations show high probabilities of reproduction year-round. The southernmost reproductive areas estimated for the Palearctic-African migratory pool reach equatorial latitudes from December to February. We thus propose a potential distribution for the winter ‘missing generations' that would expand the V. cardui migration cycle to encompass about 15 000 km in latitude, from northernmost Europe to equatorial Africa. In summer, Europe represents the major temporary resource for V. cardui, while January and February show the lowest overall suitability values, and they are potentially the most vulnerable period for the species to suffer yearly bottlenecks. In summary, we demonstrate the potential of the proposed niche modelling strategy to investigate migratory movements of insects., This work was funded by the Committee for Research and Exploration of the National Geographic Society (grant no. WW1-300R-18), the British Ecological Society (grant no. LRB16/1015), the PRIC grants of the Fundació Barcelona Zoo, and by project CGL2016-76322 (AEI/FEDER, UE) to G.T. G.T. is supported by the MINECO programme Juan de la Cierva Incorporación (IJCI-2016-29083), the Marie Curie Actions FP7-People-2013 IOF (project 622716) and ‘La Caixa' Foundation (grant LCF/BQ/PR19/11700004).

Understanding the dynamics of biodiversity, including the spatial distribution of genetic diversity, is critical for predicting responses to environmental changes, as well as for effective conservation measures. This task requires tracking changes in biodiversity at large spatial scales and correlating with species functional traits. We provide three comprehensive resources to understand the determinants for mitochondrial DNA differentiation represented by (a) 15,609 COI sequences and (b) 14 traits belonging to 307 butterfly species occurring in Western-Central Europe and (c) the first multi-locus phylogenetic tree of all European butterfly species. By applying phylogenetic regressions we show that mitochondrial DNA spatial differentiation (as measured with G, G′, D and D) is negatively correlated with species traits determining dispersal capability and colonization ability. Thanks to the high spatial resolution of the COI data, we also provide the first zoogeographic regionalization maps based on intraspecific genetic variation. The overall pattern obtained by averaging the spatial differentiation of all Western-Central European butterflies shows that the paradigm of long-term glacial isolation followed by rapid pulses of post-glacial expansion has been a pervasive phenomenon in European butterflies. The results and the extensive data sets we provide here constitute the basis for genetically-informed conservation plans for a charismatic group in a continent where flying insects are under alarming decline., Ministerio de Economía y Competitividad. Grant Number: CGL2013-48277-P and CGL2016-76322-P Marie Skłodowska‐Curie. Grant Numbers: 609402-2020, 625997.

The small cabbage white butterfly, Pieris rapae, is a major agricultural pest of cruciferous crops and has been introduced to every continent except South America and Antarctica as a result of human activities. In an effort to reconstruct the near-global invasion history of P. rapae, we developed a citizen science project, the “Pieris Project,” and successfully amassed thousands of specimens from 32 countries worldwide. We then generated and analyzed nuclear (double-digest restriction site-associated DNA fragment procedure [ddRAD]) and mitochondrial DNA sequence data for these samples to reconstruct and compare different global invasion history scenarios. Our results bolster historical accounts of the global spread and timing of P. rapae introductions. We provide molecular evidence supporting the hypothesis that the ongoing divergence of the European and Asian subspecies of P. rapae (∼1,200 y B.P.) coincides with the diversification of brassicaceous crops and the development of human trade routes such as the Silk Route (Silk Road). The further spread of P. rapae over the last ∼160 y was facilitated by human movement and trade, resulting in an almost linear series of at least 4 founding events, with each introduced population going through a severe bottleneck and serving as the source for the next introduction. Management efforts of this agricultural pest may need to consider the current existence of multiple genetically distinct populations. Finally, the international success of the Pieris Project demonstrates the power of the public to aid scientists in collections-based research addressing important questions in invasion biology, and in ecology and evolutionary biology more broadly., This research was funded by US Department of Agriculture-National Institute of Food and Agriculture Postdoctoral Fellowship Grant 2017-67012-26999 (to S.F.R.). A.E. is supported by a National Science Foundation Graduate Research Fellowship under Grant NSF-1447692. R.V. was supported by project CGL2016-76322-P (Spanish Agencia Estatal de Investigación and European Union Regional Development Fund). G.T. is supported by Ministerio de Economia y Competitividad Programme IJCI-2016-29083 and by the National Geographic Society (Grant WW1-300R-18). E.A.H. was supported by Marie Curie Actions International Outgoing Fellowship 330136.

Insects account for a large portion of Earth's biodiversity and are key players for ecosystems, notably as pollinators. While insect migration is suspected to represent a natural phenomenon of major importance, remarkably little is known about it, except for a few flagship species. The reason for this situation is mainly due to technical limitations in the study of insect movement. Here, we propose using metabarcoding of pollen carried by insects as a method for tracking their migrations. We developed a flexible and simple protocol allowing efficient multiplexing and not requiring DNA extraction, one of the most time¿consuming part of metabarcoding protocols, and apply this method to the study of the long¿distance migration of the butterfly Vanessa cardui, an emerging model for insect migration. We collected 47 butterfly samples along the Mediterranean coast of Spain in spring and performed metabarcoding of pollen collected from their bodies to test for potential arrivals from the African continent. In total, we detected 157 plant species from 23 orders, most of which (82.8%) were insect¿pollinated. Taxa present in Africa¿Arabia represented 73.2% of our data set, and 19.1% were endemic to this region, strongly supporting the hypothesis that migratory butterflies colonize southern Europe from Africa in spring. Moreover, our data suggest that a northwards trans¿Saharan migration in spring is plausible for early arrivals (February) into Europe, as shown by the presence of Saharan floristic elements. Our results demonstrate the possibility of regular insect¿mediated transcontinental pollination, with potential implications for ecosystem functioning, agriculture and plant phylogeography., GT is supported by the Marie Curie Actions FP7‐People‐2013 IOF (project 622716) and by the MINECO programme Juan de la Cierva Incorporación (IJCI‐2016‐29083). Funding was provided by the statutory funds of W. Szafer Institute of Botany, Polish Academy of Sciences to TS and MR, by the British Ecological Society (grant LRB16/1015) to GT, by project CGL2016‐76322 (AEI/FEDER, UE) and by the Committee for Research and Exploration of the National Geographic Society (grant WW1‐300R‐18) to GT and RV.

A major goal of invasion and climate change biology research is to understand the ecological and evolutionary responses of organisms to anthropogenic disturbance, especially over large spatial and temporal scales. One significant, and sometimes unattainable, challenge of these studies is garnering sufficient numbers of relevant specimens, especially for species spread across multiple continents. We developed a citizen science project, “Pieris Project”, to successfully amass thousands of specimens of the invasive agricultural pestPieris rapae, the small cabbage white butterfly, from 32 countries worldwide. We then generated and analyzed genomic (ddRAD) and mitochondrial DNA sequence data for these samples to reconstruct and compare different global invasion history scenarios. Our results bolster historical accounts of the global spread and timing ofP. rapaeintroductions. The spread ofP. rapaeover the last ∼160 years followed a linear series of at least four founding events, with each introduced population serving as the source for the next. We provide the first molecular evidence supporting the hypothesis that the ongoing divergence of the European and Asian subspecies ofP. rapae(∼1,200 yrBP) coincides with the domestication of brassicaceous crops. Finally, the international success of the Pieris Project allowed us to nearly double the geographic scope of our sampling (i.e., add >1,000 specimens from 13 countries), demonstrating the power of the public to aid scientists in collections-based research addressing important questions in ecology and evolutionary biology.Non-technical summaryWe provide genetic evidence that the success of the small cabbage white butterfly—its rise to one of the most widespread and abundant butterflies on the planet— was largely facilitated by human activities, through the domestication of its food plants and the accidental movement of the butterfly by means of trade and human movement (migration). Through an international citizen science project—Pieris Project—people from around the world helped to unravel the global invasion history of this agricultural pest butterfly by collecting samples for DNA analysis. The success of this citizen science project demonstrates the power of the public to aid in collections-based research that address important questions related to ecology and evolutionary biology.

The role of specialization in diversification can be explored along two geological axes in the butterfly family Lycaenidae. In addition to variation in host-plant specialization normally exhibited by butterflies, the caterpillars of most Lycaenidae have symbioses with ants ranging from no interactions through to obligate and specific associations, increasing niche dimensionality in ant-associated taxa. Based on mitochondrial sequences from 8282 specimens from 967 species and 249 genera, we show that the degree of ecological specialization of lycaenid species is positively correlated with genetic divergence, haplotype diversity and an increase in isolation by distance. Nucleotide substitution rate is higher in carnivorous than phytophagous lycaenids. The effects documented here for both micro- and macroevolutionary processes could result from increased spatial segregation as a consequence of reduced connectivity in specialists, niche-based divergence or a combination of both. They could also provide an explanation for the extraordinary diversity of the Lycaenidae and, more generally, for diversity in groups of organisms with similar multi-dimensional ecological specialization.

[Aim] Biogeographically puzzling taxa represent an opportunity to understand the processes that have shaped current species distributions. The systematic placement and biogeographical history of Elkalyce cogina, a small lycaenid butterfly endemic to Brazil and neighbouring Argentina, are long-standing puzzles. We use molecular tools and novel biogeographical and life history data to clarify the taxonomy and distribution of this butterfly., [Location] South America, with emphasis on the Atlantic Rain Forest and Cerrado biomes (Brazil and Argentina)., [Methods] We gathered a data set of 71 Polyommatini (Lycaenidae) samples, including representatives of all described subtribes and/or sections. Among these, we contributed new sequences for E. cogina and four additional relevant taxa in the target subtribes Everina, Lycaenopsina and Polyommatina. We inferred a molecular phylogeny based on three mitochondrial genes and four nuclear markers to assess the systematic position and time of divergence of E. cogina. Ancestral geographical ranges were estimated with the R package BioGeoBEARS. To investigate heterogeneity in clade diversification rates, we used Bayesian analysis of macroevolutionary mixtures (bamm)., [Results] Our results confirm the hypothesis that E. cogina belongs to the subtribe Everina and not Lycaenopsina, but unexpectedly recovered it as the sister group to the rest of Everina, with an estimated divergence time of approximately 10 Ma. Ancestral geographical range reconstruction points to an old colonization from Asia, the centre of diversity for the Everina, to the New World. The Neotropical Polyommatina lineage diversified to produce almost 100 species in multiple genera, whereas the E. cogina lineage did not diversify at all. Such lack of diversification is unique among the seven Everina/Polyommatina lineages that colonized the New World. We also show that the larvae of E. cogina feed on Fabaceae, supporting the identification of this host-plant family as the ancestral state for the whole group., [Main conclusions] The age and biogeographical reconstruction of the Elkalyce lineage are similar to those of the Neotropical lineage of Polyommatina and suggest that both travelled via the route proposed by Vladimir Nabokov (Asia-Beringia-North America-South America). This coincidence suggests that the climatic conditions at c. 10 Ma favoured dispersal from Asia to the Neotropics and that later events may have erased traces of these butterfly lineages in North America., Funding was provided by the Spanish Ministerio de Economía y Competitividad (project CGL2013-48277-P). G.T. is supported by the grant BP-A00275 (AGAUR-Generalitat de Catalunya), Marie Curie Actions FP7-PEOPLE-2013-IOF (project 622716) and the grant 1.50.1617.2013 at Saint Petersburg State University; L.A.K. was supported by CAPES (3200-14-0); A.V.L.F. thanks ICMBio for research permits (SISBIO no. 10802-5), CNPq (fellowship 302585/2011-7 and grant 564954/2010-1), RedeLep-SISBIOTA-Brasil/CNPq (563332/2010-7), the National Science Foundation (DEB-1256742), BR-BoL (MCT/CNPq/FNDCT 50/2010) and FAPESP (grant 2012/50260-6 and BIOTA-FAPESP Programs 2011/50225-3 and 2013/50297-0).

Of the four most diverse insect orders, Lepidoptera contains remarkably few predatory and parasitic species. Although species with these habits have evolved multiple times in moths and butterflies, they have rarely been associated with diversification. The wholly aphytophagous subfamily Miletinae (Lycaenidae) is an exception, consisting of nearly 190 species distributed primarily throughout the Old World tropics and subtropics. Most miletines eat Hemiptera, although some consume ant brood or are fed by ant trophallaxis. A well-resolved phylogeny inferred using 4915 bp from seven markers sampled from representatives of all genera and nearly one-third the described species was used to examine the biogeography and evolution of biotic associations in this group. Biogeographic analyses indicate that Miletinae likely diverged from an African ancestor near the start of the Eocene, and four lineages dispersed between Africa and Asia. Phylogenetic constraint in prey selection is apparent at two levels: related miletine species are more likely to feed on related Hemiptera, and related miletines are more likely to associate with related ants, either directly by eating the ants, or indirectly by eating hemipteran prey that are attended by those ants. These results suggest that adaptations for host ant location by ovipositing female miletines may have been retained from phytophagous ancestors that associated with ants mutualistically.

Summary Butterflies (Papilionoidea), with over 18,000 described species [1], have captivated naturalists and scientists for centuries. They play a central role in the study of speciation, community ecology, biogeography, climate change, and plant-insect interactions and include many model organisms and pest species [2, 3]. However, a robust higher-level phylogenetic framework is lacking. To fill this gap, we inferred a dated phylogeny by analyzing the first phylogenomic dataset, including 352 loci (> 150,000 bp) from 207 species representing 98% of tribes, a 35-fold increase in gene sampling and 3-fold increase in taxon sampling over previous studies [4]. Most data were generated with a new anchored hybrid enrichment (AHE) [5] gene kit (BUTTERFLY1.0) that includes both new and frequently used (e.g., [6]) informative loci, enabling direct comparison and future dataset merging with previous studies. Butterflies originated around 119 million years ago (mya) in the late Cretaceous, but most extant lineages diverged after the Cretaceous-Paleogene (K-Pg) mass-extinction 65 mya. Our analyses support swallowtails (Papilionidae) as sister to all other butterflies, followed by skippers (Hesperiidae) + the nocturnal butterflies (Hedylidae) as sister to the remainder, indicating a secondary reversal from diurnality to nocturnality. The whites (Pieridae) were strongly supported as sister to brush-footed butterflies (Nymphalidae) and blues + metalmarks (Lycaenidae and Riodinidae). Ant association independently evolved once in Lycaenidae and twice in Riodinidae. This study overturns prior notions of the taxon's evolutionary history, as many long-recognized subfamilies and tribes are para- or polyphyletic. It also provides a much-needed backbone for a revised classification of butterflies and for future comparative studies including genome evolution and ecology.

Butterflies of the Aricia species group represent a paradigm of unresolved taxonomy, both at the genus and species levels. We studied phylogenetic relationships, biogeography, and systematics based on genetic - nuclear and mitochondrial - and morphometric - external (wings) and internal (genitalia) - data. We show that Aricia is a monophyletic genus comprising the taxa Pseudoaricia, Ultraaricia and Umpria, which are here considered junior synonyms of Aricia. The taxa allous, inhonora, issekutzi, mandzhuriana, myrmecias and transalaica, which have often been raised to species rank, are shown to probably represent subspecies or synonyms. We show that montensis is likely a good species that is sister to all A. artaxerxes populations across the Palearctic region. The species A. anteros and A. morronensis are shown to display deep intraspecific divergences and they may harbor cryptic species. We also discovered that A. cramera and A. agestis exhibit a pattern of mutual exclusion on islands, and a parapatric distribution in mainland with a narrow contact zone where potential hybrids were detected. The lack of a prezygotic barrier that prevents their coexistence could explain this phenomenon. This study will hopefully contribute to the stability of the systematics of Aricia, a group with potential for the study of the link between speciation and biogeography. © 2012 Elsevier Inc., Support for this research was provided by the Spanish Ministerio de Ciencia e Innovación (Project CGL2010-21226/BOS and predoctoral fellowship BES-2008-002054 to GT), by Fundación BBVA (BIOCON08_021) and by the Wenner-Gren Foundation (postdoctoral fellowship to VD).