The Afrotropical breeding grounds of the Palearctic-African migratory painted lady butterflies (Vanessa cardui).

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.

Transposable Element Insertions Are Associated with Batesian Mimicry in the Pantropical Butterfly Hypolimnas misippus.

Hypolimnas misippus is a Batesian mimic of the toxic African Queen butterfly (Danaus chrysippus). Female H. misippus butterflies use two major wing patterning loci (M and A) to imitate three color morphs of D. chrysippus found in different regions of Africa. In this study, we examine the evolution of the M locus and identify it as an example of adaptive atavism. This phenomenon involves a morphological reversion to an ancestral character that results in an adaptive phenotype. We show that H. misippus has re-evolved an ancestral wing pattern present in other Hypolimnas species, repurposing it for Batesian mimicry of a D. chrysippus morph. Using haplotagging, a linked-read sequencing technology, and our new analytical tool, Wrath, we discover two large transposable element insertions located at the M locus and establish that these insertions are present in the dominant allele responsible for producing mimetic phenotype. By conducting a comparative analysis involving additional Hypolimnas species, we demonstrate that the dominant allele is derived. This suggests that, in the derived allele, the transposable elements disrupt a cis-regulatory element, leading to the reversion to an ancestral phenotype that is then utilized for Batesian mimicry of a distinct model, a different morph of D. chrysippus. Our findings present a compelling instance of convergent evolution and adaptive atavism, in which the same pattern element has independently evolved multiple times in Hypolimnas butterflies, repeatedly playing a role in Batesian mimicry of diverse model species.

The opposed forces of differentiation and admixture across glacial cycles in the butterfly Aglais urticae.

Glacial cycles lead to periodic population interbreeding and isolation in warm-adapted species, which impact genetic structure and evolution. However, the effects of these processes on highly mobile and more cold-tolerant species are not well understood. This study aims to shed light on the phylogeographic history of Aglais urticae, a butterfly species with considerable dispersal ability, and a wide Palearctic distribution reaching the Arctic. Through the analysis of genomic data, four main genetic lineages are identified: European, Sierra Nevada, Sicily/Calabria/Peloponnese, and Eastern. The results indicate that the Sardo-Corsican endemic taxon ichnusa is a distinct species. The split between the relict lineages in southern Europe and the main European lineage is estimated to have happened 400-450 thousand years ago, with admixture observed during the Quaternary glacial cycles, and still ongoing, albeit to a much smaller extent. These results suggest that these lineages may be better treated as subspecific parapatric taxa. Ecological niche modelling supported the existence of both Mediterranean and extra-Mediterranean refugia during the glacial periods, with the main one located on the Atlantic coast. Nevertheless, gene flow between populations was possible, indicating that both differentiation and admixture have acted continuously across glacial cycles in this cold-tolerant butterfly, generally balancing each other but producing differentiated lineages in the southern peninsulas. We conclude that the population dynamics and the processes shaping the population genetic structure of cold-adapted species during the Quaternary ice ages may be different than those classically accepted for warm-adapted species.

A hydrogen isoscape for tracing the migration of herbivorous lepidopterans across the Afro-Palearctic range.

RATIONALE: Many insect species undertake multigenerational migrations in the Afro-tropical and Palearctic ranges, and understanding their migratory connectivity remains challenging due to their small size, short life span and large population sizes. Hydrogen isotopes (δ2 H) can be used to reconstruct the movement of dispersing or migrating insects, but applying δ2 H for provenance requires a robust isotope baseline map (i.e. isoscape) for the Afro-Palearctic. METHODS: We analyzed the δ2 H in the wings (δ2 Hwing ) of 142 resident butterflies from 56 sites across the Afro-Palearctic. The δ2 Hwing values were compared to the predicted local growing-season precipitation δ2 H values (δ2 HGSP ) using a linear regression model to develop an insect wing δ2 H isoscape. We used multivariate linear mixed models and high-resolution and time-specific remote sensing climate and environmental data to explore the controls of the residual δ2 Hwing variability. RESULTS: A strong linear relationship was found between δ2 Hwing and δ2 HGSP values (r2 = 0.53). The resulting isoscape showed strong patterns across the Palearctic but limited variation and high uncertainty for the Afro-tropics. Positive residuals of this relationship were correlated with dry conditions for the month preceding sampling whereas negative residuals were correlated with more wet days for the month preceding sampling. High intra-site δ2 Hwing variance was associated with lower relative humidity for the month preceding sampling and higher elevation. CONCLUSION: The δ2 Hwing isoscape is applicable for tracing herbivorous lepidopteran insects that migrate across the Afro-Palearctic range but has limited geolocation potential in the Afro-tropics. The spatial analysis of uncertainty using high-resolution climatic data demonstrated that many African regions with highly variable evaporation rates and relative humidity have δ2 Hwing values that are less related to δ2 HGSP values. Increasing geolocation precision will require new modeling approaches using more time-specific environmental data and/or independent geolocation tools.

Environmental stress during larval development induces DNA methylation shifts in the migratory painted lady butterfly (Vanessa cardui).

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 unsuitable conditions by migrating. Despite a growing understanding of migration in the life cycles of some butterfly species, it remains 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-monotonic 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.

Migratory behaviour is positively associated with genetic diversity in butterflies.

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.

DNA Barcodes Combined with Multilocus Data of Representative Taxa Can Generate Reliable Higher-Level Phylogenies.

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. [Birabiro; DNA barcoding; incertae sedis; Kipepeo; Lycaenidae; missing data; phylogenomic; phylogeny; Polyommatina; supermatrix; systematics; taxonomy].

Linkage mapping and genome annotation give novel insights into gene family expansions and regional recombination rate variation in the painted lady (Vanessa cardui) butterfly.

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.

Global invasion history of the agricultural pest butterfly Pieris rapae revealed with genomics and citizen science.

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.

Evolutionary trade-offs between male secondary sexual traits revealed by a phylogeny of the hyperdiverse tribe Eumaeini (Lepidoptera: Lycaenidae).

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.

Two ways to be endemic. Alps and Apennines are different functional refugia during climatic cycles.

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".

Recent diversification of Chrysoritis butterflies in the South African Cape (Lepidoptera: Lycaenidae).

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.

Spatio-temporal ecological niche modelling of multigenerational insect migrations.

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.

Ecological specialization is associated with genetic structure in the ant-associated butterfly family Lycaenidae.

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.

Round-trip across the Sahara: Afrotropical Painted Lady butterflies recolonize the Mediterranean in early spring.

The Palearctic-African migratory circuit has been typically associated with birds. Very few insects are known to endure annual trans-Saharan circuits, but the Painted Lady butterfly (Vanessa cardui) is an exception. While it was demonstrated that this species massively migrates from Europe to the Afrotropics during the autumn, the existence of a reverse migration from the Afrotropics to Europe in the early spring remains hypothetical. Here, we analysed wing stable hydrogen isotope values (δ2H) of V. cardui migrants collected from February to April across the circum-Mediterranean region. We assessed their region of natal origin by comparing their wing isotope signature predicted δ2H values (isoscape). The results unambiguously demonstrate a sub-Saharan origin for many individuals, especially those collected in February, representing the first tangible evidence for a reverse northwards trans-Saharan migration in spring. This work supports the view that the Afrotropics (mostly exploited from September to February) is key in the V. cardui Palearctic-African population dynamics. This species relies on both temperate and tropical habitats to complete their multigenerational cycle, an unprecedented adaptation for butterflies and for most migratory insects. Such a migratory circuit has strong parallelisms with those of migratory birds.

Integrative analyses unveil speciation linked to host plant shift in Spialia butterflies.

Discovering cryptic species in well-studied areas and taxonomic groups can have profound implications in understanding eco-evolutionary processes and in nature conservation because such groups often involve research models and act as flagship taxa for nature management. In this study, we use an array of techniques to study the butterflies in the Spialia sertorius species group (Lepidoptera, Hesperiidae). The integration of genetic, chemical, cytogenetic, morphological, ecological and microbiological data indicates that the sertorius species complex includes at least five species that differentiated during the last three million years. As a result, we propose the restitution of the species status for two taxa often treated as subspecies, Spialia ali (Oberthür, 1881) stat. rest. and Spialia therapne (Rambur, 1832) stat. rest., and describe a new cryptic species Spialia rosae Hernández-Roldán, Dapporto, Dinca, Vicente & Vila sp. nov. Spialia sertorius (Hoffmannsegg, 1804) and S. rosae are sympatric and synmorphic, but show constant differences in mitochondrial DNA, chemical profiles and ecology, suggesting that S. rosae represents a case of ecological speciation involving larval host plant and altitudinal shift, and apparently associated with Wolbachia infection. This study exemplifies how a multidisciplinary approach can reveal elusive cases of hidden diversity.

Long-distance autumn migration across the Sahara by painted lady butterflies: exploiting resource pulses in the tropical savannah.

The painted lady, Vanessa cardui, is a migratory butterfly that performs an annual multi-generational migration between Europe and North Africa. Its seasonal appearance south of the Sahara in autumn is well known and has led to the suggestion that it results from extremely long migratory flights by European butterflies to seasonally exploit the Sahel and the tropical savannah. However, this possibility has remained unproven. Here, we analyse the isotopic composition of butterflies from seven European and seven African countries to provide new support for this hypothesis. Each individual was assigned a geographical natal origin, based on its wing stable hydrogen isotope (δ2Hw) value and a predicted δ2Hw basemap for Europe and northern Africa. Natal assignments of autumn migrants collected south of the Sahara confirmed long-distance movements (of 4000 km or more) starting in Europe. Samples from Maghreb revealed a mixed origin of migrants, with most individuals with a European origin, but others having originated in the Sahel. Therefore, autumn movements are not only directed to northwestern Africa, but also include southward and northward flights across the Sahara. Through this remarkable behaviour, the productive but highly seasonal region south of the Sahara is incorporated into the migratory circuit of V. cardui.

Ancient Neotropical origin and recent recolonisation: Phylogeny, biogeography and diversification of the Riodinidae (Lepidoptera: Papilionoidea).

We present the first dated higher-level phylogenetic and biogeographic analysis of the butterfly family Riodinidae. This family is distributed worldwide, but more than 90% of the c. 1500 species are found in the Neotropics, while the c. 120 Old World species are concentrated in the Southeast Asian tropics, with minor Afrotropical and Australasian tropical radiations, and few temperate species. Morphologically based higher classification is partly unresolved, with genera incompletely assigned to tribes. Using 3666bp from one mitochondrial and four nuclear markers for each of 23 outgroups and 178 riodinid taxa representing all subfamilies, tribes and subtribes, and 98 out of 145 described genera of riodinids, we estimate that Riodinidae split from Lycaenidae about 96Mya in the mid-Cretaceous and started to diversify about 81Mya. The Riodinidae are monophyletic and originated in the Neotropics, most likely in lowland proto-Amazonia. Neither the subfamily Euselasiinae nor the Nemeobiinae are monophyletic as currently constituted. The enigmatic, monotypic Neotropical genera Styx and Corrachia (most recently treated in Euselasiinae: Corrachiini) are highly supported as derived taxa in the Old World Nemeobiinae, with dispersal most likely occurring across the Beringia land bridge during the Oligocene. Styx and Corrachia, together with all other nemeobiines, are the only exclusively Primulaceae-feeding riodinids. The steadily increasing proliferation of the Neotropical Riodininae subfamily contrasts with the decrease in diversification in the Old World, and may provide insights into factors influencing the diversification rate of this relatively ancient clade of Neotropical insects.

Protocol to sequence markers from pollen grains carried by long-range migratory butterflies using metabarcoding.

Molecular identification of pollen carried by insects informs about their history of visited plants. For migratory butterflies, it can be used to trace long-range movements enduring days of flight over thousands of kilometers. Here, we present a protocol to (1) isolate pollen grains from butterfly bodies and (2) prepare metabarcoding libraries for their identification using the internal transcribed spacer 2 fragment, a common barcode used to identify plants. This protocol would be applicable to other insect groups and metabarcoding markers. For complete details on the use and execution of this protocol, please refer to Suchan et al.1 and Gorki et al.2.