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Butterflies and moths (Lepidoptera) are one of the major superradiations of insects, comprising nearly 160,000 described extant species. As herbivores, pollinators, and prey, Lepidoptera play a fundamental role in almost every terrestrial ecosystem. Lepidoptera are also indicators of environmental change and serve as models for research on mimicry and genetics. They have been central to the development of coevolutionary hypotheses, such as butterflies with flowering plants and moths’ evolutionary arms race with echolocating bats. However, these hypotheses have not been rigorously tested, because a robust lepidopteran phylogeny and timing of evolutionary novelties are lacking. To address these issues, we inferred a comprehensive phylogeny of Lepidoptera, using the largest dataset assembled for the order (2,098 orthologous protein-coding genes from transcriptomes of 186 species, representing nearly all superfamilies), and dated it with carefully evaluated synapomorphy-based fossils. The oldest members of the Lepidoptera crown group appeared in the Late Carboniferous (∼300 Ma) and fed on nonvascular land plants. Lepidoptera evolved the tube-like proboscis in the Middle Triassic (∼241 Ma), which allowed them to acquire nectar from flowering plants. This morphological innovation, along with other traits, likely promoted the extraordinary diversification of superfamily-level lepidopteran crown groups. The ancestor of butterflies was likely nocturnal, and our results indicate that butterflies became day-flying in the Late Cretaceous (∼98 Ma). Moth hearing organs arose multiple times before the evolutionary arms race between moths and bats, perhaps initially detecting a wide range of sound frequencies before being co-opted to specifically detect bat sonar. Our study provides an essential framework for future comparative studies on butterfly and moth evolution.

Single nucleotide polymorphisms (SNPs) are valuable tools for ecological and evolutionary studies. In non-model species, the use of SNPs has been limited by the number of markers available. However, new technologies and decreasing technology costs have facilitated the discovery of a constantly increasing number of SNPs. With hundreds or thousands of SNPs potentially available, there is interest in comparing and developing methods for evaluating SNPs to create panels of high-throughput assays that are customized for performance, research questions, and resources. Here we use five different methods to rank 43 new SNPs and 71 previously published SNPs for sockeye salmon: F(ST), informativeness (I(n)), average contribution to principal components (LC), and the locus-ranking programs BELS and WHICHLOCI. We then tested the performance of these different ranking methods by creating 48- and 96-SNP panels of the top-ranked loci for each method and used empirical and simulated data to obtain the probability of assigning individuals to the correct population using each panel. All 96-SNP panels performed similarly and better than the 48-SNP panels except for the 96-SNP BELS panel. Among the 48-SNP panels, panels created from F(ST), I(n), and LC ranks performed better than panels formed using the top-ranked loci from the programs BELS and WHICHLOCI. The application of ranking methods to optimize panel performance will become more important as more high-throughput assays become available.

Each year new exotic species are transported across the world through global commerce, causing considerable economic and ecological damage. An important component of managing invasion pathways is to identify source populations. Some of the most widespread exotic species are haplodiploid ambrosia beetles. The ability to mate with siblings (inbreed) and their transportable food source (symbiotic fungus) have enabled them to colonize most of the world and become pests of plant nurseries, lumber, and forests. One of the fastest spreading ambrosia beetles is Xylosandrus crassiusculus. In order to discover the source populations of this globally invasive species, track its movement around the world, and test biogeographical scenarios, we combined restriction site‐associated DNA sequencing (RADseq) with comprehensive sampling across the species native and introduced range. From 1,365 genotyped SNP loci across 198 individuals, we determined that in its native range, X. crassiusculus is comprised of a population in Southeast Asia that includes mainland China, Thailand, and Taiwan, and a second island population in Japan. North America and Central America were colonized from the island populations, while Africa and Oceania were colonized from the mainland Asia, and Hawaii was colonized by both populations. Populations of X. crassiusculus in North America were genetically diverse and highly structured, suggesting (1) numerous, repeated introductions; (2) introduction of a large founding population; or (3) both scenarios with higher than expected outcrossing. X. crassiusculus, other wood‐boring insects, and indeed many other pests with unusual genetic structure continue to spread around the world. We show that contemporary genetic methods offer a powerful tool for understanding and preventing pathways of future biosecurity threats. One of the fastest spreading pests in the ambrosia beetles is Xylosandrus crassiusculus. Using restriction site‐associated DNA sequencing (RADseq) combined with comprehensive sampling across the species native and introduced range, its global expansion history has been traced. Populations of X. crassiusculus in North America were genetically diverse and highly structured, suggesting (1) numerous, repeated introductions; (2) introduction of a large founding population; or (3) both scenarios with higher than expected outcrossing.

The sky islands of southeastern Arizona (AZ) mark a major transition zone between tropical and temperate biota and are considered a neglected biodiversity hotspot. Dispersal ability and host plant specificity are thought to impact the history and diversity of insect populations across the sky islands. We aimed to investigate the population structure and phylogeography of two pine‐feeding pierid butterflies, the pine white (Neophasia menapia) and the Mexican pine white (Neophasia terlooii), restricted to these “islands” at this transition zone. Given their dependence on pines as the larval hosts, we hypothesized that habitat connectivity affects population structure and is at least in part responsible for their allopatry. We sampled DNA from freshly collected butterflies from 17 sites in the sky islands and adjacent high‐elevation habitats and sequenced these samples using ddRADSeq. Up to 15,399 SNPs were discovered and analyzed in population genetic and phylogenetic contexts with Stacks and pyRAD pipelines. Low genetic differentiation in N. menapia suggests that it is panmictic. Conversely, there is strong evidence for population structure within N. terlooii. Each sky island likely contains a population of N. terlooii, and clustering is hierarchical, with populations on proximal mountains being more related to each other. The N. menapia habitat, which is largely contiguous, facilitates panmixia, while the N. terlooii habitat, restricted to the higher elevations on each sky island, creates distinct population structure. Phylogenetic results corroborate those from population genetic analyses. The historical climate‐driven fluxes in forest habitat connectivity have implications for understanding the biodiversity of fragmented habitats. The high‐throughput sequencing approach of ddRADSeq was used to investigate the phylogeography and population structure of two closely related butterfly species restricted to mountain habitats. Despite their flight capabilities and the proximity between some mountain ranges (56 km apart), there was distinct population structure among butterflies in the sky islands of southeastern Arizona. Butterflies in the largely connected belt of pine forest from east–central Arizona northwestward to the Kaibab Plateau exhibited panmixia.

Viruses encoding a replication-associated protein (Rep) within a covalently closed, single-stranded (ss)DNA genome are among the smallest viruses known to infect eukaryotic organisms, including economically valuable agricultural crops and livestock. Although circular Rep-encoding ssDNA (CRESS DNA) viruses are a widespread group for which our knowledge is rapidly expanding, biased sampling toward vertebrates and land plants has limited our understanding of their diversity and evolution. Here, we screened terrestrial arthropods for CRESS DNA viruses and report the identification of 44 viral genomes and replicons associated with specimens representing all three major terrestrial arthropod lineages, namely Euchelicerata (spiders), Hexapoda (insects), and Myriapoda (millipedes). We identified virus genomes belonging to three established CRESS DNA viral families ( Circoviridae , Genomoviridae , and Smacoviridae ); however, over half of the arthropod-associated viral genomes are only distantly related to currently classified CRESS DNA viral sequences. Although members of viral and satellite families known to infect plants ( Geminiviridae , Nanoviridae , Alphasatellitidae ) were not identified in this study, these plant-infecting CRESS DNA viruses and replicons are transmitted by hemipterans. Therefore, members from six out of the seven established CRESS DNA viral families circulate among arthropods. Furthermore, a phylogenetic analysis of Reps, including endogenous viral sequences, reported to date from a wide array of organisms revealed that most of the known CRESS DNA viral diversity circulates among invertebrates. Our results highlight the vast and unexplored diversity of CRESS DNA viruses among invertebrates and parallel findings from RNA viral discovery efforts in undersampled taxa.

Advances in nondestructive genetic sampling techniques continue to offer new opportunities for studying organisms, particularly those of conservation concern where more traditional invasive sampling methods are often not available. As part of a proof-of-concept, we investigated the effectiveness of using the chorion from residual butterfly egg debris as a source of viable genetic material for analysis. Laboratory material from a captive breeding population of the federally endangered Miami blue butterfly (Cyclargus thomasi bethunebakeri) was used to test efficacy and refine the methodology. The resulting best practices were subsequently evaluated using field-collected material from extant north Florida populations of the at-risk frosted elfin butterfly (Callophyrs irus). Our results demonstrated that it is possible to extract DNA of sufficiently high quantity and quality for successful gene sequencing. We additionally describe a simple, low-cost, and reliable method of collecting and storing egg debris samples that can be consistently adopted for field or laboratory work as well as deployed with projects that have a larger geographic scope and/or involve citizen scientists. Potential limitations related to field sample collection are discussed as well as needs for future evaluation.

Understanding the evolutionary mechanisms governing the uneven distribution of species richness across the tree of life is a great challenge in biology. Scientists have long argued that sexual conflict is a key driver of speciation. This hypothesis, however, has been highly debated in light of empirical evidence. Recent advances in the study of macroevolution make it possible to test this hypothesis with more data and increased accuracy. In the present study, we use phylogenomics combined with four different diversification rate analytical approaches to test whether sexual conflict is a driver of speciation in brush-footed butterflies of the tribe Acraeini. The presence of a sphragis, an external mating plug found in most species among Acraeini, was used as a proxy for sexual conflict. Diversification analyses statistically rejected the hypothesis that sexual conflict is associated with shifts in diversification rates in Acraeini. This result contrasts with earlier studies and suggests that the underlying mechanisms driving diversification are more complex than previously considered. In the case of butterflies, natural history traits acting in concert with abiotic factors possibly play a stronger role in triggering speciation than does sexual conflict.

Each year new exotic species are transported across the world through global commerce, causing considerable economic and ecological damage. An important component of managing invasion pathways is to identify source populations. Some of the most widespread exotic species are haplodiploid ambrosia beetles. The ability to mate with siblings (inbreed) and their transportable food source (symbiotic fungus) have enabled them to colonize most of the world and become pests of plant nurseries, lumber, and forests. One of the fastest spreading ambrosia beetles is Xylosandrus crassiusculus . In order to discover the source populations of this globally invasive species, track its movement around the world, and test biogeographical scenarios, we combined restriction site‐associated DNA sequencing ( RAD seq) with comprehensive sampling across the species native and introduced range. From 1,365 genotyped SNP loci across 198 individuals, we determined that in its native range, X. crassiusculus is comprised of a population in Southeast Asia that includes mainland China, Thailand, and Taiwan, and a second island population in Japan. North America and Central America were colonized from the island populations, while Africa and Oceania were colonized from the mainland Asia, and Hawaii was colonized by both populations. Populations of X. crassiusculus in North America were genetically diverse and highly structured, suggesting (1) numerous, repeated introductions; (2) introduction of a large founding population; or (3) both scenarios with higher than expected outcrossing. X. crassiusculus , other wood‐boring insects, and indeed many other pests with unusual genetic structure continue to spread around the world. We show that contemporary genetic methods offer a powerful tool for understanding and preventing pathways of future biosecurity threats.

Australia was predominantly tropical for most of the Early Cenozoic, then transitioned to a cooler and drier climate in the Oligocene. In response to this increasing aridity, some lineages either adapted to xeric ecosystems, contracted to increasingly fragmented mesic refugia, or went extinct. However, the lack of macroevolutionary studies at a continental scale precludes a better understanding of Australian biodiversity patterns and processes during the Cenozoic. Here, we infer a robust, dated phylogenomic tree for a radiation of Australian endemic butterflies, the Trapezitinae skippers, to test the impact of biotic and abiotic drivers on Cenozoic diversification dynamics in Australia. These butterflies originated during the Eocene (c. 42 Mya) in the mesic biome of Australia. Trapezitinae exploded in diversity during a cool, dry period in the Late Oligocene and Early Miocene, then experienced a sharp deceleration in speciation. Xeric ecosystems appear to have been colonized more recently, supporting the hypothesis of arid and semi-arid biomes as evolutionary sinks. Temperature-dependent and phytophagy-dependent diversification models received little support. Instead, we find evidence for diversity-dependent processes with declining diversification in Trapezitinae likely linked to limited ecological opportunities following a rapid initial burst of diversification.

Viruses encoding a replication-associated protein (Rep) within a covalently closed, single-stranded (ss)DNA genome are among the smallest viruses known to infect eukaryotic organisms, including economically valuable agricultural crops and livestock. Although circular Rep-encoding ssDNA (CRESS DNA) viruses are a widespread group for which our knowledge is rapidly expanding, biased sampling towards vertebrates and land plants has limited our understanding of their diversity and evolution. Here we screened terrestrial arthropods for CRESS DNA viruses and report the identification of 44 viral genomes and replicons associated with specimens representing all three major terrestrial arthropod lineages, namely Euchelicerata (spiders), Hexapoda (insects), and Myriapoda (millipedes). We identified virus genomes belonging to three established CRESS DNA viral families (Circoviridae, Genomoviridae, and Smacoviridae); however, over half of the arthropod-associated virus genomes are only distantly related to currently classified CRESS DNA viral sequences. Although members of viral and satellite families known to infect plants (Geminiviridae, Nanoviridae, Alphasatellitidae) were not identified in this study, these plant-infecting CRESS DNA viruses and replicons are transmitted by hemipterans. Therefore, members from six out of the seven established CRESS DNA viral families circulate among arthropods. Furthermore, a phylogenetic analysis of Reps, including endogenous viral sequences, reported to date from a wide array of organisms revealed that most of the known CRESS DNA viral diversity circulates among invertebrates. Our results highlight the vast and unexplored diversity of CRESS DNA viruses among invertebrates and parallel findings from RNA viral discovery efforts in undersampled taxa.