Large-scale genomic data reveal the phylogeny and evolution of owlet moths (Noctuoidea).

The owlet moths (Noctuoidea; ~43-45K described species) are one of the most ecologically diverse and speciose superfamilies of animals. Moreover, they comprise some of the world's most notorious pests of agriculture and forestry. Despite their contributions to terrestrial biodiversity and impacts on ecosystems and economies, the evolutionary history of Noctuoidea remains unclear because the superfamily lacks a statistically robust phylogenetic and temporal framework. We reconstructed the phylogeny of Noctuoidea using data from 1234 genes (946.4 kb nucleotides) obtained from the genome and transcriptome sequences of 76 species. The relationships among the six families of Noctuoidea were well resolved and consistently recovered based on both concatenation and gene coalescence approaches, supporting the following relationships: Oenosandridae + (Notodontidae + (Erebidae + (Nolidae + (Euteliidae + Noctuidae)))). A Yule tree prior with three unlinked molecular clocks was identified as the preferred BEAST analysis using marginal-likelihood estimations. The crown age of Noctuoidea was estimated at 74.5 Ma, with most families originating before the end of the Paleogene (23 Ma). Our study provides the first statistically robust phylogenetic and temporal framework for Noctuoidea, including all families of owlet moths, based on large-scale genomic data.

A global phylogeny of butterflies reveals their evolutionary history, ancestral hosts and biogeographic origins.

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.

Deploying Community Scientists to Conduct Nondestructive Genetic Sampling of Rare Butterfly Populations.

Global insect declines continue to accelerate. Effective genetic sampling is critically needed to advance the understanding of many taxa and address existing knowledge gaps. This protocol represents a demonstrated method for nondestructively sampling rare butterflies for population genetic structure or DNA barcoding analyses. It uses the chorion of hatched butterfly ovae to yield sufficiently high quantity and quality DNA for successful gene sequencing to confirm species identity and quantify genetic variation. It may be particularly useful when other tissue sampling techniques are impractical or unavailable. While developed for a lepidopteran, it nonetheless could easily be adapted for use with other insect species. It was specifically designed with ease of use as a goal to help maximize broad implementation by individuals of varying experience and skill levels, such as community scientists, conservation practitioners, and students, and for use over large geographic areas to facilitate broad population sampling. The data generated can help inform taxonomic and listing decisions, conservation and management actions, and enhance basic ecological research.

De Novo Genome Assembly and Annotation of an Andean Caddisfly, Atopsyche davidsoni Sykora, 1991, a Model for Genome Research of High-Elevation Adaptations.

We sequence, assemble, and annotate the genome of Atopsyche davidsoni Sykora, 1991, the first whole-genome assembly for the caddisfly family Hydrobiosidae. This free-living and predatory caddisfly inhabits streams in the high-elevation Andes and is separated by more than 200 Myr of evolutionary history from the most closely related caddisfly species with genome assemblies available. We demonstrate the promise of PacBio HiFi reads by assembling the most contiguous caddisfly genome assembly to date with a contig N50 of 14 Mb, which is more than 6× more contiguous than the current most contiguous assembly for a caddisfly (Hydropsyche tenuis). We recover 98.8% of insect BUSCO genes indicating a high level of gene completeness. We also provide a genome annotation of 12,232 annotated proteins. This new genome assembly provides an important new resource for studying genomic adaptation of aquatic insects to harsh, high-altitude environments.

Long-read HiFi sequencing correctly assembles repetitive heavy fibroin silk genes in new moth and caddisfly genomes.

Insect silk is a versatile biomaterial. Lepidoptera and Trichoptera display some of the most diverse uses of silk, with varying strength, adhesive qualities, and elastic properties. Silk fibroin genes are long (>20 Kbp), with many repetitive motifs that make them challenging to sequence. Most research thus far has focused on conserved N- and C-terminal regions of fibroin genes because a full comparison of repetitive regions across taxa has not been possible. Using the PacBio Sequel II system and SMRT sequencing, we generated high fidelity (HiFi) long-read genomic and transcriptomic sequences for the Indianmeal moth (Plodia interpunctella) and genomic sequences for the caddisfly Eubasilissa regina. Both genomes were highly contiguous (N50  = 9.7 Mbp/32.4 Mbp, L50  = 13/11) and complete (BUSCO complete  = 99.3%/95.2%), with complete and contiguous recovery of silk heavy fibroin gene sequences. We show that HiFi long-read sequencing is helpful for understanding genes with long, repetitive regions.

First Annotated Genome of a Mandibulate Moth, Neomicropteryx cornuta, Generated Using PacBio HiFi Sequencing.

We provide a new, annotated genome assembly of Neomicropteryx cornuta, a species of the so-called mandibulate archaic moths (Lepidoptera: Micropterigidae). These moths belong to a lineage that is thought to have split from all other Lepidoptera more than 300 Ma and are consequently vital to understanding the early evolution of superorder Amphiesmenoptera, which contains the order Lepidoptera (butterflies and moths) and its sister order Trichoptera (caddisflies). Using PacBio HiFi sequencing reads, we assembled a highly contiguous genome with a contig N50 of nearly 17 Mb. The assembled genome length of 541,115,538 bp is about half the length of the largest published Amphiesmenoptera genome (Limnephilus lunatus, Trichoptera) and double the length of the smallest (Papilio polytes, Lepidoptera). We find high recovery of universal single copy orthologs with 98.1% of BUSCO genes present and provide a genome annotation of 15,643 genes aided by resolved isoforms from PacBio IsoSeq data. This high-quality genome assembly provides an important resource for studying ecological and evolutionary transitions in the early evolution of Amphiesmenoptera.

De novo genome assemblies of butterflies.

BACKGROUND: The availability of thousands of genomes has enabled new advancements in biology. However, many genomes have not been investigated for their quality. Here we examine quality trends in a taxonomically diverse and well-known group, butterflies (Papilionoidea), and provide draft, de novo assemblies for all available butterfly genomes. Owing to massive genome sequencing investment and taxonomic curation, this is an excellent group to explore genome quality. FINDINGS: We provide de novo assemblies for all 822 available butterfly genomes and interpret their quality in terms of completeness and continuity. We identify the 50 highest quality genomes across butterflies and conclude that the ringlet, Aphantopus hyperantus, has the highest quality genome. Our post-processing of draft genome assemblies identified 118 butterfly genomes that should not be reused owing to contamination or extremely low quality. However, many draft genomes are of high utility, especially because permissibility of low-quality genomes is dependent on the objective of the study. Our assemblies will serve as a key resource for papilionid genomics, especially for researchers without computational resources. CONCLUSIONS: Quality metrics and assemblies are typically presented with annotated genome accessions but rarely with de novo genomes. We recommend that studies presenting genome sequences provide the assembly and some metrics of quality because quality will significantly affect downstream results. Transparency in quality metrics is needed to improve the field of genome science and encourage data reuse.

Is Sexual Conflict a Driver of Speciation? A Case Study With a Tribe of Brush-footed Butterflies.

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. [Acraeini butterflies; arms race; exon capture phylogenomics; Lepidoptera macroevolution; sexual selection; sphragis.].

Phylogeography and population genetics of pine butterflies: Sky islands increase genetic divergence.

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.

Phylogenomics reveals the evolutionary timing and pattern of butterflies and moths.

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.

Using Noninvasive Genetic Sampling to Survey Rare Butterfly Populations.

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.

Anchored hybrid enrichment phylogenomics resolves the backbone of erebine moths.

The subfamily Erebinae (Lepidoptera, Erebidae) includes approximately 10,000 species with many still undescribed. It is one of the most diverse clades within the moth superfamily Noctuoidea and encompasses a diversity of ecological habits. Erebine caterpillars feed on a broad range of host plants including several economically important crops. Adults possess a unique array of adaptations for predator defense, including some of the most sensitive hearing organs (tympana) across the Lepidoptera and striking wing coloration to startle visual predators. Despite the relevance of these moths to agriculture and ecological research, a robust phylogenetic framework is lacking. Here we used anchored hybrid enrichment, a relatively new approach in phylogenomics, to resolve relationships among the subfamily. Using the recently developed Lep1 anchored hybrid enrichment probe set, 658 gene fragments with an average length of 320 bp were captured from an exemplar set of 75 erebine species, representing 73 genera and 23 tribes. While the total number of erebine tribes is not firmly established, this represents at least 75% of known tribal level diversity. Anchored hybrid enrichment data were partitioned by locus and by codon position for maximum likelihood phylogenetic analysis and coalescent-based species-tree approaches. Results from our study provided strong nodal support (BP ≥ 95) for nearly all nodes in the partitioned ML tree, solidifying many relationships that were previously uncertain or moderately supported based on morphology or a smaller number of gene fragments. Likelihood analyses confidently resolved the placement of Acantholipini as a sister tribe to Sypnini and all other Erebinae. The remaining tribes were placed in a single, strongly supported clade split into two major subclades. Additionally, 25 tropical species that did not have previous tribal assignments are confidently placed on the phylogeny. Statistical comparisons with Shimodaira-Hasegawa (SH) tests found that our maximum likelihood trees were significantly more likely than alternative hypotheses. This study demonstrates the utility of anchored phylogenomics for resolving relationships within subfamilies of Lepidoptera.

Virus discovery in all three major lineages of terrestrial arthropods highlights the diversity of single-stranded DNA viruses associated with invertebrates.

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.

Phylogenetics of moth-like butterflies (Papilionoidea: Hedylidae) based on a new 13-locus target capture probe set.

The Neotropical moth-like butterflies (Hedylidae) are perhaps the most unusual butterfly family. In addition to being species-poor, this family is predominantly nocturnal and has anti-bat ultrasound hearing organs. Evolutionary relationships among the 36 described species are largely unexplored. A new, target capture, anchored hybrid enrichment probe set ('BUTTERFLY2.0') was developed to infer relationships of hedylids and some of their butterfly relatives. The probe set includes 13 genes that have historically been used in butterfly phylogenetics. Our dataset comprised of up to 10,898 aligned base pairs from 22 hedylid species and 19 outgroups. Eleven of the thirteen loci were successfully captured from all samples, and the remaining loci were captured from ≥94% of samples. The inferred phylogeny was consistent with recent molecular studies by placing Hedylidae sister to Hesperiidae, and the tree had robust support for 80% of nodes. Our results are also consistent with morphological studies, with Macrosoma tipulata as the sister species to all remaining hedylids, followed by M. semiermis sister to the remaining species in the genus. We tested the hypothesis that nocturnality evolved once from diurnality in Hedylidae, and demonstrate that the ancestral condition was likely diurnal, with a shift to nocturnality early in the diversification of this family. The BUTTERFLY2.0 probe set includes standard butterfly phylogenetics markers, captures sequences from decades-old museum specimens, and is a cost-effective technique to infer phylogenetic relationships of the butterfly tree of life.

Cryptic genetic variation in an inbreeding and cosmopolitan pest, Xylosandrus crassiusculus, revealed using ddRADseq.

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.

Wallacellus is Euwallacea: molecular phylogenetics settles generic relationships (Coleoptera: Curculionidae: Scolytinae: Xyleborini).

Euwallacea Hopkins and Wallacellus Hulcr & Cognato are ambrosia beetle genera within the tribe Xyleborini (Coleoptera: Curculionidae: Scolytinae). Several species have recently received attention due to their establishment in non-native regions with serious ecological and economic consequences. To clarify generic placement of these species, we tested reciprocal monophyly of the two genera and the placement of several species using molecular phylogenetics. We sequenced, or re-used published sequences of, three markers (COI mtDNA, 28S nuclear rDNA and ArgK single-copy nuclear) from representatives of Euwallacea, Wallacellus, the Ambrosiodmus clade, and the clade containing Xyleborus s. str., and inferred their relationships with a Bayesian approach. We also tested explicit alternative topologies, and examined taxonomic utility of characters used for the delimitation of the genera.        All species of Euwallacea, Wallacellus, and two species of Xyleborus were monophyletic with high phylogenetic support. Based on the analysis and shared morphological characters, we transferred the following species to Euwallacea: Xyleborus declivispinatus (Schedl), Wallacellus piceus (Motschulsky), Xyleborus posticus (Eichhoff), Wallacellus similis (Ferrari), and Wallacellus striatulus (Browne). The genus Wallacellus was made a junior synonym of Euwallacea and morphological diagnosis of Euwallacea was updated. The results demonstrated that Euwallacea has a pantropical distribution.

Rank and order: evaluating the performance of SNPs for individual assignment in a non-model organism.

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.