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Recent advances in both genomics and ecological modelling present new, multidisciplinary opportunities for resolving species boundaries and understanding the mechanisms that maintain their integrity in regions of contact. Here, we use a combination of high-throughput DNA sequencing and ecological niche modelling to resolve species boundaries and niche divergence within the Speyeria atlantis-hesperis (Lepidoptera: Nymphalidae) complex, a confusing group of North American butterflies. This complex is notorious for its muddled species delimitations, morphological ambiguity, and extensive mitonuclear discordance. Our admixture and multispecies coalescent-based analyses of single nucleotide polymorphisms identified substantial divergences between S. atlantis and S. hesperis in areas of contact, as well as between distinct northern and southern lineages within S. hesperis. Our results also provide evidence of past introgression relating to another species, S. zerene, which previous work has shown to be more distantly related to the S. atlantis-hesperis complex. We then used ecological models to predict habitat suitability for each of the three recovered genomic lineages in the S. atlantis-hesperis complex and assessed their pairwise niche divergence. These analyses resolved that these three lineages are significantly diverged in their respective niches and are not separated by discontinuities in suitable habitat that might present barriers to gene flow. We therefore infer that ecologically-mediated selection resulting in disparate habitat associations is a principal mechanism reinforcing their genomic integrity. Overall, our results unambiguously support significant evolutionary and ecological divergence between the northern and southern lineages of S. hesperis, sufficient to recognize the southern evolutionary lineage as a distinct species, called S. nausicaa based on taxonomic priority.

Conservation biologists have long debated the value of subspecies, which are morphologically and geographically identifiable but not necessarily evolutionarily distinctive. One example of a controversial subspecies is Cicindela formosa gibsoni, a tiger beetle that is nationally listed as threatened in Canada and whose taxonomic status is based primarily on its unique elytral (forewing) color pattern. To determine whether C. f. gibsoni represents one or more genetically distinctive units, we sampled 14 populations within or near this subspecies’ disjunct North American range and assessed their genetic differentiation from neighboring and phenotypically distinctive populations of C. f. formosa and C. f. fletcheri. Instead of clustering by color pattern, analyses of mitochondrial and nuclear markers recovered three geographically structured genetic groupings: a northern cluster from Canada, a southwestern cluster from northwestern Colorado, and a southeastern cluster of US populations east of the Continental Divide. These data, coupled with previously documented differences in larval morphology, suggest that populations of C. formosa in western Canada and northwestern Colorado may have independently evolved similar color patterns. Thus, we consider C. f. gibsoni to be endemic to Canada and describe the novel subspecies C. f. gaumeri ssp. nov. from northwestern Colorado. Both subspecies are evolutionarily significant units, and each deserves consideration for conservation listing. Collectively, our results reveal general congruence between mitochondrial and nuclear genetic data but conflict with color pattern, the conventional basis for subspecies designations in tiger beetles.

The mountain pine beetle (Dendroctonus ponderosae Hopkins) (Coleoptera: Curculionidae) is a significant destructive force in the pine forests of western Canada and has the capacity to spread east into a novel host tree species, jack pine (Pinaceae). New populations have been documented in central Alberta, Canada, but the source populations for these outbreaks have yet to be identified. In this study, we use genetic data to identify parent populations for recent outbreak sites near Slave Lake, Lac La Biche, and Hinton, Alberta. We found the northern population cluster that entered Alberta near Grande Prairie was the source of the most eastern established population near Lac La Biche, and the range expansion to this leading-edge population has been too rapid to establish evidence of population structure. However, some dispersal from a population in the Jasper and Hinton area has been detected as far north and east as Slave Lake, Alberta. We also identified two potential source populations for the current outbreak in Hinton: most beetles appear to be from Jasper National Park, Alberta, but some also originated from the northern population cluster. These findings demonstrate the dynamic dispersal capabilities of mountain pine beetle across the Alberta landscape and the potential hazard of increased dispersal to newly established leading-edge populations.

The mega-diversity of herbivorous insects is attributed to their co-evolutionary associations with plants. Despite abundant studies on insect-plant interactions, we do not know whether host-plant shifts have impacted both genomic adaptation and species diversification over geological times. We show that the antagonistic insect-plant interaction between swallowtail butterflies and the highly toxic birthworts began 55 million years ago in Beringia, followed by several major ancient host-plant shifts. This evolutionary framework provides a valuable opportunity for repeated tests of genomic signatures of macroevolutionary changes and estimation of diversification rates across their phylogeny. We find that host-plant shifts in butterflies are associated with both genome-wide adaptive molecular evolution (more genes under positive selection) and repeated bursts of speciation rates, contributing to an increase in global diversification through time. Our study links ecological changes, genome-wide adaptations and macroevolutionary consequences, lending support to the importance of ecological interactions as evolutionary drivers over long time periods., Arms races between herbivores and plants have likely affected their evolutionary histories, which could have led to their high diversity. Allio et al. find that butterflies shifting to new host plants have more adaptive molecular signatures across their genomes and show repeated bursts of speciation rates.

Insects have developed various adaptations to survive harsh winter conditions. Among freeze-intolerant species, some produce “antifreeze proteins” (AFPs) that bind to nascent ice crystals and inhibit further ice growth. Such is the case of the spruce budworm, Choristoneura fumiferana (Lepidoptera: Tortricidae), a destructive North American conifer pest that can withstand temperatures below −30°C. Despite the potential importance of AFPs in the adaptive diversification of Choristoneura, genomic tools to explore their origins have until now been limited. Here, we present a chromosome-scale genome assembly for C. fumiferana, which we used to conduct comparative genomic analyses aimed at reconstructing the evolutionary history of tortricid AFPs. The budworm genome features 16 genes homologous to previously reported C. fumiferana AFPs (CfAFPs), 15 of which map to a single region on chromosome 18. Fourteen of these were also detected in five congeneric species, indicating Choristoneura AFP diversification occurred before the speciation event that led to C. fumiferana. Although budworm AFPs were previously considered unique to the genus Choristoneura, a search for homologs targeting recently sequenced tortricid genomes identified seven CfAFP-like genes in the distantly related Notocelia uddmanniana. High structural similarity between Notocelia and Choristoneura AFPs suggests a common origin, despite the absence of homologs in three related tortricids. Interestingly, one Notocelia AFP formed the C-terminus of a “zonadhesin-like” protein, possibly representing the ancestral condition from which tortricid AFPs evolved. Future work should clarify the evolutionary path of AFPs between Notocelia and Choristoneura and assess the role of the “zonadhesin-like” protein as precursor of tortricid AFPs.

Allochronic isolation can be a strong mechanism for reproductive isolation and speciation. However, imperfect allochrony and the expression of phenological plasticity can erode temporal barriers to gene flow and result in hybridization between divergent lineages. Here, we combine behavioural ecology and genomics to investigate this scenario in two closely related species of grigs in the genus Cyphoderris. These species exhibit a unique mating system whereby females feed on the fleshy hind wings of the male during copulation, and copulation with conspecific males is more likely in food-restricted females than in well-fed females. In western Canada, Cyphoderris buckelli and Cyphoderris monstrosa are sympatric but largely allochronically separated, with C. buckelli breeding earlier. However, their breeding seasons can overlap, leading to potential for older C. buckelli females to mate with young C. monstrosa males to obtain resources via sexual cannibalism. We used behavioural assays to test whether female feeding status affects the propensity for interspecific mating between C. buckelli females and C. monstrosa males. We then tested for hybridization and gene exchange in wild populations of both species, using morphology, mitochondrial DNA and genome-wide nuclear markers. We found that interspecific courtship and mating can occur, but the relationship between food restriction and increased propensity for hybridization was not significant. Although we observed intraspecific population genetic structure in both species, we found no signatures of hybridization in the morphological or genetic datasets, which suggests that postmating reproductive barriers might be preventing successful hybridization in the wild.

The spruce budworm, Choristoneura fumiferana, is presumed to be panmictic across vast regions of North America. We examined the extent of panmixia by genotyping 3,650 single nucleotide polymorphism (SNP) loci in 1975 individuals from 128 collections across the continent. We found three spatially structured subpopulations: Western (Alaska, Yukon), Central (southeastern Yukon to the Manitoba–Ontario border), and Eastern (Manitoba–Ontario border to the Atlantic). Additionally, the most diagnostic genetic differentiation between the Central and Eastern subpopulations was chromosomally restricted to a single block of SNPs that may constitute an island of differentiation within the species. Geographic differentiation in the spruce budworm parallels that of its principal larval host, white spruce (Picea glauca), providing evidence that spruce budworm and spruce trees survived in the Beringian refugium through the Last Glacial Maximum and that at least two isolated spruce budworm populations diverged with spruce/fir south of the ice sheets. Gene flow in the spruce budworm may also be affected by mountains in western North America, habitat isolation in West Virginia, regional adaptations, factors related to dispersal, and proximity of other species in the spruce budworm species complex. The central and eastern geographic regions contain individuals that assign to Eastern and Central subpopulations, respectively, indicating that these barriers are not complete. Our discovery of previously undetected geographic and genomic structure in the spruce budworm suggests that further population modelling of this ecologically important insect should consider regional differentiation, potentially co‐adapted blocks of genes, and gene flow between subpopulations.

Genome sequencing methods and assembly tools have improved dramatically since the 2013 publication of draft genome assemblies for the mountain pine beetle, Dendroctonus ponderosae Hopkins (Coleoptera: Curculionidae). We conducted proximity ligation library sequencing and scaffolding to improve contiguity, and then used linkage mapping and recent bioinformatic tools for correction and further improvement. The new assemblies have dramatically improved contiguity and gaps compared to the originals: N50 values increased 26- to 36-fold, and the number of gaps were reduced by half. Ninety percent of the content of the assemblies is now contained in 12 and 11 scaffolds for the female and male assemblies, respectively. Based on linkage mapping information, the 12 largest scaffolds in both assemblies represent all 11 autosomal chromosomes and the neo-X chromosome. These assemblies now have nearly chromosome-sized scaffolds and will be instrumental for studying genomic architecture, chromosome evolution, population genomics, functional genomics, and adaptation in this and other pest insects. We also identified regions in two chromosomes, including the ancestral-X portion of the neo-X chromosome, with elevated differentiation between northern and southern Canadian populations.

Dispersal flights by the mountain pine beetle have allowed range expansion and major damage to pine stands in western Canada. We asked what the genetic and transcriptional basis of mountain pine beetle dispersal capacity is. Using flight mills, RNA-seq and a targeted association study, we compared strong-flying, weak-flying, and non-flying female beetles from the recently colonized northern end of their range. Nearly 3,000 genes were differentially expressed between strong and weak flying beetles, while weak fliers and nonfliers did not significantly differ. The differentially expressed genes were mainly associated with lipid metabolism, muscle maintenance, oxidative stress response, detoxification, endocrine function, and flight behavior. Three variant loci, two in the coding region of genes, were significantly associated with flight capacity but these genes had no known functional link to flight. Several differentially expressed gene systems may be important for sustained flight, while other systems are downregulated during dispersal and likely to conserve energy before host colonization. The candidate genes and SNPs identified here will inform further studies and management of mountain pine beetle, as well as contribute to understanding the mechanisms of insect dispersal flights.

The mountain pine beetle (MPB; Dendroctonus ponderosae Hopkins, 1902) forms beneficial symbiotic associations with fungi. Here we explored the fine-scale spatial genetic structure of three of those fungi using single nucleotide polymorphism. We found that single mated pairs of beetles carry not only multiple fungal species, but also multiple genotypes of each species into their galleries. We observed genetic diversity at a fine spatial scale. Most of the diversity was found within and among galleries with nonsignificant diversity among trees. We observed clonal propagation almost exclusively within galleries. Ophiostoma montium (Rumbold) Arx possessed a larger expected number of multilocus genotypes and lower linkage disequilibrium than Grosmannia clavigera (Rob.-Jeffr. & R.W. Davidson) Zipfel, Z.W. de Beer & M.J. Wingf. and Leptographium longiclavatum S.W. Lee, J.J. Kim & C. Breuil. More than 80% of fungal samples were genetically unrelated, a result that parallels what has been observed in the beetles. The proportion of genetically related samples within galleries was higher in O. montium (40%) than in G. clavigera (20%) or L. longiclavatum (6%), likely the consequence of within-gallery sexual recombination in O. montium. The underlying genetic diversity reported here and the differences among fungal species could enable the symbiont community to quickly respond to new environmental conditions or changes in the host, enhancing the maintenance of this multipartite relationship and allowing the MPB to colonize new habitats.

Environmental change is altering forest insect dynamics worldwide. As these systems change, they pose significant ecological, social, and economic risk through, for example, the loss of valuable habitat, green space, and timber. Our understanding of such systems is often limited by the complexity of multiple interacting taxa. As a consequence, studies assessing the ecology, physiology, and genomics of each key organism in such systems are increasingly important for developing appropriate management strategies. Here we summarize the genetic and genomic contributions made by the TRIA project — a long-term study of the mountain pine beetle (Dendroctonus ponderosae Hopkins) system encompassing beetle, fungi, and pine. Contributions include genetic and genomic resources for species identification, sex determination, detection of selection, functional genetic analysis, mating system confirmation, hybrid stability tests, and integrated genetic studies of multiple taxa. These resources and subsequent findings have accelerated our understanding of the mountain pine beetle system, facilitating improved management strategies (e.g., enhancements to stand susceptibility indices and predictive models) and highlighting mechanisms for promoting resilient forests. Further, work from the TRIA project serves as a model for the increasing number and severity of invasive and native forest insect outbreaks globally (e.g., Dutch elm disease and thousand cankers disease).

Butterflies have played a pivotal role in our understanding of animal movement, but little is known about landscape-scale movement in highly vagile species with large ranges and open population structure. We investigate the effect of environment and landscape on both inter- and intraspecific genetic differentiation and population structure in the Papilio machaon group of swallowtail butterflies in Alberta, Canada. These butterflies exhibit strong hilltopping behaviour, where individuals congregate on topographical prominences to mate, and thus we expect topography to influence population connectivity. We use redundancy analysis to explore the relationship between two genetic datasets (mitochondrial DNA and microsatellites) and topographic, climatic and habitat-related environmental variables at multiple spatial scales. Shared variation across these environmental variables explained a large portion of both mitochondrial (≤ 75%) and microsatellite genetic variation (≤ 40%). Individual variables, such as elevation, terrain profile, solar radiation and precipitation, had moderate explanatory power; however, the water component of land cover had the greatest explanatory power of the individual variables and acted as a surrogate variable for the larval habitat of P. machaon. Despite high levels of retained ancestral polymorphism in the mitochondrial dataset, we found similar relationships between environmental characteristics and genetic variation. Finally, the spatial scale of our analyses greatly influenced our results, and without exploring the effect of scale we would have missed or discounted the effects of several environmental characteristics. This point emphasizes the importance of exploratory analysis in systems where there is little prior knowledge of movement dynamics and the influence of the environment.

Patterns in the genetic variation of species can be used to infer their specific demographic and evolutionary history and provide insight into the general mechanisms underlying population divergence and speciation. The Macoun’s Arctic (Oeneis macounii (W.H. Edwards, 1885); MA) butterfly occurs across Canada and parts of the northern United States in association with jack pine (Pinus banksiana Lamb.) and lodgepole pine (Pinus contorta Douglas ex Loudon). MA’s current distribution is highly fragmented, and the extent of reproductive isolation among allopatric populations is unknown. Furthermore, although MA is biennial, adults emerge every year in some populations. These populations presumably consist of two alternate-year cohorts, providing the opportunity for sympatric divergence via allochronic isolation. Using mitochondrial DNA (mtDNA) and amplified fragment length polymorphism (AFLP) markers, we analyzed MA’s genetic structure to determine the current and historical role of allopatric and allochronic isolation in MA population divergence. Both markers revealed high diversity and a low, but significant, degree of spatial structure and pattern of isolation by distance. Phylogeographic structure was generally absent, with low divergence among mtDNA haplotypes. MA likely exhibits low dispersal and gene flow among most allopatric populations; however, there was no evidence of differentiation resulting from allochronic isolation for sympatric cohorts.

The island species area relationship (ISAR) is an important tool for measuring variation in species diversity in variety of insular systems, from true-island archipelagoes to fragmented terrestrial landscapes. However, it suffers from several limitations. For example, due to the sample-area effect, positive relationships between species and area cannot be directly interpreted as evidence for deterministic effects of area per se. Additionally, richness-based analyses may obscure species-level responses to area and isolation that may better inform conservation practice. Here, we use random placement models to control for variation in abundance, occupancy and richness associated with the sample-area effect, allowing deterministic effects of area and isolation, and how they vary with species' functional traits, to be resolved using linear mixed effects models. We demonstrate the utility of this approach using a butterfly assemblage persisting on a naturally fragmented landscape of lake islands. The ISAR did not significantly deviate from random placement in relation to island area, isolation or habitat diversity, supporting stochastic assembly consistent with the sample-area effect. Such inferences support the habitat amount hypothesis, which prioritizes preserving the maximum amount of habitat irrespective of its degree of fragmentation. However, species-level analyses demonstrated that species' abundances were significantly lower on both smaller and more isolated islands than what is predicted by the sample-area effect. Moreover, effects of area per se were significantly greater for smaller, less mobile and rare species. Species' occurrences also significantly deviated from predictions of the sample-area effect in relation to island isolation. Thus, our approach illustrates that richness-based analyses not only result in incorrect inferences on mechanisms underlying ISARs, but also obscure important effects of area per se and isolation on individual species that vary with functional traits. We therefore suggest that these effects should not be solely inferred from richness-based analyses, but rather evaluated on a species-by-species basis.

Lyme disease-causing Borrelia burgdorferi has been reported in 10–19% of Ixodes ticks from Alberta, Canada, where the tick vector Ixodes scapularis is at the northwestern edge of its range. However, the presence of Borrelia has not been verified independently, and the bacterial microbiome of these ticks has not been described. We performed 16S rRNA bacterial surveys on female I. scapularis from Alberta that were previously qPCR-tested in a Lyme disease surveillance program. Both 16S and qPCR methods were concordant for the presence of Borrelia. The 16S studies also provided a profile of associated bacteria that showed the microbiome of I. scapularis in Alberta was similar to other areas of North America. Ticks that were qPCR-positive for Borrelia had significantly greater bacterial diversity than Borrelia-negative ticks, on the basis of generalized linear model testing. This study adds value to ongoing tick surveillance and is a foundation for deeper understanding of tick microbial ecology and disease transmission in a region where I. scapularis range expansion, induced by climate and land use changes, is likely to have increasing public health implications.

Lyme disease-causing Borrelia burgdorferi has been reported in 10–19% of Ixodes ticks from Alberta, Canada, where the tick vector Ixodes scapularis is at the northwestern edge of its range. However, the presence of Borrelia has not been verified independently, and the bacterial microbiome of these ticks has not been described. We performed 16S rRNA bacterial surveys on female I. scapularis from Alberta that were previously qPCR-tested in a Lyme disease surveillance program. Both 16S and qPCR methods were concordant for the presence of Borrelia. The 16S studies also provided a profile of associated bacteria that showed the microbiome of I. scapularis in Alberta was similar to other areas of North America. Ticks that were qPCR-positive for Borrelia had significantly greater bacterial diversity than Borrelia-negative ticks, on the basis of generalized linear model testing. This study adds value to ongoing tick surveillance and is a foundation for deeper understanding of tick microbial ecology and disease transmission in a region where I. scapularis range expansion, induced by climate and land use changes, is likely to have increasing public health implications.

Previous work in landscape genetics suggests that geographic isolation is of greater importance to genetic divergence than variation in environmental conditions. This is intuitive when configurations of suitable habitat are a dominant factor limiting dispersal and gene flow, but has not been thoroughly examined for habitat specialists with strong dispersal capability. Here, we evaluate the effects of geographic and environmental isolation on genetic divergence for a vagile invertebrate with high habitat specificity and a discrete dispersal life stage: Dod's Old World swallowtail butterfly, Papilio machaon dodi. In Canada, P. m. dodi are generally restricted to eroding habitat along major river valleys where their larval host plant occurs. A series of causal and linear mixed effects models indicate that divergence of genome-wide single nucleotide polymorphisms is best explained by a combination of environmental isolation (variation in summer temperatures) and geographic isolation (Euclidean distance). Interestingly, least-cost path and circuit distances through a resistance surface parameterized as the inverse of habitat suitability were not supported. This suggests that, although habitat associations of many butterflies are specific due to reproductive requirements, habitat suitability and landscape permeability are not equivalent concepts due to considerable adult vagility. We infer that divergent selection related to variation in summer temperatures has produced two genetic clusters within P. m. dodi, differing in voltinism and diapause propensity. Within the next century, temperatures are predicted to rise by amounts greater than the present-day difference between regions of the genetic clusters, potentially affecting the persistence of the northern cluster under continued climate change.

Shads of the genus Alosa are essential to commercial fisheries across North America and Europe, but in some areas their species boundaries remain controversial. Traditional morphology-based taxonomy of Alosa spp. has relied heavily on the number of gill rakers and body proportions, but these can be highly variable. We use mitochondrial (mt)DNA (coI and cytb) and genome-wide single nucleotide polymorphisms (SNP) along with morphological characters to assess differentiation among endemic Ponto-Caspian shads in the Sea of Azov. Morphological species assignments based on gill-raker number were not congruent with genetic lineages shown by mtDNA and SNPs. Iterative analysis revealed that genetic lineages were associated with sampling location and several other morphometric traits (caudal peduncle depth, pre-anal length and head length). Phylogenetic analysis of the genus placed Ponto-Caspian Alosa spp. in the same evolutionary lineage as endangered Alosa spp. endemic to Greece, highlighting the importance of these findings to conservation management. We conclude that gill-raker number is not reliable for delimiting species of Alosa. This taxonomic uncertainty should be addressed by examining type material to provide a robust integrative classification for these commercially important fishes.

Widely known for pest species that include major modulators of temperate forests, the genus Choristoneura is part of the species-rich tribe Archipini of leafroller moths (Tortricidae). Delimitation of the genus has remained unresolved because no phylogeny has included species endemic to Africa and studies have often omitted the type species of the genus. Further taxonomic confusion has been generated by the transfer of Archips occidentalis (Walsingham) to Choristoneura, creating a homonym with Choristoneura occidentalis Freeman, an important defoliator of North American forests. To define the limits of the genus, we reconstructed a phylogeny using DNA sequences for mitochondrial cytochrome oxidase subunit I and nuclear ribosomal 28S genes. Our ingroup included 23 Choristoneura species-level taxa, complemented by a large sample of outgroups comprising 82 species of Archipini and other Tortricidae. We generated a time-calibrated tree using fossil and secondary calibrations and we inferred biogeographic and diversification processes in Choristoneura. Our analysis recovered the genus as polyphyletic, with Archips occidentalis, Choristoneura simonyi and Choristoneura evanidana excluded from the main clade. Based on the recovered phylogenies and a redefinition, we restrict Choristoneura primarily to species with a northern hemisphere distribution. Our analysis supports A. occidentalis as the sister group of Cacoecimorpha pronubana, C. simonyi as the sister of ‘Xenotemna’ pallorana, and C. evanidana as the sister of Archips purpurana. A new combination is proposed: Archips evanidana comb.n.; the availability of ‘Xenotemna’ as a valid name is discussed and A. occidentalis is considered as an orphaned name within the Archipini. We found support for a Holarctic origin of Choristoneura about 23 Ma, followed by early divergence in the Palearctic region. The main divergence occurred at 16Ma, with one clade in the Nearctic and another in the Palearctic. Subsequent cladogenetic events were synchronous and related to herbivorous specialization, with each clade divided into coniferophagous and polyphagous lineages. Their specialization as conifer feeders temporally matched the expansion of boreal forest during the Miocene.

Sampling markers throughout a genome with restriction enzymes emerged in the 2000s as reduced representation shotgun sequencing (RRS). Rapid advances in sequencing technology have since spurred modifications of RRS, giving rise to many derivatives with unique names, such as RADseq. But naming conventions have often been more creative than consistent, with unclear criteria for recognition as a unique method resulting in a proliferation of names characterized by ambiguity. We conducted a literature review to assess methodological and etymological relationships among 36 restriction enzyme-based methods, as well as rates of correct referencing of commonly-used methods. We identify several instances of methodological convergence or misattribution in the literature, and note that many published derivatives have modified only minor elements of parent protocols. We urge greater restraint in naming derivative methods, to strike a better balance between clarity, recognition of scientific innovation, and correct attribution.

Conservation geneticists have argued that evolutionarily significant units (ESUs) must be both genetically distinct and adaptively significant to be recognized for conservation protection. High-throughput DNA approaches can greatly increase the power to identify genetic distinctiveness, even if inferring adaptive significance remains a challenge. Here we present the first genomic evaluation of Lange’s metalmark, Apodemia mormo langei (Lepidoptera: Riodinidae), a U.S. federally endangered subspecies restricted to sand dune habitats in a single National Wildlife Refuge in California. Previous work based on very few genetic markers detected little genetic distinction for Lange’s metalmark. We use several thousand genome-wide single nucleotide polymorphisms to characterize the population structure of the A. mormo complex across California and determine if Lange’s metalmark qualifies as an ESU. We found that Lange’s metalmark is genetically identifiable, but is no more distinct than many other isolated populations across the study area. It remains unclear whether this genetic variation is adaptive, and so conservation efforts would benefit from more ecological characterization to determine conservation priorities.

Genome structure characterization can contribute to a better understanding of processes such as adaptation, speciation, and karyotype evolution, and can provide useful information for refining genome assemblies. We studied the genome of an important North American boreal forest pest, the spruce budworm, Choristoneura fumiferana, through a combination of molecular cytogenetic analyses and construction of a high-density linkage map based on single nucleotide polymorphism (SNP) markers obtained through a genotyping-by-sequencing (GBS) approach. Cytogenetic analyses using fluorescence in situ hybridization methods confirmed the haploid chromosome number of n = 30 in both sexes of C. fumiferana and showed, for the first time, that this species has a WZ/ZZ sex chromosome system. Synteny analysis based on a comparison of the Bombyx mori genome and the C. fumiferana linkage map revealed the presence of a neo-Z chromosome in the latter species, as previously reported for other tortricid moths. In this neo-Z chromosome, we detected an ABC transporter C2 (ABCC2) gene that has been associated with insecticide resistance. Sex-linkage of the ABCC2 gene provides a genomic context favorable to selection and rapid spread of resistance against Bacillus thuringiensis serotype kurstaki (Btk), the main insecticide used in Canada to control spruce budworm populations. Ultimately, the linkage map we developed, which comprises 3586 SNP markers distributed over 30 linkage groups for a total length of 1720.41 cM, will be a valuable tool for refining our draft assembly of the spruce budworm genome.

Swallowtail butterflies (Lepidoptera: Papilionidae) have been instrumental in understanding many foundational concepts in biology; despite this, a resolved and robust phylogeny of the group has been a major impediment to elucidating patterns and processes of their ecological and evolutionary history. This study presents a mitogenomic, time-calibrated phylogeny for all swallowtail genera. A shotgun sequencing approach was performed to obtain 32 complete mitogenomes that were added to available butterfly mitogenomes, resulting in a dataset including 142 butterfly taxa (and four outgroups) representing all butterfly families. Phylogenetic analyses were carried out under maximum likelihood (ML) and Bayesian inferences (BIs) with alternative partitioning strategies and the mixture (CAT) model. To test competing hypotheses about the systematics of Papilionidae, such as the enigmatic position of Baronia brevicornis or the status of the tribe Teinopalpini, we estimated the marginal likelihood of alternative topologies and computed Bayes factors. Estimates of divergence times were assessed using a Bayesian relaxed-clock approach calibrated with six fossils while testing for the number of clocks. The results recovered a well-resolved and supported phylogeny confirming that Baroniinae is sister to Parnassiinae + Papilioninae, both recovered as monophyletic. It also laid the foundations for classification at tribe and genus level, suggesting that the tribe Teinopalpini only contains the genus Teinopalpus (Meandrusa being sister to Papilio). The number of molecular clocks in dating analyses had a significant impact on divergence times. A single clock recovered an origin of butterflies in the Cretaceous (98, 66–188 Ma) and also for swallowtails (85, 55–163 Ma), while partitioning the clocks yielded an origin of Papilionoidea in the very Late Cretaceous (71, 64–86 Ma), and all butterfly families originated in the aftermath of the Cretaceous–Paleogene extinction. These results challenge previous studies suggesting that butterflies appeared in the Early Cretaceous, 110 Ma, concurrently with the rise of angiosperms.

Dispersal flights by the mountain pine beetle have allowed range expansion and major damage to pine stands in western Canada. We asked what the genetic and transcriptional basis of mountain pine beetle dispersal capacity is. Using flight mills, RNA-seq and a targeted association study, we compared strong-flying, weak-flying, and non-flying female beetles from the recently colonized northern end of their range. Nearly 3,000 genes were differentially expressed between strong and weak flying beetles, while weak fliers and nonfliers did not significantly differ. The differentially expressed genes were mainly associated with lipid metabolism, muscle maintenance, oxidative stress response, detoxification, endocrine function, and flight behavior. Three variant loci, two in the coding region of genes, were significantly associated with flight capacity but these genes had no known functional link to flight. Several differentially expressed gene systems may be important for sustained flight, while other systems are downregulated during dispersal and likely to conserve energy before host colonization. The candidate genes and SNPs identified here will inform further studies and management of mountain pine beetle, as well as contribute to understanding the mechanisms of insect dispersal flights.

Populations are often exposed to multiple sources of gene flow, but accounts are lacking of the population genetic dynamics that result from these interactions or their effects on local evolution. Using a genomic clines framework applied to 1195 SNPs, we documented genome-wide, locus-specific patterns of introgression between Choristoneura occidentalis biennis spruce budworms and two ecologically divergent relatives, C. o. occidentalis and C. fumiferana, that it interacts with at alternate boundaries of its range. We observe contrasting hybrid indexes between these hybrid zones, no overlap in ‘gene flow outliers’ (clines showing relatively extreme extents or rates of locus-specific introgression), and variable linkage disequilibrium among those outliers. At the same time, correlated genome-wide rates of introgression between zones suggest the presence of processes common to both boundaries. These findings highlight the contrasting population genetic dynamics that can occur at separate frontiers of a single population, while also suggesting that shared patterns may frequently accompany cases of divergence-with-gene-flow that involve a lineage in common. Our results point to potentially complex evolutionary outcomes for populations experiencing multiple sources of gene flow. This article is protected by copyright. All rights reserved.

High-throughput sequencing methods for genotyping genome-wide markers are being rapidly adopted for phylogenetics of non-model organisms in conservation and biodiversity studies. However, the reproducibility of SNP genotyping and degree of marker overlap or compatibility between datasets from different methodologies have not been tested in non-model systems. Using double-digest restriction site associated DNA sequencing, we sequenced a common set of 22 specimens from the butterfly genus Speyeria on two different Illumina platforms, using two variations of library preparation. We then used a de novo approach to bioinformatic locus assembly and SNP discovery for subsequent phylogenetic analyses. We found a high rate of locus recovery despite differences in library preparation and sequencing platforms, as well as overall high levels of data compatibility after data processing and filtering. These results provide the first application of NGS methods for phylogenetic reconstruction in Speyeria, and support the use and long-term viability of SNP genotyping applications in non-model systems. This article is protected by copyright. All rights reserved.

Ticks vector diverse pathogenic bacteria that are important to identify in public health and veterinary contexts. Technological advances in high throughput sequencing have given an unprecedented opportunity to comprehensively characterize bacterial associates of ticks, but recent studies have used different 16S rRNA variable regions and sequence read lengths with little consideration of whether they reveal the same bacterial diversity. We compare the effectiveness of bacterial surveys using three library preparations across nine 16S variable regions and a set of 12 tick specimens (Acari: Ixodidae). We identify the bacterial assemblages present in extractions from wild-collected Ixodes scapularis from two regions of Canada, and provide the first microbiome survey for Ixodes angustus. Four bacterial families accounted for most diversity, with Rickettsiaceae being replaced as most common by Enterobacteriaceae or Pseudomonadaceae in some I. scapularis, and Francisellaceae being most abundant in I. angustus. The commercially available Ion 16S kit, based on 6 amplicons representing 16S regions V2, V3, V4, V67, V8 and V9, gave the most comprehensive estimates of bacterial families, with the Ion V4 amplicon generally giving the highest estimated diversity. Sequencing of the V4 amplicon by the MR DNA commercial service also provided cost effective assays of tick microbiomes that were within the range of results from the Ion 16S kit. Subtraction of the number of reads found in an extraction control sample lowered estimates of the number of bacterial families by approximately half. Our study shows that diversity patterns obtained from 16S microbiome surveys depend on the amplicon and protocol used, demonstrating that more than one marker region is needed to provide reliable inferences.

Bark beetles form multipartite symbiotic associations with blue stain fungi (Ophiostomatales, Ascomycota). These fungal symbionts play an important role during the beetle's life cycle by providing nutritional supplementation, overcoming tree defences and modifying host tissues to favour brood development. The maintenance of stable multipartite symbioses with seemingly less competitive symbionts in similar habitats is of fundamental interest to ecology and evolution. We tested the hypothesis that the coexistence of three fungal species associated with the mountain pine beetle is the result of niche partitioning and adaptive radiation using SNP genotyping coupled with genotype-environment association analysis and phenotypic characterization of growth rate under different temperatures. We found that genetic variation and population structure within each species is best explained by distinct spatial and environmental variables. We observed both common (temperature seasonality and the host species) and distinct (drought, cold stress, precipitation) environmental and spatial factors that shaped the genomes of these fungi resulting in contrasting outcomes. Phenotypic intraspecific variations in Grosmannia clavigera and Leptographium longiclavatum, together with high heritability, suggest potential for adaptive selection in these species. By contrast, Ophiostoma montium displayed narrower intraspecific variation but greater tolerance to extreme high temperatures. Our study highlights unique phenotypic and genotypic characteristics in these symbionts that are consistent with our hypothesis. By maintaining this multipartite relationship, the bark beetles have a greater likelihood of obtaining the benefits afforded by the fungi and reduce the risk of being left aposymbiotic. Complementarity among species could facilitate colonization of new habitats and survival under adverse conditions.

The spruce budworm, Choristoneura fumiferana, is presumed to be panmictic across vast regions of North America. We examined the extent of panmixia by genotyping 3,650 single nucleotide polymorphism (SNP) loci in 1975 individuals from 128 collections across the continent. We found three spatially structured subpopulations: Western (Alaska, Yukon), Central (southeastern Yukon to the Manitoba–Ontario border), and Eastern (Manitoba–Ontario border to the Atlantic). Additionally, the most diagnostic genetic differentiation between the Central and Eastern subpopulations was chromosomally restricted to a single block of SNPs that may constitute an island of differentiation within the species. Geographic differentiation in the spruce budworm parallels that of its principal larval host, white spruce (Picea glauca), providing evidence that spruce budworm and spruce trees survived in the Beringian refugium through the Last Glacial Maximum and that at least two isolated spruce budworm populations diverged with spruce/fir south of the ice sheets. Gene flow in the spruce budworm may also be affected by mountains in western North America, habitat isolation in West Virginia, regional adaptations, factors related to dispersal, and proximity of other species in the spruce budworm species complex. The central and eastern geographic regions contain individuals that assign to Eastern and Central subpopulations, respectively, indicating that these barriers are not complete. Our discovery of previously undetected geographic and genomic structure in the spruce budworm suggests that further population modelling of this ecologically important insect should consider regional differentiation, potentially co‐adapted blocks of genes, and gene flow between subpopulations., We examined the population genomic structure of the spruce budworm, Choristoneura fumiferana, and found three spatially structured subpopulations: Western (Alaska, Yukon), Central (southeastern Yukon to the Manitoba–Ontario border) and Eastern (Manitoba–Ontario border to the Atlantic). Geographic differentiation in the spruce budworm parallels that of its principal larval host, white spruce (Picea glauca), providing evidence that spruce budworm and spruce trees survived in the Beringian refugium through the Last Glacial Maximum and that at least two isolated spruce budworm populations diverged with spruce/fir south of the ice sheets. Gene flow in the spruce budworm may also be affected by mountains in western North America, habitat isolation in West Virginia, regional adaptations, factors related to dispersal, and proximity of other species in the spruce budworm complex.

This article was originally published as: Trevoy, S.A.L., Janes, J.K., Muirhead, K., & Sperling, F.A.H. (2019). Repurposing population genetics data to discern genomic architecture: A case study of linkage cohort detection in mountain pine beetle (Dendroctonus ponderosae). Ecology and Evolution, 9(3), 1147-1159. https://doi.org/10.1002/ece3.4803, Genetic surveys of the population structure of species can be used as resources for exploring their genomic architecture. By adjusting filtering assumptions, genomewide single‐nucleotide polymorphism (SNP) datasets can be reused to give new insights into the genetic basis of divergence and speciation without targeted resampling of specimens. Filtering only for missing data and minor allele frequency, we used a combination of principal components analysis and linkage disequilibrium network analysis to distinguish three cohorts of variable SNPs in the mountain pine beetle in western Canada, including one that was sex‐linked and one that was geographically associated. These marker cohorts indicate genomically localized differentiation, and their detection demonstrates an accessible and intuitive method for discovering potential islands of genomic divergence without a priori knowledge of a species’ genomic architecture. Thus, this method has utility for directly addressing the genomic architecture of species and generating new hypotheses for functional research.

Species delimitation requires an assessment of varied traits that can contribute to reproductive isolation, as well as of the permanence of evolutionary differentiation among closely related lineages. Integrative taxonomy, including the combination of genome-wide molecular data with ecological data, offers an effective approach to this issue. We use genotyping-by-sequencing together with a review of ecological divergence to assess the traditionally recognized species status of three closely related members of the spruce budworm species complex, Choristoneura fumiferana (Clemens), C. occidentalis Freeman (=C. freemani Razowski) and C. biennis Freeman, each of which is a major defoliator of conifer forests. We sampled a broad region of overlap between these three taxa in Alberta and British Columbia (Canada) where potential for gene flow provides a strong test of the durability of divergence among lineages. A total of 2218 single nucleotide polymorphisms (SNPs) were assayed, and patterns of differentiation were evaluated under the biological, ecological, genotypic cluster and phylogenetic species concepts. Choristoneura fumiferana was genetically distinct with substantial barriers to genetic exchange with C. occidentalis and C. biennis. Conversely, divergence between C. occidentalis and C. biennis was limited to a small subset of outlier loci and was within the range observed within any one of the taxa. Considering both population genetic and ecological patterns of divergence, C. fumiferana should continue to be recognized as a distinct species, and C. biennis (syn.n.) should be treated as a subspecies (C. occidentalis biennis Freeman, 1967) of C. occidentalis, thereby automatically establishing the nominate name C. occidentalis occidentalis Freeman, 1967 for univoltine populations of this species.

Genomics has revolutionized our understanding of hybridization and introgression, but most of the early evidence for these processes came from studies of mitochondrial introgression. To expand these evolutionary insights from mitochondrial patterns, we evaluate phylogenetic discordance across the nuclear genomes of a hybridizing system, the Papilio machaon group of swallowtail butterflies. This species group contains three hybrid lineages (P. brevicauda, P. joanae, and P. m. kahli) that are geographically disjunct across North America and have complete fixation of a mitochondrial lineage that is otherwise primarily found in P. m. hudsonianus, a boreal subspecies of the Holarctic P. machaon. Genome-wide nuclear markers place the three hybrid lineages as a monophyletic group that is sister to P. polyxenes/P. zelicaon rather than P. machaon, although ancient hybridization between a subspecies of P. machaon and the ancestor of these three lineages is also shown by their greater nuclear affinity to P. m. hudsonianus than to other subspecies of P. machaon. Individuals from contemporary hybrid swarms in Alberta, where mitochondrial DNA fixation has not occurred, were more intermediate between their respective parent species, demonstrating diversity in mito-nuclear discordance following hybrid interactions. Our new phylogenetic findings for the P. machaon species group also include: subspecific paraphyly within P. machaon itself across its Holarctic distribution; paraphyly of P. zelicaon relative to P. polyxenes; and more divergent placement of a Mediterranean species, P. hospiton. These results provide the first comprehensive genomic evaluation of relationships within this species group and provide insight into the evolutionary dynamics of hybridization and mitochondrial introgression.

Evolutionary relationships have remained unresolved in many well-studied groups, even though advances in next-generation sequencing and analysis, using approaches such as transcriptomics, anchored hybrid enrichment, or ultraconserved elements, have brought systematics to the brink of whole genome phylogenomics. Recently, it has become possible to sequence the entire genomes of numerous nonbiological models in parallel at reasonable cost, particularly with shotgun sequencing. Here, we identify orthologous coding sequences from whole-genome shotgun sequences, which we then use to investigate the relevance and power of phylogenomic relationship inference and time-calibrated tree estimation. We study an iconic group of butterflies—swallowtails of the family Papilionidae—that has remained phylogenetically unresolved, with continued debate about the timing of their diversification. Low-coverage whole genomes were obtained using Illumina shotgun sequencing for all genera. Genome assembly coupled to BLAST-based orthology searches allowed extraction of 6621 orthologous protein-coding genes for 45 Papilionidae species and 16 outgroup species (with 32% missing data after cleaning phases). Supermatrix phylogenomic analyses were performed with both maximum-likelihood (IQ-TREE) and Bayesian mixture models (PhyloBayes) for amino acid sequences, which produced a fully resolved phylogeny providing new insights into controversial relationships. Species tree reconstruction from gene trees was performed with ASTRAL and SuperTriplets and recovered the same phylogeny. We estimated gene site concordant factors to complement traditional node-support measures, which strengthens the robustness of inferred phylogenies. Bayesian estimates of divergence times based on a reduced data set (760 orthologs and 12% missing data) indicate a mid-Cretaceous origin of Papilionoidea around 99.2 Ma (95% credibility interval: 68.6–142.7 Ma) and Papilionidae around 71.4 Ma (49.8–103.6 Ma), with subsequent diversification of modern lineages well after the Cretaceous-Paleogene event. These results show that shotgun sequencing of whole genomes, even when highly fragmented, represents a powerful approach to phylogenomics and molecular dating in a group that has previously been refractory to resolution.

Sampling markers throughout a genome with restriction enzymes emerged in the 2000s as reduced representation shotgun sequencing (RRS). Rapid advances in sequencing technology have since spurred modifications of RRS, giving rise to many derivatives with unique names, such as RADseq. But naming conventions have often been more creative than consistent, with unclear criteria for recognition as a unique method resulting in a proliferation of names characterized by ambiguity. We conducted a literature review to assess methodological and etymological relationships among 36 restriction enzyme-based methods, as well as rates of correct referencing of commonly-used methods. We identify several instances of methodological convergence or misattribution in the literature, and note that many published derivatives have modified only minor elements of parent protocols. We urge greater restraint in naming derivative methods, to strike a better balance between clarity, recognition of scientific innovation, and correct attribution.

The oriental fruit moth Grapholita molesta is one of the major pests of stone and pome fruit species in Brazil. Here, we applied 1226 SNPs obtained by genotyping-by-sequencing to test whether host species associations or other factors such as geographic distance structured populations of this pest. Populations from the main areas of occurrence of G. molesta were sampled principally from peach and apple orchards. Three main clusters were recovered by neighbor-joining analysis, all defined by geographic proximity between sampling localities. Overall genetic structure inferred by a nonhierarchical amova resulted in a significant ΦST value = 0.19109. Here, we demonstrate for the first time that SNPs gathered by genotyping-by-sequencing can be used to infer genetic structure of a pest insect in Brazil; moreover, our results indicate that those markers are very informative even over a restricted geographic scale. We also demonstrate that host plant association has little effect on genetic structure among Brazilian populations of G. molesta; on the other hand, reduced gene flow promoted by geographic isolation has a stronger impact on population differentiation.

Dispersal determines the flux of individuals, energy and information and is therefore a key determinant of ecological and evolutionary dynamics. Yet, it remains difficult to quantify its importance relative to other factors. This is particularly true in cyclic populations in which demography, drift and dispersal contribute to spatio-temporal variability in genetic structure. Improved understanding of how dispersal influences spatial genetic structure is needed to disentangle the multiple processes that give rise to spatial synchrony in irruptive species. In this study, we examined spatial genetic structure in an economically important irruptive forest insect, the spruce budworm (Choristoneura fumiferana) to better characterize how dispersal, demography and ecological context interact to influence spatial synchrony in a localized outbreak. We characterized spatial variation in microsatellite allele frequencies using 231 individuals and seven geographic locations. We show that (i) gene flow among populations is likely very high (Fst ≈ 0); (ii) despite an overall low level of genetic structure, important differences exist between adult (moth) and juvenile (larvae) life stages; and (iii) the localized outbreak is the likely source of moths captured elsewhere in our study area. This study demonstrates the potential of using molecular methods to distinguish residents from migrants and for understanding how dispersal contributes to spatial synchronization. In irruptive populations, the strength of genetic structure depends on the timing of data collection (e.g. trough vs. peak), location and dispersal. Taking into account this ecological context allows us to make more general characterizations of how dispersal can affect spatial synchrony in irruptive populations.

Aim The aim was to determine processes driving the latitudinal diversity gradient (LDG) in New World swallowtail butterfly diversity. We tested three mechanisms commonly invoked to explain the LDG: ecological opportunity, evolutionary rates and biogeographical history. Location New World and Eurasia. Time period Oligocene–Present. Major taxa studied New World swallowtail butterfly clade (Papilio: Agehana, Alexanoria, Chilasa, Heraclides and Pterourus). Methods We integrated data from the most complete current phylogeny of this clade with geographical distributions of each species inferred from ecological niche models (ENMs). We tested for correlation between breadth of available abiotic ecological niche space, latitude and differential rates of diversification between tropical and non-tropical lineages. The clade's history of climatic and geographical occupancy was also reconstructed using both continuous ancestral character reconstructions and biogeographical history inferred under a dispersal–extinction–cladogenesis model. We considered both latitudinal and climatic definitions of tropicality in our reconstructions. Results There was no strong support for ecological opportunity or macroevolutionary processes as latitudinal diversity gradient drivers. Instead, we recovered discordant patterns in phylogenetic reconstructions of latitudinal geographical range and suitable abiotic climate conditions. Heraclides are likely to have originated and diversified in climatically and latitudinally tropical environments before some lineages dispersed to temperate habitats. The Alexanoria + Chilasa + Pterourus clade is likely to have originated in climatically and latitudinally temperate habitat before dispersing and diversifying; some lineages are likely to have dispersed into the latitudinal tropics via highland temperate-analogue environments. Main conclusions The LDG in New World swallowtails results from complex interactions between ecological niche evolution and biogeographical history; both out-of-the-tropics and into-the-tropics processes have contributed to the LDG. Our results present an example where temperate zones appear to be a source, instead of a sink, for biodiversity. Our results emphasize the need to consider biogeographical history not only from the perspective of shifts in geographical space, but also in terms of constraints enforced by ecological niche conservatism.