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The longhorn beetle fauna of Kentucky has long been overlooked in the literature with revisions and historic publications reporting few records from the state. Here, we document the occurrence of 222 species of Cerambycidae in Kentucky, with 140 new state records. For each species, we summarize its distribution (overall and in the state), the counties, years, and months in which it has been collected, collecting methods, what collections house the specimens, larval host plants, adult flower visitation, chemical lure attraction, recent taxonomic changes, and other pertinent information about the species. Using this dataset, the bias-corrected Chao1 species richness estimator predicted that 241 species should be found in Kentucky, indicating that our sampling is nearly comprehensive. Additionally, we provide a list of 42 species that have been found in at least one neighboring state and the distance from the closest record of the species to Kentucky; among this candidate list are 11 species known from within 50 km of the state. This checklist remedies the historical oversight of Kentucky cerambycid diversity in the literature, and we hope it will serve as a resource for future collectors, particularly the information on chemical lure attraction, which has not been summarized to this extent for any other state to date.

As dominant features of most ecosystems, insects are responsive to changes in climate, both over short temporal scales (e.g. seasonal fluctuations in abundance) and over longer evolutionary scales (e.g. decade-scale changes in patterns of biodiversity). One such taxonomic group that is sensitive to changing climate are the craneflies (Diptera: Tipulidae). Here, we used aggregated biodiversity data to examine elevational and latitudinal distributions of adult Tipulidae between 1976 and 2019 in Wales, UK, and we related these distributions to climatic patterns. Our analyses showed that species with earlier-emerging adults were most affected by weather conditions in the year before observation. Specifically, as temperature increased, observed elevation increased in high-precipitation conditions, remained stable in average-precipitation conditions and decreased in low-precipitation conditions. For species with later-emerging adults, associations were seen between elevation and weather conditions in the year of observation. Observed latitude generally exhibited a negative association with maximum temperature in the year before observation, with observations of Tipulidae trending southwards during the 43-year study period. Our results support consideration of emergence phenology, weather and habitat data when predicting species distributional changes attributable to climate change, which is vital in understanding the selection pressures that species face in a changing environment.

Population genomics is a useful tool to support integrated pest management as it can elucidate population dynamics, demography, and histories of invasion. Here, we use a restriction site-associated DNA sequencing approach combined with whole-genome amplification (WGA) to assess genomic population structure of a newly described pest of canola, the diminutive canola flower midge, Contarinia brassicola. Clustering analyses recovered little geographic structure across the main canola production region but differentiated several geographically disparate populations at edges of the agricultural zone. Given a lack of alternative hypotheses for this pattern, we suggest these data support alternative hosts for this species and thus our canola-centric view of this midge as a pest has limited our understanding of its biology. These results speak to the need for increased surveying efforts across multiple habitats and other potential hosts within Brassicaceae to improve both our ecological and evolutionary knowledge of this species and contribute to effective management strategies. We additionally found that use of WGA prior to library preparation was an effective method for increasing DNA quantity of these small insects prior to restriction site-associated DNA sequencing and had no discernible impact on genotyping consistency for population genetic analysis; WGA is therefore likely to be tractable for other similar studies that seek to randomly sample markers across the genome in small organisms.

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.

There is growing evidence for the decline of cryptic species across the planet as a result of human activities. Accurate data regarding patterns of poorly known or hard-to-find species diversity is essential for the recognition and conservation of threatened species and ecosystems. Casey’s June beetle Dinacoma caseyi is a federally listed endangered species restricted to the Coachella Valley in southern California, where rapid development is leading to habitat fragmentation. This fragmentation may be disproportionately impacting a wide-range of poorly-dispersing, cryptic species, including Casey’s June beetle, which has flightless females. We characterized 1876 single nucleotide polymorphisms from across the genome along with 1480 bp of mitochondrial DNA of all confirmed extant Dinacoma populations. We found that Dinacoma is isolated into three distinct species, including evidence for a previously undescribed species revealed during this study. Each is restricted to a small part of the inland desert region. Our results suggest unappreciated and fine scale diversity, which may be reflected in the other cryptic species of the region. Patterns of diversity in non-vagile species should guide ongoing conservation planning in the region. These results show that genetic exchange within the one remaining island of beetle habitat (Palm Canyon Wash) is not limited. However, non-vagile species, such as Casey’s June beetle, may not colonize suitable but fragmented habitat islands which presents risks to the species due to habitat loss and periodic natural events that may put the single population at risk of extirpation.

Phylogeographic patterns in phytophagous organisms are often contextualized in light of geographic isolation and ecological (host, habitat) specialization. However, assessing the relative impact of these phenomena is not straightforward, even in areas where phylogeography is well-studied, such as the California Floristic Province. Here, we use genome-wide markers to elucidate population genomic and phylgeographic patterns for a group of monophytophagous butterflies in southern California. This group is of high conservation interest because it includes the El Segundo blue, Euphilotes battoides allyni, one of the first insects listed under the U.S. Endangered Species Act, and a newly discovered population putatively assigned to E. b. allyni. Despite using the same unique host and coastal habitat, our results indicate that the newly discovered populations are not E. b. allyni and are more closely related to geographically proximate populations of the E. battoides group using a different habitat/host combination. Aside from E. b. allyni and the newly discovered populations, the rest of the group shows only fine-scale structure and apparently maintains genetic connectivity throughout southern California, across a vast range of habitats and climates, and on multiple hosts. Thus, habitat and host specialization did not elicit genetic isolation in neighboring populations suggesting that: (1) other phenomena are needed to explain the remarkable and idiosyncratic divergence of these highly restricted, proximate, taxa, and (2) fine-scale genomic markers suggest broader implications for understanding the mechanisms of speciation and reinvestigation of phylogeographic patterns in regions like the California Floristic Province.

In the context of evolutionary theory, invasion biology provides a fantastic enigma: how does a species with limited standing genetic variation survive and adapt to a novel environment? Reduced genetic diversity is typically associated with low fitness and evolutionary potential, yet some introduced species have proven to be successful invaders despite undergoing a genetic bottleneck during the early stages of colonization. Our goal in this study was to characterize population genomic and phenotype diversity of invasive Drosophila suzukii (Diptera: Drosophilidae) since colonizing the Hawaiian archipelago as early as the 1980s. Wing phenotype analysis revealed that high altitude populations possessed significantly larger wings than low altitude populations, supporting the hypothesis that insects cope with high altitude environments by developing larger wings. While we discovered low genetic diversity and differentiation in all Hawai‘i populations, three unique genetic clusters were detected with a model-free, multivariate statistical approach. We identified 23 candidate loci under selection using two complementary analyses to detect FST outliers across the genome. For 12 of these loci, predicted proteins are associated with Drosophila spp. chemosensation, amino acid and sodium ion transport, a Ras effector pathway, and cytidine deamination. Despite a genetic bottleneck, adventive D. suzukii populations are beginning to differentiate across the Hawaiian archipelago and selection for key behavioral and cellular processes are likely ongoing.

Recurrently invading pests provide unique challenges for pest management, but also present opportunities to utilize genomics to understand invasion dynamics and inform regulatory management through pathway analysis. In the southern United States, the Mexican fruit fly Anastrepha ludens is such a pest, and its incursions into Texas and California represent major threats to the agricultural systems of those regions. We developed a draft genome assembly for A. ludens, conducted range‐wide population genomics using restriction site‐associated DNA sequencing, and then developed and demonstrated a panel of highly differentiated diagnostic SNPs for source determination of intercepted flies in this system. Using 2,081 genomewide SNPs, we identified four populations across the range of A. ludens, corresponding to western Mexico, eastern Mexico/Texas, Guatemala/Belize/Honduras, and Costa Rica/Panama, with some intergradation present between clusters, particularly in Central America. From this population genomics framework, we developed a diagnostic panel of 28 highly differentiated SNPs that were able to recreate the genomewide population structure in this species. We demonstrated this panel on a set of test specimens, including specimens intercepted as part of regular trapping surveillance in Texas and California, and we were able to predict populations of origin for these specimens. This methodology presents a highly applied use of genomic techniques and can be implemented in any group of recurrently invading pests.

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.

A new species, Contarinia brassicola Sinclair (Diptera: Cecidomyiidae), which induces flower galls on canola (Brassica napus Linnaeus and Brassica rapa Linnaeus (Brassicaceae)), is described from Saskatchewan and Alberta, Canada. Larvae develop in the flowers of canola, which causes swelling and prevents opening, pod formation, and seed set. Mature larvae exit the galls, fall to the soil, and form cocoons. Depending on conditions, larvae will either pupate and eclose in the same calendar year or enter facultative diapause and emerge the following year. At least two generations of C. brassicola occur each year. Adults emerge from overwintering cocoons in the spring and lay eggs on developing canola flower buds. The galls produced by C. brassicola were previously attributed to the swede midge, Contarinia nasturtii (Kieffer) in Saskatchewan; here, we compare and list several characters to differentiate the two species.

The swede midge, Contarinia nasturtii, is a cecidomyiid fly that feeds specifically on plants within the Brassicaceae. Plants in this family employ a glucosinolate-myrosinase defense system, which can be highly toxic to nonspecialist feeders. Feeding by C. nasturtii larvae induces gall formation, which can cause substantial yield losses thus making it a significant agricultural pest. A lack of genomic resources, in particular a reference genome, has limited deciphering the mechanisms underlying glucosinolate tolerance in C. nasturtii, which is of particular importance for managing this species. Here, we present an annotated, scaffolded reference genome of C. nasturtii using linked-read sequencing from a single individual and explore systems involved in glucosinolate detoxification. The C. nasturtii genome is similar in size and annotation completeness to that of the Hessian fly, Mayetiola destructor, but has greater contiguity. Several genes encoding enzymes involved in glucosinolate detoxification in other insect pests, including myrosinases, sulfatases, and glutathione S-transferases, were found, suggesting that C. nasturtii has developed similar strategies for feeding on Brassicaceae. The C. nasturtii genome will, therefore, be integral to continued research on plant-insect interactions in this system and contribute to effective pest management strategies.

Divergence times underpin diverse evolutionary hypotheses, but conflicting age estimates across studies diminish the validity of such hypotheses. These conflicts have continued to grow as large genomics datasets become commonplace and analytical approaches proliferate. To provide more stable temporal intervals, age estimations should be interpreted in the context of both the type of data and analysis being used. Here, we use multispecies coalescent (MSC), concatenation-based, and categorical data transformation approaches on genome-wide SNP data to infer divergence ages within the Papilio glaucus group of tiger swallowtail butterflies in North America. While the SNP data supported previously recognized relationships within the group (P. multicaudata, ((P. eurymedon, P. rutulus), (P. appalachiensis, P. canadensis, P. glaucus))), estimated ages of divergence between the major lineages varied substantially among analyses. MSC produced wide credibility intervals particularly for deeper nodes, reflecting uncertainty in the coalescence times as a possible result of conflicting signal across gene trees. Concatenation, in contrast, gave narrower and more well-defined posterior distributions for the node ages; however, the higher precision of these time estimates is a likely artefact due to more simplistic underlying assumptions of this approach that do not account for conflict among gene trees. Transformed categorical data analysis gave the least precise and the most variable results, with its simple substitution model coupled with a relaxed clock tending to produce spurious results from large genome-wide datasets. While median node ages differed considerably between analyses (∼2 Mya between MSC and concatenation-based results), their corresponding credibility intervals nonetheless highlight common temporal patterns for deeper divergences in the group as well as finer-scale phylogeography. Age distributions across analyses support an origin of the group during the warm period of the early to mid-Pliocene. Late Pliocene climate aridification and cooling drove divergence between eastern and western groups that further diversified during the period of repeated Pleistocene glaciations. Our results provide a structured comparative assessment of divergence time estimates and evolutionary relationships in a well-studied group of butterflies, and support better understanding of analytical biases in divergence time estimation.

The utility of a universal DNA ‘barcode’ fragment of 658 base pairs of the Cytochrome C Oxidase I (COI) gene for the recognition of all animal species has been a widely debated topic on theoretical and practical levels. Regardless of its challenges, large amounts of COI sequence data have been produced in the last two decades. To optimally use the data towards reliable species identification will require further steps to validate the method and reference libraries. The fruit fly tribe Dacini holds about a thousand species, of which eighty are pests of economic concern, including some of the world’s foremost fruit and vegetable pests, and there are many morphologically cryptic species complexes in the tribe. Where previous studies showed limited success in using COI to identify Dacini, our results with a highly curated morphological dataset indicate high congruence between morphology and COI: 90% of the species in our 5,576 sequences, 262-species global dataset can be identified with COI alone based on a monophyly criterion. However, in some key pest species belonging to complexes that were previously thought diagnosable with COI, we found that expanded sampling and independent validation of identifications using genomic data revealed introgression of mitochondrial DNA. We find that the informative SNPs are uniformly distributed across the COI gene, and we provide recommendations for standardization. We conclude that reliable molecular identifications with COI require extensive species coverage, population sampling, and genomics-supported reference identifications before they can be validated as a “diagnostic” marker for specific groups.

Population genomics is a useful tool to support integrated pest management as it can elucidate population dynamics, demography, and histories of invasion. Here, we use a restriction site‐associated DNA sequencing approach combined with whole‐genome amplification (WGA) to assess genomic population structure of a newly described pest of canola, the diminutive canola flower midge, Contarinia brassicola. Clustering analyses recovered little geographic structure across the main canola production region but differentiated several geographically disparate populations at edges of the agricultural zone. Given a lack of alternative hypotheses for this pattern, we suggest these data support alternative hosts for this species and thus our canola‐centric view of this midge as a pest has limited our understanding of its biology. These results speak to the need for increased surveying efforts across multiple habitats and other potential hosts within Brassicaceae to improve both our ecological and evolutionary knowledge of this species and contribute to effective management strategies. We additionally found that use of WGA prior to library preparation was an effective method for increasing DNA quantity of these small insects prior to restriction site‐associated DNA sequencing and had no discernible impact on genotyping consistency for population genetic analysis; WGA is therefore likely to be tractable for other similar studies that seek to randomly sample markers across the genome in small organisms., The canola flower midge is a newly discovered pest of canola from the Canadian Prairies. The population genetic analyses presented in this study recovered little population structure across its known range, consistent with high levels of gene flow in this region. However, a few geographically disparate populations were genetically distinct, and we suggest that these results reflect bleed‐over genetic structure from an unsampled and unknown alternative host range.

This is the peer reviewed version of the following article: Cullingham, C.I., Miller, J.M., Peery, R.M., Dupuis, J.R., Malenfant, R.M., Gorrell, J.C., & Janes, J.K. (2020). Confidently identifying the correct K value using the ΔK method: When does K=2? Molecular Ecology, 29(5), 862-869, which has been published in final form at http://doi.org/10.1111/mec.15374. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions., Populations delineated based on genetic data are commonly used for wildlife conservation and management. Many studies use the program structure combined with the ΔK method to identify the most probable number of populations (K). We recently found K = 2 was identified more often when studies used ΔK compared to studies that did not. We suggested two reasons for this: hierarchical population structure leads to underestimation, or the ΔK method does not evaluate K = 1 causing an overestimation. The present contribution aims to develop a better understanding of the limits of the method using one, two and three population simulations across migration scenarios. From these simulations we identified the “best K” using model likelihood and ΔK. Our findings show that mean probability plots and ΔK are unable to resolve the correct number of populations once migration rate exceeds 0.005. We also found a strong bias towards selecting K = 2 using the ΔK method. We used these data to identify the range of values where the ΔK statistic identifies a value of K that is not well supported. Finally, using the simulations and a review of empirical data, we found that the magnitude of ΔK corresponds to the level of divergence between populations. Based on our findings, we suggest researchers should use the ΔK method cautiously; they need to report all relevant data, including the magnitude of ΔK, and an estimate of connectivity for the research community to assess whether meaningful genetic structure exists within the context of management and conservation.

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.

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.

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.

Population genetics is a powerful tool for invasion biology and pest management, and useful for a range of questions from tracing invasion pathways to informing management decisions with inference of population demographics. Genomics greatly increases the resolution of population-scale analyses, yet outside of model species with extensive genomic resources, few studies have used population genomics in invasion biology. We use genome-wide single nucleotide polymorphisms (SNPs) to investigate population genomic structure with samples from across the range of melon fly, Bactrocera cucurbitae (Coquillett, 1849), a highly polyphagous pest of commercial produce. We then make use of a chromosome-scale genome assembly and gene set to compare signatures of selection across the melon fly’s genome, both across our sampling as a whole and in the context of two independent, established introductions. Using multiple approaches, we find support for six genetic clusters across melon fly’s distribution. Some of these agree with previously identified genetic clusters using microsatellites, but consensus of clusters in mainland and oceanic southeast Asia is confounded by variable sampling between studies. We find few adaptive signatures across the genome, and virtually no unique signatures when comparing the two independent introductions, which suggests that similar management strategies are appropriate across melon fly’s range. This is the first use of genome-wide data to characterize population structure in tephritid fruit fly pests, and our SNP dataset provides a foundation for objective and cost-effective genotyping of previously collected melon fly specimens. Future research needs to focus on truly comprehensive sampling across melon fly’s range to overcome the historic variability of range-wide estimates of population structure for this pest.

Characterizing genetic structure across geographic space is a fundamental challenge in population genetics. Multivariate statistical analyses are powerful tools for summarizing genetic variability, but geographic information and accompanying metadata are not always easily integrated into these methods in a user-friendly fashion. Here, we present a deployable Python-based web-tool, mvmapper, for visualizing and exploring results of multivariate analyses in geographic space. This tool can be used to map results of virtually any multivariate analysis of georeferenced data, and routines for exporting results from a number of standard methods have been integrated in the R package adegenet, including principal components analysis (PCA), spatial PCA, discriminant analysis of principal components, principal coordinates analysis, nonmetric dimensional scaling and correspondence analysis. mvmapper's greatest strength is facilitating dynamic and interactive exploration of the statistical and geographic frameworks side by side, a task that is difficult and time-consuming with currently available tools. Source code and deployment instructions, as well as a link to a hosted instance of mvmapper, can be found at https://popphylotools.github.io/mvMapper/.

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.

Assessments of population genetic structure have become an increasing focus as they can provide valuable insight into patterns of migration and gene flow. structure, the most highly cited of several clustering-based methods, was developed to provide robust estimates without the need for populations to be determined a priori. structure introduces the problem of selecting the optimal number of clusters, and as a result, the ΔK method was proposed to assist in the identification of the "true" number of clusters. In our review of 1,264 studies using structure to explore population subdivision, studies that used ΔK were more likely to identify K = 2 (54%, 443/822) than studies that did not use ΔK (21%, 82/386). A troubling finding was that very few studies performed the hierarchical analysis recommended by the authors of both ΔK and structure to fully explore population subdivision. Furthermore, extensions of earlier simulations indicate that, with a representative number of markers, ΔK frequently identifies K = 2 as the top level of hierarchical structure, even when more subpopulations are present. This review suggests that many studies may have been over- or underestimating population genetic structure; both scenarios have serious consequences, particularly with respect to conservation and management. We recommend publication standards for population structure results so that readers can assess the implications of the results given their own understanding of the species biology.

International audience; Compared to traditionally used plastid or ribosomal markers, nuclear gene markers provide many advantages for molecular systematics of plants, and high-throughput sequencing technologies are making large nuclear datasets available at an unprecedented rate. We used targeted amplicon sequencing of 44 nuclear genes to construct a time-calibrated phylogeny of genus Metrosideros (Myrtaceae), evaluate recent systematic revisions, and assess whether phylogenetic signal within the Hawaiian Archipelago is correlated with island biogeography or morphological diversification. We generated a final dataset of 40 nuclear genes for 187 specimens, used multiple search heuristics and species-tree analysis to estimate a phylogeny, and incorporated new fossils for the genus to estimate divergence times across the dataset. All analyses supported the monophyly of Metrosideros, including Carpolepis and Tepualia. Hawaiian Metrosideros were monophyletic and dated to 3.1 MYA using new fossils for the genus, which is intermediate to previous estimates based on nuclear ribosomal/chloroplast loci and calibrated with island ages. Within the Hawaiian Metrosideros clade, we observed short branch lengths and unresolved relationships, and phylogenetic patterns were not concordant with biogeographic hypotheses of island progression, or the delineation of taxa or morphotypes. Average nucleotide diversity was relatively consistent across the Hawaiian Islands with the exception of slightly lower diversity on Kaua'i. These results provide a data-rich estimate of the timing of a single introduction of Metrosideros to Hawai'i and highlight the need for molecular markers with higher evolutionary rates for resolution of relationships within this recent radiation.

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.

Local adaptation can be a fundamental component of speciation, but its dynamics in relation to gene flow are not necessarily straightforward. Herbivorous taxa with localized host plant or habitat specialization across their geographic range are ideal models for investigating the patterns and constraints of local adaptation and its impact on diversification. The charismatic, day-flying moths of the Hemileuca maia species complex (Lepidoptera: Saturniidae) are such taxa, as they are geographically widespread, exhibit considerable ecological and morphological variability and host and habitat specificity, but apparently lack genetic differentiation across their range. Here, we use genomewide single nucleotide polymorphisms to assess relationships and population structure of this group across North America and investigate the scales where genomic divergence correlates with adaptive ecological characteristics. In contrast to previous genetic studies of the group, we find broad- and fine-scale genetic differentiation between lineages, which is at odds with various levels of taxonomic description and recognition of conservation units. Furthermore, ecological specialization only explains some fine-scale genetic differentiation, and across much of the group's range, local adaptation is apparently occurring in the face of strong gene flow. These results provide unprecedented insight into drivers of speciation in this group, the relationship between taxonomy and genomics-informed species boundaries and conservation management of internationally protected entities. Broadly, this system provides a model for understanding how local adaptation in an herbivore can arise and be maintained in the face of apparently strong gene flow, and the importance of geographic isolation in generating genomic divergence, despite a lack of ecological divergence.

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.

New World screwworm (NWS), Cochliomyia hominivorax (Coquerel 1858) (Diptera: Calliphoridae), is a myiasis-causing fly that can be a serious threat to the health of livestock, wildlife, and humans. Its progressive eradication from the southern United States, Mexico, and Central America from the 1950s to 2000s is an excellent example of successful pest management using sterile insect technique (SIT). In late 2016, autochthonous NWS were detected in the Florida Keys, representing this species' first invasion in the United States in >30 yr. Rapid use of quarantine and SIT was successful in eliminating the infestation by early 2017; however, the geographic source of this infestation remains unknown. Here, we use amplicon sequencing to generate mitochondrial and nuclear sequence data representing all confirmed cases of NWS from this infestation, and compare these sequences to preexisting data sets sampling the native distribution of NWS. We ask two questions regarding the FL Keys outbreak. First, is this infestation the result of a single invasion from one source, or multiple invasions from different sources? And second, what is the geographic origin of this invasion? We found virtually no sequence variation between specimens collected from the FL Keys outbreak, which is consistent with a single source of introduction. However, we also found very little geographic resolution in any of the data sets, which precludes identification of the source of this outbreak. Our lack of success in answering our second question speaks to the need for finer-scale genetic or genomic assessments of NWS population structure, which would facilitate source determination of potential future outbreaks.

High-throughput sequencing has fundamentally changed how molecular phylogenetic data sets are assembled, and phylogenomic data sets commonly contain 50- to 100-fold more loci than those generated using traditional Sanger sequencing-based approaches. Here, we demonstrate a new approach for building phylogenomic data sets using single-tube, highly multiplexed amplicon sequencing, which we name HiMAP (highly multiplexed amplicon-based phylogenomics) and present bioinformatic pipelines for locus selection based on genomic and transcriptomic data resources and postsequencing consensus calling and alignment. This method is inexpensive and amenable to sequencing a large number (hundreds) of taxa simultaneously and requires minimal hands-on time at the bench (1/2 day), and data analysis can be accomplished without the need for read mapping or assembly. We demonstrate this approach by sequencing 878 amplicons in single reactions for 82 species of tephritid fruit flies across seven genera (384 individuals), including some of the most economically important agricultural insect pests. The resulting filtered data set (150,000-bp concatenated alignment, ~20% missing character sites across all individuals and amplicons) contained40,000 phylogenetically informative characters, and although some discordance was observed between analyses, it provided unparalleled resolution of many phylogenetic relationships in this group. Most notably, we found high support for the generic status of Zeugodacus and the sister relationship between Dacus and Zeugodacus. We discuss HiMAP, with regard to its molecular and bioinformatic strengths, and the insight the resulting data set provides into relationships of this diverse insect group.

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.

High-throughput sequencing has fundamentally changed how molecular phylogenetic datasets are assembled, and phylogenomic datasets commonly contain 50-100-fold more loci than those generated using traditional Sanger-based approaches. Here, we demonstrate a new approach for building phylogenomic datasets using single tube, highly multiplexed amplicon sequencing, which we name HiMAP (Highly Multiplexed Amplicon-based Phylogenomics), and present bioinformatic pipelines for locus selection based on genomic and transcriptomic data resources and post-sequencing consensus calling and alignment. This method is inexpensive and amenable to sequencing a large number (hundreds) of taxa simultaneously, requires minimal hands-on time at the bench (150,000 bp concatenated alignment) contained >40,000 phylogenetically informative characters, and although some discordance was observed between analyses, it provided unparalleled resolution of many phylogenetic relationships in this group. Most notably, we found high support for the generic status ofZeugodacusand the sister relationship betweenDacusandZeugodacus. We discuss HiMAP, with regard to its molecular and bioinformatic strengths, and the insight the resulting dataset provides into relationships of this diverse insect group.

High throughput sequencing technologies have revolutionized the potential to reconcile incongruence between gene and species trees, and numerous approaches have been developed to take advantage of these advances. Genotyping-by-sequencing is becoming a regular tool for gathering phylogenetic data, yet comprehensive evaluations of phylogenetic methods using these data are sparse. Here we use multiple phylogenetic and population genetic methods for genotyping-by-sequencing data to assess species relationships in a group of forest insect pests, the spruce budworm (Choristoneura fumiferana) species complex. With few exceptions, all methods agree on the same relationships, most notably placing C. pinus as basal to the remainder of the group, rather than C. fumiferana as previously suggested. We found strong support for the monophyly of C. pinus, C. fumiferana, and C. retiniana, but more ambiguous relationships and signatures of introgression in a clade of western lineages, including C. carnana, C. lambertiana, C. occidentalis occidentalis, C. occidentalis biennis, and C. orae. This represents the most taxonomically comprehensive genomic treatment of the spruce budworm species group, which is further supported by the broad agreement among multiple methodologies.

Diapause that extends across multiple years is considered a bet-hedging strategy in insect species that are exposed to unfavorable environmental conditions. The dynamics of extended diapause in herbivores can be complicated when their parasitoids can also extend diapause, although our knowledge of these dynamics is based primarily on pest species. Here, we report on regional variation in the phenology and life history of Trogus parasitoids of Papilio machaon group swallowtail butterflies in western Canada. We observed flexible phenological patterns in this interaction, with both hosts and parasitoids undergoing extended diapause. Additionally, we documented parasitism of earlier instar larvae in these Trogus species, which to our knowledge has not been observed in North America. Our observations provide a foundation for studying the potential evolutionary consequences of extended diapause in these multispecies interactions.

Hybrid zones provide unique natural laboratories for studying mechanisms of evolution. But identification and classification of hybrid individuals (F1, F2, backcross, etc.) can be complicated by real population changes over time as well as by use of different marker types, both of which challenge documentation of hybrid dynamics. Here, we use multiple genetic markers (mitochondrial DNA, microsatellites and genomewide single nucleotide polymorphisms) to re-examine population structure in a hybrid zone between two species of swallowtail butterflies in western Canada, Papilio machaon and P. zelicaon. Our aim was to test whether their hybrid dynamics remain the same as found 30 years ago using morphology and allozymes, and we compared different genetic data sets as well as alternative hybrid identification and classification methods. Overall, we found high differentiation between the two parental species, corroborating previous research from the 1980s. We identified fewer hybrid individuals in the main zone of hybridization in recent years, but this finding depended on the genetic markers considered. Comparison of methods with simulated data sets generated from our data showed that single nucleotide polymorphisms were more powerful than microsatellites for both hybrid identification and classification. Moreover, substantial variation among comparisons underlined the value of multiple markers and methods for documenting evolutionarily dynamic systems.

Despite taxonomy’s 250-year history, the past 20 years have borne witness to remarkable advances in technology and techniques, as well as debate. DNA barcoding has generated a substantial proportion of this debate, with its proposition that a single mitochondrial sequence will consistently identify and delimit species, replacing more evidence-rich and time-intensive methods. Although mitochondrial DNA (mtDNA) has since been the focus of voluminous discussion and case studies, little effort has been made to comprehensively evaluate its success in delimiting closely related species. We have conducted the first broadly comparative literature review addressing the efficacy of molecular markers for delimiting such species over a broad taxonomic range. By considering only closely related species, we sought to avoid confusion of success rates with those due to deeply divergent taxa. We also address whether increased population-level or geographic sampling affects delimitation success. Based on the results from 101 studies, we found that all marker groups had approximately equal success rates (∼70%) in delimiting closely related species and that the use of additional loci increased average delimitation success. We also found no relationship between increased sampling of intraspecific variability and delimitation success. Ultimately, our results support a multi-locus integrative approach to species delimitation and taxonomy.

Background The Mormon Metalmark (Apodemia mormo) species complex occurs as isolated and phenotypically variable colonies in dryland areas across western North America. Lange’s Metalmark, A. m. langei, one of the 17 subspecies taxonomically recognized in the complex, is federally listed under the U.S. Endangered Species Act of 1973. Metalmark taxa have traditionally been described based on phenotypic and ecological characteristics, and it is unknown how well this nomenclature reflects their genetic and evolutionary distinctiveness. Genetic variation in six microsatellite loci and mitochondrial cytochrome oxidase subunit I sequence was used to assess the population structure of the A. mormo species complex across 69 localities, and to evaluate A. m. langei’s qualifications as an Evolutionarily Significant Unit. Results We discovered substantial genetic divergence within the species complex, especially across the Continental Divide, with population genetic structure corresponding more closely with geographic proximity and local isolation than with taxonomic divisions originally based on wing color and pattern characters. Lange’s Metalmark was as genetically divergent as several other locally isolated populations in California, and even the unique phenotype that warranted subspecific and conservation status is reminiscent of the morphological variation found in some other populations. Conclusions This study is the first genetic treatment of the A. mormo complex across western North America and potentially provides a foundation for reassessing the taxonomy of the group. Furthermore, these results illustrate the utility of molecular markers to aid in demarcation of biological units below the species level. From a conservation point of view, Apodemia mormo langei’s diagnostic taxonomic characteristics may, by themselves, not support its evolutionary significance, which has implications for its formal listing as an Endangered Species. Electronic supplementary material The online version of this article (doi:10.1186/s12862-015-0354-9) contains supplementary material, which is available to authorized users.

Despite taxonomy's 250-year history, the past 20 years have borne witness to remarkable advances in technology and techniques, as well as debate. DNA barcoding has generated a substantial proportion of this debate, with its proposition that a single mitochondrial sequence will consistently identify and delimit species, replacing more evidence-rich and time-intensive methods. Although mitochondrial DNA (mtDNA) has since been the focus of voluminous discussion and case studies, little effort has been made to comprehensively evaluate its success in delimiting closely related species. We have conducted the first broadly comparative literature review addressing the efficacy of molecular markers for delimiting such species over a broad taxonomic range. By considering only closely related species, we sought to avoid confusion of success rates with those due to deeply divergent taxa. We also address whether increased population-level or geographic sampling affects delimitation success. Based on the results from 101 studies, we found that all marker groups had approximately equal success rates (~70%) in delimiting closely related species and that the use of additional loci increased average delimitation success. We also found no relationship between increased sampling of intraspecific variability and delimitation success. Ultimately, our results support a multi-locus integrative approach to species delimitation and taxonomy.

Hybridization between distinct populations or species is increasingly recognized as an important process for generating biodiversity. However, the interaction between hybridization and speciation is complex, and the diverse evolutionary outcomes of hybridization are difficult to differentiate. Here we characterize potential hybridization in a species group of swallowtail butterflies using microsatellites, DNA sequences, and morphology, and assess whether adaptive introgression or homoploid hybrid speciation was the primary process leading to each putative hybrid lineage. Four geographically separated hybrid populations were identified in the Papilio machaon species group. One distinct mitochondrial DNA clade from P. machaon was fixed in three hybrid taxa (P. brevicauda, P. joanae, and P. m. kahli), while one hybrid swarm (P. zelicaon x machaon) exhibited this hybrid mtDNA clade as well as widespread parental mtDNA haplotypes from both parental species. Microsatellite markers and morphology showed variable admixture and intermediacy, ranging from signatures of prolonged differential introgression from the paternal species (P. polyxenes/P. zelicaon) to current gene flow with both parental species. Divergences of the hybrid lineages dated to early- to mid-Pleistocene, suggesting that repeated glaciations and subsequent range shifts of parental species, particularly P. machaon hudsonianus, facilitated initial hybridization. Although each lineage is distinct, P. joanae is the only taxon with sufficient evidence (ecological separation from parental species) to define it as a homoploid hybrid species. The repetition of hybridization in this group provides a valuable foundation for future research on hybridization, and these results emphasize the potential for hybridization to drive speciation in diverse ways.