Julian R. Dupuis

Multi-locus species delimitation in closely related animals and fungi: one marker is not enough

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.

The K=2 conundrum

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.

Genome-wide SNPs resolve phylogenetic relationships in the North American spruce budworm (Choristoneura fumiferana) species complex

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.

Repeated Reticulate Evolution in North American Papilio machaon Group Swallowtail Butterflies

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.

Genetic evaluation of the evolutionary distinctness of a federally endangered butterfly, Lange’s Metalmark

BackgroundThe 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.ResultsWe 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.ConclusionsThis 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.

Hybrid dynamics in a species group of swallowtail butterflies.

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.

HiMAP: Robust phylogenomics from highly multiplexed amplicon sequencing

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) 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 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.

Cross‐platform compatibility of de novo‐aligned SNPs in a nonmodel butterfly genus

High‐throughput sequencing methods for genotyping genome‐wide markers are being rapidly adopted for phylogenetics of nonmodel 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 nonmodel 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 nonmodel systems.

Mitochondrial phylogenomics, the origin of swallowtail butterflies, and the impact of the number of clocks in Bayesian molecular dating

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.

mvmapper: Interactive spatial mapping of genetic structures

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. mvmappers 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/.