Felix A. H. Sperling

Synergistic effects of combining morphological and molecular data in resolving the phylogeny of butterflies and skippers

Phylogenetic relationships among major clades of butterflies and skippers have long been controversial, with no general consensus even today. Such lack of resolution is a substantial impediment to using the otherwise well studied butterflies as a model group in biology. Here we report the results of a combined analysis of DNA sequences from three genes and a morphological data matrix for 57 taxa (3258 characters, 1290 parsimony informative) representing all major lineages from the three putative butterfly super-families (Hedyloidea, Hesperioidea and Papilionoidea), plus out-groups representing other ditrysian Lepidoptera families. Recently, the utility of morphological data as a source of phylogenetic evidence has been debated. We present the first well supported phylogenetic hypothesis for the butterflies and skippers based on a total-evidence analysis of both traditional morphological characters and new molecular characters from three gene regions (COI, EF-1α and wingless). All four data partitions show substantial hidden support for the deeper nodes, which emerges only in a combined analysis in which the addition of morphological data plays a crucial role. With the exception of Nymphalidae, the traditionally recognized families are found to be strongly supported monophyletic clades with the following relationships: (Hesperiidae+(Papilionidae+(Pieridae+(Nymphalidae+(Lycaenidae+Riodinidae))))). Nymphalidae is recovered as a monophyletic clade but this clade does not have strong support. Lycaenidae and Riodinidae are sister groups with strong support and we suggest that the latter be given family rank. The position of Pieridae as the sister taxon to nymphalids, lycaenids and riodinids is supported by morphology and the EF-1α data but conflicted by the COI and wingless data. Hedylidae are more likely to be related to butterflies and skippers than geometrid moths and appear to be the sister group to Papilionoidea+Hesperioidea.

DNA-based identification of forensically important Chrysomyinae (Diptera: Calliphoridae)

Identifying an insect specimen is an important first step in a forensic-entomological analysis. However, diagnostic morphological criteria are lacking for many species and life stages. We demonstrate a method for using mitochondrial DNA sequence data and phylogenetic analysis to identify any specimen of the blow fly subfamily Chrysomyinae likely to be collected from a human corpse within Canada or the USA. The reliability of the method was illustrated by analyzing specimens designed to mimic the information likely to be obtained from highly degraded specimens as well as specimens collected from widely separated geographic locations. Our sequence database may be suitable for another country provided the investigator knows the local fly fauna well enough to narrow the choice of chrysomyine species to those used in this study.

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.

Draft genome of the mountain pine beetle, Dendroctonus ponderosae Hopkins, a major forest pest

BackgroundThe mountain pine beetle, Dendroctonus ponderosae Hopkins, is the most serious insect pest of western North American pine forests. A recent outbreak destroyed more than 15 million hectares of pine forests, with major environmental effects on forest health, and economic effects on the forest industry. The outbreak has in part been driven by climate change, and will contribute to increased carbon emissions through decaying forests.ResultsWe developed a genome sequence resource for the mountain pine beetle to better understand the unique aspects of this insects biology. A draft de novo genome sequence was assembled from paired-end, short-read sequences from an individual field-collected male pupa, and scaffolded using mate-paired, short-read genomic sequences from pooled field-collected pupae, paired-end short-insert whole-transcriptome shotgun sequencing reads of mRNA from adult beetle tissues, and paired-end Sanger EST sequences from various life stages. We describe the cytochrome P450, glutathione S-transferase, and plant cell wall-degrading enzyme gene families important to the survival of the mountain pine beetle in its harsh and nutrient-poor host environment, and examine genome-wide single-nucleotide polymorphism variation. A horizontally transferred bacterial sucrose-6-phosphate hydrolase was evident in the genome, and its tissue-specific transcription suggests a functional role for this beetle.ConclusionsDespite Coleoptera being the largest insect order with over 400,000 described species, including many agricultural and forest pest species, this is only the second genome sequence reported in Coleoptera, and will provide an important resource for the Curculionoidea and other insects.

DNA-based identification and molecular systematics of forensically important Sarcophagidae (Diptera).

Sarcophagid flies have many characteristics that make them ideal forensic indicators. However, their utility is severely limited because it is difficult or impossible to determine the species of a sarcophagid larva, and in many instances an adult specimen, based on anatomy. We developed a database of mitochondrial DNA sequence data that makes it possible to identify all sarcophagid species likely to be found feeding on a human corpse at an urban location in Canada or the USA. Analyses were based on a 783 base pair region of the gene for cytochrome oxidase subunit one (COI). The species analyzed, including some of no forensic importance that were included for purposes of phylogenetic comparisons, were members of the genera Sarcophaga, Peckia, Blaesoxipha, Rovinia, Wohlfahrtia, Brachicoma (all Sarcophagidae), and Musca (Muscidae).

Population structure and species boundary delimitation of cryptic Dioryctria moths: an integrative approach

Accurate delimitation of species boundaries is especially important in cryptic taxa where one or more character sources are uninformative or are in conflict. Rather than relying on a single marker to delimit species, integrative taxonomy uses multiple lines of evidence such as molecular, morphological, behavioural and geographic characters to test species limits. We examine the effectiveness of this approach by testing the delimitation of two cryptic Nearctic species of Dioryctria (Lepidoptera: Pyralidae) using three independent molecular markers [cytochrome c oxidase I (COI), second internal transcribed spacer unit (ITS2), and elongation factor 1α (EF1α)], forewing variation and larval host plant association. Although mitochondrial DNA (mtDNA) haplotypes do not form reciprocally monophyletic clades, restricted gene flow between COI haplotype groups, and concordance with ITS2 genotypes, forewing variation and host plant associations support delimitation of two Nearctic species: eastern Dioryctria reniculelloides and western Dioryctria pseudotsugella. Conversely, EF1α genotype variation was incongruent with the two previous markers. A case of discordance between COI and ITS2 was detected, suggesting either introgression due to hybridization or retained ancestral polymorphism due to incomplete coalescence. This study is consistent with other similar literature where molecular loci in closely related species progress from shared to fixed haplotypes/alleles, and from polyphyletic to reciprocally monophyletic relationships, although loci may vary in these characteristics despite maintenance of genomic integrity between distinct species. In particular, mtDNA in other studies generally showed a lower rate of fixation of differences than did X‐linked or autosomal loci, reinforcing the need to use an integrative approach for delimiting species.

Multilocus species identification and fungal DNA barcoding: insights from blue stain fungal symbionts of the mountain pine beetle

There is strong community‐wide interest in applying molecular techniques to fungal species delimitation and identification, but selection of a standardized region or regions of the genome has not been finalized. A single marker, the ribosomal DNA internal transcribed spacer region, has frequently been suggested as the standard for fungi. We used a group of closely related blue stain fungi associated with the mountain pine beetle (Dendroctonus ponderosae Hopkins) to examine the success of such single‐locus species identification, comparing the internal transcribed spacer with four other nuclear markers. We demonstrate that single loci varied in their utility for identifying the six fungal species examined, while use of multiple loci was consistently successful. In a literature survey of 21 similar studies, individual loci were also highly variable in their ability to provide consistent species identifications and were less successful than multilocus diagnostics. Accurate species identification is the essence of any molecular diagnostic system, and this consideration should be central to locus selection. Moreover, our study and the literature survey demonstrate the value of using closely related species as the proving ground for developing a molecular identification system. We advocate use of a multilocus barcode approach that is similar to the practice employed by the plant barcode community, rather than reliance on a single locus.

Integrating morphology and mitochondrial DNA for species delimitation within the spruce budworm (Choristoneura fumiferana) cryptic species complex (Lepidoptera: Tortricidae)

Species in cryptic complexes tend to be very difficult, if not impossible, to identify using morphological characters. One such complex is the spruce budworm (Choristoneura fumiferana Clemens, 1865) species group, an economically important group of Nearctic forest pests. Morphological, ecological, behavioural and genetic characters have been studied to try to understand the taxonomy of this group better, but diagnostic character states differ in frequency rather than being complete replacements between each species. We used mitochondrial DNA (mtDNA), together with a new morphology‐based character system that focuses on forewing colour components, to determine if one or a combination of character sources can be used for species diagnoses within the spruce budworm complex. We characterized 47 forewing morphometric measurements and sequenced a 470 bp region of cytochrome c oxidase I mtDNA for 111 ingroup individuals comprising five taxa within the complex. Larval host association and coloration or adult pheromone attraction were used as the prior method for grouping individuals. Our results showed that linear discriminant analysis of morphometric wing characters gave unique clusters for all species on the first and second canonical axes, except for a partial overlap between C. fumiferana and C. biennis, which are not sympatric in nature. In contrast, mtDNA distinguished C. fumiferana, C. pinus pinus Freeman, 1953 and a group of western species, but the three western species (C. occidentalis Freeman, 1967 , C. biennis Freeman, 1967 and C. lambertiana Busck, 1915) shared mtDNA haplotypes. On the basis of the linear discriminant analysis of the combined character set, this study supports the application of both morphology and mtDNA within a framework of integrative taxonomy as the most accurate method for species identification. Furthermore, it demonstrates the utility of quantitative colour analysis, which may be particularly helpful for groups in which colour characters are difficult to divide into discrete units due to intergrading hues.

Widespread decoupling of mtDNA variation and species integrity in Grammia tiger moths (Lepidoptera: Noctuidae)

Abstract.   We investigate the diversity of the North American tiger moth genus Grammia Rambur (Lepidoptera: Noctuidae) by comparing mitochondrial DNA (mtDNA) ‘barcode’ fragments of cytochrome oxidase I with non‐molecular characters such as morphology, ecology, behaviour and distribution. Mitochondrial DNA genealogy is strikingly at odds with morpho‐species taxonomy for most of the 28 sampled species, as haplotypic polyphyly not only is taxonomically widespread, but involves multiple shared haplotypes among two to four species. Morpho‐ecological traits show that those species sharing haplotypes are often not closely related. Furthermore, high mtDNA divergences occur within species. Haplotypic variation is highly discordant with species taxonomy, but variation at a continental scale reveals significant geographic structuring of haplogroups, transcending morpho‐species boundaries. A nested clade analysis and comparison of non‐molecular with mtDNA data indicate that most discordance between mtDNA and taxonomy in Grammia is explained best by taxonomically and geographically widespread ongoing hybridization events resulting in mtDNA introgression. We hypothesize that broad areas of sympatry, interspecifically compatible genitalic structure, and species overlap in pheromone components facilitate hybridization, with disparate interspecies abundances promoting mitochondrial introgression. The molecular evolution of Grammia challenges the view that interspecific gene exchange occurs rarely and is restricted to recently diverged species. These results show the value of mtDNA in detecting cryptic hybridization, while highlighting the inherent dangers of drawing taxonomic conclusions based solely on mtDNA.

Spatial Genetic Structure of a Symbiotic Beetle-Fungal System: Toward Multi-Taxa Integrated Landscape Genetics

Spatial patterns of genetic variation in interacting species can identify shared features that are important to gene flow and can elucidate co-evolutionary relationships. We assessed concordance in spatial genetic variation between the mountain pine beetle (Dendroctonus ponderosae) and one of its fungal symbionts, Grosmanniaclavigera, in western Canada using neutral genetic markers. We examined how spatial heterogeneity affects genetic variation within beetles and fungi and developed a novel integrated landscape genetics approach to assess reciprocal genetic influences between species using constrained ordination. We also compared landscape genetic models built using Euclidean distances based on allele frequencies to traditional pair-wise Fst. Both beetles and fungi exhibited moderate levels of genetic structure over the total study area, low levels of structure in the south, and more pronounced fungal structure in the north. Beetle genetic variation was associated with geographic location while that of the fungus was not. Pinevolume and climate explained beetle genetic variation in the northern region of recent outbreak expansion. Reciprocal genetic relationships were only detectedin the south where there has been alonger history of beetle infestations. The Euclidean distance and Fst-based analyses resulted in similar models in the north and over the entire study area, but differences between methods in the south suggest that genetic distances measures should be selected based on ecological and evolutionary contexts. The integrated landscape genetics framework we present is powerful, general, and can be applied to other systems to quantify the biotic and abiotic determinants of spatial genetic variation within and among taxa.