Lisa M. Lumley

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.

Utility of microsatellites and mitochondrial DNA for species delimitation in the spruce budworm (Choristoneura fumiferana) species complex (Lepidoptera: Tortricidae)

Species identifications have been historically difficult in the Choristoneura fumiferana group, an important insect pest complex. We examined the utility of simple sequence repeats (SSRs, also referred to as microsatellites) and mitochondrial DNA (mtDNA) for delimiting and identifying eight currently recognized species sampled across North America. Four of these species formed discrete clusters using SSRs, while only two species were delimited with mtDNA. There was evidence for hybridization or incomplete lineage sorting between several species pairs. An integrative approach, using both phenotypic traits and molecular markers, allowed for the discrimination of more biologically relevant species units than did the use of molecular markers alone. As species are currently identified using putatively adaptive phenotypic traits, the differences observed between recognized species and neutral SSRs or mtDNA suggests that these species (or evolutionary significant units) have diverged via natural selection in spite of some gene flow.

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.

Life-history traits maintain the genomic integrity of sympatric species of the spruce budworm (Choristoneura fumiferana) group on an isolated forest island.

Identification of widespread species collected from islands can be challenging due to the potential for local ecological and phenotypic divergence in isolated populations. We sought to determine how many species of the spruce budworm (Choristoneura fumiferana) complex reside in Cypress Hills, an isolated remnant coniferous forest in western Canada. We integrated data on behavior, ecology, morphology, mitochondrial DNA, and simple sequence repeats, comparing Cypress Hills populations to those from other regions of North America to determine which species they resembled most. We identified C. fumiferana, C. occidentalis, C. lambertiana, and hybrid forms in Cypress Hills. Adult flight phenology and pheromone attraction were identified as key life-history traits involved in maintaining the genomic integrity of species. Our study highlights the importance of extensive sampling of both specimens and a variety of characters for understanding species boundaries in biodiversity research.

Two's company, three's a crowd: new insights on spruce budworm species boundaries using genotyping-by-sequencing in an integrative species assessment (Lepidoptera: Tortricidae)

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

Linking adaptation, delimitation of evolutionarily significant units (ESUs), and gene function: a case study using hemlock looper ecotypes

Developing genetic markers for the identification of recently diverged groups, such as ecotypes or species complexes, remains difficult due to challenges with incomplete lineage sorting, hybridization and introgression. Genome‐wide scans of single nucleotide polymorphisms (SNPs) have proven useful for inferring patterns of genetic differentiation at the population level. In combination with a new analytical technique, the discriminant analysis of principal components (DAPC), and within the framework of iterative taxonomy, it may also be possible to extract a combination of SNPs as markers for the delimitation of closely related groups. In addition, since DAPC identifies the loci contributing the most to group clustering, it may be possible to link putative biological function to differences that define group boundaries. We tested this technique on two ecotypes of the hemlock looper (Lambdina fiscellaria), which differ in terms of number of larval stadia, developmental rate and fecundity. It was possible to separately cluster the two ecotypes with 95% correct assignment using 27 SNPs. We also determined that a storage hexamerin carried eight of these SNPs, including the two highest contributing loci, of which the top contributor was nonsynonymous. Other studies have found this protein to be highly expressed just before metamorphosis, pointing to a possible connection between its role in clustering ecotypes and its biological function. These SNP markers can now be further developed for high throughput delimitation of individuals of unknown ecotype identity.

Life‐stage differences in spatial genetic structure in an irruptive forest insect: implications for dispersal and spatial synchrony

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

Insights into the Structure of the Spruce Budworm (Choristoneura fumiferana) Genome, as Revealed by Molecular Cytogenetic Analyses and a High-Density Linkage Map

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

Distinct sources of gene flow produce contrasting population genetic dynamics at different range boundaries of a Choristoneura budworm

Populations are often exposed to multiple sources of gene flow, but accounts are lacking of the population genetic dynamics that result from these interactions or their effects on local evolution. Using a genomic clines framework applied to 1,195 single nucleotide polymorphisms, we documented genomewide, locus‐specific patterns of introgression between Choristoneura occidentalis biennis spruce budworms and two ecologically divergent relatives, C. o. occidentalis and Choristoneura fumiferana, that it interacts with at alternate boundaries of its range. We observe contrasting hybrid indexes between the two hybrid zones, no overlap in “gene‐flow outliers” (clines showing relatively extreme extents or rates of locus‐specific introgression) and variable linkage disequilibrium among those outliers. At the same time, correlated genomewide rates of introgression between zones suggest the presence of processes common to both boundaries. These findings highlight the contrasting population genetic dynamics that can occur at separate frontiers of a single population, while also suggesting that shared patterns may frequently accompany cases of divergence‐with‐gene‐flow that involve a lineage in common. Our results point to potentially complex evolutionary outcomes for populations experiencing multiple sources of gene flow.