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.     

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.     

Mitochondrial DNA sequence variation in the spruce budworm species complex (Choristoneura: Lepidoptera)

A combination of polymerase-chain-reaction amplification and automated DNA sequencing was used to survey variation in a species complex of pest insects, the spruce budworms (Choristoneura fumiferana species group), and an outgroup species, C. rosaceana. We sequenced an mtDNA region of 1,573 bp that extends from the middle of cytochrome oxidase subunit I (COI) through tRNA leucine (UUR) to the end of cytochrome oxidase subunit II. In addition, we examined levels of intraspecific variation within a 470-bp region of the COI gene. Choristoneura fumiferana clearly represented the oldest lineage within its species group, with 2.7%-2.9% sequence divergence from the other species. In contrast, the four remaining species (C. pinus, C. biennis, C. occidentalis, and C. orae) had closely related or identical mtDNA, with < 1% divergence among most of their haplotypes. Despite its older lineage and widespread geographic distribution, C. fumiferana showed significantly lower intraspecific genetic diversity than did C. occidentalis. Choristoneura orae shared haplotypes with C. occidentalis and C. biennis, and species-level separation of these three species was not supported. Two divergent, uncommon haplotypes were also found in C. occidentalis and C. biennis. The divergent haplotype in C. biennis had an unusually high number of inferred amino acid replacements, suggesting selective differences between mitochondrial DNA haplotypes. Transition:transversion ratios in Choristoneura paralleled those found in Drosophila; transition:transversion ratios were highest in closely related sequences but decreased with increasing sequence divergence. Nucleotide composition showed an A+T bias that was near the high end of the range known for insects. This work illustrates the potential utility of direct DNA sequencing in assessing population structures, species limits, and phylogenetic relationships among organisms that have not previously been subjected to DNA analysis.      

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.     

Wing wear of adult Choristoneura fumiferana (Lepidoptera: Tortricidae) in relation to age, sex, sex ratio, and presence of host plant

Wing wear of adult butterflies has been used to record age-related demographic parameters in hundreds of studies, but this technique has surprisingly been rarely used in moths and never in the context of pest management. A method for scoring wing wear of eastern spruce budworm, Choristoneura fumiferana (Clemens), the most severe pest in eastern North American boreal forests, is proposed based on the proportion of forewings covered with scales. Studies conducted in the laboratory reveal a higher level of wing damage for males than females, for 4-day-old individuals than 2-day-old individuals, and for adults that are in contact with host plant material. Males provided with mating opportunities had a lower incidence of wing damage than males deprived of mating opportunity, whereas wing wear of females was independent of the presence or absence of males. In combination with other variables, wing wear of adult spruce budworms may help to identify and forecast migration events.     

Morphological variation versus genetic divergence: a taxonomic implication for Mogannia species (Cicadidae: Cicadinae)

Species showing intraspecific morphological variation tend to be very difficult to identify using morphological characters. One such example is the cicada genus Mogannia where some species show considerable intraspecific variation mainly exhibited by wing pattern and body colouration. Thirty-one variants covering different putative species of Mogannia were recognized and illustrated in the present paper. Molecular data of mitochondrial COI and Cytb sequences were employed to test the level of variation and phylogeny of them. The existence of a ‘barcoding gap’ between intraspecific and interspecific genetic divergences and the reciprocally monophyletic clades indicate that all the closely related variants represent a single species, and that all these variants correspond to six species, respectively. However, the evolutionary relationships of intraspecific variants are not resolved possibly due to insufficient genetic variation among them. Our results indicated that some morphological characters, especially the wing pattern and body colouration, and even the number of apical processes of the aedeagus in a couple of related species, must be used with great caution in delimiting Mogannia species and their relatives. The factors responsible for intraspecific morphological variation and phylogeny of Mogannia spp. are preliminarily discussed.     

Risk of dispersal in western spruce budworm

1 Western spruce budworm Choristoneura occidentalis Free. larvae emerge in the spring before buds have expanded and spend a variable period of time foraging on branches and mining needles.
2 Losses of dispersing budworms during this needle-mining period are related directly to the severity of defoliation in previous years and inversely related to foliage biomass in the study plot and to temperature and rainfall during the needle-mining period.
3 Losses can be interpreted in terms of risk of dispersal, which is the product of the propensity of early-stage budworms to disperse in search of resources and the consequences of this behaviour for survival under variable ecological conditions.
4 A comparison of the species-specific nature of risk of dispersal in three conifer-feeding budworm systems of North America may elucidate the common nature but variable features of their respective population dynamics.     

Methods for species delimitation in bumblebees (Hymenoptera,Apidae, Bombus): towards an integrative approach

Delimitation of closely related species is often hindered by the lack of discrete diagnostic morphological characters. This is exemplified in bumblebees (genus Bombus). There have been many attempts to clarify bumblebee taxonomy by using alternative features to discrete morphological characters such as wing shape, DNA, or eco‐chemical traits. Nevertheless each approach has its own limitations. Recent studies have used a multisource approach to gather different lines of speciation evidence in order to draw a strongly supported taxonomic hypothesis in bumblebees. Yet, the resulting taxonomic status is not independent of selected evidence and of consensus methodology (i.e. unanimous procedure, majority, different weighting of evidence). In this article, we compare taxonomic conclusions for a group of taxonomically doubtful species (the Bombus lapidarius‐group) obtained from the four commonly used lines of evidence for species delimitation in bumblebees (geometric morphometric of wing shape, genetic differentiation assessment, sequence‐based species delimitation methods and differentiation of cephalic labial gland secretions). We ultimately aim to assess the usefulness of these lines of evidence as components of an integrative decision framework to delimit bumblebee species. Our results show that analyses based on wing shape do not delineate any obvious cluster. In contrast, nuclear/mitochondrial, sequence‐based species delimitation methods, and analyses based on cephalic labial gland secretions are congruent with each other. This allows setting up an integrative decision framework to establish strongly supported species and subspecies status within bumblebees.     

Hybridization and transgressive exploration of colour pattern and wing morphology in Heliconius butterflies

Hybridization can generate novel phenotypes distinct from those of parental lineages, a phenomenon known as transgressive trait variation. Transgressive phenotypes might negatively or positively affect hybrid fitness, and increase available variation. Closely related species of Heliconius butterflies regularly produce hybrids in nature, and hybridization is thought to play a role in the diversification of novel wing colour patterns despite strong stabilizing selection due to interspecific mimicry. Here, we studied wing phenotypes in first‐ and second‐generation hybrids produced by controlled crosses between either two co‐mimetic species of Heliconius or between two nonmimetic species. We quantified wing size, shape and colour pattern variation and asked whether hybrids displayed transgressive wing phenotypes. Discrete traits underlain by major‐effect loci, such as the presence or absence of colour patches, generate novel phenotypes. For quantitative traits, such as wing shape or subtle colour pattern characters, hybrids only exceed the parental range in specific dimensions of the morphological space. Overall, our study addresses some of the challenges in defining and measuring phenotypic transgression for multivariate traits and our data suggest that the extent to which transgressive trait variation in hybrids contributes to phenotypic diversity depends on the complexity and the genetic architecture of the traits.     

Pattern analysis of eastern spruce budworm Choristoneura fumiferana dispersal

Dispersal has been proposed as an important mechanism in the broad‐scale synchronisation of insect outbreaks by linking spatially disjunct populations. Evidence suggests that dispersal is influenced by landscape structure, phenology, temperature, and air currents; however, the details remain unclear due to the difficulty of quantifying dispersal. In this study, we used data on the abundance and distribution of spruce budworm Choristoneura fumiferana larvae (potential dispersers) and adult male moths (dispersers) to make inference on the effects of air currents and host‐species abundance on dispersal. Hierarchical‐Bayesian and inverse modeling was used to explore 4 dispersal models: 1) isotropic dispersal; 2) directional‐dispersal; 3) directional‐and‐host‐species dispersal; and 4) host‐species dispersal. Despite their strong dependence on balsam fir Abies balsamea and spruce species Picea spp., the mapped basal area of these host species did not influence the pattern of dispersed moths. The model that best fit the data was the directional‐dispersal model, which showed that the prevailing dispersal direction was from the northwest (328°). We infer that the strong pattern of directional dispersal was due to a prevailing wind from the same direction. Our interpretation was corroborated by independent wind data during the period of active adult male budworm flight, particularly in the region with high larval abundance. Our results indicate that there was a relatively high probability of individuals flying at least 48 km with the wind where larvae abundance at source locations was also high. Such findings emphasize the importance of long‐distance dispersal on spatial distribution of adult male spruce budworms. Insight into the population‐level consequences of such dispersal patterns requires additional research.     

Molecular phylogeny and species recognition in the mealybug genus Ferrisia Fullaway (Hemiptera: Pseudococcidae)

Previous allozyme and DNA nucleotide sequence studies of the mealybug genus Ferrisia Fullaway (Hemiptera: Pseudococcidae), although limited, have suggested greater species diversity than is recognized by the current morphology‐based taxonomy. Here we analyse nucleotide sequence data from one mitochondrial (cytochrome oxidase I) and two nuclear (EF‐1α and 28S D₂D₃) genes and recover ten well‐supported groups that allow us to reassess the taxonomic utility of morphological characters used for species recognition. We report on previously used morphological characters for which states are highly variable within genetic groups and identify new characters (of the wax‐exuding cuticular ducts and pores) with taxonomically informative states. The widespread pest species F. virgata (Cockerell), commonly called the striped mealybug, should be diagnosed more narrowly. From samples identified as F. virgata, we recover six clades that we equate with species and that can be distinguished with the newly identified morphological characters. We determine that five of the ‘electrophoretic species' identified informally by the late Uzi Nur based on electrophoretic mobility of 20 enzymes correlate with four of our genetic groups. This matching of Nur's putative species with ours was possible only because some of Nur's slide‐mounted voucher specimens were deposited in a museum and thus available for morphological study. Species confused with F. virgata are either new to science or were placed erroneously in synonymy with F. virgata by earlier authors: they will be described elsewhere. The most important characters of the adult female for distinguishing these species from F. virgata are the positions and characteristics of minute discoidal pore(s) associated both with the ventral oral‐collar tubular ducts and with the sclerotized area surrounding each dorsal enlarged tubular duct, and the number of sizes of the ventral oral‐collar tubular ducts. In addition, we determine that adult females of F. gilli Gullan from different populations on different host plants vary substantially in the number and size of clusters of small ventral oral‐collar ducts on the body margins – features previously suspected to indicate distinct species.     

The phenological window for western spruce budworm: seasonal decline in resource quality

• 1 Western spruce budworm Choristoneura occidentalis Free. (Lepidoptera: Tortricidae) emerge in the spring before budburst and then face a rapidly deteriorating host quality each season.
• 2 Measures of fitness, survival and fecundity, were made on cohorts of final‐instar spruce budworms deployed on host trees at several times during the season in four field locations in coastal and interior British Columbia, Canada.
• 3 Survival and fecundity were strongly correlated throughout the season and varied as much as four‐fold from maxima at mid‐season to minima at the end of the season.
• 4 Fitness values overall were greatest in the coastal compared with interior locations. Among interior locations, fitness was greatest at the highest elevation and least at the lowest elevation. Both cohort and sample‐based estimates of survival of wild, final‐instar budworms were relatively high in these outbreak populations.
• 5 The influence of the phenological window and degree of synchrony with the host plant on herbivore abundance often depends on other processes affecting population rates of change.

     

Oral exudate as a mediator of behavior in larval eastern and western spruce budworms (Lepidoptera: Tortricidae)

The production of oral exudate by larval eastern and western spruce budworms,Choristoneura fumiferana (Clem.) andChoristoneura occidentalis Free., respectively, was investigated in the laboratory. All larvae except those entering into a molt exhibited aggressive behavior and produced exudate in response to handling or intraspecific encounters. Larvae could be induced to produce exudate up to four times over 2–3 min and produced an average of 1.92±0.04 µl (X ± SE) per induction. Larvae on foliage spent much of their time maintaining their silken feeding tunnel, including spinning and combing silk and removing frass. Exposure to conspecific oral exudate deposited inside the tunnel, or released by agitated larvae inside the tunnel, increased the proportion of larvae that dispersed away from the tunnels and, apparently, increased the larval sensitivity to disturbances. The behavior induced by the oral exudate indicates that it acts as an epideictic (spacing) pheromone.     

A study of wing morphology and fluctuating asymmetry in interspecific hybrids between Drosophila buzzatii and D. koepferae

In this work we investigate the effect of interspecific hybridization on wing morphology using geometric morphometrics in the cactophilic sibling species D. buzzatii and D. koepferae. Wing morphology in F₁ hybrids exhibited an important degree of phenotypic plasticity and differs significantly from both parental species. However, the pattern of morphological variation between hybrids and the parental strains varied between wing size and wing shape, across rearing media, sexes, and crosses, suggesting a complex genetic architecture underlying divergence in wing morphology. Even though there was significant fluctuating asymmetry for both, wing size and shape in F₁ hybrids and both parental species, there was no evidence of an increased degree of fluctuating asymmetry in hybrids as compared to parental species. These results are interpreted in terms of developmental stability as a function of a balance between levels of heterozygosity and the disruption of coadaptation as an indirect consequence of genomic divergence.     

Variation in budburst phenology of Douglas-fir related to western spruce budworm (Lepidoptera: Tortricidae) fitness

Variation in budburst phenology among individual trees of interior Douglas-fir (Pseudotsuga menziesii var. glauca [Beissn.] Franco) may influence their susceptibility to western spruce budworm (Choristoneura occidentalis Freeman) defoliation. We tested the hypothesis that phenological asynchrony between Douglas-fir and the western spruce budworm is a mechanism of resistance using clones derived from parent trees that showed resistance versus susceptibility to C. occidentalis defoliation in the field. Susceptible clones had earlier budburst phenology compared with resistant clones when they were grown in a common greenhouse environment, demonstrating a genetic basis for parallel phenological differences exhibited by the parent trees. We tested the importance of phenological asynchrony as a factor influencing fitness of C. occidentalis using two different greenhouse bioassay experiments. One experiment compared western spruce budworm performance on equivalent phenological stages of susceptible and resistant clones by matching larval feeding to the columnar (fourth) bud development stage of each clone. Larvae reared on resistant clones had greater realized fitness (i.e., number of F1 offspring produced) than those reared on susceptible clones when the influence of variation in budburst phenology was minimized. In the other experiment, western spruce budworm larvae were placed on all trees on the same date when approximately 50% of all terminal buds in the population were in the yellow (second) budburst stage. Larvae reared on susceptible clones had greater realized fitness than those reared on resistant clones when the influence of phenological asynchrony was expressed. Our results suggest that resistant phenotypes of Douglas-fir have negative effects on survival and reproduction of C. occidentalis under the natural conditions that insects and trees experience in the field. Genetic variation among trees in budburst phenology has an important influence on interactions between the western spruce budworm and Douglas-fir.     

Genetic constraints on wing pattern variation in Lycaeides butterflies: A case study on mapping complex,multifaceted traits in structured populations

Patterns of phenotypic variation within and among species can be shaped and constrained by trait genetic architecture. This is particularly true for complex traits, such as butterfly wing patterns, that consist of multiple elements. Understanding the genetics of complex trait variation across species boundaries is difficult, as it necessitates mapping in structured populations and can involve many loci with small or variable phenotypic effects. Here, we investigate the genetic architecture of complex wing pattern variation in Lycaeides butterflies as a case study of mapping multivariate traits in wild populations that include multiple nominal species or groups. We identify conserved modules of integrated wing pattern elements within populations and species. We show that trait covariances within modules have a genetic basis and thus represent genetic constraints that can channel evolution. Consistent with this, we find evidence that evolutionary changes in wing patterns among populations and species occur in the directions of genetic covariances within these groups. Thus, we show that genetic constraints affect patterns of biological diversity (wing pattern) in Lycaeides, and we provide an analytical template for similar work in other systems.     

Continent‐wide population genomic structure and phylogeography of North America’s most destructive conifer defoliator,the spruce budworm (Choristoneura fumiferana)

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

Discordance between morphological and molecular species boundaries among Caribbean species of the reef sponge Callyspongia

Sponges are among the most species‐rich and ecologically important taxa on coral reefs, yet documenting their diversity is difficult due to the simplicity and plasticity of their morphological characters. Genetic attempts to identify species are hampered by the slow rate of mitochondrial sequence evolution characteristic of sponges and some other basal metazoans. Here we determine species boundaries of the Caribbean coral reef sponge genus Callyspongia using a multilocus, model‐based approach. Based on sequence data from one mitochondrial (COI), one ribosomal (28S), and two single‐copy nuclear protein‐coding genes, we found evolutionarily distinct lineages were not concordant with current species designations in Callyspongia. While C. fallax, C. tenerrima, and C. plicifera were reciprocally monophyletic, four taxa with different morphologies (C. armigera, C. longissima, C. eschrichtii, and C. vaginalis) formed a monophyletic group and genetic distances among these taxa overlapped distances within them. A model‐based method of species delimitation supported collapsing these four into a single evolutionary lineage. Variation in spicule size among these four taxa was partitioned geographically, not by current species designations, indicating that in Callyspongia, these key taxonomic characters are poor indicators of genetic differentiation. Taken together, our results suggest a complex relationship between morphology and species boundaries in sponges.