Amanda D. Roe

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.

Population structure and migration pattern of a conifer pathogen, Grosmannia clavigera, as influenced by its symbiont, the mountain pine beetle

We investigated the population structure of Grosmannia clavigera (Gc), a fungal symbiont of the mountain pine beetle (MPB) that plays a crucial role in the establishment and reproductive success of this pathogen. This insect–fungal complex has destroyed over 16 million ha of lodgepole pine forests in Canada, the largest MPB epidemic in recorded history. During this current epidemic, MPB has expanded its range beyond historically recorded boundaries, both northward and eastward, and has now reached the jack pine of Alberta, potentially threatening the Canadian boreal forest. To better understand the dynamics between the beetle and its fungal symbiont, we sampled 19 populations in western North America and genotyped individuals from these populations with eight microsatellite markers. The fungus displayed high haplotype diversity, with over 250 unique haplotypes observed in 335 single spore isolates. Linkage equilibria in 13 of the 19 populations suggested that the fungus reproduces sexually. Bayesian clustering and distance analyses identified four genetic clusters that corresponded to four major geographical regions, which suggested that the epidemic arose from multiple geographical sources. A genetic cluster north of the Rocky Mountains, where the MPB has recently become established, experienced a population bottleneck, probably as a result of the recent range expansion. The two genetic clusters located north and west of the Rocky Mountains contained many fungal isolates admixed from all populations, possibly due to the massive movement of MPB during the epidemic. The general agreement in north–south differentiation of MPB and G. clavigera populations points to the fungal pathogen’s dependence on the movement of its insect vector. In addition, the patterns of diversity and the individual assignment tests of the fungal associate suggest that migration across the Rocky Mountains occurred via a northeastern corridor, in accordance with meteorological patterns and observation of MPB movement data. Our results highlight the potential of this pathogen for both expansion and sexual reproduction, and also identify some possible barriers to gene flow. Understanding the ecological and evolutionary dynamics of this fungus–beetle association is important for the modelling and prediction of MPB epidemics.

Comparative phylogeography, genetic differentiation and contrasting reproductive modes in three fungal symbionts of a multipartite bark beetle symbiosis

Multipartite symbioses are complex symbiotic relationships involving multiple interacting partners. These types of partnerships provide excellent opportunities in which to apply a comparative approach to identify common historical patterns of population differentiation and species‐specific life history traits. Using three symbiotic blue‐stain fungal species (Ophiostomatacea) associated with outbreaking populations of the mountain pine beetle (Dendroctonus ponderosae Hopkins) in western Canada, we applied phylogenetic, population genetic and demographic approaches to clarify phylogeographic patterns among the three fungal species. Broadly, the three species showed significant population differentiation, forming northern and southern populations, despite dramatic differences in haplotype diversity. Finer structuring and population demographic patterns were less consistent, showing some interspecific incongruence. By contrasting these species simultaneously, we were able to identify differences in recombination rate and ecological traits that can explain the observed patterns of incongruence among the fungal species. By applying a comparative approach to partners of a multipartite symbiosis, we were able to distinguish congruent population structuring and species‐specific differences that help us to understand the complexity and evolution of this symbiotic system.

Spatial Community Structure of Mountain Pine Beetle Fungal Symbionts Across a Latitudinal Gradient

Symbiont redundancy in obligate insect–fungal systems is thought to buffer the insect host against symbiont loss and to extend the environmental conditions under which the insect can persist. The mountain pine beetle is associated with at least three well-known and putatively obligate ophiostomatoid fungal symbionts that vary in their environmental tolerances. To better understand the spatial variation in beetle–fungal symbiotic associations, we examined the community composition of ophiostomatoid fungi associated with the mountain pine beetle as a function of latitude and elevation. The region investigated represents the leading edge of a recent outbreak of mountain pine beetle in western Canada. Using regression and principal components analysis, we identified significant spatial patterns in fungal species abundances that indicate symmetrical replacement between two of the three fungi along a latitudinal gradient and little variation in response to elevation. We also identified significant variation in the prevalence of pair-wise species combinations that occur within beetle galleries. Frequencies of pair-wise combinations were significantly different from what was expected given overall species abundances. These results suggest that complex processes of competitive exclusion and coexistence help determine fungal community composition and that the consequences of these processes vary spatially. The presence of three fungal symbionts in different proportions and combinations across a wide range of environmental conditions may help explain the success of mountain pine beetle attacks across a broad geographic range.

Identification of Dioryctria (Lepidoptera: Pyralidae) in a Seed Orchard at Chico, California

Abstract Species of Dioryctria Zeller (Lepidoptera: Pyralidae) are important pests of conifers, particularly in seed orchards, and accurate species identification is needed for effective monitoring and control. Variable forewing morphology and lack of species-specific genitalic features hinder identification, prompting the search for additional diagnostic characters. Mitochondrial DNA (mtDNA) sequences from the cytochrome c oxidase I and II genes (COI and COII) were obtained from specimens collected at lights, pheromone traps, and host plants in the Pacific Northwest, focusing on a U.S. Forest Service seed orchard in Chico, CA. A 475-bp fragment of COI was used to identify eight distinct genetic lineages from 180 Dioryctria specimens, and these were identified as eight described species. Comparisons among mtDNA variation, adult morphology, larval host association, and pheromone attraction were used to assign individuals to species groups and to identify diagnostic characters for species identification. A 2.3-kb fragment of COI-COII was sequenced for 14 specimens to increase resolution of phylogenetic relationships. Species groups were well resolved using both the 475-bp and “DNA barcode” subsets of the 2.3-kb sequences, with the 475-bp fragment generally showing lower divergences. The zimmermani and ponderosae species groups were sister groups and had similar male genitalic morphology and larval feeding habits. The pentictonella group was sister to the zimmermani + ponderosae group clade, and all species have raised scales and a Pinus sp. larval host (where known). Combining molecular characters with morphological and behavioral characters improved identification of Dioryctria species and supported previous species group relationships.

Phylogeographic insights into an irruptive pest outbreak

Irruptive forest insect pests cause considerable ecological and economic damage, and their outbreaks have been increasing in frequency and severity. We use a phylogeographic approach to understand the location and progression of an outbreak by the MPB (Dendroctonus ponderosae Hopkins), an irruptive bark beetle that has caused unprecedented damage to lodgepole pine forests in western North America and is poised to expand its range across the boreal forest. We sampled MPB populations across British Columbia and Alberta and used phylogeographic methods to describe lineage diversification, characterize population structure, investigate expansion dynamics, and identify source populations of the outbreak. Using 1181 bp of mitochondrial DNA sequence from 267 individuals, we found high haplotype diversity, low nucleotide diversity, and limited lineage diversification. The overall pattern was consistent with isolation by distance at a continental scale, and with reduced diversity and population structure in the northerly, outbreak regions. Post-Pleistocene expansion was detected, however more recent expansion signals were not detected, potentially due to the size and rapid rate of range expansion. Based on the limited genetic structure, there were likely multiple source populations in southern British Columbia, although the magnitude of the demographic expansion and rate of spread have obscured the signature of these source populations. Our data highlight the need for caution in interpreting phylogeographic results for species with similar demographics.

Isolation and characterization of 16 microsatellite loci in the mountain pine beetle, Dendroctonus ponderosae Hopkins (Coleoptera: Curculionidae: Scolytinae)

We isolated 16 polymorphic microsatellite loci in the mountain pine beetle (Dendroctonus ponderosae Hopkins) and developed conditions for amplifying these markers in four multiplex reactions. Three to 14 alleles were detected per locus across two sampled populations. Observed and expected heterozygosities ranged from 0.000 to 0.902 and from 0.100 to 0.830, respectively. Three loci deviated from Hardy–Weinberg equilibrium in one sampled population. One of these loci may be sex linked. These markers will be useful in the study of population structure in this important pest species.

Population Structure of Mountain Pine Beetle Symbiont Leptographium longiclavatum and the Implication on the Multipartite Beetle-Fungi Relationships

Over 18 million ha of forests have been destroyed in the past decade in Canada by the mountain pine beetle (MPB) and its fungal symbionts. Understanding their population dynamics is critical to improving modeling of beetle epidemics and providing potential clues to predict population expansion. Leptographium longiclavatum and Grosmannia clavigera are fungal symbionts of MPB that aid the beetle to colonize and kill their pine hosts. We investigated the genetic structure and demographic expansion of L. longiclavatum in populations established within the historic distribution range and in the newly colonized regions. We identified three genetic clusters/populations that coincide with independent geographic locations. The genetic profiles of the recently established populations in northern British Columbia (BC) and Alberta suggest that they originated from central and southern BC. Approximate Bayesian Computation supports the scenario that this recent expansion represents an admixture of individuals originating from BC and the Rocky Mountains. Highly significant correlations were found among genetic distance matrices of L. longiclavatum, G. clavigera, and MPB. This highlights the concordance of demographic processes in these interacting organisms sharing a highly specialized niche and supports the hypothesis of long-term multipartite beetle-fungus co-evolutionary history and mutualistic relationships.

Biology and management of North American cone-feeding Dioryctria species

Abstract Coneworms, Dioryctria Zeller (Lepidoptera: Pyralidae), are destructive pests of conifers across North America, and members of several different species groups present significant pestmanagement challenges in conifer seed orchards. Dioryctria abietivorella Grote (abietella group) is the most pestiferous Dioryctria species in Canada. Despite this status, control tactics are currently limited to broad-spectrum pesticides that threaten non-target species and may result in pesticide resistance. The development of integrated pest management programs targeting Dioryctria species will benefit from a conceptual framework on which to base future research. To create this structure, we review the systematics, evolutionary ecology, and management of cone-feeding North American Dioryctria species. Current research suggests that many species boundaries are in need of further revision. Major gaps in our understanding of Dioryctria ecology impede the development of integrated pest management tactics. For example, host-generated semiochemicals are important in Dioryctria reproduction, although the uses of these cues in host-finding and host acceptance remain unknown. Future research should identify factors that mediate population distribution at landscape (e.g., migration), local (e.g., feeding stimulants), and temporal (e.g., development thresholds) scales.

Modeling Landscape-Level Spatial Variation in Sex Ratio Skew in the Mountain Pine Beetle (Coleoptera: Curculionidae)

Abstract Through their influence on effective population sizes, sex ratio skew affects population dynamics. We examined spatial variation in female-biased sex ratios in the mountain pine beetle (Dendroctonus ponderosae Hopkins) outbreak in western Canada to better understand how environmental context affects sex ratio skew. Our specific objectives were to: 1) characterize spatial variation in mountain pine beetle sex ratio; 2) test previously asserted hypotheses that beetle sex ratio varies with tree diameter and year in outbreak; and 3) develop predictive models of sex ratio skew for larval and adult populations. Using logistic regression, we modeled the probability that an individual beetle (n = 2,369) was female as a function of multiple environmental variables across 34 stands in British Columbia and Alberta, Canada. We identified a consistent female-biased sex ratio with significantly greater skew in adults (2: 1, n = 713) than in larvae (1.76: 1, n = 1,643). We found that the proportion of larval females increased with decreasing tree size and with outbreak age. However, adults did not respond to tree size and larvae did not respond to outbreak age. Predictive models differed between larvae and adults. All identified models perform well and included predictors related to weather, tree diameter, and year in outbreak. Female-biased sex ratios appear to originate from differential male mortality during development rather than from sex-biased oviposition, suggesting sex ratio skew is not the cause of outbreaks, but rather a consequence.