Paired acoustic recordings and point count surveys reveal Clark's nutcracker and whitebark pine associations across Glacier National Park.

Global declines in tree populations have led to dramatic shifts in forest ecosystem composition, biodiversity, and functioning. These changes have consequences for both forest plant and wildlife communities, particularly when declining species are involved in coevolved mutualisms. Whitebark pine (Pinus albicaulis) is a declining keystone species in western North American high-elevation ecosystems and an obligate mutualist of Clark's nutcracker (Nucifraga columbiana), an avian seed predator and disperser. By leveraging traditional point count surveys and passive acoustic monitoring, we investigated how stand characteristics of whitebark pine in a protected area (Glacier National Park, Montana, USA) influenced occupancy and vocal activity patterns in Clark's nutcracker. Using Bayesian spatial occupancy models and generalized linear mixed models, we found that habitat use of Clark's nutcracker was primarily supported by greater cone density and increasing diameter of live whitebark pine. Additionally, we demonstrated the value of performing parallel analyses with traditional point count surveys and passive acoustic monitoring to provide multiple lines of evidence for relationships between Clark's nutcracker and whitebark pine forest characteristics. Our findings allow managers to gauge the whitebark pine conditions important for retaining high nutcracker visitation and prioritize management efforts in whitebark pine ecosystems with low nutcracker visitation.

Fidelity or love the one you're with? Biotic complexity and tradeoffs can drive strategy and specificity in beetle-fungus by-product mutualisms.

By-product mutualisms are ubiquitous yet seldom considered in models of mutualism. Most models represent conditional mutualisms that shift between mutualism and antagonism in response to shifts in costs and benefits resulting from changes in environmental quality. However, in by-product mutualisms, benefits arise as a part of normal life processes that may be costly to produce but incur little-to-no additional costs in response to the interaction. Without costs associated with the interaction, they do not have antagonistic alternate states. Here, we present a conceptual model that differs from traditional conditional models in three ways: (1) partners exchange by-product benefits, (2) interactions do not have alternate antagonistic states, and (3) tradeoffs are allowed among factors that influence environmental quality (rather than all factors that contribute to environmental quality being combined into a single gradient ranging from high to low). We applied this model to bark and ambrosia beetles (Curculionidae: Scolytinae), a diverse group that associates with fungi and that has repeatedly developed two distinct pathways to by-product mutualism. We used independent axes for each major factor influencing environmental quality in these systems, including those that exhibit tradeoffs (tree defense and nutritional quality). For these symbioses, tradeoffs in these two factors are key to which mutualism pathway is taken.

Context Dependency in Bark Beetle-Fungus Mutualisms Revisited: Assessing Potential Shifts in Interaction Outcomes Against Varied Genetic, Ecological, and Evolutionary Backgrounds.

Context dependency occurs when biological interactions shift in sign or magnitude depending upon genetic, abiotic, and biotic context. Most models of mutualism address systems where interaction outcomes slide along a mutualism-antagonism continuum as environmental conditions vary altering cost-benefit relationships. However, these models do not apply to the many mutualisms that involve by-product benefits and others that do not have antagonistic alternate states. The ubiquity of such mutualisms indicates a need for different approaches and models to understand how environmental variability influences their strength, stability, and ecological roles. In this paper, we apply the concept of context dependency to mutualisms among bark beetles and fungi that span a variety of life strategies and exposures to environmental variability. Bark beetles and their mutualist fungi co-construct a niche based on by-product benefits that allows them to exist in a resource that is otherwise intractable or inaccessible. For the closest of these partnerships, this has resulted in some of the most influential agents of forest mortality in conifer forests worldwide. Understanding these symbioses is key to understanding their influence on forest structure and dynamics and responses to change. We found no evidence that bark beetle mutualisms change in sign as conditions vary, only in magnitude, and that the "closest" (and most environmentally influential) of these partnerships have evolved behaviors and mechanisms to reduce context-dependency and stabilize benefit delivery. The bark beetle-fungus symbioses most likely to slide along a mutualism-antagonism continuum are those involving loosely associated facultative symbionts that may provide benefits under some circumstances and that are horizontally transmitted by the beetle host. Additionally, some symbiotic fungi are never mutualists - these "third party" fungi are exploiters and may shift from commensalism to antagonism depending on environmental context. Our assessment indicates that a careful differentiation between bark beetle-fungus partnerships is crucial to understanding how they influence forests and respond to environmental variability.

Niche construction theory can link bark beetle-fungus symbiosis type and colonization behavior to large scale causal chain-effects.

Bark beetles form a variety of symbioses with fungi. Recent studies reveal how the fungi influence beetle nutrition and detoxify tree defenses and provide insight into why these symbioses vary so greatly in their outcomes, not only for host and symbiont, but also for the forest ecosystems within which they exist. Here, I review recent advances in our knowledge of these systems. I then introduce how niche construction theory can provide a framework to use this knowledge to better understand how different symbiosis types result in a gradient of ecosystem effects ranging from massive and durable to those of little ecological consequence.

Bark Beetle Population Dynamics in the Anthropocene: Challenges and Solutions.

Tree-killing bark beetles are the most economically important insects in conifer forests worldwide. However, despite >200 years of research, the drivers of population eruptions and crashes are still not fully understood and the existing knowledge is thus insufficient to face the challenges posed by the Anthropocene. We critically analyze potential biotic and abiotic drivers of population dynamics of an exemplary species, the European spruce bark beetle (ESBB) (Ips typographus) and present a multivariate approach that integrates the many drivers governing this bark beetle system. We call for hypothesis-driven, large-scale collaborative research efforts to improve our understanding of the population dynamics of this and other bark beetle pests. Our approach can serve as a blueprint for tackling other eruptive forest insects.

Are Survivors Different? Genetic-Based Selection of Trees by Mountain Pine Beetle During a Climate Change-Driven Outbreak in a High-Elevation Pine Forest.

Increased mortality of forest trees, driven directly or indirectly by climate change, is occurring around the world. In western North America, whitebark pine, a high elevation keystone species, and lodgepole pine, a widespread ecologically and economically important tree, have experienced extensive mortality in recent climate-driven outbreaks of the mountain pine beetle. However, even in stands experiencing high levels of mortality, some mature trees have survived. We hypothesized that the outbreak acted as a natural selection event, removing trees most susceptible to the beetle and least adapted to warmer drier conditions. If this was the case, genetic change would be expected at loci underlying beetle resistance. Given we did not know the basis for resistance, we used inter-simple sequence repeats to compare the genetic profiles of two sets of trees, survivors (mature, living trees) and general population (trees just under the diameter preferred by the beetles and expected to approximate the genetic structure of each tree species at the site without beetle selection). This method detects high levels of polymorphism and has often been able to detect patterns associated with phenotypic traits. For both whitebark and lodgepole pine, survivors and general population trees mostly segregated independently indicating a genetic basis for survivorship. Exceptions were a few general population trees that segregated with survivors in proportions roughly reflecting the proportion of survivors versus beetle-killed trees. Our results indicate that during outbreaks, beetle choice may result in strong selection for trees with greater resistance to attack. Our findings suggest that survivorship is genetically based and, thus, heritable. Therefore, retaining survivors after outbreaks to act as primary seed sources could act to promote adaptation. Further research will be needed to characterize the actual mechanism(s) of resistance.

Novel ophiostomatalean fungi from galleries of Cyrtogenius africus (Scolytinae) infesting dying Euphorbia ingens.

Euphorbia ingens trees have been dying in large numbers in the Limpopo Province of South Africa for approximately 15 years. The ambrosia beetle Cyrtogenius africus is often found infesting diseased and dying trees. The aim of this study was to identify the ophiostomatoid fungi occurring in the galleries of C. africus. Logs infested with this beetle were collected from the KwaZulu-Natal, Limpopo, Mpumalanga, and North West Provinces of South Africa. Fungi belonging to the Ophiostomatales were identified based on morphology and comparison of sequence data for the ß-tubulin, ITS1-5.8S-ITS2 and LSU gene regions. A novel species of Ophiostoma and a novel genus in the Ophiostomatales were identified. Inoculation studies with these fungi produced lesions in the branches of healthy E. ingens trees.

Experimental evidence of bark beetle adaptation to a fungal symbiont.

The importance of symbiotic microbes to insects cannot be overstated; however, we have a poor understanding of the evolutionary processes that shape most insect-microbe interactions. Many bark beetle (Coleoptera: Curculionidae, Scolytinae) species are involved in what have been described as obligate mutualisms with symbiotic fungi. Beetles benefit through supplementing their nutrient-poor diet with fungi and the fungi benefit through gaining transportation to resources. However, only a few beetle-fungal symbioses have been experimentally manipulated to test whether the relationship is obligate. Furthermore, none have tested for adaptation of beetles to their specific symbionts, one of the requirements for coevolution. We experimentally manipulated the western pine beetle-fungus symbiosis to determine whether the beetle is obligately dependent upon fungi and to test for fine-scale adaptation of the beetle to one of its symbiotic fungi, Entomocorticium sp. B. We reared beetles from a single population with either a natal isolate of E. sp. B (isolated from the same population from which the beetles originated), a non-natal isolate (a genetically divergent isolate from a geographically distant beetle population), or with no fungi. We found that fungi were crucial for the successful development of western pine beetles. We also found no significant difference in the effects of the natal and non-natal isolate on beetle fitness parameters. However, brood adult beetles failed to incorporate the non-natal fungus into their fungal transport structure (mycangium) indicating adaption by the beetle to particular genotypes of symbiotic fungi. Our results suggest that beetle-fungus mutualisms and symbiont fidelity may be maintained via an undescribed recognition mechanism of the beetles for particular symbionts that may promote particular associations through time.

Severe White Pine Blister Rust Infection in Whitebark Pine Alters Mountain Pine Beetle (Coleoptera: Curculionidae) Attack Density, Emergence Rate, and Body Size.

Exotic tree pathogens can cause devastating ecological effects on forests that can be exacerbated when infections increase the likelihood of attack by insects. Current high rates of mortality of whitebark pine (Pinus albicaulis Engelm.) are due to white pine blister rust caused by the exotic fungus, Cronartium ribicola J.C. Fisch, and the native mountain pine beetle (Dendroctonus ponderosae Hopkins). These two mortality agents interact in whitebark pine; mountain pine beetle preferentially selects white pine blister rust-infected whitebark pine over healthy trees, and likelihood of attack has been observed to increase with infection severity. We examined attack and emergence rates, and size and sex ratio of mountain pine beetle in whitebark pines exhibiting varying white pine blister rust infection severities. Mountain pine beetle attack density was lowest on the most severely infected trees, but emergence rates and size of beetles from these trees were greater than those from uninfected and less severely infected trees. Low attack rates on severely infected whitebark pine may indicate these trees have lower defenses and that fewer beetle attacks are needed to kill them. Higher beetle emergence rates from severely infected trees may be due to low intraspecific competition resulting from low attack rates or differences in nutrient quality.

Effects of Temperature on Growth, Sporulation, and Competition of Mountain Pine Beetle Fungal Symbionts.

The mountain pine beetle, Dendroctonus ponderosae, depends on two fungi, Grosmannia clavigera and Ophiostoma montium, to augment a nutrient-poor woody food resource. Because the two fungi exert differential effects on the host beetle, temperature-driven differences in fungal growth and competition outcomes have a strong potential to influence host population dynamics. Weisolated fungi from beetles and wood from three locations in Montana and Utah, USA, and measured their growth rates and sporulation between 5 and 35 °C on artificial media. We also measured growth rates and percent resource capture for each fungus at 10, 15, 21, and 25 °C during inter- and intra-specific competition. G. clavigera excelled at resource capture at most temperatures. Its optimal growth temperature occurs around 20 °C while that of O. montium occurs near 30 °C. There was no effect of collection site on growth or sporulation; however, O. montium exhibited greater variability in response to temperature than did G. clavigera. Sporulation of G. clavigera was greatest at 30 °C while O. montium sporulated at low levels across all temperatures. During competition experiments, G. clavigera captured more resources than O. montium at most temperatures and captured a greater percentage of resources at a greater rate during inter-specific competition than during intra-specific competition. In contrast, O. montium captured a greater percentage of resources during intra-specific competition. These results demonstrate that temperature can differentially affect growth, sporulation, and resource capture of the two symbionts, indicating that it may be an important factor influencing the composition and dynamics of the symbiosis.

Integrating models to investigate critical phenological overlaps in complex ecological interactions: the mountain pine beetle-fungus symbiosis.

The fates of individual species are often tied to synchronization of phenology, however, few methods have been developed for integrating phenological models involving linked species. In this paper, we focus on mountain pine beetle (MPB, Dendroctonus ponderosae) and its two obligate mutualistic fungi, Grosmannia clavigera and Ophiostoma montium. Growth rates of all three partners are driven by temperature, and their idiosyncratic responses affect interactions at important life stage junctures. One critical phase for MPB-fungus symbiosis occurs just before dispersal of teneral (new) adult beetles, when fungi are acquired and transported in specialized structures (mycangia). Before dispersal, fungi must capture sufficient spatial resources within the tree to ensure contact with teneral adults and get packed into mycangia. Mycangial packing occurs at an unknown time during teneral feeding. We adapt thermal models predicting fungal growth and beetle development to predict overlap between the competing fungi and MPB teneral adult feeding windows and emergence. We consider a spectrum of mycangial packing strategies and describe them in terms of explicit functions with unknown parameters. Rates of growth are fixed by laboratory data, the unknown parameters describing various packing strategies, as well as the degree to which mycangial growth is slowed in woody tissues as compared to agar, are determined by maximum likelihood and two years of field observations. At the field location used, the most likely fungus acquisition strategy for MPB was packing mycangia just prior to emergence. Estimated model parameters suggested large differences in the relative growth rates of the two fungi in trees at the study site, with the most likely model estimating that G. clavigera grew approximately twenty-five times faster than O. montium under the bark, which is completely unexpected in comparison with observed fungal growth on agar.

Broadscale specificity in a bark beetle-fungal symbiosis: a spatio-temporal analysis of the mycangial fungi of the western pine beetle.

Whether and how mutualisms are maintained through ecological and evolutionary time is a seldom studied aspect of bark beetle-fungal symbioses. All bark beetles are associated with fungi and some species have evolved structures for transporting their symbiotic partners. However, the fungal assemblages and specificity in these symbioses are not well known. To determine the distribution of fungi associated with the mycangia of the western pine beetle (Dendroctonus brevicomis), we collected beetles from across the insect's geographic range including multiple genetically distinct populations. Two fungi, Entomocorticium sp. B and Ceratocystiopsis brevicomi, were isolated from the mycangia of beetles from all locations. Repeated sampling at two sites in Montana found that Entomocorticium sp. B was the most prevalent fungus throughout the beetle's flight season, and that females carrying that fungus were on average larger than females carrying C. brevicomi. We present evidence that throughout the flight season, over broad geographic distances, and among genetically distinct populations of beetle, the western pine beetle is associated with the same two species of fungi. In addition, we provide evidence that one fungal species is associated with larger adult beetles and therefore might provide greater benefit during beetle development. The importance and maintenance of this bark beetle-fungus interaction is discussed.

The bark beetle holobiont: why microbes matter.

All higher organisms are involved in symbioses with microbes. The importance of these partnerships has led to the concept of the holobiont, defined as the animal or plant with all its associated microbes. Indeed, the interactions between insects and symbionts form much of the basis for the success and diversity of this group of arthropods. Insects rely on microbes to perform basic life functions and to exploit resources and habitats. By "partnering" with microbes, insects access new genomic variation instantaneously allowing the exploitation of new adaptive zones, influencing not only outcomes in ecological time, but the degree of innovation and change that occurs over evolutionary time. In this review, I present a brief overview of the importance of insect-microbe holobionts to illustrate how critical an understanding of the holobiont is to understanding the insect host and it interactions with its environment. I then review what is known about the most influential insect holobionts in many forest ecosystems-bark beetles and their microbes-and how new approaches and technologies are allowing us to illuminate how these symbioses function. Finally, I discuss why it will be critical to study bark beetles as a holobiont to understand the ramifications and extent of anthropogenic change in forest ecosystems.

Abundance and dynamics of filamentous fungi in the complex ambrosia gardens of the primitively eusocial beetle Xyleborinus saxesenii Ratzeburg (Coleoptera: Curculionidae, Scolytinae).

Insect fungus gardens consist of a community of interacting microorganisms that can have either beneficial or detrimental effects to the farmers. In contrast to fungus-farming ants and termites, the fungal communities of ambrosia beetles and the effects of particular fungal species on the farmers are largely unknown. Here, we used a laboratory rearing technique for studying the filamentous fungal garden community of the ambrosia beetle, Xyleborinus saxesenii, which cultivates fungi in tunnels excavated within dead trees. Raffaelea sulfurea and Fusicolla acetilerea were transmitted in spore-carrying organs by gallery founding females and established first in new gardens. Raffaelea sulfurea had positive effects on egg-laying and larval numbers. Over time, four other fungal species emerged in the gardens. Prevalence of one of them, Paecilomyces variotii, correlated negatively with larval numbers and can be harmful to adults by forming biofilms on their bodies. It also comprised the main portion of garden material removed from galleries by adults. Our data suggest that two mutualistic, several commensalistic and one to two pathogenic filamentous fungi are associated with X. saxesenii. Fungal diversity in gardens of ambrosia beetles appears to be much lower than that in gardens of fungus-culturing ants, which seems to result from essential differences in substrates and behaviours.

New species of Gondwanamyces from dying Euphorbia trees in South Africa.

Gondwanamyces and its Custingophora anamorphs were first described from Protea infructescences in South Africa. Subsequently these unusual fungi were also found on Cecropia in Central America. During an investigation into the decline and death of native Euphorbia trees in South Africa, several fungal isolates resembling the anamorph state of Gondwanamyces were obtained from diseased tissues. In this study these isolates are identified based on morphology and comparisons of DNA sequences. Two previously unknown Gondwanamyces species were identified, both were associated with damage caused by beetles (Cossonus sp.). Inoculation studies showed that the new species of Gondwanamyces are pathogenic on Euphorbia ingens and may contribute to the decline of these trees.

Ecological and Evolutionary Determinants of Bark Beetle -Fungus Symbioses.

Ectosymbioses among bark beetles (Curculionidae, Scolytinae) and fungi (primarily ophiostomatoid Ascomycetes) are widespread and diverse. Associations range from mutualistic to commensal, and from facultative to obligate. Some fungi are highly specific and associated only with a single beetle species, while others can be associated with many. In addition, most of these symbioses are multipartite, with the host beetle associated with two or more consistent partners. Mycangia, structures of the beetle integument that function in fungal transport, have evolved numerous times in the Scolytinae. The evolution of such complex, specialized structures indicates a high degree of mutual dependence among the beetles and their fungal partners. Unfortunately, the processes that shaped current day beetle-fungus symbioses remain poorly understood. Phylogeny, the degree and type of dependence on partners, mode of transmission of symbionts (vertical vs. horizontal), effects of the abiotic environment, and interactions among symbionts themselves or with other members of the biotic community, all play important roles in determining the composition, fidelity, and longevity of associations between beetles and their fungal associates. In this review, I provide an overview of these associations and discuss how evolution and ecological processes acted in concert to shape these fascinating, complex symbioses.

Fungal associates of the lodgepole pine beetle, Dendroctonus murrayanae.

Bark beetles are well known vectors of ophiostomatoid fungi including species of Ophiostoma, Grosmannia and Ceratocystis. In this study, the most common ophiostomatoid fungi associated with the lodgepole pine beetle, Dendroctonus murrayanae, were characterized. Pre-emergent and post-attack adult beetles were collected from lodgepole pines at four sites in British Columbia, Canada. Fungi were isolated from these beetles and identified using a combination of morphology and DNA sequence comparisons of five gene regions. In all four populations, Grosmannia aurea was the most common associate (74-100% of all beetles) followed closely by Ophiostoma abietinum (29-75%). Other fungi isolated, in order of their relative prevalence with individual beetles were an undescribed Leptographium sp. (0-13%), Ophiostoma ips (0-15%), Ophiostoma piliferum (0-11%), a Pesotum sp. (0-11%) and Ophiostoma floccosum (0-1%). Comparisons of the DNA sequences of Leptographium strains isolated in this study, with ex-type isolates of G. aurea, Grosmannia robusta, Leptographium longiclavatum, and Leptographium terebrantis, as well as with sequences from GenBank, revealed a novel lineage within the Grosmannia clavigera complex. This lineage included some of the D. murrayane isolates as well as several isolates from previous studies referred to as L. terebrantis. However, the monophyly of this lineage is not well supported and a more comprehensive study will be needed to resolve its taxonomic status as one or more novel taxa.

Gene genealogies reveal cryptic species and host preferences for the pine fungal pathogen Grosmannia clavigera.

Grosmannia clavigera is a fungal pathogen of pine forests in western North America and a symbiotic associate of two sister bark beetles: Dendroctonus ponderosae and D. jeffreyi. This fungus and its beetle associate D. ponderosae are expanding in large epidemics in western North America. Using the fungal genome sequence and gene annotations, we assessed whether fungal isolates from the two beetles inhabiting different species of pine in epidemic regions of western Canada and the USA, as well as in localized populations outside of the current epidemic, represent different genetic lineages. We characterized nucleotide variations in 67 genomic regions and selected 15 for the phylogenetic analysis. Using concordance of gene genealogies and distinct ecological characteristics, we identified two sibling phylogenetic species: Gc and Gs. Where the closely related Pinus ponderosa and P. jeffreyi are infested by localized populations of their respective beetles, Gc is present. In contrast, Gs is an exclusive associate of D. ponderosae mainly present on its primary host-tree P. contorta; however, in the current epidemic areas, it is also found in other pine species. These results suggest that the host-tree species and the beetle population dynamics may be important factors associated with the genetic divergence and diversity of fungal partners in the beetle-tree ecosystems. Gc represents the original G. clavigera holotype, and Gs should be described as a new species.

The role of phytopathogenicity in bark beetle-fungus symbioses: a challenge to the classic paradigm.

The idea that phytopathogenic fungi associated with tree-killing bark beetles are critical for overwhelming tree defenses and incurring host tree mortality, herein called the classic paradigm (CP), has driven research on bark beetle-fungus symbiosis for decades. It has also strongly influenced our views of bark beetle ecology. We discuss fundamental flaws in the CP, including the lack of consistency of virulent fungal associates with tree-killing bark beetles, the lack of correspondence between fungal growth in the host tree and the development of symptoms associated with a successful attack, and the ubiquity of similar associations of fungi with bark beetles that do not kill trees. We suggest that, rather than playing a supporting role for the host beetle (tree killing), phytopathogenicity performs an important role for the fungi. In particular, phytopathogenicity may mediate competitive interactions among fungi and support survival and efficient resource capture in living, defensive trees.