Cascading speciation among mutualists and antagonists in a tree-beetle-fungi interaction.

Cascading speciation is predicted to occur when multiple interacting species diverge in parallel as a result of divergence in one species promoting adaptive differentiation in other species. However, there are few examples where ecological interactions among taxa have been shown to result in speciation that cascades across multiple trophic levels. Here, we test for cascading speciation occurring among the western pine beetle (Dendroctonus brevicomis), its primary host tree (Pinus ponderosa), and the beetle's fungal mutualists (Ceratocystiopsis brevicomi and Entomocorticium sp. B). We assembled genomes for the beetle and a fungal symbiont and then generated reduced representation genomic data (RADseq) from range-wide samples of these three interacting species. Combined with published data for the host tree, we present clear evidence that the tree, the beetle, and the fungal symbionts are all genetically structured into at least two distinct groups that have strongly codiverged with geographical isolation. We then combine our genomic results with diverse population and laboratory-based data to show evidence for reproductive isolation at each level of the cascade and for coevolution of both antagonistic and mutualistic species interactions within this complex network.

The role of temperature variability in stabilizing the mountain pine beetle-fungus mutualism.

As global climate patterns continue to change and extreme weather events become increasingly common, it is likely that many ecological interactions will be affected. One such interaction is the multipartite symbiosis that exists between the mountain pine beetle and two species of fungi, Grosmannia clavigera and Ophiostoma montium. In this mutualism, the fungi provide nutrition to the beetle, while the fungi benefit by being dispersed to new host trees. Multi-partite mutualisms are predicted to be unstable due to strong direct competition among symbionts or natural selection for superior over inferior mutualists. However, this mutualism has remained stable over long periods of evolutionary time. In this paper, we developed a temperature-based model for the spread of fungi within a tree and connected it to an existing model for mountain pine beetle development. Using this integrated model for fungal growth, we explored the possibility that temperature variability is a stabilizing mechanism for the mountain pine beetle-fungi mutualism. Of the three types of temperature variability we tested: intra-year, inter-year and variability due to transitioning between different thermal habitats (thermal migration), we found that thermal migration was the most robust stabilizing mechanism. Additionally, we found that the MPB attack density or spacing between fungal lesions also had a significant effect on the stability of the system. High attack densities or close lesion spacings also tended to stabilize the system, regardless of temperature.

Competition and coexistence in a multi-partner mutualism: interactions between two fungal symbionts of the mountain pine beetle in beetle-attacked trees.

Despite overlap in niches, two fungal symbionts of the mountain pine beetle (Dendroctonus ponderosae), Grosmannia clavigera and Ophiostoma montium, appear to coexist with one another and their bark beetle host in the phloem of trees. We sampled the percent of phloem colonized by fungi four times over 1 year to investigate the nature of the interaction between these two fungi and to determine how changing conditions in the tree (e.g., moisture) affect the interaction. Both fungi colonized phloem at similar rates; however, G. clavigera colonized a disproportionately larger amount of phloem than O. montium considering their relative prevalence in the beetle population. High phloem moisture appeared to inhibit fungal growth shortly after beetle attack; however, by 1 year, low phloem moisture likely inhibited fungal growth and survival. There was no inverse relationship between the percent of phloem colonized by G. clavigera only and O. montium only, which would indicate competition between the species. However, the percent of phloem colonized by G. clavigera and O. montium together decreased after 1 year, while the percent of phloem from which no fungi were isolated increased. A reduction in living fungi in the phloem at this time may have significant impacts on both beetles and fungi. These results indicate that exploitation competition occurred after a year when the two fungi colonized the phloem together, but we found no evidence of strong interference competition. Each species also maintained an exclusive area, which may promote coexistence of species with similar resource use.

Effects of water potential and solute on the growth and interactions of two fungal symbionts of the mountain pine beetle.

We investigated the effect of water potential (WP) on the growth of, and interaction between, two ophiostomatoid fungi, Grosmannia clavigera and Ophiostoma montium, associated with the mountain pine beetle (Dendroctonus ponderosae). The WP of malt extract agar was amended by adding potassium chloride (KCl) or sucrose. Growth of both fungi decreased with WP on KCl-amended media. Growth of G. clavigera also decreased with WP on sucrose-amended media, although growth was stimulated on these media compared to unamended treatments. Growth of O. montium remained relatively constant on sucrose-amended media, confounding the effect of WP on this species. Both fungi were able to colonize media occupied by the other species, but at a slower rate than on unoccupied media, indicating competition. In most treatments, G. clavigera grew faster than O. montium and colonized a greater area when the two fungi were inoculated concurrently but distant to one another on a Petri dish. However, when each fungus was inoculated adjacent to a 10-d-old well-established colony of the other species, O. montium colonized occupied media more effectively than G. clavigera considering the growth rate of each species alone. Thus, G. clavigera dominated primary (uncolonized) resources on most media, whereas O. montium was more effective in colonizing secondary (occupied) resources. The differential response of the two fungi to sucrose indicates that they may use different carbon sources, or use different carbon sources at different rates, in the tree. Fine-scale resource partitioning, differences in primary and secondary resource capture abilities, and the non-equilibrium dynamics in an attacked tree over time, could all act to promote the co-existence of two unit-restricted dispersers on a discontinuous resource.

Temperature determines symbiont abundance in a multipartite bark beetle-fungus ectosymbiosis.

In this study, we report evidence that temperature plays a key role in determining the relative abundance of two mutualistic fungi associated with an economically and ecologically important bark beetle, Dendroctonus ponderosae. The symbiotic fungi possess different optimal temperature ranges. These differences determine which fungus is vectored by dispersing host beetles as temperatures fluctuate over a season. Grosmannia clavigera is the predominant fungus carried by dispersing beetles during cool periods but decreases in prevalence as daily maximum temperatures approach 25 degrees C, and becomes extremely rare when temperatures reach or exceed 32 degrees C. In contrast, Ophiostoma montium increases in prevalence as temperatures approach 25 degrees C, and becomes the predominant symbiont dispersed when temperatures reach or exceed 32 degrees C. The possession of different optimal growth temperatures may facilitate the stable coexistence of the two fungi by supporting growth of each fungus at different times, minimizing direct competition. Furthermore, the beetle may reduce its risk of being left aposymbiotic by exploiting not one, but two symbionts, whose combined growth optima span a wide range of environmental conditions. The possession of multiple symbionts with different temperature tolerances may allow the beetle to occupy highly variable habitats over a wide geographic range. Such temperature-driven symbiont shifts are likely to have major consequences for both the host and its symbionts under current temperature regimes and those predicted to occur because of climate change.

Dietary benefits of fungal associates to an eruptive herbivore: potential implications of multiple associates on host population dynamics.

We used the mountain pine beetle (Dendroctonus ponderosae Hopkins) and its two fungal associates, Grosmannia clavigera and Ophiostoma montium, to study potential nutritional benefits of fungi to bark beetles. We tested for potential effects of feeding on phloem colonized by fungi on beetle performance in field and laboratory studies. The fungi increased nitrogen levels in the phloem of attacked trees by 40%, indicating that it may be an important source of dietary nitrogen for mountain pine beetles. However, nitrogen levels of phloem inoculated with fungi in the laboratory were similar to uncolonized phloem, indicating that the fungi may redistribute nitrogen from the sapwood to the phloem rather than increase absolute levels of nitrogen. Beetles emerging from attacked trees carrying G. clavigera were larger than beetles carrying O. montium, which in turn were larger than beetles lacking fungi. Results of experimental laboratory studies varied, likely because of differences in the growth and sporulation of fungi under artificial conditions. Results indicate that the two fungi may offer complementary benefits to the mountain pine beetle because larvae preferentially fed on phloem colonized by both fungi together over phloem colonized by one fungus or uncolonized phloem. Teneral adults preemergence fed on spores in pupal chambers when they were produced and consumed little phloem before emerging. Teneral adults mined extensively in the phloem before emerging when spores were not produced in the pupal chamber. Our results provide evidence for a nutritional role of fungi in the diet of bark beetles and show that multiple associates may differentially affect beetle performance, which could have important implications for bark beetle population dynamics.

Genetic Diversity and the Presence of Two Distinct Groups in Ophiostoma clavigerum Associated with Dendroctonus ponderosae in British Columbia and the Northern Rocky Mountains.

ABSTRACT The sapstaining fungal pathogen Ophiostoma clavigerum is associated with the mountain pine beetle (Dendroctonus ponderosae), which is currently the most destructive forest pest in North America. The genetic diversity of O. clavigerum populations collected from five sites in Canada and two sites in the United States was estimated with amplified fragment length polymorphism (AFLP) analysis. Genomic DNA from 170 O. clavigerum isolates was digested with EcoRI and PstI and amplified with six primer sets. A total of 469 AFLP markers consisting of 243 monomorphic and 226 polymorphic loci were scored. The overall genetic diversity of the O. clavigerum population was low (Hs = 0.0531) and the differentiation of the seven O. clavigerum populations was moderate (Phi = 0.143). Genetic distances among the populations were not significantly correlated with geographic distance (r = 0.3235, P = 0.074). Two genetically distinct groups in the O. clavigerum populations were shown by unique AFLP profiles and the unweighted pair group method with arithmetic averages. Further work to characterize biological differences between the two groups will be needed to confirm whether cryptic species are present in the O. clavigerum population.