Phylogenetic and ecological analyses of soil and sporocarp DNA sequences reveal high diversity and strong habitat partitioning in the boreal ectomycorrhizal genus Russula (Russulales; Basidiomycota).

SUMMARY: *Although critical for the functioning of ecosystems, fungi are poorly known in high-latitude regions. Here, we provide the first genetic diversity assessment of one of the most diverse and abundant ectomycorrhizal genera in Alaska: Russula. *We analyzed internal transcribed spacer rDNA sequences from sporocarps and soil samples using phylogenetic methods, operational taxonomic unit (OTU) delimitations and ordinations to compare species composition in various types of boreal forest. *The genus Russula is highly diverse in Alaska, with at least 42 nonsingleton OTUs (soil) and 50 phylogroups (soil + sporocarp). Russula taxa showed strong habitat preference to one of the two major forest types in the sampled regions (black spruce and birch-aspen-white spruce), and some preference for soil horizon. *Our results show that the vast majority of Russula species are present in the soil samples, although some additional taxa are expected to be found with extended sampling. OTU diversity in black spruce forests was only one-third of the diversity observed in mixed upland forests. Our findings suggest that some of the diversity is niche based, especially along host and successional axes, because most OTUs predictably occurred in specific habitats, regardless of geographical location.

Mycorrhizal networks counteract competitive effects of canopy trees on seedling survival.

The dynamics of forest ecosystems depend largely on the survival of seedlings in their understories, but seedling survival is known to be limited by preemption of light and soil resources by overstory trees. It has been hypothesized that "common mycorrhizal networks," wherein roots of seedlings are linked to overstory trees by symbiotic mycorrhizal fungi, offset some or all of the negative effects of trees on seedlings. Here we report the results of an unambiguous experimental test of this hypothesis in a monodominant Pinus radiata forest. We also tested the hypothesis that adaptive differentiation among plant populations causes local plant genotypes to respond more positively to mycorrhizal networks than nonlocal plant genotypes. Our results demonstrate large positive effects of overstory mycorrhizal networks on seedling survival, along with simultaneous negative effects of tree roots, regardless of whether plant genotypes were locally derived. Physiological and leaf-chemistry measurements suggest that seedlings connected to common mycorrhizal networks benefited from increased access to soil water. The similar magnitude of the positive and negative overstory effects on seedlings and the ubiquity of mycorrhizal networks in forests suggest that mycorrhizal networks fundamentally influence the demographic and community dynamics of forest trees.

Molecular phylogenetic biodiversity assessment of arctic and boreal ectomycorrhizal Lactarius Pers. (Russulales; Basidiomycota) in Alaska, based on soil and sporocarp DNA.

Despite the critical roles fungi play in the functioning of ecosystems, especially as symbionts of plants and recyclers of organic matter, their biodiversity is poorly known in high-latitude regions. In this paper, we discuss the molecular diversity of one of the most diverse and abundant groups of ectomycorrhizal fungi: the genus Lactarius Pers. We analysed internal transcribed spacer rDNA sequences from both curated sporocarp collections and soil polymerase chain reaction clone libraries sampled in the arctic tundra and boreal forests of Alaska. Our genetic diversity assessment, based on various phylogenetic methods and operational taxonomic unit (OTU) delimitations, suggests that the genus Lactarius is diverse in Alaska, with at least 43 putative phylogroups, and 24 and 38 distinct OTUs based on 95% and 97% internal transcribed spacer sequence similarity, respectively. Some OTUs were identified to known species, while others were novel, previously unsequenced groups. Non-asymptotic species accumulation curves, the disparity between observed and estimated richness, and the high number of singleton OTUs indicated that many Lactarius species remain to be found and identified in Alaska. Many Lactarius taxa show strong habitat preference to one of the three major vegetation types in the sampled regions (arctic tundra, black spruce forests, and mixed birch-aspen-white spruce forests), as supported by statistical tests of UniFrac distances and principal coordinates analyses (PCoA). Together, our data robustly demonstrate great diversity and nonrandom ecological partitioning in an important boreal ectomycorrhizal genus within a relatively small geographical region. The observed diversity of Lactarius was much higher in either type of boreal forest than in the arctic tundra, supporting the widely recognized pattern of decreasing species richness with increasing latitude.

Increasing ecological inference from high throughput sequencing of fungi in the environment through a tagging approach.

High throughput sequencing methods are widely used in analyses of microbial diversity, but are generally applied to small numbers of samples, which precludes characterization of patterns of microbial diversity across space and time. We have designed a primer-tagging approach that allows pooling and subsequent sorting of numerous samples, which is directed to amplification of a region spanning the nuclear ribosomal internal transcribed spacers and partial large subunit from fungi in environmental samples. To test the method for phylogenetic biases, we constructed a controlled mixture of four taxa representing the Chytridiomycota, Zygomycota, Ascomycota and Basidiomycota. Following cloning and colony restriction fragment length polymorphism analysis, we found no significant difference in representation in 19 of the 23 tested primers. We also generated a clone library from two soil DNA extracts using two primers for each extract and compared 456 clone sequences. Community diversity statistics and contingency table tests applied to counts of operational taxonomic units revealed that the two DNA extracts differed significantly, while the pairs of tagged primers from each extract were indistinguishable. Similar results were obtained using UniFrac phylogenetic comparisons. Together, these results suggest that the pig-tagged primers can be used to increase ecological inference in high throughput sequencing projects on fungi.