Little to no genetic structure in the ectomycorrhizal basidiomycete Suillus spraguei (Syn. S. pictus) across parts of the Northeastern USA.

Population genetic studies of ectomycorrhizal fungi at scales larger than 100 km are still relatively rare with highly variable results. In this study, we determined the population genetic structure of the ectomycorrhizal basidiomycete Suillus spraguei over distances up to 600 km in northeastern USA forests. S. spraguei associates exclusively with five-needled pines and only with white pine (Pinus strobus) in the eastern USA. We used six microsatellite loci to assess the genetic structure between eight sites sampled in the Adirondack Park of New York and seven sites sampled in other forests of New York, Pennsylvania, and Massachusetts. Except for one site, little to no genetic differentiation was detected in pairwise comparisons of the sites (F ST = 0 to 0.05). Only one site was moderately differentiated from most other sites (F ST = 0.02 to 0.15). The Mantel test showed no significant correlation between genetic and geographic distances (isolation by distance; R (2) = 0.003, P = 0.3). The STRUCTURE analysis also supported the presence of a single cluster (K = 1).

Within-population genetic structure differs between two sympatric sister-species of ectomycorrhizal fungi, Rhizopogon vinicolor and R. vesiculosus.

Using spatial autocorrelation analysis, we examined the within-population genetic structure of Rhizopogon vinicolor and R. vesiculosus, two hypogeous ectomycorrhizal (EM) species that are sympatric sister taxa known to differ in their clonal structure. We collected 121 sporocarps and 482 tuberculate EM of both species from a 20 ha forest stand dominated by Douglas-fir (Pseudotsuga menziesii). Field collections were identified to species with restriction fragment length polymorphism analysis of the nuclear ribosomal internal transcribed spacer. Five and six microsatellite markers were used to characterize the genetic diversity of EM and sporocarp samples from R. vesiculosus and R. vinicolor respectively. After correcting for genet structure, spatial autocorrelation analyses of the EM samples were used to test the null hypothesis that multilocus genotypes characterized from each species were randomly distributed within the study area. We detected positive and statistically significant fine-scale genetic structure up to 120 m within the R. vesiculosus sample. In contrast, no spatial genetic structure was evident for R. vinicolor, indicating that the genotypes characterized for this species were randomly distributed throughout the study area. Differences in statistical power or the nuclear count of basidiospores are unlikely agents of the genetic patterns observed. Our results suggest that differences in reproductive output or competitive ability may act individually or in combination to create clusters of similar genotypes for R. vesiculosus throughout the study area.

Bioretention column study of bacteria community response to salt-enriched artificial stormwater.

Cold climate cities with green infrastructure depend on soil bacteria to remove nutrients from road salt-enriched stormwater. Our research examined how bacterial communities in laboratory columns containing bioretention media responded to varying concentrations of salt exposure from artificial stormwater and the effect of bacteria and salt on column effluent concentrations. We used a factorial design with two bacteria treatments (sterile, nonsterile) and three salt concentrations (935, 315, and 80 ppm), including a deionized water control. Columns were repeatedly saturated with stormwater or deionized and then drained throughout 5 wk, with the last week of effluent analyzed for water chemistry. To examine bacterial communities, we extracted DNA from column bioretention media at time 0 and at week 5 and used molecular profiling techniques to examine bacterial community changes. We found that bacterial community taxa changed between time 0 and week 5 and that there was significant separation between taxa among salt treatments. Bacteria evenness was significantly affected by stormwater treatment, but there were no differences in bacterial richness or diversity. Soil bacteria and salt treatments had a significant effect on the effluent concentration of NO, PO, Cu, Pb, and Zn based on ANOVA tests. The presence of bacteria reduced effluent NO and Zn concentrations by as much as 150 and 25%, respectively, while having a mixed effect on effluent PO concentrations. Our results demonstrate how stormwater can affect bacterial communities and how the presence of soil bacteria improves pollutant removal by green infrastructure.

Analysis of genet size and local gene flow in the ectomycorrhizal basidiomycete Suillus spraguei (synonym S. pictus).

Several recent fine-scale genetic structure studies of ectomycorrhizal fungi have reported significant spatial clustering of genets with similar genotypes, supporting locally restricted gene flow. In this study we used genotype data from microsatellite markers and spatial autocorrelation analysis to examine local gene flow in Suillus spraguei at distances up to 2 km. Previously developed microsatellite markers for S. spraguei from Japan were unsuccessful at amplifying DNA isolated from sporocarps found in New York state, and other research suggested that both are disjunct species. Novel microsatellite markers therefore were developed with New York specimens. We identified nine polymorphic microsatellite loci and developed primer sets to amplify these regions. We tested the efficiency of the primer sets on 50 sporocarps collected from a natural Pinus strobus stand. The majority of the markers were in Hardy-Weinberg and linkage equilibrium. The location of all sampled sporocarps was recorded and used along with multilocus genotype data to create a genet map. The distance between sporocarps with the same multilocus genotype was small (≤ 7.65 m) and the majority of sporocarps collected were genetically unique, suggesting frequent spore establishment and sexual recombination on this site. Spatial autocorrelation analysis did not support clustering of similar genotypes, suggesting few restrictions to gene flow within this local population.

Architecture of the wood-wide web: Rhizopogon spp. genets link multiple Douglas-fir cohorts.

*The role of mycorrhizal networks in forest dynamics is poorly understood because of the elusiveness of their spatial structure. We mapped the belowground distribution of the fungi Rhizopogon vesiculosus and Rhizopogon vinicolor and interior Douglas-fir trees (Pseudotsuga menziesii var. glauca) to determine the architecture of a mycorrhizal network in a multi-aged old-growth forest. *Rhizopogon spp. mycorrhizas were collected within a 30 x 30 m plot. Trees and fungal genets were identified using multi-locus microsatellite DNA analysis. Tree genotypes from mycorrhizas were matched to reference trees aboveground. Two trees were considered linked if they shared the same fungal genet(s). *The two Rhizopogon species each formed 13-14 genets, each colonizing up to 19 trees in the plot. Rhizopogon vesiculosus genets were larger, occurred at greater depths, and linked more trees than genets of R. vinicolor. Multiple tree cohorts were linked, with young saplings established within the mycorrhizal network of Douglas-fir veterans. A strong positive relationship was found between tree size and connectivity, resulting in a scale-free network architecture with small-world properties. *This mycorrhizal network architecture suggests an efficient and robust network, where large trees play a foundational role in facilitating conspecific regeneration and stabilizing the ecosystem.

Bacterial communities associated with tuberculate ectomycorrhizae of Rhizopogon spp.

We have previously reported the design of a new PCR primer pair that allows amplification of a broad range of eubacterial 16S rDNA sequences from ectomycorrhizae (ECM) without co-amplification of plastid or mitochondrial sequences. Here, we report using a similar primer combination to generate three small 16S rDNA libraries from tuberculate ECM of Rhizopogon spp., two from R. vinicolor ECM (libraries Rvi18 and Rvi24) and one from R. vesiculosus ECM (library Rve13). At the class level, libraries were dominated by sequences from the Alphaproteobacteria, Gammaproteobacteria, and Acidobacteria, with some Sphingobacteria, Actinobacteria, Planctomycetacia, and Verrucomicrobiae present as well. Based on the parsimony test implemented in TreeClimber, libraries Rvi18 and Rvi24 were significantly different from Rve13 at the alpha = 0.05 level, while they were only borderline significantly different from each other (p = 0.07). Differences between Rvi and Rve libraries were primarily due to differences in the number of Alphaproteobacteria sequences and specifically sequences from the Rhizobiales, which were more common in the Rve13 library. It is currently unknown what drives these differences between eubacterial communities. Amplification success for eubacterial 16S rDNA sequences was generally low in this study indicating low abundance of bacteria on tuberculate ECM. Attempts to amplify nitrogenase reductase (nifH) sequences were unsuccessful.

Molecular analysis of bacterial communities associated with the roots of Douglas fir (Pseudotsuga menziesii) colonized by different ectomycorrhizal fungi.

We studied the effect of ectomycorrhizal fungi on bacterial communities colonizing roots of Douglas fir (Pseudotsuga menziesii). Mycorrhizal tips were cleaned of soil and separated based on gross morphological characteristics. Sequencing of the internal transcribed spacers of the nuclear rRNA gene cluster indicated that the majority of the tips were colonized by fungi in the Russulaceae, with the genera Russula and Lactarius comprising 70% of the tips. Because coamplification of organellar 16S rRNA genes can interfere with bacterial community analysis of root tips, we developed and tested a new primer pair that permits amplification of bacterial 16S rRNA genes but discriminates more effectively against organellar sequences than commonly used bacterial primer sets. We then used terminal restriction fragment length polymorphism (T-RFLP) and sequence analysis of the 16S rRNA gene to examine differences in bacterial communities associated with the mycorrhizal tips. Cluster analysis of T-RFLP profiles indicated that there were different bacterial communities among the root tips; however, the communities did not seem to be affected by the taxonomic identity of the ectomycorrhizal fungi. Terminal restriction fragment profiling and sequencing of cloned partial 16S rRNA genes indicated that most bacteria on the ectomycorrhizal tips were related to the Alphaproteobacteria and the Bacteroidetes group.

Soil bacterial diversity in a loblolly pine plantation: influence of ectomycorrhizas and fertilization.

We studied the effect of ectomycorrhizas and fertilization on soil microbial communities associated with roots of 10-year-old loblolly pine. Ectomycorrhizas were identified using a combination of community terminal restriction fragment profiling and matching of individual terminal restriction fragments to those produced from ectomycorrhizal clones and sequences recovered from roots and sporocarps. Differences between bacterial communities were initially determined using cluster analysis on community terminal restriction fragment profiles and through subsequent recovery of 16S rDNA clones. Analysis of bacterial clones revealed that terminal restriction fragment length was often shared between taxonomically dissimilar bacterial types. Consequently, we could not reliably infer the identity of peaks in the bacterial community profile with some exceptions, notably chloroplast rDNA that generated an approximate peak size of 80.2 bp. Fertilization increased the frequency of a Piloderma-like ectomycorrhiza. However, we did not detect clear effects of fertilization or the presence of viable ectomycorrhizas on bacterial communities. Bacterial communities seemed to be determined largely by the carbon and nitrogen content of soil. These results suggest that important soil microbial groups respond differently to soil conditions and management practices, with ectomycorrhizal communities reflecting past nutrient conditions and bacterial communities reflecting current environmental conditions of soil microsites.

Patterns of vegetative growth and gene flow in Rhizopogon vinicolor and R. vesiculosus (Boletales, Basidiomycota).

We have collected sporocarps and tuberculate ectomycorrhizae of both Rhizopogon vinicolor and Rhizopogon vesiculosus from three 50 x 100 m plots located at Mary's Peak in the Oregon Coast Range (USA); linear map distances between plots ranged from c. 1 km to c. 5.5 km. Six and seven previously developed microsatellite markers were used to map the approximate size and distribution of R. vinicolor and R. vesiculosus genets, respectively. Genetic structure within plots was analysed using spatial autocorrelation analyses. No significant clustering of similar genotypes was detected in either species when redundant samples from the same genets were culled from the data sets. In contrast, strong clustering was detected in R. vesiculosus when all samples were analysed, but not in R. vinicolor. These results demonstrate that isolation by distance does not occur in either species at the intraplot sampling scale and that clonal propagation (vegetative growth) is significantly more prevalent in R. vesiculosus than in R. vinicolor. Significant genetic differentiation was detected between some of the plots and appeared greater in the more clonal species R. vesiculosus with Phi(ST) values ranging from 0.010 to 0.078*** than in R. vinicolor with Phi(ST) values ranging from -0.002 to 0.022** (*P < 0.05, **P < 0.01, ***P < 0.001). When tested against the null hypothesis of no relationship between individuals, parentage analysis detected seven likely parent/offspring pairs in R. vinicolor and four in R. vesiculosus (alpha = 0.001). Of these 11 possible parent/offspring pairs, only two R. vinicolor pairs were still supported as parent/offspring when tested against the alternative hypothesis of being full siblings (alpha = 0.05). In the latter two cases, parent and offspring were located at approximately 45 m and 28 m from each other. Challenges to parentage analysis in ectomycorrhizal fungi are discussed.

Taxonomy of the Rhizopogon vinicolor species complex based on analysis of ITS sequences and microsatellite loci.

We are re-addressing species concepts in the Rhizopogon vinicolor species complex (Boletales, Basidiomycota) using sequence data from the internal-transcribed spacer (ITS) region of the nuclear ribosomal repeat, as well as genotypic data from five microsatellite loci. The R. vinicolor species complex by our definition includes, but is not limited to, collections referred to as R. vinicolor Smith, R. diabolicus Smith, R. ochraceisporus Smith, R. parvulus Smith or R. vesiculosus Smith. Holo- and/or paratype material for the named species is included. Analyses of both ITS sequences and microsatellite loci separate collections of the R. vinicolor species complex into two distinct clades or clusters, suggestive of two biological species that subsequently are referred to as R. vinicolor sensu Kretzer et al and R. vesiculosus sensu Kretzer et al. Choice of the latter names, as well as morphological characters, are discussed.