Arbutus menziesii (Ericaceae) facilitates regeneration dynamics in mixed evergreen forests by promoting mycorrhizal fungal diversity and host connectivity.

PREMISE OF STUDY: In the mixed evergreen forests in the western United States, Arbutus menziesii is able to quickly resprout following disturbance and, as such, act as a nurse tree during forest regeneration. The mechanism for this nurse tree effect has frequently been ascribed to mycorrhizal fungi, but no detailed molecular-based studies of the mycorrhizal fungal communities associated with A. menziesii roots have yet been conducted. • METHODS: We examined the structure of the mycorrhizal fungal communities associated with A. menziesii in varying forest types and seasons and assessed the potential for common mycelial networks between A. menziesii and Pinaceae hosts, particularly Pseudotsuga menziesii. Study sites were located in the Klamath-Siskyou region in southern Oregon, United States. Molecular approaches were used to identify the mycorrhizal fungi (ITS rDNA) and plant hosts (trnL cDNA). • KEY RESULTS: Arbutus menziesii hosts a highly diverse mycorrhizal fungal community with similar composition to communities found on other angiosperm and Pinaceae hosts. Phylogenetic analyses of the mycorrhizal genus Piloderma revealed that host species and geographic location had little effect on fungal taxon relatedness. Multihost fungal taxa were significantly more frequent and abundant than single-host fungal taxa, and there was high potential for the formation of common mycelial networks with P. menziesii. • CONCLUSIONS: Our results suggest A. menziesii is a major hub of mycorrhizal fungal diversity and connectivity in mixed evergreen forests and plays an important role in forest regeneration by enhancing belowground resilience to disturbance.

Protecting rare, little known, old-growth forest-associated fungi in the Pacific Northwest USA: a case study in fungal conservation.

In 1994, 234 fungal species were listed for protection under the Survey and Manage Programme (SMP) guidelines of the Northwest Forest Plan (NWFP), an area encompassing 9.7 Mha of federal land in the states of Washington, Oregon, and northern California. The fungal species were presumed rare, associated with late-successional old-growth forests, and in need of protection not afforded by the major elements of the NWFP, including a vast system of forest reserves. The SMP guidelines thus called for protecting known sites while gathering information through surveys to learn more about species rarity, distribution, habitat requirements, and persistence concerns. If new information revealed that a species was not rare, not associated with late-successional old-growth forests, or that other aspects of the NWFP guidelines provided for their persistence (e.g. adequate protection provided by forest reserves), the species could be removed from the programme. The first assemblage of known site records from fungal herbaria yielded approximately 3500 records for all listed species. After 12 y of survey the total number of records increased four-fold to approximately 14,400 records. Fifty-five percent of species were found at 20 or fewer sites and considered rare; 42% were found at ten or fewer sites. Over the life of the programme, 39 species were removed from the programme primarily because they were no longer considered rare; many were found at several hundred sites throughout the NWFP area. Mapped distributions of known sites varied among species. When viewed across species, however, known sites were well distributed throughout the NWFP area, thus indicating the importance of the entire NWFP area in maintaining this diverse array of fungi. The NWFP relies on a system of late-successional forest reserves to act as a coarse-filter conservation approach to provide protection for late-successional species. Ninety percent of fungal species had some portion of their known sites within reserves, but only 34% of total sites occurred within reserves. Thus, for the rarest species, applying a fine-filter conservation approach that protects known sites outside of reserves becomes an important aspect of species protection. The SMP became a costly and controversial aspect of the NWFP and underwent several administrative revisions including attempts to end the programme in 2004 and 2007. Regardless of costs and controversy, this conservation programme represents an unprecedented attempt to conserve rare fungal species at a regional scale. One of the more important lessons learned is the absolute need for professional mycologists to develop long-term partnerships with resource managers and other scientists, and apply mycological expertise to complex species and habitat conservation issues in an interdisciplinary setting.

Forest stand age and the occurrence of chanterelle (Cantharellus) species in Oregon's central Cascade Mountains.

We describe watershed-scale habitat associations of three Cantharellus species with respect to stand age. During the 1998 autumn fruiting season we collected chanterelle sporocarps from 18 forest stands in and adjacent to the H.J. Andrews experimental forest in the central Cascade Mountains of Oregon. Sampled stands represented two age categories: old growth ( approximately 350+ y) and 40-60-y-old second growth naturally regenerated from clear-cut harvest. Old growth and second growth stands were spatially paired to reduce the chance of spurious habitat relationships caused by unmeasured correlated variables. We found stand age to be a good predictor of the distribution of C. subalbidus and C. formosus, but only marginally useful for predicting the occurrence of C. cascadensis. The odds that a randomly located chanterelle sporocarp will be C. subalbidus, compared to other chanterelles, are 3-23.5 times higher in old growth than in second growth. Alternatively, there is only a 4-38% chance that a randomly located sporocarp will be C. formosus in old growth. C. cascadensis was found to be uncommon throughout the study area and showed no significant habitat associations. The abundance of C. cascadensis increased substantially with decreasing elevation indicating that landscape features other than stand age may be more useful in predicting its occurrence.

Spatial analysis of within-population microsatellite variability reveals restricted gene flow in the Pacific golden chanterelle (Cantharellus formosus).

We examined the within-population genetic structure of the Pacific golden chanterelle (Cantharellus formosus) in a 50 y old forest stand dominated by Douglas-fir (Pseudotsuga menziesii) and western hemlock (Tsuga heterophylla) with spatial autocorrelation analysis. We tested the null hypothesis that multilocus genotypes possessed by chanterelle genets were randomly distributed within the study area. Fruit bodies from 203 C. formosus genets were collected from a 50 ha study plot. One hundred six unique multilocus genotypes were identified after scoring these collections at five microsatellite loci. Statistically significant positive spatial autocorrelation was detected indicating the presence of fine-scale genetic structure within the area. Repeated autocorrelation analyses with varied minimum distance classes (50-500 m) detected positive spatial genetic structure up to 400 m. Therefore nonrandom evolutionary processes (e.g., isolation by distance) can cause fine-scale genetic structure in C. formosus. The implications of this research for future broad-scale population studies of this species are that population samples should be separated by at least 400 m to be considered statistically independent. Sampling designs that account for fine-scale genetic structure will better characterize heterogeneity distributed across the landscape by avoiding the effects of pseudo replication.

Protecting rare, old-growth, forest-associated species under the Survey and Manage Program guidelines of the Northwest Forest Plan.

The Survey and Manage Program of the Northwest Forest Plan (NWFP) represents an unparalleled attempt to protect rare, little-known species associated with late-successional and old-growth forests on more than 9.7 million ha of federal lands. Approximately 400 species of amphibians, bryophytes, fungi, lichens, mollusks, vascular plants, arthropod functional groups, and one mammal were listed under this program because viability evaluations indicated the plan's network of reserve land allocations might not sustain the species over time. The program's standards and guidelines used an adaptive approach, protecting known sites and collecting new information to address concerns for species persistence and to develop management strategies. Since implementation in 1994, approximately 68,000 known sites have been recorded at an expense of several tens of millions of dollars. New knowledge from surveys reduced concern for nearly 100 species, and they were removed from the protection list. Although successful in protecting hundreds of rare species not typically considered in most conservation programs, some of the enacted conservation measures created conflicts in meeting other management objectives of the plan, particularly timber harvest. The program accrued important gains in knowledge, reduced uncertainty about conservation of a number of species, and developed new methods of species inventory that will be useful in future management planning and implementation at many scales. The program, however was not completed because of changes in land-management philosophy. Ongoing litigation regarding its termination and potential changes to the plan cast further uncertainty on how the original goal of maintaining persistence of late-successional and old-growth species will be met and measured. The outcomes, controversies, and management frustrations of the program exemplify the inherent difficulties in balancing broad, regional conservation goals with social and economic goals of the NWFP Defining acceptable trade-offs to reach that balance and developing practical conservation solutions remain challenges for the science and management communities. Lessons learned from the program provide a valuable biological and managerial reference to benefit future discussion on meeting those challenges.

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.

Analysis of nrDNA sequences and microsatellite allele frequencies reveals a cryptic chanterelle species Cantharellus cascadensis sp. nov. from the American Pacific Northwest.

In the Pacific Northwest, yellow chanterelles have long been referred to as Cantharellus cibarius, synonymous with the European yellow chanterelle. Broad scale genetic surveys of North American chanterelles with C. cibarius-like morphology have demonstrated that the nrDNA internal transcribed spacer exhibits length variability, suggesting that this common morphology masks a species complex. Recently researchers have used morphological and genetic data to identify the yellow chanterelle most frequently harvested from American Pacific Northwest forests as C. formosus, a species once thought to be rare in the region. We present three genetic data sets and one morphological data set that characterize a previously undescribed, species of yellow chanterelle from the central Cascade Mountains of Oregon. Phylogenetic analyses of the nrDNA large subunit and ITS regions show that C. cascadensis sp. nov., along with two other yellow chanterelle taxa (C. cibarius var. roseocanus and European C. cibarius), are more closely related to white chanterelles (C. subalbidus) than they are to C. formosus. Data from five microsatellite loci provide evidence that C. formosus, C. subalbidus, and C. cascadensis sp. nov. do not interbreed when they co-occur spatially and temporally in Douglas fir-western hemlock forests. This demonstrates that these three sympatric chanterelles are biological species with boundaries congruent with those delineated by nrDNA phylogenetic clades. Morphological data indicate that the colour of the pileus and shape of the stipe can be used to separate fresh collections of the two yellow species now known to co-occur in Douglas fir-western hemlock forests in Oregon.

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.

Biology of the ectomycorrhizal genus Rhizopogon. VI. Re-examination of infrageneric relationships inferred from phylogenetic analyses of ITS sequences.

Rhizopogon (Basidiomycota, Boletales) is a genus of hypogeous fungi that form ectomycorrhizal associations mostly with members of the Pinaceae. This genus comprises an estimated 100(+) species, with the greatest diversity found in coniferous forests of the Pacific northwestern United States. Maximum parsimony analyses of 54 nuclear ribosomal DNA internal transcribed spacer (ITS) sequences including 27 Rhizopogon and 10 Suillus species were conducted to test sectional relationships in Rhizopogon and examine phylogenetic relationships with the closely related epigeous genus, Suillus. Sequences from 10 Rhizopogon type collections were included in these analyses. Rhizopogon and Suillus were both monophyletic. Rhizopogon section Rhizopogon is not monophyletic and comprised two clades, one of which consisted of two well supported lineages characterized by several long insertions. Rhizopogon sections Amylopogon and Villosuli formed well supported groups, but certain species concepts within these sections were unresolved. Four species from section Fulviglebae formed a strongly supported clade within section Villosuli. Subgeneric taxonomic revisions are presented.

Biology of the ectomycorrhizal genus, Rhizopogon: III. Influence of co-cultured conifer species on mycorrhizal specificity with the arbutoid hosts Arctostaphylos uva-ursi and Arbutus menziesii.

Seedlings of Pseudotsuga menziesii (Mirb.) Franco, Pinus ponderosa Dougl. ex Laws, Arbutus menziesii Pursh., and cuttings of Arclast apliylos uva-ursi (L.) Spreng were grown in monoculture and in conifer-hardwood dual-culture combinations in the glasshouse and inoculated with spore slurries of six Rhizopogon species. The primary objectives were to assess and compare the pattern of host specificity between symbionts and to study the influence of co-cultured plants on ecromycorrhiza development. The Rhizopogon spp. ranged from genus-specific to multiple-host compatible. In monoculture, four Rhisopogon sp, (R. ellenae Smith, R. occidentalis Zeller & Dodge, R. smithii Hosfnrd and R. subcaerulescens Smith) formed ectomycorrhizas with Pinus ponderosa, and two Rhizopogon sp. (R. parksii Smith and R. vinicolor Smith) formed ectomycorrhizas with Pseudotsuga menziesii. None of the fungi tested developed ectomycorrhizas on Arbutus menziesii or Arctostaphylos uvaursi in monoculture. In dual culture, three of the four Rhizopogon species (R. ellenae, R. occidentalis and R. subcaerulescens) that formed ectomycorrhizas on Pinus ponderosa, formed some ectomycorrhizas on Arbutus menziesii and Arrtastaphvlos uva-ursi. Rhizopogon parksii and R. tinicoior only formed ectomycorrhizas on Pseudotsuga mensiesti.

Reciprocal transfer of carbon isotopes between ectomycorrhizal Betula papyrifera and Pseudotsuga menziesii.

Interspecific C transfer was studied in laboratory microcosms containing pairs of 6-month-old Betula papyrifera Marsh, and Pseudotsuga menziesii (Mirb.) Franca seedlings growing in individual, root-restrictive (28µm pore size) pouches filled with field soil. Interspecific transfer was examined by reciprocal labelling of seedlings with 13 CO2(gas) and 14 CO2(gas) . At the time of labelling, the root zones of ectomycorrhizal (EM) B. papyrifera and P. menziesii were interconnected by an extensive network of EM mycelium. Carbon transferred through EM connections was distinguished from that through soil pathways by comparing microcosms where interconnecting hyphae were left intact vs. those where they were severed immediately before labelling. Transfer was bidirectional, and represented 5 % of total isotope uptake by both B. papyrifera and P. menziesii together. P. menziesii received on average 50% more 14 C and 66% more 13 C from paper birch than vice versa, however, differences between species were not statistically significant. Neither net nor bidirectional transfer differed between severing treatments, leaving in question the relative importance of EM hyphae versus soil transfer pathways. The tendency for P. menziesii to receive more isotope than B. papyrifera corresponded with a 10-fold greater net photosynthetic rate per seedling and two-fold greater foliar N concentration of B. papyrifera than P. menziesii.

Occurrence of ectomycorrhizas on ericaceous and coniferous seedlings grown in soils from the Oregon Coast Range.

Seedlings of Gaultheria shallon, Pseudotsuga menziesii, Rhododendron macrophyllum, and Tsuga heterophylla were grown together in the greenhouse in soils from three young managed Douglas fir forests in the Oregon Coast Range. The objective of the study was to evaluate the ability of ericaceous plants and overstory conifers to share compatible mycorrhizal fungi in order to assess potential mycorrhizal linkages. Ericoid mycorrhizal fungi were examined in Gaultheria and Rhododendron to assess their assumed presence in soils of the Pacific Northwestern region of the United States. Nine ectomycorrhizal types were recognized on the conifers and two on the Ericaceae. All nine ectomycorrhizal types occurred on both conifer species, and the two ectomycorrhizal types on the ericaceous hosts resembled types associated with the conifer hosts. Ectomycorrhizal fungi occurred on all the conifer seedlings and in trace amounts on 26% of the ericaceous seedlings in the study. Ericoid mycorrhiza developed on all the ericaceous seedlings.