Symbiosis in the Pacific Ring of Fire : evolutionary-biology of Rhizopogon subgenus Villosuli as mutualists of Pseudotsuga

Rhizopogon is a large genus of ectomycorrhizal (EM) fungi that grows in association with host trees of the family Pinaceae. We have conducted a series of studies investigating the ecology and evolutionary biology of the EM symbiosis shared between R. subgenus Villosuli and trees of the genus Pseudotsuga. Two members of R. subgenus Villosuli, the sister taxa R. vinicolor and R. vesiculosus, are the primary focus of the first two investigations presented in this dissertation. These two species share a sympatric range in the Pacific Northwest where they share a host association with Pseudotsuga menziesii (Douglas fir). In Chapter two we perform a series of container plant experiments, which manipulate substrate stratification and competitive conditions, in order to investigate ecological factors influencing vertical resource partitioning between these species. From the findings of this work we conclude that priority effects and substrate heterogeneity allow R. vinicolor to maintain EM root tips colonized during independent mating events in the face of competition from individuals of R. vesiculosus, which compete for root tips through vegetative expansion. The need for high rates of mating in R. vinicolor populations to successfully compete with R. vesiculosus may be driving microevolutionary adaptation in these species and our second investigation seeks to elucidate the genetic mechanisms that underlie this adaptation. In Chapter three we report the draft genomes of R. vinicolor and R. vesiculosus, which were sequenced in order to characterize the structure and gene content of the loci responsible for mating-type recognition in these fungi. The functional traits of mating-type loci can have dramatic effects upon the population structure and adaptability of fungi and we have sought to characterize these loci to better understand the role of mating in competitive interactions between these species. We have found that the differential life history strategies that shape the population structure of R. vinicolor and R. vesiculosus correlate with differential structure of the mating type B-locus between these species. The B-locus is involved in recognition of compatible mates through the production of lipopeptide pheromones and complementary pheromone receptors. R. vinicolor possesses a greater diversity of both pheromones and receptors than R. vesiculosus and this property is likely reinforced by R. vinicolor's need for frequent mating events to compete successfully with R. vesiculosus. The loss of diversity in this genomic region is a derived trait in R. vesiculosus, which competes successfully through vegetative expansion of a few individuals independent of the frequency of mating. Rhizopogon species possess one of the highest rates of host specificity of any EM fungus and we hypothesize that R. subgenus Villosuli has undergone a process of comigration and cospeciation with their Pseudotsuga hosts. Chapter four seeks to test these hypotheses by conducting a genome-scale phylogeographical analysis of Rhizopogon species growing in association with Pseudotsuga species. We have performed a multinational and intercontinental sampling of Rhizopogon species from the Pacific Rim distribution of all Pseudotsuga species and sequenced low coverage genomes of Rhizopogon species from across this range. Phylogenetic analysis of a 1.97 mbp sequence alignment dataset mined from these genomes demonstrates that members of R. subgenus Villosuli have undergone a process of comigration and cospeciation with their Pseudotsuga hosts. Our work presents macroevolutionary evidence that the EM symbiosis between these genera evolved only once between the common ancestor of R. subgenus Villosuli and the common ancestor of Pseudotsuga in Western North America.Keywords: Vertical Partitioning, Fungal Mating, Competition, Rhizopogon, Pseudotsuga, Ectomycorrhizae, Cospeciation, Phylogeography, Comigratio

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.This is the publisher’s final pdf. The published article is copyrighted by the Mycological Society of America and can be found at: http://www.msafungi.org/Keywords: genet, Rhizopogon, ectomycorrhizal, genetic structure, spatial autocorrelation, fung

Co-invasive ectomycorrhizal fungi alter native soil fungal communities

Purpose: Pinaceae (pine family) trees are native to the Northern Hemisphere and their invasion into the Southern Hemisphere is a growing problem threatening biological diversity. Pinaceae are ectomycorrhizal (ECM) and their invasions are facilitated by non-native and co-invasive ECM fungi. Nothofagaceae species (southern beeches) are dominant overstory trees across large swaths of the Southern Hemisphere and are the only widespread ECM trees native to southern South America (SSA). This observational study investigates the in situ impact of Pinaceae invasions upon native soil fungi associated with Nothofagaceae hosts in SSA. Methods: We performed soil nutrient testing and metabarcode sequencing of fungi in the rhizosphere of Nothofagus antarctica and Nothofagus dombeyi invaded by Pinaceae trees to determine whether co-invasive fungi might impact native soil fungi. Sampling transects extended from invasions into adjacent Nothofagus stands without invasive Pinaceae. Results: The fungal community composition of the Nothofagaceae rhizosphere was dominated by plant-associated Mortierellaceae OTUs in metabarcode data. Mortierellaceae OTU relative abundance was significantly reduced near invasions of Pinus contorta (Pinaceae). Invasions of Pseudotsuga menziesii (Pinaceae) and Pinus contorta were associated with reduced relative abundance of Nothofagus-associated ECM OTUS in the Nothofagus rhizosphere. Pinus contorta invasions were also associated with reduced soil organic matter, total carbon, total phosphorus, and total nitrogen. Conclusion: Further empirical study is warranted to investigate the hypothesis that Mortierellaceae and Pinaceae-specific /suillus-rhizopogon ECM fungi compete for nutrients bound in soil organic matter. Such competition may have potential long-term legacy effects upon post-invasion restoration efforts and implications for Pinaceae invasions globally.Fil: Mujic, Alija Bajro. California State University; Estados UnidosFil: Policelli, Nahuel. Boston University; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Patagonia Norte. Instituto de Investigaciones en Biodiversidad y Medioambiente. Universidad Nacional del Comahue. Centro Regional Universidad Bariloche. Instituto de Investigaciones en Biodiversidad y Medioambiente; ArgentinaFil: Nuñez, Martin Andres. University Of Houston; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Patagonia Norte. Instituto de Investigaciones en Biodiversidad y Medioambiente. Universidad Nacional del Comahue. Centro Regional Universidad Bariloche. Instituto de Investigaciones en Biodiversidad y Medioambiente; ArgentinaFil: Truong, Camille. University of Florida; Estados UnidosFil: Smith, Matthew E.. University of Florida; Estados Unido

Unveiling new sequestrate Cortinarius species from northern Patagonian Nothofagaceae forests based on molecular and morphological data

Because of systematic sampling campaigns in the northern Patagonian Nothofagaceae forests of Argentina, several specimens of sequestrate fungi were collected. Some of those collections showed phylogenetic affinities and morphological similarities to members of the formerly recognized sequestrate genus Thaxterogaster, currently a synonym of Cortinarius on the basis of molecular data. Comparisons of macro- and micromorphological features and sequences of nuc rDNA internal transcribed spacer (ITS) regions have revealed that these collections belong to formerly undescribed species. The sequences of the four new taxa presented here, Cortinarius flavopurpureus, C. translucidus, C. nahuelhuapensis, and C. infrequens, were combined into a data set including additional sequences generated from herbarium collections and retrieved from public gene databases and analyzed by maximum likelihood and Bayesian inference methods. The four new species were resolved as distinct clades with strong support; at the same time, they showed unique morphological characteristics (hypogeous to subhypogeous habit, complete gasteromycetation, and spore shape and ornamentation) that separate them from previously described Cortinarius species. In addition, several undescribed and/or not previously sequenced species from these forests were detected through phylogenetic analysis of ectomycorrhizal root tip sequences. A key of characters to identify the sequestrate Cortinarius from Patagonia is provided.Fil: Pastor, Nicolás. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto Multidisciplinario de Biología Vegetal. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas Físicas y Naturales. Instituto Multidisciplinario de Biología Vegetal; ArgentinaFil: Chiapella, Jorge Oscar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto Multidisciplinario de Biología Vegetal. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas Físicas y Naturales. Instituto Multidisciplinario de Biología Vegetal; ArgentinaFil: Kuhar, José Francisco. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto Multidisciplinario de Biología Vegetal. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas Físicas y Naturales. Instituto Multidisciplinario de Biología Vegetal; ArgentinaFil: Mujic, Alija Bajro. University of Florida; Estados UnidosFil: Crespo, Esteban María. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnol.conicet - San Luis. Unidad de Adm.territorial; ArgentinaFil: Nouhra, Eduardo Ramon. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto Multidisciplinario de Biología Vegetal. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas Físicas y Naturales. Instituto Multidisciplinario de Biología Vegetal; Argentin

Comparative Genomics of the Ectomycorrhizal Sister Species Rhizopogon vinicolor and Rhizopogon vesiculosus (Basidiomycota: Boletales) Reveals a Divergence of the Mating Type B Locus.

Divergence of breeding system plays an important role in fungal speciation. Ectomycorrhizal fungi, however, pose a challenge for the study of reproductive biology because most cannot be mated under laboratory conditions. To overcome this barrier, we sequenced the draft genomes of the ectomycorrhizal sister species Rhizopogon vinicolor Smith and Zeller and R. vesiculosus Smith and Zeller (Basidiomycota, Boletales)-the first genomes available for Basidiomycota truffles-and characterized gene content and organization surrounding their mating type loci. Both species possess a pair of homeodomain transcription factor homologs at the mating type A-locus as well as pheromone receptor and pheromone precursor homologs at the mating type B-locus. Comparison of Rhizopogon genomes with genomes from Boletales, Agaricales, and Polyporales revealed synteny of the A-locus region within Boletales, but several genomic rearrangements across orders. Our findings suggest correlation between gene content at the B-locus region and breeding system in Boletales with tetrapolar species possessing more diverse gene content than bipolar species. Rhizopogon vinicolor possesses a greater number of B-locus pheromone receptor and precursor genes than R. vesiculosus, as well as a pair of isoprenyl cysteine methyltransferase genes flanking the B-locus compared to a single copy in R. vesiculosus Examination of dikaryotic single nucleotide polymorphisms within genomes revealed greater heterozygosity in R. vinicolor, consistent with increased rates of outcrossing. Both species possess the components of a heterothallic breeding system with R. vinicolor possessing a B-locus region structure consistent with tetrapolar Boletales and R. vesiculosus possessing a B-locus region structure intermediate between bipolar and tetrapolar Boletales

Comparative Genomics of the Ectomycorrhizal Sister Species Rhizopogon vinicolor and Rhizopogon vesiculosus (Basidiomycota: Boletales) Reveals a Divergence of the Mating Type B Locus.

Divergence of breeding system plays an important role in fungal speciation. Ectomycorrhizal fungi, however, pose a challenge for the study of reproductive biology because most cannot be mated under laboratory conditions. To overcome this barrier, we sequenced the draft genomes of the ectomycorrhizal sister species Rhizopogon vinicolor Smith and Zeller and R. vesiculosus Smith and Zeller (Basidiomycota, Boletales)-the first genomes available for Basidiomycota truffles-and characterized gene content and organization surrounding their mating type loci. Both species possess a pair of homeodomain transcription factor homologs at the mating type A-locus as well as pheromone receptor and pheromone precursor homologs at the mating type B-locus. Comparison of Rhizopogon genomes with genomes from Boletales, Agaricales, and Polyporales revealed synteny of the A-locus region within Boletales, but several genomic rearrangements across orders. Our findings suggest correlation between gene content at the B-locus region and breeding system in Boletales with tetrapolar species possessing more diverse gene content than bipolar species. Rhizopogon vinicolor possesses a greater number of B-locus pheromone receptor and precursor genes than R. vesiculosus, as well as a pair of isoprenyl cysteine methyltransferase genes flanking the B-locus compared to a single copy in R. vesiculosus Examination of dikaryotic single nucleotide polymorphisms within genomes revealed greater heterozygosity in R. vinicolor, consistent with increased rates of outcrossing. Both species possess the components of a heterothallic breeding system with R. vinicolor possessing a B-locus region structure consistent with tetrapolar Boletales and R. vesiculosus possessing a B-locus region structure intermediate between bipolar and tetrapolar Boletales