Kazuhide Nara

Towards global patterns in the diversity and community structure of ectomycorrhizal fungi

Global species richness patterns of soil micro-organisms remain poorly understood compared to macro-organisms. We use a global analysis to disentangle the global determinants of diversity and community composition for ectomycorrhizal (EcM) fungi-microbial symbionts that play key roles in plant nutrition in most temperate and many tropical forest ecosystems. Host plant family has the strongest effect on the phylogenetic community composition of fungi, whereas temperature and precipitation mostly affect EcM fungal richness that peaks in the temperate and boreal forest biomes, contrasting with latitudinal patterns of macro-organisms. Tropical ecosystems experience rapid turnover of organic material and have weak soil stratification, suggesting that poor habitat conditions may contribute to the relatively low richness of EcM fungi, and perhaps other soil biota, in most tropical ecosystems. For EcM fungi, greater evolutionary age and larger total area of EcM host vegetation may also contribute to the higher diversity in temperate ecosystems. Our results provide useful biogeographic and ecological hypotheses for explaining the distribution of fungi that remain to be tested by involving next-generation sequencing techniques and relevant soil metadata.

ECTOMYCORRHIZAL FUNGI ON ESTABLISHED SHRUBS FACILITATE SUBSEQUENT SEEDLING ESTABLISHMENT OF SUCCESSIONAL PLANT SPECIES

Ectomycorrhizal (ECM) fungi are symbiotic microorganisms that can significantly improve the water and nutrient uptake of host plants. With reference to ECM fungi, we studied the effects of established shrubs on the subsequent seedling establishment of three woody plant species in a volcanic desert on Mt. Fuji, Japan, during early primary succession. Salix reinii, an alpine dwarf willow, is the pioneer ECM plant at this research site, and many ECM fungi have colonized established willow shrubs. ECM formation was found to be high on S. reinii seedlings that had been transplanted near established willow shrubs. The growth and nitrogen content of the seedlings increased significantly with the numbers of associated ECM fungal species and ECM root tips. ECM formation on transplanted seedlings of Betula ermanii and Larix kaempferi, two subsequent successional tree species, was also higher near the established willow shrubs. Molecular identification showed that almost all of the ECM fungi on transplanted seedlings of the three plant species were of the same species as those observed on the established willow shrubs. These results indicate that ECM fungi associated with established willow shrubs are essential in facilitating seedling establishment of successional plant species in the early successional volcanic desert.

Biogeography of ectomycorrhizal fungi associated with alders (Alnus spp.) in relation to biotic and abiotic variables at the global scale

· Much of the macroecological information about microorganisms is confounded by the lack of standardized methodology, paucity of metadata and sampling effect of a particular substrate or interacting host taxa. · This study aims to disentangle the relative effects of biological, geographical and edaphic variables on the distribution of Alnus-associated ectomycorrhizal (ECM) fungi at the global scale by using comparable sampling and analysis methods. · Ribosomal DNA sequence analysis revealed 146 taxa of ECM fungi from 22 Alnus species across 96 sites worldwide. Use of spatial and phylogenetic eigenvectors along with environmental variables in model selection indicated that phylogenetic relations among host plants and geographical links explained 43 and 10%, respectively,in ECM fungal community composition, whereas soil calcium concentration positively influenced taxonomic richness. · Intrageneric phylogenetic relations among host plants and regional processes largely account for the global biogeographic distribution of Alnus-associated ECM fungi. The biogeography of ECM fungi is consistent with ancient host migration patterns from Eurasia to North America and from southern Europe to northern Europe after the last glacial maximum, indicating codispersal of hosts and their mycobionts.

Genetic structure and reproduction dynamics of Salix reinii during primary succession on Mount Fuji, as revealed by nuclear and chloroplast microsatellite analysis

The early stage of volcanic desert succession is underway on the southeastern slope of Mount Fuji. We used markers of nuclear microsatellites (simple sequence repeats; SSR) and chloroplast microsatellites (cpSSR) to investigate the population genetic structure and reproduction dynamics of Salix reinii, one of the dominant pioneer shrubs in this area. The number of S. reinii genets in a patch and the area of the largest genet within the patch increased with patch area, suggesting that both clonal growth and seedling recruitment are involved in the reproduction dynamics of S. reinii. Five polymorphic cpSSR markers were developed for S. reinii by sequencing the noncoding regions between universal sequences in the chloroplast genome. Nineteen different cpSSR haplotypes were identified, indicating that S. reinii pioneer genets were created by the long‐distance dispersal of seeds originating from different mother genets around the study site, where all vegetation was destroyed during the last eruption. Furthermore, the clustered distributions of different haplotypes within each patch or plot suggested that newly colonized genets tended to be generated from seeds dispersed near the initially established mother genets. These results revealed that the establishment of the S. reinii population on the southeastern slope of Mount Fuji involved two sequential modes of seed dispersal: long‐distance dispersal followed by short‐distance dispersal.

Historical biogeography and diversification of truffles in the Tuberaceae and their newly identified southern hemisphere sister lineage.

Truffles have evolved from epigeous (aboveground) ancestors in nearly every major lineage of fleshy fungi. Because accelerated rates of morphological evolution accompany the transition to the truffle form, closely related epigeous ancestors remain unknown for most truffle lineages. This is the case for the quintessential truffle genus Tuber, which includes species with socio-economic importance and esteemed culinary attributes. Ecologically, Tuber spp. form obligate mycorrhizal symbioses with diverse species of plant hosts including pines, oaks, poplars, orchids, and commercially important trees such as hazelnut and pecan. Unfortunately, limited geographic sampling and inconclusive phylogenetic relationships have obscured our understanding of their origin, biogeography, and diversification. To address this problem, we present a global sampling of Tuberaceae based on DNA sequence data from four loci for phylogenetic inference and molecular dating. Our well-resolved Tuberaceae phylogeny shows high levels of regional and continental endemism. We also identify a previously unknown epigeous member of the Tuberaceae – the South American cup-fungus Nothojafnea thaxteri (E.K. Cash) Gamundí. Phylogenetic resolution was further improved through the inclusion of a previously unrecognized Southern hemisphere sister group of the Tuberaceae. This morphologically diverse assemblage of species includes truffle (e.g. Gymnohydnotrya spp.) and non-truffle forms that are endemic to Australia and South America. Southern hemisphere taxa appear to have diverged more recently than the Northern hemisphere lineages. Our analysis of the Tuberaceae suggests that Tuber evolved from an epigeous ancestor. Molecular dating estimates Tuberaceae divergence in the late Jurassic (∼156 million years ago), with subsequent radiations in the Cretaceous and Paleogene. Intra-continental diversification, limited long-distance dispersal, and ecological adaptations help to explain patterns of truffle evolution and biodiversity.

Competition between ectomycorrhizal fungi colonizing Pinus densiflora

Abstract Interactive competition of Pisolithus tinctorius (Pers.) Coker et Couch with an unidentified species Tanashi 01 and Suillus luteus (L.: Fr.) S. F. Gray was investigated using a rhizobox. Pinus densiflora Sieb. et Zucc. was used as the host plant and mycelia were distinguished by hyphal color. The speed of mycelial spread differed between the fungi;P. tinctorius and Tanashi 01 grew faster than S. luteus. A P. tinctorius mycorrhizal seedling and a Tanashi 01 mycorrhizal seedling were transplanted on opposite sides of the rhizobox. The mycelia and mycorrhizae of P. tinctorius were overgrown by Tanashi 01 hyphae and development of P. tinctorius was gradually inhibited. The areas occupied by mycelia and mycorrhiza of P. tinctorius decreased by 52% and 37%, respectively, 154 days after transplantation relative to that at 91 days. In the overlap area of P. tinctorius and Tanashi 01, the latter fungus infected new root tips emerging from P. tinctorius mycorrhiza, which lacked a mantle of P. tinctorius hyphae, and formed a composite mycorrhizal structure. P. tinctorius mycorrhizae were progressively replaced by Tanashi 01 mycorrhizae. Mycelial spread of P. tinctorius and S. luteus were naturally inhibited but there was no interaction in mycorrhizal formation.

Germination and infectivity of ectomycorrhizal fungal spores in relation to their ecological traits during primary succession

The spores of ectomycorrhizal fungi (EMF) play critical roles in the population and community development of EMF. Here, the germination and infectivity of EMF spores are examined with reference to the ecological traits of the EMF species. Spores were collected from 12 EMF species, whose successional patterns have been studied in the volcanic desert on Mount Fuji, Japan. Spore germination experiments were conducted with host plants (Salix reinii), with nonhost plants (Polygonum cuspidatum), and without plants. The mycorrhizal formation ability of spores was also examined in seven EMF using spore inoculation experiments. To determine the effects of the spore preservation period, both experiments were repeated up to 1 yr after spore collection. Spore germination was very low in the absence of host plants. In the presence of hosts, even 30 d after spore collection, spore germination was significantly enhanced in all pioneer EMF (c. 20%) but less so in late-stage EMF (< 5%), except in Hebeloma species. Mycorrhizal formation from spores was also greater in pioneer EMF but was significantly reduced by 1 yr of spore preservation. High spore germination and infectivity of pioneer EMF should enable these species to colonize disturbed and isolated areas in accordance with their ecological traits.

Extensive gene flow over Europe and possible speciation over Eurasia in the ectomycorrhizal basidiomycete Laccaria amethystina complex

Biogeographical patterns and large‐scale genetic structure have been little studied in ectomycorrhizal (EM) fungi, despite the ecological and economic importance of EM symbioses. We coupled population genetics and phylogenetic approaches to understand spatial structure in fungal populations on a continental scale. Using nine microsatellite markers, we characterized gene flow among 16 populations of the widespread EM basidiomycete Laccaria amethystina over Europe (i.e. over 2900 km). We also widened our scope to two additional populations from Japan (104 km away) and compared them with European populations through microsatellite markers and multilocus phylogenies, using three nuclear genes (NAR, G6PD and ribosomal DNA) and two mitochondrial ribosomal genes. European L. amethystina populations displayed limited differentiation (average FST = 0.041) and very weak isolation by distance (IBD). This panmictic European pattern may result from effective aerial dispersal of spores, high genetic diversity in populations and mutualistic interactions with multiple hosts that all facilitate migration. The multilocus phylogeny based on nuclear genes confirmed that Japanese and European specimens were closely related but clustered on a geographical basis. By using microsatellite markers, we found that Japanese populations were strongly differentiated from the European populations (FST = 0.416), more than expected by extrapolating the European pattern of IBD. Population structure analyses clearly separated the populations into two clusters, i.e. European and Japanese clusters. We discuss the possibility of IBD in a continuous population (considering some evidence for a ring species over the Northern Hemisphere) vs. an allopatric speciation over Eurasia, making L. amethystina a promising model of intercontinental species for future studies.

Extensive gene flow over Europe and possible speciation

Biogeographical patterns and large‐scale genetic structure have been little studied in ectomycorrhizal (EM) fungi, despite the ecological and economic importance of EM symbioses. We coupled population genetics and phylogenetic approaches to understand spatial structure in fungal populations on a continental scale. Using nine microsatellite markers, we characterized gene flow among 16 populations of the widespread EM basidiomycete Laccaria amethystina over Europe (i.e. over 2900 km). We also widened our scope to two additional populations from Japan (104 km away) and compared them with European populations through microsatellite markers and multilocus phylogenies, using three nuclear genes (NAR, G6PD and ribosomal DNA) and two mitochondrial ribosomal genes. European L. amethystina populations displayed limited differentiation (average FST = 0.041) and very weak isolation by distance (IBD). This panmictic European pattern may result from effective aerial dispersal of spores, high genetic diversity in populations and mutualistic interactions with multiple hosts that all facilitate migration. The multilocus phylogeny based on nuclear genes confirmed that Japanese and European specimens were closely related but clustered on a geographical basis. By using microsatellite markers, we found that Japanese populations were strongly differentiated from the European populations (FST = 0.416), more than expected by extrapolating the European pattern of IBD. Population structure analyses clearly separated the populations into two clusters, i.e. European and Japanese clusters. We discuss the possibility of IBD in a continuous population (considering some evidence for a ring species over the Northern Hemisphere) vs. an allopatric speciation over Eurasia, making L. amethystina a promising model of intercontinental species for future studies.

The mid-domain effect in ectomycorrhizal fungi: range overlap along an elevation gradient on Mount Fuji, Japan

Mid-domain effect (MDE) models predict that the random placement of species’ ranges within a bounded geographical area leads to increased range overlap and species richness in the center of the bounded area. These models are frequently applied to study species-richness patterns of macroorganisms, but the MDE in relation to microorganisms is poorly understood. In this study, we examined the characteristics of the MDE in richness patterns of ectomycorrhizal (EM) fungi, an ecologically important group of soil symbionts. We conducted intensive soil sampling to investigate overlap among species ranges and the applicability of the MDE to EM fungi in four temperate forest stands along an elevation gradient on Mount Fuji, Japan. Molecular analyses using direct sequencing revealed 302 EM fungal species. Of 73 EM fungal species found in multiple stands, 72 inhabited a continuous range along the elevation gradient. The maximum overlap in species range and the highest species richness occurred at elevations in the middle of the gradient. The observed richness pattern also fit within the 95% confidence interval of the mid-domain null model, supporting the role of the MDE in EM fungal richness. Deviation in observed richness from the mean of the mid-domain null estimation was negatively correlated with some environmental factors, including precipitation and soil C/N, indicating that unexplained richness patterns could be driven by these environmental factors. Our results clearly support the existence of microbial species’ ranges along environmental gradients and the potential applicability of the MDE to better understand microbial diversity patterns.