Reproductive isolation among cryptic species in the ectomycorrhizal genus Strobilomyces: population-level CAPS marker-based genetic analysis

Among the higher fungi, reproductively isolated cryptic species exist that are morphologically difficult to distinguish owing to a lack of taxonomically useful morphological characters. Mating tests are helpful for detecting reproductive isolation between cryptic species, but are often difficult to perform for higher fungi, especially ectomycorrhizal fungi. In order to identify cryptic species of the ectomycorrhizal genus Strobilomyces more efficiently, lineages were defined based on the nucleotide sequence of two mitochondrial genes. Then the gene flow among lineages was measured using cleaved amplified polymorphic sequences (CAPS) markers designed for single copy nuclear genes. No heterozygosity was observed between different lineages, but within the same lineage heterozygosity was present at the ratio expected given Hardy Weinberg equilibrium. These results show that the mtDNA lineages are separate Mendelian populations, possibly cryptic species that are reproductively isolated from each other.     

Genome wide AFLP markers support cryptic species in Coniophora (Boletales)

Numerous fungal morphospecies include cryptic species that routinely are detected by sequencing a few unlinked DNA loci. However, whether the patterns observed by multi-locus sequencing are compatible with genome wide data, such as amplified fragment length polymorphisms (AFLPs), is not well known for fungi. In this study we compared the ability of three DNA loci and AFLP data to discern between cryptic fungal lineages in the three morphospecies Coniophora olivacea, Coniophora arida, and Coniophora puteana. The sequences and the AFLP data were highly congruent in delimiting the morphotaxa into multiple cryptic species. However, while the DNA sequences indicated introgression or hybridization between some of the cryptic lineages the AFLP data did not. We conclude that as few as three polymorphic DNA loci was sufficient to recognize cryptic lineages within the studied Coniophora taxa. However, based on analyses of a few (three) sequenced loci the hybridization could not easily be distinguished from incomplete lineage sorting. Hence, great caution should be taken when concluding about hybridization based on data from just a few loci.     

Habitat association in two genetic groups of the insect-pathogenic fungus metarhizium anisopliae: uncovering cryptic species?

Strains of insect-pathogenic fungi with high virulence toward certain pest insects have great potential for commercial biological control applications. Identifying such strains has been a central theme in using fungi for biological control. This theme is supported by a persistent paradigm in insect pathology which suggests that the host insect is the predominant influence on the population genetics of insect-pathogenic fungi. In this study, a population genetics analysis of the insect-pathogenic fungus Metarhizium anisopliae from forested and agricultural habitats in Ontario, Canada, showed a nonrandom association of alleles between two distinct, reproductively isolated groups (index of multilocus association = 1.2). Analyses of the mitochondrial DNA showed no differences between the groups. The two groups were associated with different habitat types, and associations with insect hosts were not found. The group from forested areas showed an ability for cold-active growth (i.e., 8 degrees C), while the group from the agricultural area showed an ability for growth at high temperatures (i.e., 37 degrees C) and resilience to UV exposure. These results represent a significant paradigm shift; habitat selection, not host insect selection, drives the population structure of these insect-pathogenic deuteromycetous fungi. With each group we observed recombining population structures as well as clonally reproducing lineages. We discuss whether these groups may represent cryptic species. Worldwide, M. anisopliae may be an assembly of cryptic species, each adapted to certain environmental conditions. The association of fungal genotypes with habitat but not with host insects has implications on the criteria for utility of this, and perhaps other, fungal biocontrol agents.     

Open relationships in the castles of clay: high diversity and low host specificity of Termitomyces fungi associated with fungus-growing termites in Africa

In the African and Asian tropics, termites of the subfamily Macrotermitinae play a major role in the decomposition of dead plant material. Their ecological success lies in the obligate mutualism of the termites with fungi of the genus Termitomyces. Before the advent of molecular studies, the interaction with these fungi was poorly understood. Here, we combined available ITS sequence data from West, Central, and South Africa with data of 39 new samples from East Africa to achieve the most comprehensive view of the diversity and host specificity of Termitomyces symbionts across Africa to date. A high amount of sequence divergence in the ITS sequences was found; 11 different Termitomyces lineages in East Africa and >30 lineages across Africa were identified, and the expected diversity is estimated to be about 41 lineages. The fungal lineages belong to four major clades, each almost exclusively associated with one termite host genus. Analysis of molecular variance revealed that 40% of the ITS sequence variation occurred between host genera, indicating close co-evolution at this level. However, within host genera, fungal lineages and haplotypes were frequently shared among host species and sampling localities, except for fungal symbionts of Odontotermes. Horizontal transmission of fungal symbionts may facilitate the transfer of haplotypes and species among hosts. However, at present, we have little understanding of the maintenance of specificity at the genus level. Possible explanations range from substrate specificity of fungi to an active selection of fungi by termites.     

Diversity and host specificity of endophytic Rhizoctonia-like fungi from tropical orchids

All orchids have an obligate relationship with mycorrhizal symbionts. Most orchid mycorrhizal fungi are classified in the form-genus Rhizoctonia. This group includes anamorphs of Tulasnella, Ceratobasidium, and Thanatephorus. Rhizoctonia can be classified according to the number of nuclei in young cells (multi-, bi-, and uninucleate). From nine Puerto Rican orchids we isolated 108 Rhizoctonia-like fungi. Our isolates were either bi- or uninucleate, the first report of uninucleate Rhizoctonia-like fungi as orchid endophytes. We sequenced the internal transcribed spacer (ITS) region of nuclear ribosomal DNA from 26 isolates and identified four fungal lineages, all related to Ceratobasidium spp. from temperate regions. Most orchid species hosted more than one lineage, demonstrating considerable variation in mycorrhizal associations even among related orchid species. The uninucleate condition was not a good phylogenetic character in mycorrhizal fungi from Puerto Rico. All four lineages were represented by fungi from Tolumnia variegata, but only one lineage included fungi from Ionopsis utricularioides. Tropical epiphytic orchids appear to vary in degree of specificity in their mycorrhizal interactions more than previously thought.     

Habitat Association in Two Genetic Groups of the Insect-Pathogenic Fungus Metarhizium anisopliae: Uncovering Cryptic Species?

Strains of insect-pathogenic fungi with high virulence toward certain pest insects have great potential for commercial biological control applications. Identifying such strains has been a central theme in using fungi for biological control. This theme is supported by a persistent paradigm in insect pathology which suggests that the host insect is the predominant influence on the population genetics of insect-pathogenic fungi. In this study, a population genetics analysis of the insect-pathogenic fungus Metarhizium anisopliae from forested and agricultural habitats in Ontario, Canada, showed a nonrandom association of alleles between two distinct, reproductively isolated groups (index of multilocus association = 1.2). Analyses of the mitochondrial DNA showed no differences between the groups. The two groups were associated with different habitat types, and associations with insect hosts were not found. The group from forested areas showed an ability for cold-active growth (i.e., 8°C), while the group from the agricultural area showed an ability for growth at high temperatures (i.e., 37°C) and resilience to UV exposure. These results represent a significant paradigm shift; habitat selection, not host insect selection, drives the population structure of these insect-pathogenic deuteromycetous fungi. With each group we observed recombining population structures as well as clonally reproducing lineages. We discuss whether these groups may represent cryptic species. Worldwide, M. anisopliae may be an assembly of cryptic species, each adapted to certain environmental conditions. The association of fungal genotypes with habitat but not with host insects has implications on the criteria for utility of this, and perhaps other, fungal biocontrol agents.     

Community composition and diversity of Neotropical root‐associated fungi in common and rare trees

Interactions between plants and root‐associated fungi can affect the assembly, diversity, and relative abundances of tropical plant species. Host–symbiont compatibility and some degree of host specificity are prerequisites for these processes to occur, and these prerequisites may vary with host abundance. However, direct assessments of whether specificity of root‐associated fungi varies with host abundance are lacking. Here, in a diverse tropical forest in Los Tuxtlas, Mexico, we couple DNA metabarcoding with a sampling design that controls for host phylogeny, host age, and habitat variation, to characterize fungal communities associated with the roots of three confamilial pairs of host species that exhibit contrasting (high and low) relative abundances. We uncovered a functionally and phylogenetically diverse fungal community composed of 1,038 OTUs (operational taxonomic units with 97% genetic similarity), only 14 of which exhibited host specificity. Host species was a significant predictor of fungal community composition only for the subset of OTUs composed of putatively pathogenic fungi. We found no significant difference in the number of specialists associating with common versus rare trees, but we found that host abundance was negatively correlated with the diversity of root fungal communities. This latter result was significant for symbiotrophs (mostly arbuscular mycorrhizal fungi) and, to a lesser extent, for pathotrophs (mostly plant pathogens). Thus, root fungal communities differ between common and rare trees, which may impact the strength of conspecific negative density dependence. Further studies from other tropical sites and host lineages are warranted, given the role of root‐associated fungi in biodiversity maintenance.     

Evaluating multilocus Bayesian species delimitation for discovery of cryptic mycorrhizal diversity

The increasing availability of DNA sequence data enables exciting new opportunities for fungal ecology. However, it amplifies the challenge of how to objectively classify the diversity of fungal sequences into meaningful units, often in the absence of morphological characters. Here, we test the utility of modern multilocus Bayesian coalescent-based methods for delimiting cryptic fungal diversity in the orchid mycorrhiza morphospecies Serendipita vermifera. We obtained 147 fungal isolates from Caladenia, a speciose clade of Australian orchids known to associate with Serendipita fungi. DNA sequence data for 7 nuclear and mtDNA loci were used to erect competing species hypotheses by clustering isolates based on: (a) ITS sequence divergence, (b) Bayesian admixture analysis, and (c) mtDNA variation. We implemented two coalescent-based Bayesian methods to determine which species hypothesis best fitted our data. Both methods found strong support for eight species of Serendipita among our isolates, supporting species boundaries reflected in ITS divergence. Patterns of host plant association showed evidence for both generalist and specialist associations within the host genus Caladenia. Our findings demonstrate the utility of Bayesian species delimitation methods and suggest that wider application of these techniques will readily uncover new species in other cryptic fungal lineages.     

Contrasting ectomycorrhizal fungal communities on the roots of co-occurring oaks (Quercus spp.) in a California woodland

Plant host species is considered an important factor influencing ectomycorrhizal (EM) communities. To gain insights into the role of host species in structuring EM communities, EM communities on sympatric oak (Quercus) species were compared in the Sierra Nevada foothills of California. Using molecular methods (polymerase chain reaction, cloning, restriction fragment length polymorphism and DNA sequencing), EM fungi on roots of deciduous Quercus douglasii and evergreen Quercus wislizeni trees were identified from 64 soil cores. The total EM species richness was 140, of which 40 taxa were detected on both oak hosts. Greater diversity and frequency of EM fungi with epigeous fruiting habit were found on Q. wislizeni, while taxa in the Ascomycota were more frequent and diverse on Q. douglasii. Using ordination, it was determined that both soil extractable phosphorus and oak host species explained a significant proportion of the variation in EM species distribution. These results indicate that plant host species can be an important factor influencing EM fungal community composition, even within congeneric trees.     

Cryptic diversity and significant cophylogenetic signal detected by DNA barcoding the rust fungi (Pucciniaceae) of Cyperaceae–Juncaceae

Plants play important roles as habitat and food for a tremendous diversity of specialist animals and fungi. The disappearance of any plant species can lead to extinction cascades of its associated biota. In consequence, documenting the diversity and specificity of plant-associated organisms is of high practical relevance in biodiversity conservation. Here, we present the first large-scale molecular investigation into the diversity, host specificity, and cophylogenetic congruence of an especially rich plant–fungal association, the rust fungi (Pucciniaceae) of Cyperaceae and Juncaceae. Using the largest rust fungi DNA barcoding dataset published to date (252 sequences, 82 taxa), we reject the presence of a global ITS2-28S barcode gap, but find a local gap in Cyperaceae–Juncaceae rusts, and suggest the existence of many cryptic species in North America, with some broadly circumscribed species possibly corresponding to >10 cryptic species. We test previous hypotheses of correlations between the phylogenies of rust fungi and their Cyperaceae–Juncaceae hosts using a combination of global-fit and event-based cophylogenetic methods. A significant cophylogenetic signal is detected between rusts and their hosts, but the small number of cospeciations argues for preferential host jumps as the driving process behind these correlations. In addition, temporal congruence between the origin of major Carex clades and their rusts suggests that host diversification may have promoted parasite diversification. Finally, we discuss the relevance of rust infection patterns to the systematics of Cyperaceae, highlight some taxonomic problems uncovered by the analyses, and call attention to the promise of DNA barcoding for bridging knowledge gaps in poorly studied plant-associated microorganisms.     

High richness of ectomycorrhizal fungi and low host specificity in a coastal sand dune ecosystem revealed by network analysis

Ectomycorrhizal (EM) fungi are ubiquitous in temperate and boreal forests, comprising over 20,000 species forming root symbiotic associations with Pinaceae and woody angiosperms. As much as 100 different EM fungal species can coexist and interact with the same tree species, forming complex multispecies networks in soils. The degree of host specificity and structural properties of these interaction networks (e.g., nestedness and modularity) may influence plant and fungal community assembly and species coexistence, yet their structure has been little studied in northern coniferous forests, where trees depend on EM fungi for nutrient acquisition. We used high‐throughput sequencing to characterize the composition and diversity of bulk soil and root‐associated fungal communities in four co‐occurring Pinaceae in a relic foredune plain located at Îles de la Madeleine, Québec, Canada. We found high EM fungal richness across the four hosts, with a total of 200 EM operational taxonomic units (OTUs), mainly belonging to the Agaricomycetes. Network analysis revealed an antinested pattern in both bulk soil and roots EM fungal communities. However, there was no detectable modularity (i.e., subgroups of interacting species) in the interaction networks, indicating a low level of specificity in these EM associations. In addition, there were no differences in EM fungal OTU richness or community structure among the four tree species. Limited shared resources and competitive exclusion typically restrict the number of taxa coexisting within the same niche. As such, our finding of high EM fungal richness and low host specificity highlights the need for further studies to determine the mechanisms enabling such a large number of EM fungal species to coexist locally on the same hosts.     

Soil propagule banks of ectomycorrhizal fungi share many common species along an elevation gradient

We conducted bioassay experiments to investigate the soil propagule banks of ectomycorrhizal (EM) fungi in old-growth forests along an elevation gradient and compared the elevation pattern with the composition of EM fungi on existing roots in the field. In total, 150 soil cores were collected from three forests on Mt. Ishizuchi, western Japan, and subjected to bioassays using Pinus densiflora and Betula maximowicziana. Using molecular analyses, we recorded 23 EM fungal species in the assayed propagule banks. Eight species (34.8 %) were shared across the three sites, which ranged from a warm–temperate evergreen mixed forest to a subalpine conifer forest. The elevation pattern of the assayed propagule banks differed dramatically from that of EM fungi on existing roots along the same gradient, where only a small proportion of EM fungal species (3.5 %) were shared across sites. The EM fungal species found in the assayed propagule banks included many pioneer fungal species and composition differed significantly from that on existing roots. Furthermore, only 4 of 23 species were shared between the two host species, indicating a strong effect of bioassay host identity in determining the propagule banks of EM fungi. These results imply that the assayed propagule bank is less affected by climate compared to EM fungal communities on existing roots. The dominance of disturbance-dependent fungal species in the assayed propagule banks may result in higher ecosystem resilience to disturbance even in old-growth temperate forests.     

A molecular phylogeny of Basidiophora reveals several apparently host-specific lineages on Astereae

The genus Basidiophora has long been thought to contain only two species, Basidiophora entospora and Basidiophora kellermanii, the latter of which was transferred to a newly described monotypic genus, Benua, at the end of the twentieth century, leaving Basidiophora monotypic, despite its vast host range, including a member of the Eupatoriae and several genera in the subfamily Asteroideae of the Asteraceae. Using historic herbarium specimens, we demonstrate that while Benua kellermanii is genetically highly homogenous, at least seven distinct phylogenetic lineages exist within Basidiophora, which, based on sequence divergence, most likely constitute hitherto overlooked cryptic species. As the specimens from Symphyotrichum novae-angliae formed a well-supported clade with little variation, we consider Peronospora simplex described on this host as an independent species, which is transferred to the genus Basidiophora in this study. The phylogeny of the pathogens corresponds well to the phylogeny of the respective hosts, which is unusual in downy mildews and might hint at clade-limited colonisation and subsequent radiation to closely related hosts of Astereae or even suggest a co-evolution scenario. Our findings provide further evidence that species with assumed broad host ranges should be thoroughly evaluated with respect to their phylogenetic relationships, especially in biotrophic genera with only limited morphological diversity. In some cases, host specificity of genetically divergent lineages might be the only phenotypic trait remaining for species delimitation. Future detailed morphological comparisons are needed to reveal if the seemingly cryptic species of Basidiophora can be distinguished based on subtle morphological characteristics.     

Host associations and beta diversity of fungal endophyte communities in New Guinea rainforest trees

Processes shaping the distribution of foliar fungal endophyte species remain poorly understood. Despite increasing evidence that these cryptic fungal symbionts of plants mediate interactions with pathogens and herbivores, there remain basic questions regarding the extent to which dispersal limitation and host specificity might shape fungal endophyte community composition in rainforests. To assess the relative importance of spatial pattern and host specificity, we isolated fungi from a sample of mapped trees in lowland Papua New Guinea. Sequences of the internal transcribed spacer (ITS) region were obtained for 2079 fungal endophytes from three sites and clustered into molecular operational taxonomic units (MOTUs) at 95% similarity. Multivariate analyses suggest that host affinity plays a significant role in structuring endophyte community composition whereas there was no evidence of endophyte spatial pattern at the scale of tens to hundreds of metres. Differences in endophyte communities between sampled trees were weakly correlated with variation in foliar traits but not with tree species relatedness. The dominance of relatively few generalist endophytes and the presence of a large number of rare MOTUs was a consistent observation at three sites separated by hundreds of kilometres and regional turnover was low. Host specificity appears to play a relatively weak but more important role than dispersal limitation in shaping the distribution of fungal endophyte communities in New Guinea forests. Our results suggest that in the absence of strong ecological gradients and host turnover, beta diversity of endophyte communities could be low in large areas of contiguous forest.     

Phylogenetic constrains on mycorrhizal specificity in eight Dendrobium(Orchidaceae) species

Plant phylogeny constrains orchid mycorrhizal(OrM) fungal community composition in some orchids. Here, we investigated the structures of the OrM fungal communities of eight Dendrobium species in one niche to determine whether similarities in the OrM fungal communities correlated with the phylogeny of the host plants and whether the Dendrobium-OrM fungal interactions are phylogenetically conserved. A phylogeny based on DNA data was constructed for the eight coexisting Dendrobium species,and the OrM fungal communities were characterized by their roots. There were 31 different fungal lineages associated with the eight Dendrobium species. In total, 82.98% of the identified associations belonging to Tulasnellaceae, and a smaller proportion involved members of the unknown Basidiomycota(9.67%). Community analyses revealed that phylogenetically related Dendrobium tended to interact with a similar set of Tulasnellaceae fungi. The interactions between Dendrobium and Tulasnellaceae fungi were significantly influenced by the phylogenetic relationships among the Dendrobium species. Our results provide evidence that the mycorrhizal specificity in the eight coexisting Dendrobium species was phylogenetically conserved.     

Host plant genus-level diversity is the best predictor of ectomycorrhizal fungal diversity in a Chinese subtropical forest

Microbial diversity is generally far higher than plant diversity, but the relationship between microbial diversity and plant diversity remains enigmatic. To shed light on this problem, we examined the diversity of a key guild of root-associated microbes, that is, ectomycorrhizal (EM) fungi along a plant diversity gradient in a Chinese subtropical forest. The results indicated that EM fungal diversity was positively correlated with host plant diversity. Furthermore, this relationship was best predicted by host genus-level diversity, rather than species-level diversity or family-level diversity. The generality of this finding was extended beyond our study system through the analyses of 100 additional studies of EM fungal communities from tropical and temperate forests. Here as well, EM fungal lineage composition was significantly affected by EM plant diversity levels, and some EM fungal lineages were co-associated with some host plant genera. These results suggest a general diversity maintenance mechanism for host-specific microbes based on higher order host plant phylogenetic diversity.     

The diversity of ant-associated black yeasts: insights into a newly discovered world of symbiotic interactions

Based on pure culture studies and DNA phylogenetic analyses, black yeasts (Chaetothyriales, Ascomycota) are shown to be widely distributed and important components of numerous plant-ant-fungus networks, independently acquired by several ant lineages in the Old and New World. Data from ITS and LSU nu rDNA demonstrate that a high biodiversity of fungal species is involved. There are two common ant-fungus symbioses involving black yeasts: (1) on the carton walls of ant nests and galleries, and (2) the fungal mats growing within non-pathogenic naturally hollow structures (so-called domatia) provided by myrmecophytic plants as nesting space for ants (ant-plant symbiosis). Most carton- and domatia-inhabiting fungi stem from different phylogenetic lineages within Chaetothyriales, and almost all of the fungi isolated are still undescribed. Despite being closely related, carton and domatia fungi are shown to differ markedly in their morphology and ecology, indicating that they play different roles in these associations. The carton fungi appear to improve the stability of the carton, and several species are commonly observed to co-occur on the same carton. Carton fungi commonly have dark-walled monilioid hyphae, colouring the carton blackish and apparently preventing other fungi from invading the carton. Despite the simultaneous presence of usually several species of fungi, forming complex associations on the carton, little?overlap is observed between carton fungi from different ant species, even those that co-occur in nature, indicating at least some host specificity of fungi. Most fungi present on carton belong to Chaetothyriales, but in a few samples, Capnodiales are also an important component. Carton fungi are difficult to assign to anamorph genera, as most lack conidiation. The domatia fungi are more specific. In domatia, usually only one or two fungal species co-occur, producing a dense layer on living host plant tissue in domatia. They have hyaline or light brown thin-walled hyphae, and are commonly sporulating. In both carton and domatia, the fungal species seem to be specific to each ant-plant symbiosis. Representative examples of carton and domatia ant-fungus symbioses are illustrated. We discuss hypotheses on the ecological significance of the Chaetothyriales associated with ants.