Akifumi S. Tanabe

High-coverage ITS primers for the DNA-based identification of ascomycetes and basidiomycetes in environmental samples.

The kingdom Fungi is estimated to include 1.5 million or more species, playing key roles as decomposers, mutualists, and parasites in every biome on the earth. To comprehensively understand the diversity and ecology of this huge kingdom, DNA barcoding targeting the internal transcribed spacer (ITS) region of the nuclear ribosomal repeat has been regarded as a prerequisite procedure. By extensively surveying ITS sequences in public databases, we designed new ITS primers with improved coverage across diverse taxonomic groups of fungi compared to existing primers. An in silico analysis based on public sequence databases indicated that the newly designed primers matched 99% of ascomycete and basidiomycete ITS taxa (species, subspecies or varieties), causing little taxonomic bias toward either fungal group. Two of the newly designed primers could inhibit the amplification of plant sequences and would enable the selective investigation of fungal communities in mycorrhizal associations, soil, and other types of environmental samples. Optimal PCR conditions for the primers were explored in an in vitro investigation. The new primers developed in this study will provide a basis for ecological studies on the diversity and community structures of fungi in the era of massive DNA sequencing.

Two New Computational Methods for Universal DNA Barcoding: A Benchmark Using Barcode Sequences of Bacteria, Archaea, Animals, Fungi, and Land Plants

Taxonomic identification of biological specimens based on DNA sequence information (a.k.a. DNA barcoding) is becoming increasingly common in biodiversity science. Although several methods have been proposed, many of them are not universally applicable due to the need for prerequisite phylogenetic/machine-learning analyses, the need for huge computational resources, or the lack of a firm theoretical background. Here, we propose two new computational methods of DNA barcoding and show a benchmark for bacterial/archeal 16S, animal COX1, fungal internal transcribed spacer, and three plant chloroplast (rbcL, matK, and trnH-psbA) barcode loci that can be used to compare the performance of existing and new methods. The benchmark was performed under two alternative situations: query sequences were available in the corresponding reference sequence databases in one, but were not available in the other. In the former situation, the commonly used “1-nearest-neighbor” (1-NN) method, which assigns the taxonomic information of the most similar sequences in a reference database (i.e., BLAST-top-hit reference sequence) to a query, displays the highest rate and highest precision of successful taxonomic identification. However, in the latter situation, the 1-NN method produced extremely high rates of misidentification for all the barcode loci examined. In contrast, one of our new methods, the query-centric auto-k-nearest-neighbor (QCauto) method, consistently produced low rates of misidentification for all the loci examined in both situations. These results indicate that the 1-NN method is most suitable if the reference sequences of all potentially observable species are available in databases; otherwise, the QCauto method returns the most reliable identification results. The benchmark results also indicated that the taxon coverage of reference sequences is far from complete for genus or species level identification in all the barcode loci examined. Therefore, we need to accelerate the registration of reference barcode sequences to apply high-throughput DNA barcoding to genus or species level identification in biodiversity research.

Community composition of root-associated fungi in a Quercus-dominated temperate forest: “codominance” of mycorrhizal and root-endophytic fungi

In terrestrial ecosystems, plant roots are colonized by various clades of mycorrhizal and endophytic fungi. Focused on the root systems of an oak-dominated temperate forest in Japan, we used 454 pyrosequencing to explore how phylogenetically diverse fungi constitute an ecological community of multiple ecotypes. In total, 345 operational taxonomic units (OTUs) of fungi were found from 159 terminal-root samples from 12 plant species occurring in the forest. Due to the dominance of an oak species (Quercus serrata), diverse ectomycorrhizal clades such as Russula, Lactarius, Cortinarius, Tomentella, Amanita, Boletus, and Cenococcum were observed. Unexpectedly, the root-associated fungal community was dominated by root-endophytic ascomycetes in Helotiales, Chaetothyriales, and Rhytismatales. Overall, 55.3% of root samples were colonized by both the commonly observed ascomycetes and ectomycorrhizal fungi; 75.0% of the root samples of the dominant Q. serrata were so cocolonized. Overall, this study revealed that root-associated fungal communities of oak-dominated temperate forests were dominated not only by ectomycorrhizal fungi but also by diverse root endophytes and that potential ecological interactions between the two ecotypes may be important to understand the complex assembly processes of belowground fungal communities.

Diversification of endosymbiosis: replacements, co-speciation and promiscuity of bacteriocyte symbionts in weevils

The processes and mechanisms underlying the diversification of host–microbe endosymbiotic associations are of evolutionary interest. Here we investigated the bacteriocyte-associated primary symbionts of weevils wherein the ancient symbiont Nardonella has experienced two independent replacement events: once by Curculioniphilus symbiont in the lineage of Curculio and allied weevils of the tribe Curculionini, and once by Sodalis-allied symbiont in the lineage of grain weevils of the genus Sitophilus. The Curculioniphilus symbiont was detected from 27 of 36 Curculionini species examined, the symbiont phylogeny was congruent with the host weevil phylogeny, and the symbiont gene sequences exhibited AT-biased nucleotide compositions and accelerated molecular evolution. These results suggest that the Curculioniphilus symbiont was acquired by an ancestor of the tribe Curculionini, replaced the original symbiont Nardonella, and has co-speciated with the host weevils over evolutionary time, but has been occasionally lost in several host lineages. By contrast, the Sodalis-allied symbiont of Sitophilus weevils exhibited no host–symbiont co-speciation, no AT-biased nucleotide compositions and only moderately accelerated molecular evolution. These results suggest that the Sodalis-allied symbiont was certainly acquired by an ancestor of the Sitophilus weevils and replaced the original Nardonella symbiont, but the symbiotic association must have experienced occasional re-associations such as new acquisitions, horizontal transfers, replacements and/or losses. We detected Sodalis-allied facultative symbionts in populations of the Curculionini weevils, which might represent potential evolutionary sources of the Sodalis-allied primary symbionts. Comparison of these newcomer bacteriocyte-associated symbiont lineages highlights potential evolutionary trajectories and consequences of novel symbionts after independent replacements of the same ancient symbiont.

How are plant and fungal communities linked to each other in belowground ecosystems? A massively parallel pyrosequencing analysis of the association specificity of root-associated fungi and their host plants.

In natural forests, hundreds of fungal species colonize plant roots. The preference or specificity for partners in these symbiotic relationships is a key to understanding how the community structures of root-associated fungi and their host plants influence each other. In an oak-dominated forest in Japan, we investigated the root-associated fungal community based on a pyrosequencing analysis of the roots of 33 plant species. Of the 387 fungal taxa observed, 153 (39.5%) were identified on at least two plant species. Although many mycorrhizal and root-endophytic fungi are shared between the plant species, the five most common plant species in the community had specificity in their association with fungal taxa. Likewise, fungi displayed remarkable variation in their association specificity for plants even within the same phylogenetic or ecological groups. For example, some fungi in the ectomycorrhizal family Russulaceae were detected almost exclusively on specific oak (Quercus) species, whereas other Russulaceae fungi were found even on “non-ectomycorrhizal” plants (e.g., Lyonia and Ilex). Putatively endophytic ascomycetes in the orders Helotiales and Chaetothyriales also displayed variation in their association specificity and many of them were shared among plant species as major symbionts. These results suggest that the entire structure of belowground plant–fungal associations is described neither by the random sharing of hosts/symbionts nor by complete compartmentalization by mycorrhizal type. Rather, the colonization of multiple types of mycorrhizal fungi on the same plant species and the prevalence of diverse root-endophytic fungi may be important features of belowground linkage between plant and fungal communities.

Sharing of Diverse Mycorrhizal and Root-Endophytic Fungi among Plant Species in an Oak-Dominated Cool- Temperate Forest

Most terrestrial plants interact with diverse clades of mycorrhizal and root-endophytic fungi in their roots. Through belowground plant–fungal interactions, dominant plants can benefit by interacting with host-specific mutualistic fungi and proliferate in a community based on positive plant–mutualistic fungal feedback. On the other hand, subordinate plant species may persist in the community by sharing other sets (functional groups) of fungal symbionts with each other. Therefore, revealing how diverse clades of root-associated fungi are differentially hosted by dominant and subordinate plant species is essential for understanding plant community structure and dynamics. Based on 454-pyrosequencing, we determined the community composition of root-associated fungi on 36 co-occurring plant species in an oak-dominated forest in northern Japan and statistically evaluated the host preference phenotypes of diverse mycorrhizal and root-endophytic fungi. An analysis of 278 fungal taxa indicated that an ectomycorrhizal basidiomycete fungus in the genus Lactarius and a possibly endophytic ascomycete fungus in the order Helotiales significantly favored the dominant oak (Quercus) species. In contrast, arbuscular mycorrhizal fungi were generally shared among subordinate plant species. Although fungi with host preferences contributed to the compartmentalization of belowground plant–fungal associations, diverse clades of ectomycorrhizal fungi and possible root endophytes were associated not only with the dominant Quercus but also with the remaining plant species. Our findings suggest that dominant-ectomycorrhizal and subordinate plant species can host different subsets of root-associated fungi, and diverse clades of generalist fungi can counterbalance the compartmentalization of plant–fungal associations. Such insights into the overall structure of belowground plant–fungal associations will help us understand the mechanisms that facilitate the coexistence of plant species in natural communities.

Ericaceous plant–fungus network in a harsh alpine–subalpine environment

In terrestrial ecosystems, plant species and diverse root‐associated fungi form complex networks of host–symbiont associations. Recent studies have revealed that structures of those below‐ground plant–fungus networks differ between arbuscular mycorrhizal and ectomycorrhizal symbioses. Nonetheless, we still remain ignorant of how ericaceous plant species, which dominate arctic and alpine tundra, constitute networks with their root‐associated fungi. Based on a high‐throughput DNA sequencing data set, we characterized the statistical properties of a network involving 16 ericaceous plant species and more than 500 fungal taxa in the alpine–subalpine region of Mt. Tateyama, central Japan. While all the 16 ericaceous species were associated mainly with fungi in the order Helotiales, they varied remarkably in association with fungi in other orders such as Sebacinales, Atheliales, Agaricales, Russulales and Thelephorales. The ericaceous plant–fungus network was characterized by high symbiont/host preferences. Moreover, the network had a characteristic structure called ‘anti‐nestedness’, which has been previously reported in ectomycorrhizal plant–fungus networks. The results lead to the hypothesis that ericaceous plants in harsh environments can host unexpectedly diverse root‐associated fungal taxa, constituting networks whose structures are similar to those of previously reported ectomycorrhizal networks but not to those of arbuscular mycorrhizal ones.

Network modules and hubs in plant-root fungal biomes.

Terrestrial plants host phylogenetically and functionally diverse groups of below-ground microbes, whose community structure controls plant growth/survival in both natural and agricultural ecosystems. Therefore, understanding the processes by which whole root-associated microbiomes are organized is one of the major challenges in ecology and plant science. We here report that diverse root-associated fungi can form highly compartmentalized networks of coexistence within host roots and that the structure of the fungal symbiont communities can be partitioned into semi-discrete types even within a single host plant population. Illumina sequencing of root-associated fungi in a monodominant south beech forest revealed that the network representing symbiont–symbiont co-occurrence patterns was compartmentalized into clear modules, which consisted of diverse functional groups of mycorrhizal and endophytic fungi. Consequently, terminal roots of the plant were colonized by either of the two largest fungal species sets (represented by Oidiodendron or Cenococcum). Thus, species-rich root microbiomes can have alternative community structures, as recently shown in the relationships between human gut microbiome type (i.e. ‘enterotype’) and host individual health. This study also shows an analytical framework for pinpointing network hubs in symbiont–symbiont networks, leading to the working hypothesis that a small number of microbial species organize the overall root–microbiome dynamics.

Diet disparity among sympatric herbivorous cichlids in the same ecomorphs in Lake Tanganyika: amplicon pyrosequences on algal farms and stomach contents

BackgroundLake Tanganyika, an ancient lake in the Great Rift Valley, is famous for the adaptive radiation of cichlids. Five tribes of the Cichlidae family have acquired herbivory, with five ecomorphs: grazers, browsers, scrapers, biters and scoopers. Sixteen species of the herbivorous cichlids coexist on a rocky littoral slope in the lake. Seven of them individually defend feeding territories against intruding herbivores to establish algal farms. We collected epiphyton from these territories at various depths and also gathered fish specimens. Algal and cyanobacteria community structures were analysed using the amplicon-metagenomic method.ResultsBased on 454-pyrosequencing of SSU rRNA gene sequences, we identified 300 phototrophic taxa, including 197 cyanobacteria, 57 bacillariophytes, and 31 chlorophytes. Algal farms differed significantly in their composition among cichlid species, even in the same ecomorph, due in part to their habitat-depth segregation. The algal species composition of the stomach contents and algal farms of each species differed, suggesting that cichlids selectively harvest their farms. The stomach contents were highly diverse, even between species in the same tribe, in the same feeding ecomorph.ConclusionsIn this study, the amplicon-metagenomic approach revealed food niche separation based on habitat-depth segregation among coexisting herbivorous cichlids in the same ecomorphs in Lake Tanganyika.

Contrasting Diversity and Host Association of Ectomycorrhizal Basidiomycetes versus Root- Associated Ascomycetes in a Dipterocarp Rainforest

Root-associated fungi, including ectomycorrhizal and root-endophytic fungi, are among the most diverse and important belowground plant symbionts in dipterocarp rainforests. Our study aimed to reveal the biodiversity, host association, and community structure of ectomycorrhizal Basidiomycota and root-associated Ascomycota (including root-endophytic Ascomycota) in a lowland dipterocarp rainforest in Southeast Asia. The host plant chloroplast ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) region and fungal internal transcribed spacer 2 (ITS2) region were sequenced using tag-encoded, massively parallel 454 pyrosequencing to identify host plant and root-associated fungal taxa in root samples. In total, 1245 ascomycetous and 127 putative ectomycorrhizal basidiomycetous taxa were detected from 442 root samples. The putative ectomycorrhizal Basidiomycota were likely to be associated with closely related dipterocarp taxa to greater or lesser extents, whereas host association patterns of the root-associated Ascomycota were much less distinct. The community structure of the putative ectomycorrhizal Basidiomycota was possibly more influenced by host genetic distances than was that of the root-associated Ascomycota. This study also indicated that in dipterocarp rainforests, root-associated Ascomycota were characterized by high biodiversity and indistinct host association patterns, whereas ectomycorrhizal Basidiomycota showed less biodiversity and a strong host phylogenetic preference for dipterocarp trees. Our findings lead to the working hypothesis that root-associated Ascomycota, which might be mainly represented by root-endophytic fungi, have biodiversity hotspots in the tropics, whereas biodiversity of ectomycorrhizal Basidiomycota increases with host genetic diversity.