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Environmental filtering and host identity collectively shape root-associated microbiomes of Ericaceae and ectomycorrhizal plants in fumarole fields

Akifumi Murata, Mikihito NoguchiiD, Ibuki HayashiiD, Keitaro FukushimaiD, Hirokazu TojuiD

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Abstract

Background Symbiosis with microbes is a key strategy that has enabled plants to colonize extreme environments. Since the benefits conferred by root-associated microbes depend on both environmental conditions and host-microbe combinations, plant adaptation to harsh environments is closely linked to the assembly of root microbial communities. Understanding how environmental and host filtering jointly shape these communities is therefore fundamental to elucidating the mechanisms underlying plant adaptation to extreme environments. Results In this study, we investigated the differentiation of root-associated prokaryotic and fungal communities and individual operational taxonomic units (OTUs) across two contrasting habitats surrounding fumaroles, solfatara-field and forest-edge habitats, and six dominant Ericaceae and ectomycorrhizal plant taxa. Prokaryotic and fungal OTUs rarely exhibited strong preferences for both habitat and host identity. Instead, many of prokaryotic and fungal OTUs specialized to one of these niches, collectively generating root microbial communities differentiated by both factors. Nonetheless, striking specializations in habitat and host niches were observed in the fungal family Hyaloscyphaceae (Helotiales). To gain insight into the evolutionary basis of microbial specialization, we examined phylogenetic signals in preference phenotypes. The resulting weak phylogenetic signals in these preference phenotypes further suggest that this fungal clade has undergone substantial ecological divergence. Conclusion Overall, our findings indicate that root-associated microbial communities in extreme environments are assembled through the accumulation of microbial taxa specialized to either habitat or host, and that strong ecological specialization in fungi can arise with little phylogenetic constraint.

In-depth summary

Claude Opus 5.5 · xhigh effort · 8 Nov 2026 · from the full text of version 1 (26 Sept 2026) · CC BY 4.0

Few root-associated microbial operational taxonomic units (OTUs) in Japanese fumarole fields prefer both a habitat and a host; most specialize on one axis, and Hyaloscyphaceae preferences lack significant phylogenetic signal.

Previous work has mostly compared whole root microbial communities across environments and hosts, which cannot reveal the taxon-level responses behind those differences, and in nature host composition tends to shift along environmental gradients, confounding the two filters. Fumarole fields, where the same Ericaceae and ectomycorrhizal hosts grow across soils that change sharply within meters, let the authors score habitat and host preference separately for each OTU.

Key results

  1. ShownHabitat and host identity each structured root prokaryotic and fungal communities, explaining similar and largely non-overlapping shares of variation; host explained slightly more even when fitted after habitat.

    Sequential permutational multivariate ANOVA (PERMANOVA) on Sørensen dissimilarities, 206 root samples: habitat R² = 0.052 (prokaryotes) and 0.066 (fungi), host R² = 0.066 and 0.083, all P < 0.001; habitat × host interaction only for fungi (R² = 0.029, P < 0.05); variation partitioning showed negligible overlap; dispersion (PERMDISP) differed among hosts (P < 0.01) but not habitats (P > 0.05). · Community structural differentiation across…; Community differentiation; Preparation of datasets for downstream analyses

  2. ShownSpecialized OTUs mostly specialized on one axis only: dual habitat-and-host specialists were rare (two prokaryotic, no fungal OTUs), and the strengths of the two specializations were uncorrelated. Only fungi included OTUs preferring the solfatara field.

    Prokaryotes: 23 OTUs with habitat preference (all toward forest edge), 9 with host preference, 2 with both. Fungi: 2 solfatara-preferring and 13 forest-edge-preferring OTUs, 12 host-preferring, none with both. Spearman r = 0.035 (P = 0.607) for prokaryotes, 0.006 (P = 0.949) for fungi. Tested OTUs: 222 prokaryotic and 137 fungal found in three or more root samples. · OTU-level preferences for habitats and host…; OTU-level habitat and host specialization; Figure legends

  3. SuggestedWithin the fungal family Hyaloscyphaceae, closely related OTUs differed in habitat and host preference, and neither preference showed a significant phylogenetic signal.

    Neighbor-joining tree of 24 Hyaloscyphaceae OTU sequences plus 14 NCBI references resolved three clades. Habitat preference: Blomberg's K = 0.233 (P = 0.609), Pagel's λ = 0.071 (P = 0.578). Host preference: K = 0.288 (P = 0.250), λ < 0.001 (P = 1.000). · Phylogenetic signals in habitat and host…; Phylogenetic signals in habitat and host…

  4. ShownRoots held far fewer OTUs than the surrounding soil. Root richness was lower in solfatara-field than in forest-edge habitats, while soil richness did not differ significantly between habitats.

    Negative binomial generalized linear mixed model (GLMM): soil richness 3.70-fold that of roots for prokaryotes (95% CI 2.89–4.73) and a soil/root ratio of 4.96 for fungi (95% CI 3.92–6.29), both P < 0.001. Roots, solfatara vs forest edge: rank-biserial r = −0.44 and −0.67 (prokaryotes), −0.28 and −0.71 (fungi), FDR < 0.001; soil FDR > 0.05. · Community composition and diversity patterns; Community differentiation

  5. ShownSolfatara-field soils were less acidic than forest-edge soils but depleted of exchangeable base and metal cations, so the two habitats differ not only in stress intensity but also in the type of soil chemical constraint.

    PERMANOVA on pH and cations: R² = 0.28, P < 0.001. pH near 3.0 in both, higher in solfatara field (rank-biserial r = 0.43 and 0.72, FDR < 0.001). At Arayu-Jigoku, Ca²⁺, Mg²⁺, K⁺, Al³⁺, Mn²⁺ and Zn²⁺ were lower in solfatara soils (r = 0.59–0.84, FDR < 0.05); none significant at Ofukasawa; dispersion also differed between habitats (PERMDISP P < 0.001), with forest-edge soils more heterogeneous. · Soil chemical characterization in solfatara…; Statistical analysis for soil chemical profiling

Methods, data and reproducibility

Roots of 304 plants from two habitats at two fumarole fields were sampled and profiled by 16S (V5–V6) and fungal internal transcribed spacer 1 (ITS1) metabarcoding, with hosts identified by plant ITS2; 206 samples from six focal host taxa were analysed. Per-OTU habitat and host preferences were randomization z-scores; Blomberg's K and Pagel's λ tested their phylogenetic signal in Hyaloscyphaceae.

  • Root-endosphere prokaryotic 16S rRNA (V5–V6) amplicons from surface-sterilized root tips, solfatara-field and forest-edge habitats at Arayu-Jigoku and Ofukasawa (Onikobe, Miyagi, Japan) · prokaryotes (585 of 612 OTUs bacterial) · n = 206 samples from 105 sampling positions; 612 OTUs · DDBJ PRJDB42913 (to be released after acceptance)
  • Root-endosphere fungal ITS1 amplicons from the same root tips · fungi · n = 206 samples from 105 sampling positions; 484 OTUs · DDBJ PRJDB42913 (to be released after acceptance)
  • Bulk soil prokaryotic 16S and fungal ITS1 amplicons from representative sampling positions · soil prokaryotes and fungi · n = 20 samples; 490 prokaryotic and 471 fungal OTUs · DDBJ PRJDB42913 (to be released after acceptance)
  • Plant ITS2 amplicons from roots plus a reference leaf ITS2 library, used to assign host identity; six focal host taxa retained · Ericaceae (4 taxa), Betula ermanii, Pinus parviflora · n = 304 individuals of 15 plant species sampled · DDBJ PRJDB42913 (to be released after acceptance)
  • Soil pH (portable meter, in the field at each plant patch) and exchangeable cations (inductively coupled plasma optical emission spectrometry, 28 representative points) · n = 28 cation sampling points (25 in multivariate analyses)
Methods & tools
Illumina MiSeq, DADA2 v1.18.0, Cutadapt v4.4, Claident v0.9.2022.01.26, SILVA v138.2, UNITE v10.0, VSEARCH v2.15.2, NCBI BLAST+ v2.17.0+, vegan v2.7-2, bipartite v2.23, phytools v2.5.2, MEGA12.1
Software & environment
R v4.3.1 for read processing and R v4.5.3 for statistics; package versions are stated (e.g. DADA2 v1.18.0, decontam v1.30.0, vegan v2.7-2, bipartite v2.23, glmmTMB v1.1.14, DHARMa v0.5.0, phytools v2.5.2, ape v5.8, ggtree v3.14.0). No container or lock file is mentioned.
Reproducibility gaps
Sequence data are deposited at DDBJ under PRJDB42913 but will be released only after the manuscript is accepted. Rarefaction used a fixed random seed whose value is not given in the text.
Limitations (6)
  • Sampling is a single time point (June 2024); root microbiomes probably vary with space, season and host developmental stage, so long-term, geographically replicated studies are needed. · stated by the authors
  • Metabarcoding shows composition and associations, not microbial activity or function; the proposed roles of Hyaloscyphaceae OTUs in host nutrition and stress tolerance are speculative. · stated by the authors
  • The phylogenetic-signal test covered one fungal family, chosen because its preferences varied strongly, so whether weak phylogenetic constraint holds for root fungi generally is untested. · stated by the authors
  • Habitat and host explained only a small share of community variation; unmeasured factors such as fine-scale soil chemistry, microclimate, microbe-microbe interactions and drift probably contribute. · stated by the authors
  • The phylogenetic test used at most 24 Hyaloscyphaceae OTUs on a neighbor-joining tree; with so few tips, non-significant K and λ have limited power to rule out conservatism. · noted in this summary
  • The soil comparison rests on 20 metabarcoded and 28 chemistry samples; at Ofukasawa (n = 5 per habitat) no individual cation differed significantly, so the chemical contrast is clear mainly at one site. · noted in this summary

Read this if you study how root microbiomes assemble in extreme or volcanic soils, or want a randomization-based way to separate habitat preference from host preference for individual OTUs.

Citations
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