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Pyrola japonica, a partially mycoheterotrophic Ericaceae, has mycorrhizal preference for russulacean fungi in central Japan

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Abstract

Mycorrhizal symbiosis often displays low specificity, except for mycoheterotrophic plants that obtain carbon from their mycorrhizal fungi and often have higher specificity to certain fungal taxa. Partially mycoheterotrophic (or mixotrophic, MX) plant species tend to have a larger diversity of fungal partners, e.g., in the genus Pyrola (Monotropoideae, Ericaceae). Preliminary evidence however showed that the Japanese Pyrola japonica has preference for russulacean fungi based on direct sequencing of the fungal internal transcribed spacer (ITS) region from a single site. The present study challenges this conclusion using (1) sampling of P. japonica in different Japanese regions and forest types and (2) fungal identification by ITS cloning. Plants were sampled from eight sites in three regions, in one of which the fungal community on tree ectomycorrhizal (ECM) tips surrounding P. japonica was also analyzed. In all, 1512 clone sequences were obtained successfully from 35 P. japonica plants and 137 sequences from ECM communities. These sequences were collectively divided into 74 molecular operational taxonomic units (MOTUs) (51 and 33 MOTUs, respectively). MOTUs from P. japonica involved 36 ECM taxa (96 % of all clones), and 17 of these were Russula spp. (76.2 % of all clones), which colonized 33 of the 35 sampled plants. The MOTU composition significantly differed between P. japonica and ECM tips, although shared species represented 26.3 % of the ECM tips community in abundance. This suggests that P. japonica has a preference for russulacean fungi.

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Acknowledgments

We thank Mr. S Iio for habitat information regarding the Hashirida shrine, Iyama shrine, and Makiba Mizunosaka West Hill and the Aichi prefecture government office for permission to access the sites. We also thank T. Chikada, Life Science Research Center, Center for Molecular Biology and Genetics, Mie University, for helping out with DNA analyses, the members of the Laboratory of Forest Pathology and Mycology, Graduate School of Bioresources, Mie University, for their help in field sampling, and D. Marsh for correcting our English. This study was partly supported by KAKENHI (22688011, 23658124, 25304026) to YM and the Fondation de France (fond Ars Cuttoli & Appel) to M-AS.

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Authors and Affiliations

  1. Laboratory of Forest Mycology, Graduate School of Bioresources, Mie University, Kurimamachiya 1577, Tsu, Mie, 514-8507, Japan

    Takashi Uesugi, Keisuke Obase & Yosuke Matsuda

  2. Faculty of Bioresources, Mie University, Kurimamachiya 1577, Tsu, Mie, 514-8507, Japan

    Miho Nakano

  3. Institut de Systématique, Évolution, Biodiversité (ISYEB - UMR 7205 – CNRS, MNHN, UPMC, EPHE), Muséum national d’Histoire naturelle, Sorbonne Universités, 57 rue Cuvier, CP50, 75005, Paris, France

    Marc-André Selosse

  4. Department of Plant Taxonomy and Nature Conservation, University of Gdansk, Wita Stwosza 59, 80-308, Gdansk, Poland

    Marc-André Selosse

  5. Department of Forest Microbiology, Forestry and Forest Products Research Institute, Matsunosato 1, Tsukuba, Ibaraki, 305-8687, Japan

    Keisuke Obase

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  1. Takashi Uesugi
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  2. Miho Nakano
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Correspondence to Yosuke Matsuda.

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Table S1

BLAST results for ITS consensus sequences of each MOTU. (DOC 92 kb)

Table S2

Fungal taxa mycorrhizal on Pyrola japonica. Putative trophic status: ECM, ectomycorrhizal; ERM, ericoid; END, endophytic; SAP, saprotrophic; NEM, nematophagous. Asterisk indicates MOTUs that were detected from Pyrola japonica and tree roots but not identical soil cores. (DOC 130 kb)

Table S3

Fungal taxa detected from Pyrola japonica and tree roots in the same cores. MOTUs shared between Pyrola japonica and ECM tips in one core are enclosed. Hosts, Py: Pyrola japonica, Q: Quercus glauca, P: Pinus densiflora, C: Castanopsis cuspidata. (DOC 130 kb)

Fig. S1

Images of each study site: (a) Aichi, (b) Mie and (c) Kyoto, (d) flowering Pyrola japonica, (e) root fragment, (f) light and (g) fluorescent microscopic views of P. japonica roots made by hand cross section. Arrows in (e), (f) and (g) indicate hyphal coils, and no fungal mantles were formed on epidermal cells. Double arrowheads in (e) and (f) indicate plant cells that contained no hyphal coils. Bars 1 mm for (e) and (f) and 100 μm for (g). (DOC 728 kb)

Fig. S2

Rarefaction curves of (a) MOTUs in Pyrola japonica collected at all study sites (white circle), Aichi (black square) and Mie (black up-pointing triangle) and (b) Simpson’s diversity of clones derived from each plant collected. Plants collected at Kyoto were not analyzed owing to the lack of replicates (n = 3). Plant ID is shown with the combination of site codes, overstory trees (P for Pinus and Q for Quercus) and replicates. (DOC 81 kb)

Fig. S3

A non-metric multidimensional scaling plot of the mycorrhizal community of Pyrola japonica. Clone abundance data of each site were used for ordination with Horn dissimilarity index (a). White up-pointing triangle, Aichi sites A1 to A3; black square, Mie site M1 to M4; white circle, Kyoto unique site K1. Grouping according to region was not significant (PERMANOVA, p = 0.1). Eight variables were tested for significant correlation with mycorrhizal community dissimilarities (b). Significantly correlated variables are shown in the plot as arrows. The length of the arrow is proportional to the strength of the correlation with the ordination (*p < 0.05). PCNM is the principal coordinates of neighborhood matrix and see for “Materials and methods” in details. (DOC 61 kb)

Fig. S4

Rarefaction curves of MOTUs in Pyrola japonica (white circle) and its surrounding ectomycorrhizal roots (black circle) collected in Aichi. (DOC 43 kb)

Fig. S5

Neighbor-joining phylogeny of russulacean MOTUs recovered in this study. Bootstrap tests (1000 replicates) larger than 90 % are shown next to the branches. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Maximum Composite Likelihood method. MOTUs detected in this study are in bold, and when they were derived from ECM tips denoted by an ‘e’ following the MOTU names. (DOC 103 kb)

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Uesugi, T., Nakano, M., Selosse, MA. et al. Pyrola japonica, a partially mycoheterotrophic Ericaceae, has mycorrhizal preference for russulacean fungi in central Japan. Mycorrhiza 26, 819–829 (2016). https://doi.org/10.1007/s00572-016-0715-2

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