Multiple Fungi May Connect the Roots of an Orchid (Cypripedium reginae) and Ash (Fraxinus nigra) in Western Newfoundland

Abstract

Showy lady's slipper (Cypripedium reginae Walter, Orchidaceae) and black ash (Fraxinus nigra Marshall, Oleaceae) often co-occur in close proximity in fens in western Newfoundland, Canada. Metabarcoding of DNA extracted from root samples of both species following surface sterilization, and others without surface sterilization was used to determine if there were shared fungal endophytes in the roots of both species that could form a common mycorrhizal network between them. A wide variety of fungi were recovered from primers amplifying the nuclear ribosomal internal transcribed spacer region (ITS2). Sixty-six fungal sequences were shared by surface-sterilized roots of both orchid and ash, among them arbuscular mycorrhizal fungi (Claroideoglomus, Dominikia, Glomus and Rhizophagus), ectomycorrhizal fungi (Inocybe and Tomentella), the broad-host root endophyte Cadophora orchidicola, along with root pathogens (Dactylonectria, Ilyonectria, Pyricularia, and Xylomyces) and fungi of unknown function. There appear to be multiple fungi that could form a common mycorrhizal network between C. reginae and F. nigra, which might explain their frequent co-occurrence. Transfer of nutrients or carbon between the orchid and ash via one or more of the shared fungal endophytes remains to be demonstrated.

Keywords: ITS2; Illumina MiSeq; black ash; common mycorrhizal network; metabarcoding; mixotrophy; mycorrhiza; showy lady's slipper orchid.

Figure 1

A sampling site in a Newfoundland fen, with showy lady's slipper (Cypripedium reginae; arrowheads labeled O) surrounding a shrubby growth of black ash (Fraxinus nigra; arrows labeled A). Photograph by Voitk (2011).

Figure 2

Approximate sampling locations in Western Newfoundland, Canada. Samples from a total of 54 plants were collected, 27 showy lady's slipper orchid (Cypripedium reginae) root samples and 27 black ash (Fraxinus nigra) root samples. Basemap provided by Google Maps through the ggmap package in R.

Figure 3

Images of Cypripedium reginae roots (AO-Orch) (A) in transverse section, (B,C) transverse section with likely condensed/collapsed pelotons as dark circles within root cells (arrows), (D) in longitudinal section, and Fraxinus nigra roots (AO-Ash), (E) in transverse section showing branched hyphae and likely vesicles (arrow), and (F) simple septate hyphae (arrow) externally associated with root tips. Roots were stained with trypan blue and images taken using a ZeissZ1 microscope with brightfield illumination.

Figure 4

Alpha diversity metrics for surface-sterilized and non-surface-sterilized black ash (Fraxinus nigra) and showy lady's slipper (Cypripedium reginae) roots: ITS2 (A) Observed richness, (B) Fisher's alpha diversity index of surface-sterilized roots, (C) Observed richness, and (D) Fisher's alpha diversity index of non-surface-sterilized roots. Orchid and ash sample groups: ANO, ash, no orchid; AO-Ash, ash near orchid; AO-Orch, orchid near ash; ONA, orchid, no ash. The same letters within each plot identify samples that are not significantly different from one another.

Figure 5

(A) PCoA and (B) NMDS of ITS2 ASVs recovered from surface-sterilized roots. All samples are identified by orchid and ash sample groups (ANO, ash, no orchid; AO-Ash, ash near orchid; AO-Orch, orchid near ash; ONA, orchid, no ash) and location. PCoA sample distances calculated using Bray-Curtis. NMDS model stress values are included. Abbreviated ASVs include: Cad. orch., Cadophora orchidicola; Hym. epi., Hymenoscyphus epiphyllus; Rhiz. intra., Rhizophagus intraradices; Rhiz. irreg., Rhizophagus irregularis; T. max., Tetracladium maxilliforme. Where present, values in square brackets after each label indicate the number of clustered vectors [e.g., Glomus (1–6) means there are six Glomus sp. vectors].

Figure 6

Heatmap of the top 10 abundant fungal genera (ITS2) found between shared (A) surface-sterilized (SS) and (B) non-surface-sterilized (NSS) roots of black ash (Fraxinus nigra) and showy lady's slipper orchid (Cypripedium reginae). Sample groups: ANO, ash, no orchid; AO-Ash, ash near orchid; AO-Orch, orchid near ash; ONA, orchid, no ash. Identical names followed by the same clade (C) number signify ASVs that are within the same clade in maximum likelihood analysis, with <0.01 evolutionary distance, and likely belong to a single organism.

Figure 7

Correlation network of fungi occurring in surface-sterilized roots of both black ash (Fraxinus nigra) and showy lady's slipper orchid (Cypripedium reginae), based on ITS2 sequence data (ASVs grouped as clades <0.01 distance in ML phylogeny). Edges between nodes represent a strong positive Spearman's correlation (ρ > +0.57). Modules are grouped by color, edge widths are scaled by correlation size, and node sizes are proportional to the number of connections. Duplicate ASV clades sharing the same genus are numbered. Shortened taxon names include: Cad. orchid., Cadophora orchidicola; Cla. claroideum, Claroideoglomus claroideum; Cup. borealis, Cuphophyllus borealis; Dac. pauc., Dactylonectria paucispora; Ily. radicicola, Ilyonectria radicicola; Ino. ochroalba, Inocybe ochroalba; Glo. macro., Glomus macrocarpum; Rem. stel., Remispora stellata; Rhi. irreg., Rhizophagus irregularis; Rhi. intra., Rhizophagus intraradices; Spir. cupreo., Spirosphaera cupreorufescens; Tet. maxi., Tetracladium maxilliforme; Tom. galzinii, Tomentella galzinii; Xyl. aquaticus, Xylomyces aquaticus.