Mucoromycotina Fine Root Endophyte Fungi Form Nutritional Mutualisms with Vascular Plants

Abstract

Fungi and plants have engaged in intimate symbioses that are globally widespread and have driven terrestrial biogeochemical processes since plant terrestrialization >500 million years ago. Recently, hitherto unknown nutritional mutualisms involving ancient lineages of fungi and nonvascular plants have been discovered, although their extent and functional significance in vascular plants remain uncertain. Here, we provide evidence of carbon-for-nitrogen exchange between an early-diverging vascular plant (Lycopodiella inundata) and Mucoromycotina (Endogonales) fine root endophyte fungi. Furthermore, we demonstrate that the same fungal symbionts colonize neighboring nonvascular and flowering plants. These findings fundamentally change our understanding of the physiology, interrelationships, and ecology of underground plant-fungal symbioses in modern terrestrial ecosystems by revealing the nutritional role of Mucoromycotina fungal symbionts in vascular plants.

Figure 1.

Land plant phylogeny and species used in this study. A, Land plant phylogeny showing key nodes alongside commonly associated fungal symbionts (Duckett and Ligrone, 1992; Duckett et al., 2006; James et al., 2006; Bidartondo et al., 2011). B, L. inundata at Thursley Common, Surrey, United Kingdom, June 2017.

Figure 2.

Carbon-for-nutrient exchange between L. inundata and Mucoromycotina fine root endophyte fungi. A, Total plant-fixed carbon transferred to Mucoromycotina FRE fungi by L. inundata. B, Percent allocation of plant-fixed carbon to Mucoromycotina FRE fungi. C and D, Total plant tissue phosphorus (³³P) and nitrogen (¹⁵N) content in nanograms (C) and tissue concentration (ng g⁻¹) of fungal-acquired ³³P and ¹⁵N in L. inundata tissue (D). In (A) and (B), n = 20; in (C) and (D), n = 10 (n indicates the number of biological replicates used during carbon-for-nutrient exchange experiments). Experiments were carried out three times. Asterisk (*) indicates where P < 0.05, Student’s t test. Error bars = means ± se.

Figure 3.

Carbon- and nitrogen-stable isotope natural abundance of L. inundata (L.i., n = 6), J. bulbosus (J.b., n = 6) and surrounding angiosperms (AM: M. caerulea, M.c., n = 6; ectomycorrhizal: P. sylvestris, P.s., n = 1; B. pendula, B.p., n = 1; ericoid mycorrhizal: C. vulgaris, C.v., n = 3, E. tetralix, E.t., n = 6) for leaf (A) and root (B) samples, respectively. Values = means ± sds. One-tailed Kruskal–Wallis test, followed by Dunn’s post hoc procedure, found significant differences (P > 0.05) among L. inundata, J. bulbosus, and surrounding angiosperms as references in leaf carbon- and nitrogen-stable isotope natural abundance and in root nitrogen-stable isotope natural abundance.

Figure 4.

Light micrographs of trypan-blue–stained tissues. A and B, Branching fine hyphae with small swellings/vesicles in thallus cells (A) and rhizoid (B) of the liverwort F. foveolata (from Thursley Common) colonized by both Mucoromycotina FREs and Glomeromycotina; in (B) also note the coarse hyphae (arrowhead). C to E, Fine hyphae with small swellings/vesicles in the root hairs and root cells of the lycophyte L. inundata colonized by Mucoromycotina FREs only (field-collected specimens from Thursley Common). F, Fine hyphae with small swellings/vesicles and large vesicles in a root of the grass H. lanatus (from Lynn Crafnant, Wales) colonized by both Mucoromycotina FREs and Glomeromycotina. G and H, Roots of the grass M. caerulea (from Thursley Common) colonized by both Mucoromycotina FREs and Glomeromycotina, showing fine hyphae (G) and coarse hyphae with large vesicles (H). Scale bars = 50 μm (A and B, D–F); 100 μm (C, G, and H).

Figure 5.

SEM images. A, Fine hyphae (arrows) with a small swelling/vesicle (*) in the thallus cells of F. foveolata (from Thursley Common); also note the much coarser hyphae (arrowheads). B to G, Fungal colonization in L. inundata. Intercalary (B) and terminal (C) small swellings/vesicles on fine hyphae in the ventral cell layers of a protocorm (from Thursley Common). Centrally and above this intracellular colonization zone, the fungus becomes exclusively intercellular, as evidenced by the presence of abundant, tightly-appressed hyphae surrounding the central protocorm cells (D, arrows) and eventually completely fills the large, mucilage-filled ICSs present in this zone (E, *). Cross sections of roots of experimental plants; several cells colonized exclusively (*) by branching fine hyphae with small swellings/vesicles (F), enlarged in (G). H and I, Cross sections of roots of J. bulbosus (from Thursley Common) showing fine (arrows) and coarse (arrowhead) hyphae (H) and a fine hypha with small swellings/vesicles (I). Scale bars = 20 μm (A, D, G, and I), 10 μm (B, C, and H), and 100 μm (E); 50 μm (F).