Revealing natural relationships among arbuscular mycorrhizal fungi: culture line BEG47 represents Diversispora epigaea, not Glomus versiforme

Abstract

Background: Understanding the mechanisms underlying biological phenomena, such as evolutionarily conservative trait inheritance, is predicated on knowledge of the natural relationships among organisms. However, despite their enormous ecological significance, many of the ubiquitous soil inhabiting and plant symbiotic arbuscular mycorrhizal fungi (AMF, phylum Glomeromycota) are incorrectly classified.

Methodology/principal findings: Here, we focused on a frequently used model AMF registered as culture BEG47. This fungus is a descendent of the ex-type culture-lineage of Glomus epigaeum, which in 1983 was synonymised with Glomus versiforme. It has since then been used as 'G. versiforme BEG47'. We show by morphological comparisons, based on type material, collected 1860-61, of G. versiforme and on type material and living ex-type cultures of G. epigaeum, that these two AMF species cannot be conspecific, and by molecular phylogenetics that BEG47 is a member of the genus Diversispora.

Conclusions: This study highlights that experimental works published during the last >25 years on an AMF named 'G. versiforme' or 'BEG47' refer to D. epigaea, a species that is actually evolutionarily separated by hundreds of millions of years from all members of the genera in the Glomerales and thus from most other commonly used AMF 'laboratory strains'. Detailed redescriptions substantiate the renaming of G. epigaeum (BEG47) as D. epigaea, positioning it systematically in the order Diversisporales, thus enabling an evolutionary understanding of genetical, physiological, and ecological traits, relative to those of other AMF. Diversispora epigaea is widely cultured as a laboratory strain of AMF, whereas G. versiforme appears not to have been cultured nor found in the field since its original description.

Figure 1. Phylogenetic tree of Diversisporales computed from the core dataset of nuclear SSU-ITS-LSU rDNA sequences.

RAxML maximum likelihood analysis with bootstrap support shown at the branches; topologies with support below 50% were collapsed to polytomies. The most recent synonyms for species in Diversispora are given in brackets. The published ‘Entrophospora nevadensis‘ sequence (SSU rDNA) is short and does not allow species resolution, but clusters with high support within the Diversispora celata - D. eburnea clade. The two short, concatenated ‘Otospora bareae‘ sequences (SSU rDNA) also cluster within the genus Diversispora. The genus Redeckera comprises the species formerly published as Glomus fulvum, G. megalocarpum and G. pulvinatum. The tree is rooted with three representative sequences of the sister order Glomerales. The scale bar indicates proportional substitutions per site.

Figure 2. Phylogenetic tree of Diversisporaceae computed from the extended dataset, including environmental nuclear rDNA sequences.

RAxML maximum likelihood analysis with bootstrap support shown at the branches; topologies with support below 50% were collapsed to polytomies. The tree is rooted with representatives of the Glomerales. The scale bar indicates proportional substitutions per site. Except for very short environmental SSU rDNA sequences that distorted the tree topology, all Diversisporaceae sequences which were available from the public databases were used and have the following origins: ¹ the specimen from which this sequence was derived has Claroideoglomus etunicatum-like spore morphology; soil from a re-vegetated coal spoil heap, beneath Salix sp. and associated weeds, which included Plantago major, P. lanceolata, Fragaria vesca and various grasses; ² Fazio's Greenhouse, from M. Pfeiffer's pot culture no. 157, Building 42-2R, University of Arizona; ³ other plants reported at the soil sampling location were Alchemilla fontqueri and Senecio elodes (both endemic) and Sorbus hybrid (non-endemic); ⁴ fungus with an appearance similar to a ‘large-spored D. epigaea’, from a temperate greenhouse of Royal Botanical Garden Edinburgh, Plant No. 842581 H; ⁵ immature spores; from fern house of Botanical Garden Jena (the plant was transferred to Jena from the botanical garden of the Wilhelma, Stuttgart, Germany); ⁷ Diversispora epigaea-like spores; temperate greenhouse of Royal Botanical Garden Edinburgh, the pot also contained an Oxalis sp. as a weed; ⁶ tropical greenhouse at the USDA-ARS horticultural research station; ⁸ sporocarp from litter layer of semi natural woodland, with associated understory, including an Allium sp.; ⁹ this sequence most likely represents a species distinct from Redeckera fulvum, therefore it is annotated here as ‘R. fulvum-like’; ¹⁰ sequences annotated as ‘D. trimurales’, from the same submission as the three sequences (FJ461851,54,55) that cluster in Diversispora, but clearly falling in distinct families; ¹¹ culture published as GINCO4695rac-11G2 from the AFTOL project, but lacking further information.

Figure 3. Dimensions of spores from Glomus versiforme type collection and of Diversispora epigaea (grey: lengths and white: width).

A. Spores of the lectotype of Glomus versiforme (W4551) prepared from the Endogone versiformis type material. B. Large spore type (W4550) of an unknown species in the E. versiformis type material. C. Diversispora epigaea BEG47 (combined measurements of specimens from 49 voucher collections sampled from among 29 ex-type sub-cultures).

Figure 4. Photomicrographs of specimens from the holotype collection of Glomus versiforme (basionym Endogone versiformis).

Pale spores (G. versiforme) of W4551, dark spores (undetermined Glomus sp.) of W4550. A. Sporocarp portion of G. versiforme showing pale spores (s) and a felted, pale-coloured peridium (p). Larger, dark coloured spores of an unknown Glomus sp. can be seen out of focus in the background. B. Part of a sporocarp of Glomus sp. showing the pigmented peridial (p) and contextual (c) hyphae and embedded spores (arrows). C. The two different spore morphs in water (G. versiforme indicated by arrows), illustrating the difference in spore size and colour. D. The pale-coloured spores of G. versiforme showing clustered spores from a sporocarp. E. Sporocarp portion of the dark spored unknown Glomus sp. F. Five clustered spores of G. versiforme from a sporocarp with accompanying vesicles (ve). G. Peridial hyphae of G. versiforme showing size and colour. H. Peridial hyphae of the dark spored Glomus sp. I. Spores of G. versiforme (left) and of the dark spored Glomus sp. (centre), allowing comparison of size, shape and pigmentation. J. Thin-walled pale-coloured spore of G. versiforme. K. A thick-walled darkly coloured spore of Glomus sp. L and M. Subtending hyphae of G. versiforme. Most specimens are sessile because of breakage of the very thin subtending hyphal wall at the spore base. N and O. Subtending hyphae of the dark spored Glomus sp., broken close to the spore base and occluded by an amorphous plug in the bowl-shaped lumen (N) or persistent and occluded by spore wall thickening (O). P. Wall detail of a spore of G. versiforme showing two components in the structural spore wall (1, 2) and a questionable third component internally (3?). Q and R. Wall detail of a spore of the dark spored Glomus sp. showing three components in the structural spore wall (Q), and a possible fourth (4?) separate component (R) internally.

Figure 5. Photomicrographs of specimens from Diversispora epigaea ex-type pot cultures (including culture line BEG47).

Dark spores of W5165, pale spores of W5167 except Figure 5L, which is from W4565. A. Spore cluster, formed on roots near the surface of a pot. B. View of a spore cluster showing the undifferentiated aggregation of pale coloured and orange spores. C. Spore mass, showing pale and dark spores. D. Spores photographed in water, uncovered on a glass microscope slide. E. Spores of both colours, showing variation in size, shape and pigmentation. F. A thick-walled pigmented spore of the dark morph. G. Thin-walled, immature pale-coloured spore. H and I. Subtending hyphae of dark (H) and pale (I) spores showing occlusion by spore wall thickening and a distal septum in the dark morph. Note the difference in wall thickness. J and K. Wall structure of dark (J) and pale (K), spores showing thin outer (1), thick laminated (2), and thin inner (3) components. L. Hyphal bridging, also known as wound healing, in the somatic mycelium. M. Spore germination (g) at the base of the subtending hypha (sh). N. Germination directly through the wall. O. A septum occluding the hyphal attachment of a thin-walled spore of the pale morph close to the spore base.