A new order, Entrophosporales, and three new Entrophospora species in Glomeromycota

Abstract

As a result of phylogenomic, phylogenetic, and morphological analyses of members of the genus Claroideoglomus, four potential new glomoid spore-producing species and Entrophospora infrequens, a new order, Entrophosporales, with one family, Entrophosporaceae (=Claroideoglomeraceae), was erected in the phylum Glomeromycota. The phylogenomic analyses recovered the Entrophosporales as sister to a clade formed by Diversisporales and Glomeraceae. The strongly conserved entrophosporoid morph of E. infrequens, provided with a newly designated epitype, was shown to represent a group of cryptic species with the potential to produce different glomoid morphs. Of the four potential new species, three enriched the Entrophosporales as new Entrophospora species, E. argentinensis, E. glacialis, and E. furrazolae, which originated from Argentina, Sweden, Oman, and Poland. The fourth fungus appeared to be a glomoid morph of the E. infrequens epitype. The physical association of the E. infrequens entrophosporoid and glomoid morphs was reported and illustrated here for the first time. The phylogenetic analyses, using nuc rDNA and rpb1 concatenated sequences, confirmed the previous conclusion that the genus Albahypha in the family Entrophosporaceae sensu Oehl et al. is an unsupported taxon. Finally, the descriptions of the Glomerales, Entrophosporaceae, and Entrophospora were emended and new nomenclatural combinations were introduced.

Keywords: Claroideoglomus; arbuscular mycorrhizal fungi; four new taxa; morphology; new combinations; nuc rDNA; phylogenomic and phylogenetic taxonomy; rpb1.

FIGURE 1

Best Maximum Likelihood phylogeny inferred with IQ-TREE from a concatenated alignment of 1260 single copy orthologs shared by at least 50% of the taxa. The same topology was recovered with ASTRAL III. Values near the branches correspond to bootstrap support (BS) from the ML analysis, local posterior probabilities (LPP), and quartet support from the ASTRAL analysis (BS/LPP/quartet support). Asterisks indicate maximum support (100 or 1.0). Mucoromycota and Mortierellomycota were used as outgroups. Bar indicates 0.2 expected change per site per branch.

FIGURE 2

Evaluation of support among 1260 individual gene trees for alternative hypotheses of the phylogenetic placement of Claroideoglomeraceae. Bar graphs represent the gene tree quartet frequencies for three possible branching orders. The dashed horizontal line indicates the expectation of a hard polytomy.

FIGURE 3

A 50% majority-rule consensus tree from the Bayesian analysis of 45S nuc rDNA sequences concatenated with rpb1 sequences of Claroideoglomus 1, 2, 4 (newly described as Entrophospora argentinensis, E. glacialis, and E. furrazolae, respectively), Claroideoglomus 3 (a glomoid morph of the E. infrequens epitype), eight other species of Claroideoglomus sensu C. Walker and A. Schüßler, and three Diversispora species serving as outgroup. The former species names included in Entrophosporales are reported between brackets. The new species and the accession numbers of the sequences obtained in this study are in bold. The Bayesian posterior probabilities ≥0.90 and ML bootstrap values ≥50% are shown near the branches, respectively. Bar indicates 0.002 expected change per site per branch. The two basal branches were shortened to 20% in length to improve visibility (indicated by //).

FIGURE 4

Entrophospora infrequens. (A) Entrophosporoid morph with spore (sp) formed inside the sporiferous saccule (ss). (B) Funnel-shaped structure, continuous with spore wall 1 layer 3, supporting the sporiferous saccule wall (ssw). (C) Spore wall 1 layers (sw1l) 1–3; swl1 is almost completely sloughed off; tooth-shaped projections (tp) in cross-view are visible. (D) Spore wall 1 (sw1) and spore wall 2 layers (sw2l) 1–3. (E) Sporiferous saccule wall layers (sswl) 1 and 2, and the neck (n) of sporiferous saccule. (F) Juvenile glomoid spore (sp) with subtending hypha (sh) developed from the sporiferous saccule neck (ssn); soil debris (sd) are indicated. (G) Juvenile glomoid spore (sp) with subtending hypha (sh) developed from sporiferous saccule (ss). (H) Mycorrhiza with arbuscule (a), vesicles (v), and intraradical hyphae (ih). (A–D,F–H) Spores and mycorrhizal structures in PVLG. (E) Sporiferous saccule in PVLG+Melzer’s reagent. (A–H) Differential interference microscopy. Scale bars: (A) = 50 μm, (B–H) = 10 μm.

FIGURE 5

Entrophospora infrequens. (A) Intact glomoid spores (sp) with subtending hyphae (sh). (B–F) Spore wall layers (swl) 1 and 2 continuous with subtending hyphal wall layers (shwl) 1 and 2; a septum (s) continuous with swl2 in the lumen of the sh is indicated in (F). (G,H) Arbuscules (a), intraradical hyphae (ih), and vesicles (v) in roots of Plantago lanceolata stained in 0.1% Trypan blue. (A–D,G,H) Spores and mycorrhizal structures in PVLG. (E,F) Spores in PVLG+Melzer’s reagent. (A–H) Differential interference microscopy. Scale bars: (A) = 20 μm, (B–H) = 10 μm.

FIGURE 6

Entrophospora argentinensis. (A) Intact spores. (B–D,F) Spore wall layers (swl) 1–3; note the differences in thickness of the laminate swl2 in mature spores depicted in (B–D) and the equal thickness of swl2 in a young spore presented in (F), as well as the birefringent (b) properties of layer 2 in polarized light visible in (C). (E) Circular and ellipsoidal lighter patches (p) formed by the thinner areas of the laminate spore wall layer 2 seen in a plan view. (G,H) Subtending hyphal wall layers (shwl) 1–3 continuous with spore wall layers (swl) 1–3; note the highly deteriorated swl1 and a septum (s) in the subtending hyphal lumen formed by shwl2 and 3 continuous with swl2 and 3. (A,D–H) Spores in PVLG. (B) Spore in PVLG+Melzer’s reagent. (A–H) Differential interference microscopy. Scale bars: (A) = 50 μm, (B–H) = 10 μm.

FIGURE 7

Mycorrhizal structures of Entrophospora argentinensis in roots of Plantago lanceolata stained in 0.1% Trypan blue. (A) Arbuscule (a), coiled (c) and straight (sh) intraradical hyphae. (B) Vesicle (v) and intraradical hyphae (ih). (A,B) In PVLG. (A,B) Differential interference microscopy. Scale bars: (A) = 10 μm, (B) = 20 μm.

FIGURE 8

Entrophospora glacialis. (A) Intact spores (sp) with subtending hyphae (sh); juvenile spore (js) is indicated. (B–F) Spore wall layers (swl) 1–5. (G,H) Subtending hyphal wall layers (shwl) 1–5 continuous with spore wall layers (swl) 1–5; a septum (s) continuous with swl5 is indicated in (H). (A,E,G,H) Spores in PVLG. (B–D,F) Spores in PVLG+Melzer’s reagent. (A–H) Differential interference microscopy. Scale bars: (A) = 50 μm, (B–H) = 10 μm.

FIGURE 9

Entrophospora furrazolae. (A) Intact spores (sp) with subtending hyphae (sh). (B–F) Spore wall layers (swl) 1–4; a septum (s) continuous with swl4 is indicated in (C). (G,H) Subtending hypha (sh) with subtending hyphal wall layers (shwl) 1–3; a septum (s) continuous with swl4 is indicated in (H). (A–C) Spores in PVLG. (D–H) Spores in PVLG+Melzer’s reagent. (A–H) Differential interference microscopy. Scale bars: (A) = 20 μm, (B–H) = 10 μm.