Molecular phylogeny
The topologies of the 18S-ITS-28S and 18S-ITS-28S + RPB1 trees generated from BI and ML analyses of the alignments were similar (Fig. S1 and Fig. 1, respectively). Both BI and ML analyses indicated that our fungi, initially named Species 1 and Species 2, are new species in the genera Glomus and Dominikia, respectively (Fig. 1 and Fig. S1). Consequently, these species are newly described here as G. chinense and D. gansuensis. The intraspecific similarities of 18S-ITS-28S sequences of G. chinense and D. gansuensis were 97.2–100% and 97.2–99.5%, respectively; the similarities of RPB1 sequences of these species were 99.53–100% and 99.54–100%, respectively.
The phylogenetic analyses showed that the closest relatives of G. chinense are G. atlanticum and G. ibericum (Fig. 1 and Fig. S1), whose 18S-ITS-28S and RPB1 sequences differed by ca. 3–4% and 1.3–1.5% (G. atlanticum only), respectively, from those of the new species. Instead, the sister relative of D. gansuensis is D. glomerocarpica, whose 18S-ITS-28S and RPB1 sequence divergences were ca. 7–9% and 5.5–5.8%, respectively. In the analyses, the G. chinense clade was fully or strongly supported (18S-ITS-28S tree: BI = 1.0, ML = 99%; 18S-ITS-28S + RPB1 tree: BI = 1.0, ML = 100%). Also, the node linking this clade with the clades with G. atlanticum and G. ibericum obtained sufficient supports (18S-ITS-28S tree: BI = 1.0, ML = 84%; 18S-ITS-28S + RPB1 tree: BI = 1.0, ML = 74%). The clade with D. gansuensis was fully supported in both BI and ML analyses, as well as the node linking it with the D. glomerocarpica clade obtained full or strong supports (18S-ITS-28S tree: BI = 1.0, ML = 100%; 18S-ITS-28S + RPB1 tree: BI = 1.0, ML = 99%).
Taxonomy
Glomus chinense F. Yu, B.T. Goto, H. Feng & Y. Liu, sp. nov. Figure 2A-F
MycoBank number: MB841240
Etymology. Latin, chinense, referring to the country (China) in which this fungus was originally discovered.
Diagnosis. Differs from G. atlanticum and G. ibericum, the sister phylogenetic relatives, in the spore wall structure and the phenotypic and biochemical properties of the spore wall layers, as well as in the nucleotide composition of sequences of the 18S-ITS-28S nuclear rDNA region and RPB1 gene.
Description. Spores formed singly or occasionally in loose clusters in soil; arise blastically at tips of sporogenous hyphae continuous with extraradical mycorrhizal hyphae. Spores pale orange-yellow (0-45-100-6) to dark brown (37-76-92-51); globose to subglobose, (47–)64(–93) µm diam, rarely ovoid, 22–43 × 45–74 µm, with one subtending hypha (Fig. 2A). Spore wall consists of three layers (layers 1–3; Fig. 2A–E). Layer 1, forming the spore surface, evanescent (short lived), hyaline, slightly roughened in mature spores, (1.7–)2.0(–2.5) µm thick, staining pinkish purple (0-60-0-40) in Melzer’s reagent (Fig. 2C–E). Layer 2 laminate, permanent, orange-yellow (0-45-100-6) to dark brown (37–76–92–51), smooth, (1.1–)2.4(–3.8) µm thick, staining yellow brown (0-51-76-36) in Melzer’s reagent (Fig. 2C–E). Layer 3 light brown (0-50-75-28) to bright yellow (0-5-85-5), (0.6–)0.9(–1.6) µm thick, sometimes difficult to see, not reacting in Melzer’s reagent (Fig. 2C). Subtending hypha pale orange-yellow (0-45-100-6) to dark brown (37-76-92-51), straight or curved, cylindrical, usually constricted at the spore base, (4.4–)9.1(–14.7) µm wide at the spore base (Fig. 2A, D). Pore of subtending hyphae open. Wall of subtending hypha pale orange-yellow (0-45-100-6) to dark brown (37-76-92-51), (2.6–)3.7(–5.8) µm thick at the spore base, composed of three layers continuous with spore wall layers 1–3 (Fig. 2C). Germination unknown.
Mycorrhizal associations. In the field, G. chinense was found in mixed rhizosphere soils of Potentilla fruticose, Spiraea alpina, and Potentilla sischanensis in a shrubland located in eastern Tibetan Plateau of China. In single-species cultures with Sorghum bicolor as host plant, G. chinense formed abundant arbuscules and intraradical hyphae (Fig. 2F).
Specimens examined. Slides with glomerospores permanently mounted in PVLG and a mixture of PLVG and Melzer’s reagent. Holotype deposited in the herbarium of Lanzhou University, China (accession number: LZU_AMF_1708). Isotypes deposited in the herbarium of Lanzhou University, China (LZU_AMF_1709 to 1722) and in the UFRN herbarium, Brazil (Fungos 3388).
Distribution and habitat. The shrubland nearby the Hezuo city, Gansu province, China (35°13'8.86''N, 102°48'30.83"E; 2843 m a.s.l.) is the only site in which this fungus was physically found so far. Soil pH at this site was 7.62 ± 0.08 (mean ± SD, n = 5) and the concentration of soil available phosphorus was 10.4 ± 3.5 mg kg− 1. This type of shrubland, mainly dominated by Potentilla fruticose, is very common on the eastern Tibetan Plateau. BLAST searches showed only one environmental sequence (from an alpine meadow on the Tibetan Plateau; GenBank accession number: JX096603) suggesting conspecificity to G. chinense. Thus, G. chinense may be an endemic species on the Tibetan Plateau.
Dominikia gansuensis F. Yu, B.T. Goto, H. Feng & Y. Liu, sp. nov. Figure 3A-F
MycoBank number: MB841241
Etymology. Latin, gansuensis, referring to the Gansu Province of China, where this fungus was originally found.
Diagnosis. Differs from D. glomerocarpica, the sister phylogenetic relative, in (i) morphology of glomerocarps, (ii) the spore wall structure and the histochemical properties of the spore wall layers, as well as in (iii) the nucleotide composition of sequences of the 18S-ITS-28S nuclear rDNA region and RPB1 gene.
Description. Spores usually produced in hypogeous glomerocarps, rarely singly in soil. Glomerocarps dull yellow (8-40-83-0) to yellow-brown (0-51-76-36), 99–(198)–354 × 188–(434)–463 µm, with dozens randomly distributed spores (Fig. 3A). Spores pale yellow (0-4-29-0) to yellow-brown (0-51-76-36), globose to subglobose, (20–)47(–86) µm diam, rarely ovoid, 18–42 × 30–60 µm, with one subtending hypha (Fig. 3B). Spore wall composed of three layers (Fig. 3B–D). Layer 1, forming the spore surface, evanescent, short-lived, hyaline, (0.4–)0.8(–1.4) µm thick. Layer 2 < 0.5 µm thick, usually tightly adherent to layer 3, even in vigorously crushed spores. Layer 3 laminate, dull yellow (8-40-83-0) to yellow-brown (0-51-76-36), (0.3–)0.7(–1.2) µm thick. Only spore wall layer 1 stains royal purple (0-40-0-0) to pinkish purple (0-80-65-0) in Melzer’s reagent (Fig. 3C, D). Subtending hypha straight or recurved, cylindrical, (4.4–)6.6(–9.9) µm wide at the spore base (Fig. 3B). Pore (1.0–)2.5(–4.5) µm wide, occluded by a septum continuous with spore wall layer 3 near the spore base (Fig. 3C). Germination by a germ tube arising from the septum at the spore base and emerging through the lumen of the subtending hypha (Fig. 3E).
Mycorrhizal associations. Dominikia gansuensis was originally extracted from rhizosphere soil sampled in a forest dominated by Betula platyphylla, Euonymus alatus, and some understory grasses. However, the plant species with which D. gansuensis formed symbiosis was not determined. In the greenhouse, D. gansuensis formed abundant arbuscules in the roots of Sorghum bicolor (Fig. 3F).
Specimens examined. Permanent slides with glomerocarps/glomerospores. Holotype deposited in the herbarium of Lanzhou University, China (LZU_AMF_1727); isotypes deposited in the herbarium of Lanzhou University, China (LZU_AMF_1728 to1741) and in the UFRN herbarium, Brazil (Fungos 3389).
Distribution and habitat. So far, D. gansuensis was physically found only in a forest site nearby the Hezuo city, Gansu province, China (35°06'27.77"N, 102°51'18.78"E; 2816 m a.s.l.), where the soil pH was 6.44 ± 0.22 (mean ± SD, n = 5) and the concentration of soil available phosphorus was 11.0 ± 4.9 mg kg− 1. BLAST queries indicated several environmental sequences of an identity of > 97% to D. gansuensis 18S-ITS-28S sequences. These sequences were derived from a wetland ecosystem in southern China, a semi-mangrove forest in southern China, a grassland in northern China, and a forest in Czech Republic.