Entrophospora infrequens and the potential new species, initially named Claroideoglomus 1–4, were characterized based on spores extracted from single-species pot cultures. These cultures were established from spores extracted from trap pot cultures inoculated with field-collected mixtures of rhizosphere soils and root fragments of the following plant species. The field host of E. infrequens was Juniperus communis L. growing in a pine forest on inland sand dunes of Kampinos National Park (52^o19′ N, 20^o45′ E), Poland. The field sample was collected by J. Błaszkowski on 26 July 1986.

The field hosts of Claroideoglomus 1 were Deschampsia flexuosa (L.) Trin and Poa rigidifolia Steud. These plants grew in a steppe community located in northern Tierra del Fuego (Mendoza et al., 2011) on a complex of moraine deposits and glacial-outwash plains dominated by grasses of Chiliotrichum diffusum (G. Forst.) Kuntze, D. flexuosa, Empetrum rubrum Vahl ex Willd., Festuca gracillima Hook., and P. rigidifolia (Collantes et al., 1999).

The regional climate is influenced by the proximity of the Atlantic Ocean (Paruelo et al., 1998). The mean air temperature is about 0°C in July (winter) and reaches 10°C in January (summer). The annual rainfall ranges from about 300 to 400 mm along a NE–SW gradient and is evenly distributed throughout the year. The field sample was collected by R. Mendoza, Museo Argentino de Ciencias Naturales (MACN-CONICET) “Bernardino Rivadavia,” Buenos Aires, Argentina, during the period from 1 to 15 March 2009.

Claroideoglomus 2 was harbored in the field within an initial community consisting of cryptogamic species and the following vascular plants: F. vivipara (L.) Sm., Po. alpina L., Salix herbacea L., S. polaris Wahlenb., and Silene acaulis (L.) Jacq. This community grew in a glacier foreland of Isfallglaciarän located in Tarfala valley, N Sweden (67°54′58 N, 18°35′ E). The average annual air temperature and the annual sum of rainfall in this site are 3.4°C and 503.3 mm, respectively (The Swedish Agency for Marine and Water Management, available at: www.smhi.se). The soil contained 19.28 and 3.17% of silt and clay, respectively, and its chemical properties were as follows: pH (in H₂O), 6.3; P₂O₅ (mg/100 g), 4.63; Na (cmol/kg), 0.04; N (%), 0.00; and C (%), 0.08. The field sample was collected by Paulina Wietrzyk-Pełka and Michał Węgrzyn (Institute of Botany of the Jagiellonian University) on 23 July 2019.

The field inoculum containing Claroideoglomus 3 was collected under an initial community consisting of Limonium sinuatum (L.) Mill. This community grew in a Solonetz Gley sodic soil, which was hydromorphic, compact, and contained high levels of surface salt deposits. The community was located at Fortuna, Murcia, Spain (38°06′ N, 1°06′ W). The mean annual air temperature and the annual sum of rainfall in this site are 19.6°C and 300 mm, respectively. The soil contained 20.28 and 53% of silt and clay, respectively, and its chemical properties were as follows: pH (in H₂0), 8.5; P₂O₅ (mg/100 g), 2.1, CaCO₃ (%), 12.0; Mg₂ (cmol/kg), 2967.04; K (cmol/kg), 2955; Na (cmol/kg), 1829.4; N (%), 0.90; and C (%), 0.08. The field sample was collected by Félix Fernández in November 2009.

Claroideoglomus 4 was isolated from samples collected in the Sultanate of Oman and in Poland. In Oman, Claroideoglomus 4 was associated with roots of Prosopis cineraria (L.) Druce. It grew in an undisturbed habit adjacent to a date palm (Phoenix dactylifera L.) plantation of an experimental station (22°14′ N, 59°10′ E) belonging to the Ministry of Agriculture, Fisheries and Water Resources of Oman. Summer temperatures exceed 48°C (Glennie and Singhvi, 2002). Based on the aridity index defined by the United Nations Environmental Program (UNEP, 2006), this site is characterized as Hyperarid (AI < 0.05), with annual rainfall not exceeding 100 mm (Fisher and Membery, 1998). The soil of this site was sandy loam with the following chemical properties: pH (in H₂O), 8.2; contents of P (mg kg^–1), 41.4; N (%), < 0.1; and organic matter (%), 1.5 (Al-Yahya’ei et al., 2011). The field sample was collected by Mohamed N. Al-Yahya’ei in August 2006.

The site in Poland where Claroideoglomus 4 was found is a field near Lubliniec in the Silesian Upland (50°37′ N, 18°43′ E) from where Dominikia bonfanteae was isolated (Błaszkowski et al., 2021b). Details about plant community and the physico-chemical properties of soil are available in Malicka et al. (2020). The climate of the region, where the study material was collected, is characterized by Błaszkowski et al. (2019).

Spores of the species originally described as G. walkeri and C. hanlinii, the latter of which was also included in our analyses (see below), originated from single-species cultures grown by J. Błaszkowski.

Methods used to establish trap and single-species cultures, growing conditions, and methods of spore extraction and staining of mycorrhizal structures were as those described previously (Błaszkowski et al., 2012). Five to ten spores of uniform morphology of each AMF species were used to establish single-species cultures.

Morphological features of spores and phenotypic and histochemical characters of spore wall layers of the new species presented here were characterized based on at least 50-100 spores of each species mounted in water, lactic acid, polyvinyl alcohol/lactic acid/glycerol (PVLG, Omar et al., 1979), and a mixture of PVLG and Melzer’s reagent (1:1, v/v). The preparation of spores for study and photography was as described previously (Błaszkowski, 2012; Błaszkowski et al., 2012). The types of spore wall layers were defined by Walker (1983) and Błaszkowski (2012). Color names were from Kornerup and Wanscher (1983). The nomenclature of fungi and the authors of fungal names are from the Index Fungorum website http://www.indexfungorum.org/AuthorsOfFungalNames.htm. The term “glomerospores” was used for spores produced by AMF as proposed by Goto and Maia (2006).

Voucher specimens of the proposed new species [spores permanently mounted in PVLG and a mixture of PVLG and Melzer’s reagent (1:1, v/v) on slides] were deposited at ZT Myc (ETH Zurich, Switzerland; holotypes) and in the Laboratory of Plant Protection, Department of Shaping of Environment (LPPDSE), West Pomeranian University of Technology in Szczecin, Poland (isotypes).

Genomic DNA for obtaining 45S and rpb1 sequences was extracted from eight single spores of E. infrequens and each of the four potential new Claroideoglomus species. Details of the treatment of the spores prior to PCR, conditions and primers used for PCR, as well as cloning and sequencing of PCR products to obtain 45S sequences of the species are available in Krüger et al. (2009), Symanczik et al. (2014), and Błaszkowski et al. (2015b,c).

To obtain rpb1 sequences of these five species, as well as C. hanlinii, C. luteum, and the species originally described as G. walkeri, a new set of primers was developed. The primer set consisted of two forward primers (RPB1-3F: GTC TTC GTG CAG TTT GGG A; RPB1-4F: CTA GGC CTG ATT GGA TGA T) and two reverse primers (RPB1-5R: ACG ATT TGT TTT GGT ACC AT; RPB1-5RN: TTC ATC TCA CCA TCA A). Characteristics of the primers and the PCR conditions are available in Błaszkowski et al. (2021a). To increase the chance to obtain a successful amplification of unknown sequences, the following approach was used to design the primers. All the possible 19-21 bases oligos obtained from the rpb1 sequence available for C. claroideum were assessed according to the following criteria: (i) a perfect match with the C. etunicatum and C. claroideum sequences, (ii) at least one C/G in the 3′ end, and (iii) the highest number of matches among all the AMF rpb1 sequences available (about 200) with maximum three mismatches tolerated if not in the last five residues at the 3′ end. Candidate oligos were evaluated with OligoAnalyzer for Tm and the presence of secondary structures.

Cloning and sequencing of rpb1 PCR products were performed identically to those used to obtain 45S sequences (see above). Both 45S and rpb1 sequences were deposited in GenBank (45S: MH590060, MH590061, MT722021–MT722024, ON950363–ON950390, and MT722034–MT722038; rpb1: MT733201–MT733210 and ON938320–ON938327).

Previous genome and transcriptome data phylogenies have shown two conflicting topologies with respect to Claroideoglomeraceae (Beaudet et al., 2018; Montoliu-Nerin et al., 2021). Therefore, we compiled three different datasets based on previously published data (Supplementary Table 1). The first dataset included data used by Beaudet et al. (2018) and Montoliu-Nerin et al. (2021), the second dataset included only Beaudet et al.’s (2018) data, and the third dataset included only genomes from the Montoliu-Nerin et al. (2021) analyses. Single-copy orthologs (SCOs) were identified with OrthoMCL (Li et al., 2003) using default settings, and only SCOs that were present in at least 50% of the taxa were included in the phylogenetic analyses. Individual SCOs were aligned with MAFFT 7.407 (Katoh and Standley, 2013). Poorly aligned regions were trimmed with trimAl 1.4.1 (Capella-Gutiérrez et al., 2009) with a gap threshold of 0.1. The individual alignments were concatenated using the script geneStitcher.py (Ballesteros and Hormiga, 2016). An ML phylogeny was inferred with IQ-TREE 2.0 (Nguyen et al., 2015) using ModelFinder (Kalyaanamoorthy et al., 2017) and searching for the best partition scheme. In addition, the individual SCO alignments were used to infer individual gene trees with IQ-TREE and then a phylogenetic inference with ASTRAL-III 5.7.3 (Zhang et al., 2018) was performed. The dataset that included only genome data used by Montoliu-Nerin et al. (2021) was then used to test the hypothesis that Claroideoglomeraceae is sister to Diversisporales and Glomeraceae. For this, we evaluated the support among individual gene trees for alternative branching orders and carried out a polytomy test in ASTRAL-III (Sayyari and Mirarab, 2018).

To clarify the status of Albahypha and Entrophospora, as well as to infer the position of the four potential new species, two sequence datasets were produced. The first consisted of sequences of the 45S segment, or part thereof, of our E. infrequens and the AMF, initially recognized as new species of Claroideoglomus, as well as all other sequenced species of these genera sensu Oehl et al. (2011b). Claroideoglomus hanlinii, a species described later (Błaszkowski et al., 2015a), was also included. The second dataset contained rpb1 sequences available for the species of the first dataset and sequences from the genome of C. candidum. The sequences covered part of the fourth and fifth exons of the rpb1 gene and the intron between them. In each of these datasets, the outgroup was represented by Diversispora species, and the datasets were aligned separately using MAFFT 7 with the E-INS-i strategy. Subsequently, the two alignments were used to manually produce a concatenated alignment, 45S+rpb1. The 45S sequences lacking the rpb1 part were not removed from the alignment. An additional alignment was produced as follows: 45S+rpb1_G with three Glomus instead of Diversispora species in the outgroup to test the impact of this outgroup representing Glomeraceae, the sister family of Claroideoglomeraceae in the Glomerales sensu Schüßler and Walker (2010), on the topology and measures used in comparison of the trees generated in the analyses (described below).

The phylogenetic position of Albahypha, E. infrequens, and the four potential new species was reconstructed based on Bayesian inference (BI) and maximum likelihood (ML) phylogenetic analyses of the 45S, rpb1, 45S+rpb1, and 45S+rpb1_G alignments, performed via CIPRES Science Gateway 3.1 (Miller et al., 2010). The 45S alignment was divided into five partitions: 18S, ITS1, 5.8S, ITS2, and 28S. Five additional rpb1 partitions were added in the alignments with the concatenated genes, as described in Błaszkowski et al. (2021b).

As suggested by Abadi et al. (2019), in both BI and ML analyses, GTR+I+G was chosen as the nucleotide substitution model for each nucleotide partition. Substitution models selected by ModelTest-NG 0.1.5 (Darriba et al., 2020) were also tested in ML analyses, but the trees obtained final loglikelihood values lower compared to those where GTR+I+G was used.

Four Markov chains were run over two million generations in MrBayes 3.2 (Ronquist et al., 2012), sampling every 2,000 generations, with a burn-in at 3,000 sampled trees. The ML phylogenetic tree inference was performed with RAxML-NG 1.0.1 (Kozlov et al., 2019) using a maximum likelihood/1,000 bootstrapping run and an ML estimated proportion of invariable sites and base frequencies. The alignments and tree files were deposited as Supplementary material.

We assumed that clades were supported when the Bayesian posterior probabilities and the ML bootstrap values were ≥0.95 and ≥70%, respectively. The generated phylogenetic trees were visualized and edited in TreeGraph 2 (Stöver and Müller, 2010). To evaluate possible conflicts between the genes, the topologies of the ML trees (collapsed at bootstrap values <70%) were compared. In addition, the trees were compared based on three measures, referred to the ingroup, which are as follows: (i) the number of species clades supported with BI ≥ 0.95 and ML ≥ 70%, (ii) mean BI and ML values for species clades when supported, (iii) mean supports of nodes with BI ≥ 0.95 and ML ≥ 70%, and (iv) the amount of resolution of each tree. The amount of resolution was calculated for the ingroup of the tree by dividing the number of significantly supported internal branches by the size of the ingroup (Thorley and Wilkinson, 2000).

To detect other possible findings of the potential new species, their 45S sequences were used as queries in BLASTn to retrieve more than 300 nucleotide sequences from GenBank. The sequences were selected according to the percentage of identity > 96.5, with at least one of the queries. The possible relatedness with any of the four species was assessed using an evolutionary placement algorithm-based (EPA) approach, performed with RAXML 8.2.10 (Berger et al., 2011). The sequences were aligned in MAFFT together with the 45S dataset, and the 45S+rpb1 ML tree was used as reference support for the phylogenetic affiliation. GTRGAMMA model, the maximum likelihood estimation for the base frequencies, and the partition scheme previously described for the 45S alignment were used for the phylogenetic inference. The jplace output was used in Gappa (Czech et al., 2020) for the placement mass (likelihood weight ratio) accumulation of the placements of each sequence upward from the reference tree with the threshold = 0.9.

The first dataset used for the phylogenomic analyses, which included data from Beaudet et al. (2018) and Montoliu-Nerin et al. (2021), contained 78 SCOs and recovered Claroideoglomeraceae as sister to Diversisporales and Glomeraceae (Supplementary Figure 1). Since Beaudet et al. (2018) showed Claroideoglomeraceae to be sister to Ambisporaceae and Paraglomeraceae based on analyses of spore transcriptomic data, we produced and analyzed a dataset that contained only these data. The analysis yielded 122 SCOs, and the phylogeny showed again Claroideoglomeraceae as sister to Diversisporales and Glomeraceae (Supplementary Figure 2). Finally, to obtain a higher number of SCOs and to perform a topology test, we excluded the Beaudet et al.’s (2018) dataset and analyzed only AM genome data used by Montoliu-Nerin et al. (2021). For this dataset, we obtained 1,260 SCOs present in at least 50% of the taxa, and the phylogenomic analysis also recovered Claroideoglomeraceae as sister to Diversisporales and Glomeraceae (Figure 1). Other phylogenomic analyses on every genome dataset consistently recovered Glomerales as polyphyletic, with Claroideoglomeraceae as sister to Diversisporales and Glomeraceae (data not shown). The quartet gene frequencies inferred with ASTRAL supported this relationship (q1 = 0.43, q2 = 0.26, q3 = 0.31) (Figure 2).

The phylogenetic position of Albahypha, Claroideoglomus, E. infrequens, and the four potential new species was reconstructed based on 115 sequences of the 45S segment or part thereof (28S) and 36 rpb1 sequences. Of these, 57 were newly obtained (39 45S, 18 rpb1). The 45S and rpb1 sequences characterized 14 and 10 species, respectively, of Albahypha/Claroideoglomus, including our four new species, as well as three Diversispora (Glomus) species that served as an outgroup. These sequences were used to prepare four alignments (45S, rpb1, 45S+rpb1, and 45S+rpb1_G) for BI and ML analyses. Data about the numbers of variable and parsimony informative sites of each alignment are presented in Table 1.

Four phylogenetic trees summarizing the BI and ML analyses were reconstructed, named 45S, rpb1, 45S+rpb1, and 45S+rpb1_G (Figure 3 and Supplementary Figures 3–5, respectively). Small differences that occurred in the topologies of the trees were described below. In all trees, the number of supported species clades and the mean support values of species clades and nodes were identical or similar (Supplementary Table 2). Overall, the trees with concatenated genes performed slightly better than the 45S tree.

In all trees, the clades with Claroideoglomus 1–4 obtained full or almost full BI (= 0.99–1.0) supports (Figure 3 and Supplementary Figures 3–5). Also, the ML supports of these species in all trees were significant (≥70%), and most of them were very high or full.

In all trees, the glomoid Claroideoglomus 3 formed a mixed clade with the entrophosporoid E. infrequens, whose 45S and rpb1 sequences were obtained from spores originating from our collection (Figure 3 and Supplementary Figures 3–5). In the 45S+rpb1 and 45S trees, the glomoid Claroideoglomus 2 also grouped with E. infrequens (Figure 3 and Supplementary Figure 3), whose 28S sequences were obtained by Oehl et al. (2011b); no rpb1 sequence of the fungus was available (pers. comm.). Hereafter, the two mixed clades will be referred to as Claroideoglomus 2 and Claroideoglomus 3.

In all trees, Claroideoglomus 1 and Claroideoglomus 3 occupied autonomous phylogenetic positions, except for the 45S tree, which showed an unsupported sister relationship of Claroideoglomus 1 with C. etunicatum (Figure 3 and Supplementary Figures 3–5). Claroideoglomus 2 formed a clade with C. candidum and six of the eleven analyzed sequences of C. claroideum in the 45S (not supported), 45S+rpb1, and 45S+rpb1_G trees. Claroideoglomus 4 was placed either in a strongly supported autonomous clade (in the 45S+rpb1 and rpb1 trees) or a BI-supported clade sister to C. drummondii (45S, 45S+rpb1_G). However, the ML supports of this sister relationship were insignificant (45S) or slight (45S+rpb1_G; ML = 76%).

Four other 28S sequences ascribed to E. infrequens formed a clade with or were accommodated in the neighborhood of C. hanlinii or clustered in an autonomous clade; none of the positions obtained support (Figure 3 and Supplementary Figures 3, 5). Although Claroideoglomus 4, C. drummondii, and C. walkeri formed a supported group corresponding to the genus Albahypha sensu Oehl et al. (2011b) in the 45S+rpb1_G tree, the clade comprising the other analyzed species was not supported (Supplementary Figure 5).

Entrophosporales Błaszk., Sánchez-García, B.T. Goto, and Magurno, ord. nov.

MycoBank MB846043

Type family: Entrophosporaceae (Oehl and Sieverd.), emend. Błaszk., Sánchez-García, B.T. Goto, and Magurno.

Diagnosis: Producing entrophosporoid and glomoid spores or only one of these morphs. Entrophosporoid spores formed singly within the necks of sporiferous saccules, in soil, rarely in roots. Spores with two spore walls. Spore wall 1, forming the spore surface, composed of two short-lived to semi-permanent layers continuous with the neck and sporiferous saccule wall layers and a permanent, laminate, pigmented layer continuous with a wall of a cylindric to a funnel-shaped structure supporting the wall of the part of the neck directly connected with the sporiferous saccule and the wall of the saccule at its base; not extending into the part of the neck located distally to the sporiferous saccule, hence spores have only one persistent cicatrix, proximal to the sporiferous saccule, created when the opening connecting the interior of the spore with the lumen of the sporiferous saccule neck is closed by a plug made of the contents of the spore when fully formed; layer 1 stains dark in Melzer’s reagent. Spore wall 2, having no physical contact with spore wall 1, composed of three, hyaline, smooth layers: two thin, flexible layers enclosing a much thicker, semi-flexible layer; none of the layers stains in Melzer’s reagent. Glomoid spores formed at tips of sporogenous hyphae either branched from the sporiferous saccule wall and/or the sporiferous saccule neck of the entrophosporoid morph or continuous with extraradical mycorrhizal hyphae, occasionally intercalarily inside hyphae, rarely in roots, usually with one subtending hypha. Extraradical spores produced singly, rarely in clusters with few spores. Spores with one spore wall consisting of two to five layers; when multilayered, the innermost layer frequently is flexible to semi-flexible, <1.0 μm thick, colorless or brightly colored, and loosely associated with the inner surface of the penultimate, usually laminate, layer. One or two spore wall layers may stain in Melzer’s reagent. Subtending hypha with a hyaline, rarely brightly colored wall, always strikingly much lighter than the spore wall in colored spores, frequently conspicuously funnel- or bill-shaped at the spore base; subtending hyphal wall composed of layers continuous with spore wall layers. Pore closed by a septum connecting the inner surfaces of either the subtending hyphal wall or the spore wall, or septa connecting both these structures, at or slightly below the spore base, and frequently additionally by a septum continuous with the innermost flexible to semi-flexible spore wall layer. Entrophosporoid and glomoid species producing mycorrhiza with arbuscules, vesicles, as well as intra- and extraradical hyphae staining dark in Trypan blue.

Glomerales J.B. Morton and Benny, emend. Błaszk., B.T. Goto, and Magurno.

Type family: Glomeraceae Piroz. and Dalpé.

Type genus: Glomus Tul. and C. Tul. emend. Oehl, G.A. Silva, and Sieverd.

Other genera: Dominikia Błaszk., Chwat, and Kovács, Microdominikia Oehl, Corazon-Guivin, and G.A. Silva, Kamienskia Błaszk., Chwat, and Kovács, Microkamieskia Corazon-Guivin, G.A. Silva, and Oehl, Orientoglomus G.A. Silva, Oehl, and Corazon-Guivin, Nanoglomus Corazon-Guivin, G.A. Silva, and Oehl, Septoglomus Sieverd., G.A. Silva, and Oehl, Rhizoglomus Sieverd., G.A. Silva, and Oehl (= Rhizophagus), Oehlia Błaszk., Kozłowska, Niezgoda, B.T. Goto, and Dalpé, Halonatospora Błaszk., Niezgoda, B.T. Goto, and Kozłowska, Sclerocarpum B.T. Goto, Błaszk., Niezgoda, Kozłowska, and Jobim, Epigeocarpum Błaszk., B.T. Goto, Jobim, Niezgoda, and Magurno, Funneliformis C. Walker and A. Schüssler emend. Oehl, G.A. Silva, and Sieverd., Funneliglomus Corazon-Guivin, G.A. Silva, and Oehl, Silvaspora Błaszk., Niezgoda, B.T. Goto, Crossay, and Magurno, Sclerocystis Berk. and Broome.

Diagnosis: Spores formed at tips of sporogenous hyphae, occasionally intercalarily inside hyphae, and in roots, usually with one subtending hypha. Extraradical spores produced singly, in clusters with few to dozens of spores, compact unorganized glomerocarps (= sporocarps) with randomly distributed spores or organized glomerocarps with spores born terminally from hyphae developed radially from a central plexus of hyphae. Glomerocarps with or without a peridium. Spores with one spore wall consisting of one laminate layer or many layers; when multilayered, the innermost layer usually is laminate and thicker than the others. Subtending hypha with a wall concolorous with or slightly lighter than the spore wall, funnel-shaped, cylindrical, or constricted at the spore base; subtending hyphal wall composed of layers continuous with spore wall layers. Pore closed by thickening spore wall and subtending hyphal wall toward the center of the subtending hyphal lumen connecting it with the spore interior, a septum connecting the inner surfaces of the subtending hyphal wall slightly below the spore base, or the innermost spore wall layer at or slightly above the spore base. Mycorrhiza with arbuscules, vesicles, as well as intra- and extraradical hyphae staining dark in Trypan blue.

Entrophosporaceae (Oehl and Sieverd.), emend. Błaszk., Sánchez-García, B.T. Goto, and Magurno. = Entrophosporaceae Oehl and Sieverd., emend. Oehl, Sieverd., Palenz., and G.A. Silva. Mycotaxon 117:306, 2011. = Claroideoglomeraceae C. Walker and A. Schüßler. The Glomeromycota – a species list:21. 2010.

Type genus: Entrophospora R.N. Ames and R.W. Schneid., emend. Błaszk., Sánchez-García, Fernández, B.T. Goto, and Magurno.

Diagnosis: As that of Entrophosporales.

Entrophospora R.N. Ames and R.W. Schneid., emend. Błaszk., Sánchez-García, Fernández, B.T. Goto, and Magurno. = Albahypha Oehl, G.A. Silva, B.T. Goto, and Sieverd., Mycotaxon 117:308. 2011. = Claroideoglomus C. Walker and A. Schüßler, emend. Oehl, Sieverd., B.T. Goto, and G.A. Silva, Mycotaxon 117:308. 2011. = Claroideoglomus C. Walker and A. Schüßler, The Glomeromycota – a species list:21. 2010.

Type species: Entrophospora infrequens (I.R. Hall) R.N. Ames and R.W. Schneid., emend. Błaszk., Sánchez-García, Fernández, B.T. Goto, and Magurno.

Epitype: Entrophosporoid morph, POLAND. Spores from a single-species culture established from spores extracted from a trap culture inoculated with a field-collected mixture of rhizosphere soil and root fragments of J. communis from a pine forest in inland sand dunes of Kampinos National Park (52^o19′ N, 20^o45′ E), July 1986, J. Błaszkowski (slide with spores no. 668–674, isoepitypes slides with spores no. 3870–3872, LPPDSE; 45S and rpb1 sequences: MT722034–MT722038 and MT733201–MT733202, respectively, GenBank).

Glomoid morph, SPAIN. FORTUNA, MURCIA. Spores from a single-species culture established from spores extracted from a trap culture inoculated with a field-collected mixture of rhizosphere soil and root fragments of L. sinuatum from an initial community (38°06’57 N, 1°06’W), November 2009, F. Fernández (epitype slide with spores no. ZT Myc 0066907, isoepitypes slides with spores no. 3839–3846, LPPDSE); 45S and rpb1 sequences: ON950364–ON950367 and ON938324–ON938325, respectively, GenBank.

Diagnosis: As that of Entrophosporales.

Entrophospora infrequens R.N. Ames and R.W. Schneid., emend. Błaszk., Sánchez-García, Fernández, B.T. Goto, and Magurno.

Diagnosis: Differs from other Entrophospora species forming glomoid spores in spore morphometric features, phenotypic and histochemical properties of spore wall layers, as well as in nucleotide composition of sequences of the 45S nuc rDNA region and the rpb1 gene.

Description: Producing entrophosporoid and glomoid glomerospores (= spores), or only one of these morphs. Entrophosporoid spores formed in soil, singly, arise blastically inside the neck of a sporogenous saccule (Figures 4A–H); golden yellow (5B8) to brownish orange (7C7); globose to subglobose; (95–)135(–175) μm diam, with two cicatrices. Spores with two spore walls having no physical contact with one another. Spore wall 1 composed of three layers. Layer 1, forming the spore surface, evanescent, hyaline, (1.5–)2.5(–4.5) μm thick, staining crayfish red (9B8) to cerise (12C8) in Melzer’s reagent, highly deteriorated or completely sloughed off in mature and older spores. Layer 2, semi-permanent, smooth, 2.8–6.0 μm thick, slowly degrading with age. Layer 3, permanent, laminate, golden yellow (5B8) to brownish orange (7C7); (1.5–)2.5(–4.5) μm thick, on the upper surface ornamented with tooth-shaped outgrowths, 1.8–3.8 × 1.0–3.0 μm, with a central depression in the upper surface. Layers 1 and 2 continuous with the neck and sporiferous saccule wall layers. Layer 3 continuous with a wall of a cylindric to funnel-shaped structure supporting the wall of the part of the neck directly connected with the sporiferous saccule and the wall of the saccule at its base; not extending into the part of the neck located distally to the sporiferous saccule. Spore wall 2 consisting of three permanent, hyaline layers. Layer 1, flexible, <0.5 μm thick, usually tightly adherent to layer 2, and, hence, frequently difficult to see. Layer 2, flexible to semi-flexible, coriaceous sensu Walker (1986), (5.5–)7.8(–10.0) μm thick. Layer 3 flexible, ∼0.5 μm thick, rarely separating from the lower surface of layer 2 in even vigorously crushed spores. None of the spore wall 2 layers 1–3 stains in Melzer’s reagent. Cicatrices. Visible as two scars in the region of contact between the spore and the saccule neck. A scar proximal to the saccule is a slightly depressed area, circular, 13.5–27.0 μm diam, to ellipsoidal, 13.5–23.0 × 16.0–27.0 μm, when seen in a plane view. A scar distal to the saccule is circular, 11.0–14.5 μm diam when observed in a plane view; it is usually completely sealed in mature spores and, therefore, invisible. Sporiferous saccule hyaline, consisting of a cylindric to slightly funnel-shaped neck, 21–33 wide, and a globose to egg-shaped, 100–170 × 130–190 μm, saccule, usually detached from mature spores. Sporiferous saccule wall composed of spore wall 1 layers 1–3. Germination unknown. Mycorrhiza. In single-species cultures with P. lanceolata and Pueraria phaseoloides (Roxb.) Benth. as the host plants, formed mycorrhiza with arbuscules, vesicles, and hyphae staining clearly in Trypan blue (Sieverding and Oehl, 2006; pers. observ.).

Glomoid spores formed in soil, singly or in loose clusters with 2–3 spores, arise blastically at tips of sporogenous hyphae (Figures 5A,B,D–F) either branched from the sporiferous saccule wall and/or the sporiferous saccule neck of the entrophosporoid morph (Figures 4F,G), or continuous with extraradical mycorrhizal hyphae, occasionally intercalarily. Spores subhyaline to pastel yellow (3A4); globose to subglobose; (20–)50(–76) μm diam; rarely ovoid; 36–64 × 41–69 μm; with one subtending hypha (Figures 4F,G, 5A–F). Spore wall composed of two permanent layers (Figures 5B–F). Layer 1, forming the spore surface, uniform (not containing visible sublayers), semi-flexible, hyaline to yellowish white (3A2), (0.6–)1.1(–2.0) μm thick, tightly adherent to the upper surface of layer 2, rarely slightly deteriorated in its upper part (Figures 5B–F). Layer 2 laminate, semi-flexible, hyaline to pastel yellow (3A4), (1.2–)2.0(–3.4) μm thick; consisting of very thin, <0.5 μm thick, laminae, tightly adherent to each other (Figures 5B-F). In Melzer’s reagent, only layer 2 turns yellow (3A6; Figures 5E,F). Subtending hypha hyaline to pastel yellow (3A4); straight or recurved, usually slightly funnel-shaped, rarely cylindrical or slightly constricted at the spore base; (5.6–)7.1(–9.2) μm wide at the spore base (Figures 5A,B,D–F); not braking in crushed spores. Wall of subtending hypha subhyaline to pastel yellow (3A4); (1.4–)1.7(–2.8) μm thick at the spore base; consisting of two layers continuous with spore wall layers 1 and 2; subtending hyphal wall layer 2 formed up to 5.2 μm below the spore base (Figures 5D–F). Pore (2.0–)3.0(–3.8) μm wide at the spore base, usually open, rarely closed by a curved septum, 0.6–1.7 μm thick, continuous with spore wall layer 2 in mature spores (Figures 5B,D–F). Spore content of hyaline oily substance. Germination unknown. Mycorrhiza. In single-species cultures with P. lanceolata as the host plant, formed mycorrhiza with arbuscules, vesicles, as well as intra- and extraradical hyphae that stained dark [pale violet (16A3) to grayish violet (16E5)] in 0.1% trypan blue (Figures 4H, 5G,H).

Ecology and distribution: Entrophosporoid morph found associated with roots of many cultivated and uncultivated plant species growing in soils highly differing in pH and the content of nutrients, organic matter, and water (Sieverding and Oehl, 2006; Błaszkowski, 2012). Glomoid morph physically connected with an entrophosporoid morph grown in a single-species culture with the host plant P. lanceolata (Figures 4F,G) and probably lived in arbuscular mycorrhizal symbiosis with roots of L. sinuatum in the field, but no molecular analysis was performed to confirm this hypothesis. Reported as C. cf. claroideum, E. infrequens, Glomeromycota sp., and uncultured Glomus from Czech Republic, France, Peru, Poland, Spain, Switzerland, and USA (Supplementary Table 3).

Entrophospora candida (Furrazola, Kaonongbua, and Bever) Błaszk., Niezgoda, B.T. Goto, and Magurno, comb. nov.

MycoBank MB836245

≡ Glomus candidum Furrazola, Kaonongbua, and Bever. Mycotaxon 113: 103. 2010 (basionym).

≡ Claroideoglomus candidum (Furrazola, Kaonongbua, and Bever) Oehl, G.A. Silva, and Sieverd. Mycotaxon 116:106. 2011.

Entrophospora claroidea (N.C. Schenck and G.S. Sm.) Błaszk., Niezgoda, B.T. Goto, and Magurno, comb. nov.

MycoBank MB836246

≡ Glomus claroideum N.C. Schenck and G.S. Sm., Mycologia 74:84. 1982 (basionym).

≡ Claroideoglomus claroideum (N.C. Schenck and G.S. Sm.) C. Walker and A. Schüßler. The Glomeromycota – a species list:21. 2010.

Entrophospora drummondii (Błaszk. and Renker) Błaszk., Niezgoda, B.T. Goto, and Magurno, comb. nov.

MycoBank MB836247

≡ Glomus drummondii Błaszk. and Renker. Mycological Research 110:559. 2006 (basionym).

≡ Claroideoglomus drummondii (Błaszk. and Renker) C. Walker and A. Schüßler. The Glomeromycota – a species list:22. 2010.

≡ Albahypha drummondii (Błaszk. and Renker) Sieverd., Oehl, B.T. Goto, and G.A. Silva. Mycotaxon 117:308. 2011.

Entrophospora etunicata (W.N. Becker and Gerd.) Błaszk., Niezgoda, B.T. Goto, and Magurno, comb. nov.

MycoBank MB836248

≡ Glomus etunicatum W.N. Becker and Gerd. Mycotaxon 6:29. 1977 (basionym).

≡ Claroideoglomus etunicatum (W.N. Becker and Gerd.) C. Walker and A. Schüßler. The Glomeromycota – a species list:22. 2010.

Entrophospora hanlinii (Błaszk., Chwat, and Góralska) Błaszk., Niezgoda, B.T. Goto, and Magurno, comb. nov.

MycoBank MB836249

≡ Claroideoglomus hanlinii Błaszk., Chwat, and Góralska. Mycological Progress 14:7. 2015 (basionym).

Entrophospora lamellosa (Dalpé, Koske, and Tews) Błaszk., Niezgoda, B.T. Goto, and Magurno, comb. nov.

MycoBank MB836250

≡ Glomus lamellosum Dalpé, Koske, and Tews. Mycotaxon 43:289. 1992 (basionym).

≡ Claroideoglomus lamellosum (Dalpé, Koske, and Tews) C. Walker and A. Schüßler. The Glomeromycota – a species list:22. 2010.

Entrophospora lutea (L.J. Kenn., J.C. Stutz, and J.B. Morton) Błaszk., Niezgoda, B.T. Goto, and Magurno, comb. nov.

MycoBank MB836251

≡ Glomus luteum L.J. Kenn., J.C. Stutz, and J.B. Morton. Mycologia 91:1090. 1999 (basionym).

≡ Claroideoglomus luteum (L.J. Kenn., J.C. Stutz, and J.B. Morton) C. Walker and A. Schüßler. The Glomeromycota – a species list:22. 2010.

Entrophospora walkeri (Błaszk. and Renker) Błaszk., Niezgoda, B.T. Goto, and Magurno, comb. nov.

MycoBank MB836252

≡ Glomus walkeri Błaszk. and Renker. Mycological Research 110:563. 2006 (basionym).

≡ Claroideoglomus walkeri (Błaszk. and Renker) C. Walker and A. Schüßler. The Glomeromycota – a species list:22. 2010.

≡ Albahypha walkeri (Błaszk. and Renker) Sieverd., Oehl, B.T. Goto, and G.A. Silva. Mycotaxon 117:309. 2011.

Entrophospora argentinensis Błaszk., B.T. Goto, Magurno, Niezgoda, and Cabello, sp. nov.

Figures 6A–H, 7A,B.

MycoBank MB836244

Typification: ARGENTINA. TIERRA DEL FUEGO: Spores from a single-species culture established from spores extracted from a trap culture inoculated with a field-collected mixture of rhizosphere soil and root fragments of D. flexuosa and Po. rigidifolia from a steppe community (Mendoza et al., 2011), 1–15 Mar 2009, R. Mendoza (holotype slide with spores no. ZT Myc 61117, isotypes slides with spores no. 3694–3699, LPPDSE); 45S and rpb1 sequences: MT722021–MT722024 and MT733203–MT733204, respectively, GenBank.

Etymology: argentinensis (Latin), referring to Argentina, where the species was originally found.

Diagnosis: Differs from other species of Entrophospora in having a laminate spore wall layer uneven in thickness in its different regions and nucleotide composition of sequences of the 45S nuc rDNA region and the rpb1 gene.

Description: Glomerospores (= spores) glomoid, formed singly in soil (Figure 6A). Spores arising blastically at the tips of sporogenous hyphae (Figures 6G,H). Spores yellowish white (3A2) to dark yellow (4C8); globose to subglobose; (45–)95(–115) μm diam, rarely ovoid; 85–115 × 100–140 μm; with one subtending hypha (Figures 6A–H). Spore wall composed of three layers (Figures 6B–D,F–H). Layer 1, forming the spore surface, evanescent, semi-flexible, smooth in young spores, becoming roughened with age, occasionally completely sloughed off in older spores, hyaline to yellowish white (4A2), (0.8–)1.1(–1.5) μm thick when intact (Figures 6B–D,F–H). Layer 2 permanent, laminate, semi-flexible, yellowish white (3A2) to dark yellow (4C8), uneven in thickness when observed in a cross view, (1.0–)3.8(–7.5) μm thick in thinner regions, (2.4–)6.0(–10.0) μm thick in thicker regions; therefore, the lower edge of this layer is wavy, when observed in a plan view; thinner regions visible as circular, (1.2–)5.0(–13.0) μm diam or ellipsoidal, 5.2–7.0 × 7.4–14.8 μm, more or less evenly distributed lighter patches (Figures 6B–H); in young spores, layer 2 frequently uniform in thickness (Figures 6F,H); layer 2 with birefringent properties in polarized light, where smaller or larger areas, despite yellow-colored, turn almost black (Figures 6C,G). Layer 3 permanent, uniform (not divided into visible sublayers), semi-flexible, hyaline to light yellow (4A4), 0.8–1.3 μm thick, usually tightly adherent to the lower surface of layer 2, occasionally separating from layer 2 in vigorously crushed spores (Figures 6F,H). Layers 1–3 do not stain in Melzer’s reagent (Figure 6B). Subtending hypha yellowish white (3A2) to dark yellow (4C8) near the spore base, hyaline below the pigmented portion; straight or recurved, cylindrical or slightly funnel-shaped, rarely slightly constricted at the spore base; (6.2–)7.8(–8.8) μm wide at the spore base (Figures 6G,H); not braking in crushed spores. Wall of subtending hypha yellowish white (3A2) to dark yellow (4C8) up to 9.2 μm below the spore base, then hyaline; (2.0–)3.4(–4.8) μm thick at the spore base; consisting of three layers continuous with spore wall layers 1–3; subtending hyphal wall layer (shwl) 1 usually highly deteriorated or completely sloughed off even in young spores; shwl 3 present only at the spore base or extending up to 2.8 μm below the spore base (Figures 6G,H). Pore (0.8–)1.5(–3.4) μm wide at the spore base, occluded by a straight or curved septum continuous with a few innermost laminae of spore wall layer 2 and spore wall layer 3; septum 0.8–3.4 μm wide, 1.0–2.0 μm thick, positioned at or up to 4.2 μm below the spore base (Figures 6G,H). Germination unknown.

Ecology and distribution: In the field, associated with roots of D. flexuosa and Po. rigidifolia in Tierra del Fuego, Argentina. Forming mycorrhiza with arbuscules, vesicles, as well as intraradical and extraradical hyphae in single-species cultures with P. lanceolata as the host (Figures 7A,B); structures stained violet white (16A2) to deep violet (16E8) in 0.1% Trypan blue. Found as Glomus cf. claroideum associated with a spore of E. infrequens grown in trap culture inoculated with soil from an agricultural grassland of Switzerland and as trap-cultured glomeromycotan spore in the USA (Supplementary Table 3).

Entrophospora glacialis Zubek, Niezgoda, B.T. Goto, Magurno, and Błaszk., sp. nov.

Figures 8A–H.

MycoBank MB846045

Typification: SWEDEN. TARFALA VALLEY: Spores from a single-species culture established from spores extracted from a trap culture inoculated with a field-collected mixture of rhizosphere soil and root fragments of F. vivipara, Po. alpina, S. herbacea, S. polaris, and Si. acaulis from a glacier foreland of Isfallglaciarän (67°54′ N, 18°35′ E), 23 July 2019, P. Wietrzyk-Pełka and M. Węgrzyn (holotype slide with spores no. ZT Myc 0066908, isotypes slides with spores no. 3847–3859, LPPDSE); 45S and rpb1 sequences: ON950374–ON950380 and ON938326–ON938327, respectively, GenBank.

Etymology: Latin, glacialis, referring to the glacial habitat, in which this new species was originally found.

Diagnosis: Differs from other Entrophospora species with glomoid spores in producing spores with a spore wall consisting of five permanent layers and nucleotide composition of sequences of the 45S nuc rDNA region and the rpb1 gene.

Description: Glomerospores (= spores) glomoid, formed singly in soil, arise blastically at tips of sporogenous hyphae (Figures 8A–H) continuous with extraradical mycorrhizal hyphae. Spores yellowish white (4A2) to yellowish brown (5E8); globose to subglobose; (40–)91(–129) μm diam; rarely ovoid; 77–101 × 90–123 μm; with one subtending hypha (Figures 8A–H). Spore wall composed of five permanent layers (Figures 8B–H). Layer 1, forming the spore surface, uniform (not containing visible sublayers), semi-flexible, hyaline to yellowish white (4A2), (0.8–)1.3(–2.0) μm thick, tightly adherent to the upper surface of layer 2, occasionally with small, local thickenings rendering the spore surface slightly wavy when observed in a cross-section (Figures 8B–H). Layer 2 uniform, semi-flexible, yellowish white (4A2), (1.0–)1.8(–2.6) μm thick (Figures 8B–H). Layer 3 uniform, semi-flexible, hyaline to yellowish white (4A2), (2.0–)4.1(–9.0) μm thick (Figures 8B–H). Layer 4 laminate, semi-flexible, yellowish white (4A2) to yellowish brown (5E8), (2.4–)3.6(–5.0) μm thick, consisting of very thin, < 0.5 μm thick, laminae, tightly adherent to each other (Figures 8B–H). Layer 5 uniform, flexible to semi-flexible, (1.0–)1.1(–1.2) μm thick, usually slightly separating from the lower surface of the laminate layer 4 (Figures 8B–H). None of the spore wall layers stains in Melzer’s reagent. Subtending hypha hyaline to yellowish white (4A2); straight or recurved, usually slightly funnel-shaped, rarely cylindrical or slightly constricted at the spore base; (8.4–)10.8(–14.2) μm wide at the spore base (Figures 8A–H); not braking in crushed spores. Wall of subtending hypha hyaline to yellowish white (4A2); (3.8–)4.8(–6.4) μm thick at the spore base; consisting of five layers continuous with spore wall layers 1–5; subtending hyphal wall layers 4 and 5 formed up to 6.1 μm below the spore base (Figures 8A–H). Pore (1.0–)1.4(–2.0) μm wide at the spore base, usually open, rarely closed by a curved septum, 0.7–1.1 μm thick, continuous with spore wall layers 4 and 5 (Figures 8G,H). Spore content of hyaline oily substance. Germination unknown.

Ecology and distribution: In the field, present under an initial community consisting of cryptogamic species, as well as F. vivipara, Po. alpina, S. herbacea, S. polaris, and Si. acaulis in a glacier foreland of Sweden. No molecular analysis was performed to reveal which of these species harbored E. glacialis. In single-species cultures with P. lanceolata as the host plant, E. glacialis formed mycorrhiza with arbuscules, vesicles, as well as intra- and extraradical hyphae that stained dark [pale violet (16A2) to deep violet (16D8)] in 0.1% trypan blue. Found as C. claroideum, C. cf. claroideum, E. infrequens, G. luteum, Glomeromycota sp., and uncultured Glomus in cultivated and uncultivated sites, including submerged and contaminated habitats, in Brazil, China, Japan, Netherlands, Norway, Poland, Switzerland, and USA (Supplementary Table 3).

Entrophospora furrazolae Magurno, Niezgoda, B.T. Goto, and Błaszk., sp. nov.

Figures 9A–H.

MycoBank MB 846048

Typification: POLAND. LUBLINIEC, SILESIAN UPLAND: Spores from a single-species culture established from spores extracted from a trap culture inoculated with a field-collected mixture of rhizosphere soil and root fragments of Po. trivialis from a plant community in the unpolluted shore of Kokotek pond II (50°37′ N, 18°43′ E), July 2016, Monika Malicka (holotype slide with spores no. ZT Myc 0066909, isotypes slides with spores no. 3860–3869, LPPDSE); 45S and rpb1 sequences: MH590060, MH590061, ON950381–ON950390, and MT733209–MT733210, respectively, GenBank.

Etymology: Latin, furrazolae, in honor of Eduardo Furrazola, in recognition of his important contribution to studies of arbuscular mycorrhizal fungi. This recognition comes after the premature death caused by COVID-19 in 2021.

Diagnosis: Differs from other Entrophospora species with glomoid spores in formation of spores with a spore wall containing three permanent layers and two layers staining in Melzer’s reagent, as well as in nucleotide composition of sequences of the 45S nuc rDNA region and the rpb1 gene.

Description: Glomerospores (=spores) glomoid, formed singly in soil, arise blastically at tips of sporogenous hyphae (Figures 9A,C,G,H) continuous with extraradical mycorrhizal hyphae. Spores hyaline to yellowish white (4A2); globose to subglobose; (60–)72(–87) μm diam; rarely ovoid; 50–57 × 60–74 μm; with one subtending hypha (Figures 9A–H). Spore wall composed of four layers (Figures 9B–F,H). Layer 1, forming the spore surface, mucilaginous, short-lived, flexible, hyaline, (0.6–)0.8(–1.0) μm thick, usually highly deteriorated or completely sloughed off in mature spores (Figures 9B–F,H). Layer 2 uniform (without visible sublayers), permanent, flexible to semi-flexible, hyaline, (0.8–)1.0(–1.6) μm thick, tightly adherent to the upper surface of layer 3, not separating from this layer in even vigorously crushed spores (Figures 9B–F,H). Layer 3 laminate, permanent, semi-flexible, hyaline to yellowish white (4A2), (2.4–)6.4(–10.2) μm thick, consisting of very thin, <0.5 μm thick, laminae, tightly adherent to each other (Figures 9B–F,H); layer 3 has birefringent properties in polarized light, in which smaller or larger colorless or yellowish white (4A2) fragments of this layer turn almost black (Figures 9B,C,E,F). Layer 4 flexible, permanent, hyaline, (0.6–)0.8(–1.0) μm thick, usually separating from the lower surface of the laminate layer 3 in even moderately crushed spores (Figures 9B–F,H). In Melzer’s reagent, spore wall layers 1 and 4 frequently stain reddish white (10A2) to grayish rose (11B4) and reddish white (8A2) to pastel pink (11A3), respectively, but these staining reactions do not appear in all spores (Figures 9D–H). Subtending hypha hyaline; straight or recurved, usually slightly funnel-shaped, rarely cylindrical or slightly constricted at the spore base; (3.6–)5.3(–7.4) μm wide at the spore base (Figures 9A,C,G,H); frequently braking at the base of crushed spores. Wall of subtending hypha hyaline; (1.6–)2.3(–3.5) μm thick at the spore base; consisting of three layers continuous with spore wall layers 1–3; subtending hyphal wall layer (shwl) 1 usually highly deteriorated or completely sloughed off in mature spores, thereby difficult to see or invisible; shwl2 usually present only at the spore base, rarely starting developing up to ca. 6 μm below the spore base; shwl3 gradually thickening beginning from ca. 9 μm below the spore base (Figure 9H). Pore (0.8–)1.0(–1.4) μm wide at the spore base, closed by a curved septum, 0.6–1.0 μm thick, continuous with spore wall layer 4; the septum usually located at the center of the laminate spore wall layer 3 when observed in a cross section (Figures 9C–H). Spore content of hyaline oily substance. Germination unknown.

Ecology and distribution: In Oman, E. furrazolae probably lived in the field in arbuscular mycorrhizal symbiosis with Pr. cineraria in a site characterized by hyper aridity, but no molecular analysis was performed to confirm this hypothesis. In Poland, E. furrazolae was associated with Po. trivialis in an unpolluted natural site, as indicated by NGS sequencing of partial 28S PCR amplicons obtained from DNA extracted from rhizosphere soil and root samples of this plant species (Malicka et al., 2022). In single-species cultures with P. lanceolata as the host plant, E. furrazolae formed mycorrhiza with arbuscules and intra- and extraradical hyphae that stained bluish-white (20A2) to light blue (20A5) in 0.1% trypan blue. Found as C. drummondii, as well as uncultured Claroideoglomeraceae, Glomus, and Glomeromycota in undisturbed and degraded soils of Czech Republic, China, Spain, and Switzerland (Supplementary Table 3).