Three new species of arbuscular mycorrhizal fungi (Glomeromycota) and Acaulospora gedanensis revised

Studies of the morphology and the 45S nuc rDNA phylogeny of three potentially undescribed arbuscular mycorrhizal fungi (phylum Glomeromycota) grown in cultures showed that one of these fungi is a new species of the genus Diversispora in the family Diversisporaceae; the other two fungi are new Scutellospora species in Scutellosporaceae. Diversispora vistulana sp. nov. came from maritime sand dunes of the Vistula Spit in northern Poland, and S. graeca sp. nov. and S. intraundulata sp. nov. originally inhabited the Mediterranean dunes of the Peloponnese Peninsula, Greece. In addition, the morphological description of spores of Acaulospora gedanensis, originally described in 1988, was emended based on newly found specimens, and the so far unknown phylogeny of this species was determined. The phylogenetic analyses of 45S sequences placed this species among Acaulospora species with atypical phenotypic and histochemical features of components of the two inner germinal walls.


Introduction
The order Diversisporales was originally created in the newly described phylum Glomeromycota (Schüßler et al., 2001) based on the phylogenetic analyses of the nuclear SSU rDNA gene sequences of members belonging to the Glomus group C sensu Schwarzott et al. (2001).The order included Diversisporaceae fam.ined., as well as the members of the families Acaulosporaceae and Gigasporaceaeboth sensu Morton and Benny (1990), the former with the genera Acaulospora and Entrophospora, and the latter with Gigaspora and Scutellospora.Walker and Schüßler (2004) validated Diversisporaceae as a new family with the type genus and species Diversispora and D. spurca, respectively, originally described as Glomus Błaszkowski et al., 2019b).However, only the 28S sequences from a curated database, as established by Delavaux et al. (2021Delavaux et al. ( , 2022)), can be used as a marker to separate closely related species in Glomeromycota.In addition, a large proportion of glomoid sporeproducing species of this order have partial sequences of the protein-coding largest subunit of the RNA polymerase II (rpb1) gene.Compared to 45S sequences, the taxonomic resolution of the rpb1 sequences is higher, and their use reduces the risk of creating potential phylogenetic artifacts due to paralogous sequences because rpb1 is a single-copy gene in arbuscular mycorrhizal fungi (Stockinger et al., 2014).Other single-copy marker loci recommended for use in reconstructing AMF phylogenies are the ß-tubulin and glomalin genes (Corradi et al., 2004a,b;Msiska and Morton, 2009;Magurno et al., 2019;Kokkoris et al., 2023).However, lower resolution and poor support at the order level of the ß-tubulin phylogenies, compared to those reconstructed from SSU, LSU, and rpb1 sequences (Msiska and Morton, 2009) and the relatively small number of sequences for the AMF glomalin gene (Magurno et al., 2019;Kokkoris et al., 2023), have made these two protein-coding loci of little use in taxonomic analyses of large numbers of Glomeromycota representatives.
It is now strongly recommended to reconstruct fungal phylogenies based on sequences derived from unlinked loci, including those encoding a protein (Chethana et al., 2021).Unfortunately, the vast majority of species of Diversisporales producing spores other than glomoid (ca.68%), are still missing rpb1 sequences, except for two Acaulospora species (out of 60 described), three Gigaspora species (9), and five species with gigasporoid spores (31).Due to the small number of mycologists involved in collecting and characterizing AMF (morphological and molecular), rpb1 sequences for most non-glomoid sporeproducing species are not expected to be obtained even in the distant future; overall, out of 278 rpb1 sequences available in the GenBank for Glomeromycota, approximately 68% were obtained by J. Błaszkowski and his collaborators.
One of the relatively "old" non-sequenced members of Diversisporales is A. gedanensis, which was classified in Acaulospora because it formed acaulosporoid spores (Błaszkowski, 1988).However, the presence of only one germinal wall in the spore subcellular structure and the atypical phenotypic and histochemical properties of layers sensu Morton et al. (1995) of this wall (Błaszkowski, 1988) clearly separated this species from all previously described Acaulospora species.The emended definition of A. gedanensis informed about the presence of two germinal walls, typical of Acaulospora species.However, the rigidity and fragility of the one-layered thin germinal wall 1, the lack of a beaded ornamentation on the upper surface of germinal wall 2 layer 1, and the non-plasticity and non-reactivity of germinal wall 2 layer 2 in PVLG and Melzer's reagent, respectively, suggested this species belonged to the genus Ambispora (Błaszkowski, 2012) in the family Ambisporaceae (Archaeosporales).We grew A. gedanensis in culture, which allowed us to re-analyze the morphology of this species and carry out phylogenetic analyses to establish its position in the Glomeromycota phylogeny.
This study had the following aims: (i) to reconstruct the phylogenies of three potentially new species in Glomeromycota, to characterize their morphologies and compare them with those of the phylogenetically most closely related species; (ii) to determine the phylogeny of A. gedanensis, show morphological differences between this species and its closest relatives; and (iii) to calculate the genetic distance of the three potentially new species and A. gedanensis from their most closely related species.

Origin of AMF isolates
The potentially new species were initially named Diversispora 448, Scutellospora 431, and Scutellospora 437 (numbers are from an AMF database maintained by J. Błaszkowski).These isolates, as well as A. gedanensis, were characterized based on spores extracted from trap cultures because numerous attempts to grow the fungi in single-species cultures failed.The trap cultures were established from field mixtures of rhizosphere soils and root fragments collected under the following plant species.The field host of Diversispora 448 was Ammophila arenaria (L.) Link.The plant colonized maritime sand dunes near Przebrno (54 • 22'30.3''N19 • 23'13.6''E)located on the Vistula Spit in northern Poland.The soil sample was collected by P. Niezgoda on July 10, 2020.The data relating to the climatic and soil chemical properties of Vistula Spit are detailed in Błaszkowski et al. (2002).Scutellospora 431 and Scutellospora 437 were hosted in the field by A. arenaria, which colonized maritime sand dunes of the Voidokoilia beach (36 • 57'46''N 21 • 39'45''E) located on the Peloponnese peninsula, Greece.The soil sample was collected by J. Błaszkowski on September 8, 2015.Data relating to the climate and soil chemical properties of the sampled site are detailed in Błaszkowski et al. (2019a).In the field, A. gedanensis was associated with the roots of Festuca rubra L. growing among trees inhabiting a moist site located near Władysławowo (54 • 47'00''N 18 • 26'17''E) on the Hel Peninsula in northern Poland.The host plant was sampled by J. Błaszkowski on July 18, 2022.The trap and single-species cultures were established and grown, spores extracted, and mycorrhizal structures stained, as described in Błaszkowski et al. (2006Błaszkowski et al. ( , 2009)).In all the attempts aimed at establishing single-species cultures, 5-15 spores of each isolate were used.

Microscopy and nomenclature
Morphological features of spores, as well as phenotypic and histochemical characters of spore wall and germinal wall layers of A. gedanensis, Diversispora 448, Scutellospora 431, and Scutellospora 437 were characterized based on at least 50-100 spores of each isolate 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).Spores for study and photography were prepared as described in Błaszkowski (2012) and Błaszkowski et al. (2012).The types of spore wall and germinal wall layers have been defined by Walker (1983), Morton (1986), Morton et al. (1995), andBłaszkowski (2012).Color names were obtained from Kornerup and Wanscher (1983).The nomenclature of fungi and the authors of fungal names were taken 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).
The 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)

DNA extraction, PCR, cloning, and DNA sequencing
Genomic DNA of A. gedanensis, Diversispora 448, Scutellospora 431, and Scutellospora 437 was extracted from single or 2-5 spores.The method of processing 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 four isolates were as those described by Błaszkowski et al. (2021).A nested PCR with the primers RPB1-4F2/RPB1-5R1 and RPB1-4F3/RPB1-5R2 was used to obtain rpb1 sequences from Diversispora 448 like in Błaszkowski et al. (2022).Both 45S and rpb1 sequences were deposited in GenBank (OR669295, OR669296, OR669014-OR669021, OR669025-OR669038).

Phylogenetic analyses
To confirm the BLAST results indicating the analyzed AMF as undescribed species of Diversisporaceae (Diversispora 448) and Scutellosporaceae (Scutellospora 431, Scutellospora 437), as well as to determine the positions of these isolates within these families and A. gedanensis among sequenced Acaulospora species, five alignments were prepared.The phylogeny of Diversispora 448 was reconstructed based on the analyses of the 45S and rpb1 sequences, while the phylogenies of the other isolates were reconstructed from the 45S sequences alone.The 45S and rpb1 sequence sets were aligned separately in MAFFT 7 with the E-INS-i option.
For Diversispora 448, the ingroup of the 45S alignment consisted of 85 sequences of 22 Diversispora species and Diversispora 448, and the outgroup was Corymbiglomus corymbiforme, Desertispora omaniana, Redeckera megalocarpum, and Sieverdingia tortuosa.The species of the outgroup represented the most closely related genera to Diversispora selected through prior analyses of members of all genera of Diversisporaceae.The 45S alignment with Scutellospora 431 and Scutellospora 437 contained 28 sequences of 6 Scutellospora species and the 2 potentially new species in the ingroup and Orbispora pernambucana in the outgroup.The ingroup of the A. gedanensis 45S alignment counted 118 sequences, representing 48 Acaulospora species, and the outgroup included Sacculospora baltica and S. felinovii sequences.
The Diversispora 448 rpb1 alignment included 36 rpb1 sequences that characterized 18 Diversispora species of the Diversispora 448 45S alignment and Diversispora 448, i.e., all Diversispora species having sequences of the two loci.The The percentage genetic distances of A. gedanensis, Diversispora 448, Scutellospora 431, and Scutellospora 437 from their closest relatives were calculated in BioEdit (Hall, 1999).All comparisons were performed on sequences of the same length.
The BI reconstruction was done in MrBayes 3.2 (Ronquist et al., 2012) based on four Markov chains run over one million generations, sampling every 1,000 generations with a burn-in at 30% of sampled trees.The ML phylogenetic tree inference was performed with RAxML-NG 1.0.1 (Kozlov et al., 2019), using a maximum likelihood/1000 bootstrapping run and ML estimated proportion of invariable sites and base frequencies.The alignments and tree files are available as online resources (Supplementary Tables 2-13).
Clades were considered supported when the Bayesian posterior probabilities and the ML bootstrap values were ≥0.95 and ≥70%, respectively.The phylogenetic trees were visualized and edited in MEGA6 (Tamura et al., 2013).
The occurrence of A. gedanensis, Diversispora 448, Scutellospora 431, and Scutellospora 437 in other ecosystems worldwide was checked by comparing 45S sequences of these species/isolates with environmental sequences deposited in GenBank and retrieved using BLASTn.It was assumed that the sequences shown could represent the same species/isolates as the four AMF discussed here when their similarity was >96%.

General data and phylogeny
Of the 45S and rpb1 sequences analyzed, 22 and 2, respectively, were newly obtained in this study.The numbers of analyzed sequences and species/isolates, as well as the numbers of base pairs, variable, and parsimony informative sites of each of the alignments studied are presented in Supplementary Table 1.
The topologies of the 45S and 45S+rpb1 trees generated by BI and ML analyses of sequences of Acaulospora, Diversispora, and Scutellospora members were identical (Figures 1, 3, 7, and Supplementary Figure S1).In the trees with 45S+rpb1 and 45S sequences (Figure 1, Supplementary Figure S1), Diversispora 448 grouped in a clade sister to D. densissima sequences, and in both these trees, the Diversispora 448 clade obtained full BI (=1.0) and very high ML (=99% and 97%, respectively) supports.Analyses of 45S sequences of Scutellospora 431 and Scutellospora 437 indicated that they were sister species and closely related to Scutellospora ovalis.Each of the potentially new species obtained full BI (=1.0) and full (=100% for Scutellospora 437) or high (=84% for Scutellospora 431) ML supports (Figure 3).
The divergence of 45S sequences of Diversispora 448 from those of D. densissima ranged from 5.0% to 6.7%.The range of differences between 45S sequences of Scutellospora 431 and Scutellospora 437 was 3.6-5.3%.
Acaulospora gedanensis was placed in a basal clade sister to a clade consisting of two subclades, one with A. brasiliensis and the second with A. pustulata (Figure 7).The A. gedanensis clade was fully (BI =1.0) and strongly (ML = 89%) supported.Also, the clades with A. brasiliensis and A. pustulata, as well as the node linking the A. gedanensis clade with the A. brasiliensis and the A. pustulata clade obtained full BI and very high ML (98%−100%) supports.
Acaulospora gedanensis and A. brasiliensis differed in terms of the nucleotide composition of the 45S sequences by 3.2%−7.4% and A. gedanensis and A. pustulata by 3.4%−4.4%.

Taxonomy
The results of phylogenetic analyses and comparisons of sequences described above confirmed our supposition resulting from the preliminary morphological and molecular studies of the three fungi considered here that they are undescribed species and showed the closest relatives of A. gedanensis.Consequently, Diversispora 448, Scutellospora 431, and Scutellospora 437 have been described below as D. vistulana sp.nov., S. graeca sp.nov., and S. intraundulata sp.nov., respectively.As for A. gedanensis, its morphology was compared with the original one characterized by Błaszkowski (1988Błaszkowski ( , 2012) ) and the morphology of the species closely related phylogenetically (Figure 3).Etymology: Latin, vistulana, referring to the Vistula Bar, where the new species was originally found.
Ecology and distribution: In the field, D. vistulana probably lived in arbuscular mycorrhizal symbiosis with roots of A. arenaria in maritime sand dunes near Przebrno (54 • 22'30,3''N 19 • 23'13,6''E) located on the Vistula Spit in northern Poland, but no molecular analyses were performed to confirm this supposition.Numerous attempts at growing this species in single-species cultures with P. lanceolata as the host plant failed.According to BLASTn search and our phylogenetic analyses, D. vistulana appears to have a disjunct distribution since only two sequences (JF439143 with 97.18% of identity and coverage = 100% from China and EU350809 with 97.36% of identity and coverage = 34% from Netherlands: Island Terschelling, Atlantic Coast) clustered in the clade with 45S sequences of the new species (data not shown).
Figures 4A-H MycoBank No. MB 851413 Etymology: Latin, graeca, referring to the country, Greece, in which the species was originally found.
Diagnosis: Differs from (A) S. intraundulata, the closest phylogenetic relative (Figure 3), in: (i) spore color, (ii) the thickness of the spore wall, (iii) having a laminate spore wall layer 2 that is uniform in thickness in its different regions (vs.often with local depressions on the lower surface), and (iv) nucleotide composition of sequences of the 45S nuc rDNA region (see Discussion for details); (B) other smooth-spored Scutellospora species, i.e., S. arenicola, S. calospora, S. dipurpurescens, and S. deformata, in (i) spore color and size, (ii) the permanence of spore wall layer 1 and the reactivity of spore wall layer 2 in Melzer's reagent, (iii) the thickness of spore wall layer 2, (iv) the composition and phenotypic and histochemical properties of germinal wall 1, (v) the composition of germinal wall 2 (Morton and Koske, 1988;Koske and Halvorson, 1989;Guillén et al., 2021; https://invam.ku.edu/ species-descriptions), and (vi) the phylogenetic position among sequenced Scutellospora species (Figure 3).
Ecology and distribution: In the field, S. graeca probably lived in arbuscular mycorrhizal symbiosis with roots of A. arenaria, but no molecular analyses were performed to confirm this supposition.GenBank searches, using BLASTn, and our phylogenetic analyses (data not shown) revealed only two sequences, which suggested the presence of AMF identical to S. gracea and S. intraundulata, the latter described below, in other localities.The sequences were GU322902 with 99.28% identity and coverage = 49% from chalk grassland soil in the Netherlands and JX096615 with 96.87% identity and coverage = 87% from Qinghai-Tibet Plateau, China.Specimens examined: GREECE.Spores from a trap culture inoculated with a field-collected mixture of the rhizosphere soil and root fragments of Ammophila arenaria inhabiting a maritime sand dune site of the beach Voidokoilia (36 • 57'N 21 • 39'E), the Peloponnese Peninsula, Greece, September 8, 2015, J. Błaszkowski (holotype slide with spores no.ZTMyc 0067009, isotype slides with spores no.3933-3950, LPPDSE).
Ecology and distribution: In the field, S. intraundulata probably lived in arbuscular mycorrhizal symbiosis with roots of A. arenaria, but no molecular analyses were performed to confirm this supposition.Many attempts at growing this species in singlespecies cultures with P. lanceolata as the host plant failed.Remarks on the possible occurrence of S. intraundulata in other sites are presented in the "Ecology and distribution" section under S. graeca (see above).Diagnosis: Differs from (A) A. brasiliensis and A. pustulata, the phylogenetically closest species (Figure 7), in: (i) that the upper surface of spore wall layer 1 of A. gedanensis is usually smooth, rarely ornamented with small outgrowths (vs.always ornamented with relatively larger outgrowths in A. brasiliensis and A. pustulata), (ii) color and size of spores, as well as the morphometric features of the spore and germinal walls and the phenotypic properties of germinal wall 1 (to A. brasiliensis), (iii) the composition and flexibility of germinal wall 1 and the lack of granular ornamentation on the upper surface of germinal wall 2 layer 1 (to A. pustulata), and (iv) nucleotide composition of sequences of the 45S nuc rDNA region (see Discussion for details); (B) A. flavopapillosa and A. tortuosa, producing acaulosporoid spores with spore wall layer 1 ornamented on the upper surface, in (i) spore size, (ii) the morphology of the ornamentation, (iii) the composition and phenotypic properties of germinal walls 1 and 2 (Palenzuela et al., 2013;Corazon-Guivin et al., 2022), and (iv) the phylogenetic position to other sequenced Acaulospora species (Figure 7).

Notes on Acaulospora gedanensis
Description: Glomerospores (= spores) are formed singly in soil, laterally, either directly on the neck of a sporiferous saccule (sessile spores) or at the top of a short branch (pedicel) of the neck; the sporiferous saccules are hyaline, globose to subglobose, 60-70 µm in diameter, with a single-layered wall, 0.8-1.0µm thick, continuous with the neck wall and spore wall layer 1, forming the spore surface; necks 36-60 µm long, tapering from a 17-20 µm diameter at the saccules to a 6-15 µm diameter at the point of spore attachments; the neck's wall is 0.8-1.0µm thick (Figures 8A-C).Spores have a slightly raised, circular thickening of layer 2 of the spore wall (cicatrix) or a cylindrical to slightly funnel-shaped pedicel, 1.3-10.0µm long, 5.0-9.4µm wide, with a wall 1.4-2.0µm thick, and continuous with spore wall layer 1 when observed in a cross view (Figures 8B, C); the cicatrix and pedicel surround a circular (2.0-3.5 µm in diameter) slight depression in layer 2 of the spore wall when seen in a plan view (Figure 8D).The sporiferous saccules usually collapse or fall off in mature spores.The spores are pale yellow (3A3) to lemon yellow (3B8), globose to subglobose, (55-)65(−75) µm in diameter, with a spore wall and two germinal walls (Figures 8A-H, 9A, B).The spore wall has three layers (spore wall layers 1-3; Figures 8E, G, H, 9A, B).Layer 1 is semi-permanent, rarely strongly deteriorated or completely sloughed off, hyaline, and (0.8-)1.3(−1.6)µm thick; the upper surface of this layer usually is smooth when intact or slightly roughened when deteriorated, rarely covered with blister-like outgrowths, 1.2-2.8µm high and 1.8-7.6 µm wide when seen in cross and plan views, respectively (Figures 8C, E, G, H, 9A, B); in a plan view, the outgrowths are circular to ellipsoidal and more or less uniformly distributed (Figure 8F).Layer 2 is permanent, laminate, smooth, semi-rigid, pale yellow (3A3) to lemon yellow (3B8), and (2.2-)3.6(−4.3)µm thick (Figures 8C, E, G, H, 9A, B).Layer 3 is uniform, smooth, flexible to semi-flexible, concolorous with layer 2, 0.8-1.0µm thick, and rarely separating from the lower surface of layer 2 in even vigorously crushed spores; therefore, it is usually difficult to detect (Figures 8G, H, 9A).The germinal wall 1 has one rigid, fragile, hyaline, 0.3-0.5 µm thick layer, often breaking in even moderately crushed spores (Figures 8E,G,H,9A,B).Germinal wall 2 consists of two flexible to semi-flexible, smooth, hyaline layers (germinal wall 2 layers 1 and 2), 1.0-1.8µm thick and 0.8-1.3µm thick, respectively, always easily separating from one another in crushed spores (Figures 8E,G,H,9A,B).None of the layers of the spore wall and germinal walls stain in Melzer's reagent (Figures 8B-E, G, H).A germination orb was not found, and germination is unknown.

Discussion
The above described phylogenetic analyses with 45S and rpb1 sequences of four AMF (i) confirmed the preliminary morphological studies and BLAST's indications that the fungus with glomoid spores and two morphotypes producing gigasporoid spores represented three new Glomeromycota species (Figures 1,  3, Supplementary Figure S1), (ii) showed their closest sequenced  relatives (Figures 1, 3, Supplementary Figure S1), and (iii) grouped A. gedanensis with sequenced Acaulospora species characterized by a distinctively atypical morphology (Figure 7).These analyses showed that the closest relative of the new glomoid sporeproducing species, described here as D. vistulana, was D. densissima in Diversisporaceae (Figure 1, Supplementary Figure S1), and the two new species with gigasporoid spores, named S. graeca and S. intraundulata, are sisters in Scutellosporaceae (Figure 3).The novelty of these species was also strongly supported by the magnitudes of divergences between the 45S sequences of D. vistulana vs. D. densissima and S. graeca vs. S. intraundulata.In addition, the morphology of the three new species clearly differed from that of their closest phylogenetic relatives.
The morphological features that most strongly separate D. vistulana from D. densissima are the number of spore wall layers and the thickness of the spore wall.Diversispora vistulana lacks the flexible to semi-flexible innermost spore wall layer 3 of D. densissima, and the spore wall of the former species is 1.9-2.4-foldthinner (Błaszkowski et al., 2022).In addition, D. densissima spores are clearly darker-colored [pale orange (5A3) to light brown (6D8)], and their subtending hypha may be up to 1.7-fold narrower.The genetic distance between these two species (5.0%−6.7%) is greater than the genetic distance between other closely related Diversispora species.For example, the distances between the D. celata AM713402 and D. eburnea AM713406 sequences and the D. alba OP195880 and D. spurca FN547639 sequences (Figure 1, Supplementary Figure S1) are 4.5% and 4.2%, respectively.
The main morphological feature distinguishing the two new Scutellospora species is the uniformness in thickness of the spore wall of S. graeca (Figures 4A-E, H) compared to the nonuniform thickness of the S. intraundulata spore wall (Figure 5D).This confirms the conclusion of Morton et al. (1995) that the morphological differences between species in Glomeromycota reside in the phenotypic characters of the spore wall.Moreover, compared to S. intraundulata spores, those of S. graeca are clearly lighter-colored, more frequently ovoid than globose (vs.usually globose), 1.1-1.3-foldsmaller when globose, and their spore wall may be up to 1.6-fold thinner (Figures 4A-E, G, H, 5A-C).
Of the species producing gigasporoid spores, the lower surface of the laminate spore wall 1 is wavy only in Dentiscutata colliculosa (Goto et al., 2010).The morphological differences clearly separating S. intraundulata and D. colliculosa occur in the spore size and color, the spore wall, the germinal wall 1, and the germination shield.The spores of S. intraundulata are approximately 2.5-fold smaller when globose and much lighter-colored (vs.red-brown to black) and do not have spore wall layer 2 of D. colliculosa.The germinal wall 1 of S. intraundulata is single-layered (vs.two-layered in D. colliculosa) and much thinner (approximately 1 µm thick vs. 3.1-5.2µm).The germination shield of S. intraundulata is colorless (vs.yellow-brown to brown) and is not dentate at the margin as in D. colliculosa.
Our phylogenetic analyses evidenced that S. ovalis is the closest relative of both S. graeca and S. intraundulata.However, the phenotypic features of the spore wall layer 1, the composition of the spore wall and two germinal walls, as well as the histochemical properties of germinal wall 1 differentiate S. ovalis from both species.In S. ovalis, the spore wall layer 1 often disappears due to sloughing off as the spores age (Crossay et al., 2018;vs.it is permanent in S. graeca and S. intraundulata; Figures 4B-E, 5B-D, G, 6A, B).The spore wall and the germinal wall 2 of S. ovalis lack the innermost layer 3 of the spore wall, and the germinal wall 2 of the two new Scutellospora species (Figures 4B-E, 5B, C, 6B), and germinal wall 1 is two-layered in S. ovalis (vs.single-layered; Figures 4D, E, 5B, C, 6B), of which none stains in Melzer's reagent (vs. it stains clearly; Figures 4D, E, 6B, C).In addition, the spore wall of S. ovalis vs. that of S. intraundulata is uniform in thickness (vs.often with local depressions on the lower surface; Figure 5D) and 1.3-1.5-foldthinner.Molecularly, the identities of S. ovalis 45S sequences differ from those of S. graeca and S. intraundulata by 4.3%−7.6%and 7.0%−7.2%,respectively.
The phylogenetic analyses showed that A. gedanensis is nested in a clade formed by other species with atypical spore morphology: (i) the lack or rare presence of a beaded layer 1 in germinal wall 2, (ii) the absence of Melzer's reaction in layer 2 of this wall, and (iii) the easy separation of these two layers from each other in even moderately crushed spores.Other species that share these features are A. brasiliensis, A. pustulata, A. colliculosa, and A. tortuosa, the first two being the closest relatives of A. gedanensis (Figure 7), except for A. colliculosa, in which layers 1 and 2 of the germinal wall 2 do not separate from each other.
Acaulospora gedanensis differs mainly from A. brasiliensis and A. pustulata in the properties of the structures ornamenting the upper surface of their spores.Krüger et al. (2011) characterized A. brasiliensis spores as having the upper surface of the spore wall ornamented with semi-permanent, large (up to 10 µm high, up to 20 × 30 µm wide) outgrowths, which rarely are highly deteriorated and difficult to see.According to Palenzuela et al. (2013), A. pustulata spores are covered with permanent, densely crowded projections that are 1.2-5.5 µm high and 1.2-9.5 µm wide at the base.In our study, most A. gedanensis spores were smooth, or the blister-like ornamentation of their surface was difficult to detect due to its small size or short duration (Figures 8E, F), even in freshly matured spores still associated with the sporiferous saccule necks (Figures 8A, B), which usually detach from older spores (Figures 8A, C-H, 9A, B).In addition, compared to A. gedanensis, the thickness of components of all three A. brasiliensis walls is 1.2-1.8-foldlower, and the single-layered germinal wall 1 is flexible.Although the germinal wall 1 of A. gedanensis is only 0.3-0.5 µm thick (vs. up to 1 µm thick in A. brasiliensis), it usually breaks in even moderately crushed spores due to its rigidity and fragility (Figures 8G, H, 9A, B)-a phenomenon not found in any other Acaulospora species.Finally, A. brasiliensis spores may be much darker-colored (yellowish brown to yellowish-red) and clearly larger (48-91 × 51-100 µm diam) when globose to subglobose (vs.pale yellow to lemon yellow; 55-75 µm in diameter in A. gedanensis).Other features that distinguish A. gedanensis from A. pustulata are the composition and phenotypic features of germinal walls 1 and 2. In A. pustulata, the germinal wall 1 consists of two semi-flexible layers (vs.one rigid layer) and the upper surface of layer 1 of the three-layered germinal wall 2 is rarely ornamented with granular excrescences (Palenzuela et al., 2013) (vs. is smooth), unlike the vast majority of Acaulospora species in which this ornamentation is present and easy to notice (da Silva et al., 2022).
alignment was created by concatenation of the 45S and rpb1 alignments.

FIGURE
FIGURE % majority-rule consensus tree from the Bayesian analysis of sequences of S nuc rDNA concatenated with rpb sequences of Diversispora vistulana, other Diversispora species, as well as Corymbiglomus corymbiforme, Desertispora omaniana, Redeckera megalocarpum, and Sieverdingia tortuosa serving as outgroup.The new species is in bold font.The Bayesian posterior probabilities ≥ .and ML bootstrap values ≥ % are shown near the branches.The bar indicates .expected change per site per branch.

FIGURE
FIGURE % majority-rule consensus tree from the Bayesian analysis of sequences of S nuc rDNA of Scutellospora graeca and S. intraundulata, six other Scutellospora species, and Orbispora pernambucana serving as outgroup.The new species are in bold font.The Bayesian posterior probabilities ≥ .and ML bootstrap values ≥ % are shown near the branches.The bar indicates .expected change per site per branch.

FIGURE
FIGURE % majority-rule consensus tree from the Bayesian analysis of sequences of S nuc rDNA of Acaulospora gedanensis, other Acaulospora species, and two Sacculospora species serving as outgroup.Acaulospora gedanensis is in bold font.The Bayesian posterior probabilities ≥ .and ML bootstrap values ≥ % are shown near the branches.The bar indicates .expected change per site per branch.
and in the Laboratory of Plant Protection, Department of Environmental Management (LPPDEM), West Pomeranian University of Technology in Szczecin, Poland (isotypes and A. gedanensis specimens).