Morphological and phylogenetic analysis of the early-diverging lineage of Glomeromycota suggest two new genera and recombinations in Archaeosporales

249 Morphological and phylogenetic analysis of the early-diverging lineage of Glomeromycota suggest two new genera and recombinations in Archaeosporales Keyvan Esmaeilzadeh-Salestani1,2* , Mariana Bessa de Queiroz3* , Vladimir Mikryukov1,4 , Sylwia Uszok5, Bruno Tomio Goto6, Leho Tedersoo1,7 , Franco Magurno5 1 Mycology and Microbiology Center, University of Tartu, Liivi 2, 50409 Tartu, Estonia 2 Institute of Technology, University of Tartu, Nooruse 1, 50411 Tartu, Estonia 3 Programa de Pós-graduação em Sistemática e Evolução, Centro de Biociências, Universidade Federal do Rio Grande do Norte, Campus Universitário, Natal 59072-970, RN, Brazil 4 Institute of Ecology and Earth Sciences, University of Tartu, Liivi 2, 50409 Tartu, Estonia 5 InstituteofBiology,BiotechnologyandEnvironmentalProtection,FacultyofNaturalSciences,UniversityofSilesiainKatowice,Jagiellońska28,40-032, Katowice, Poland 6 Departamento de Botânica e Zoologia, Universidade Federal do Rio Grande do Norte, Campus Universitário, Natal 59072-970, RN, Brazil 7 DepartmentofZoology,CollegeofScience,KingSaudUniversity,12371Riyadh,SaudiArabia Corresponding authors: Bruno Tomio Goto ([email protected]); Leho Tedersoo ([email protected]); Franco Magurno (fr[email protected]) Copyright: © Keyvan Esmaeilzadeh-Salestani et al. This is an open access article distributed under terms of the Creative Commons Attribution License (Attribution 4.0 International – CC BY 4.0). Research Article Abstract The family Archaeosporaceae (Archaeosporales), an early-diverging lineage of Glomeromycota, is currently represented by a single genus, Archaeospora, with seven species described. During the analysis of pot cultures established for the maintenance of Glomeromycota isolates, an unanticipated fungus emerged as a contaminant. Morphological and phylogenetic analyses revealed this fungus as a new species, forming an autonomous genus-level clade within Archaeosporaceae, herein proposed as Antiquispora disseminans gen. et sp. nov. Sequences for this species were obtained using the newly designed primer FULlongF in combination with FULR, both not Glomeromycota specific. Positive clones after transformation were then screened and selected using the Archaeosporaceae-specific reverse primer SpAll_Archaeo_R in combination with the vector sequencing primers. In addition, independent phylogenetic analysis using specimen-based sequences and eDNA supported the genus status of Archaeospora ecuadoriana and A. spainii, both with diagnostic morphological traits, leading to the establishment of the new genus Andinospora to accommodate Andinospora ecuadoriana comb. nov. and the genus status revalidation of Palaeospora with P. spainii. Archaeospora remains to include A. trappei, A. europaea, A. schenckii, while A. myriocarpa and A. undulata require additional analysis. Environmental sequences from the EUKARYOME database also showed that most of the genus-level clades described in Archaeosporaceae have worldwide distribution and are populated by several potential new species. Key words: Arbuscular mycorrhizal fungi, Archaeosporaceae, description of new taxa, molecular phylogeny, taxonomy Academic editor: Thorsten Lumbsch Received: 24 July 2025 Accepted: 14 October 2025 Published: 3 November 2025 Citation: Esmaeilzadeh-Salestani K, Queiroz MB, Mikryukov V, Uszok S, Goto BT, Tedersoo L, Magurno F (2025) Morphological and phylogenetic analysis of the earlydiverging lineage of Glomeromycota suggest two new genera and recombinations in Archaeosporales. MycoKeys 124: 249–273. https://doi. org/10.3897/mycokeys.124.166449 MycoKeys 124: 249–273 (2025) DOI: 10.3897/mycokeys.124.166449 * These authors contributed equally to this work. 250 MycoKeys 124: 249–273 (2025), DOI: 10.3897/mycokeys.124.166449 Keyvan Esmaeilzadeh-Salestani et al.: New genera and recombinations in Archaeosporales Introduction Arbuscular mycorrhizal fungi (AMF), mostly belonging to the phylum Glomeromycota, are ancient organisms that have coevolved with plants for over 400 million years, and associate with over 70% of terrestrial plant species (Brundrett and Tedersoo 2018). AMF plays a crucial role in several ecosystems by enhancing plant nutrient acquisition, improving water uptake, contributing to soil structure, and influencing plant community composition (Fall et al. 2022). Despite their ecological importance, their high diversity suggested by sequence-based environmental studies (Tedersoo et al. 2024a) and the recent description of several new species, only about 370 AMF species have been formally described, distributed across 52 genera and 21 families (da Silva et al. 2024, 2025; Goto et al. 2024; Błaszkowski et al. 2025a, b). The family Archaeosporaceae (Archaeosporales) represents one of the earliest-diverging clades within Glomeromycota. It was established by Morton and Redecker (2001) with only one genus, Archaeospora, based on morphological and phylogenetic analyses of three species previously assigned to the genus Acaulospora: Arc. trappei (type species), which produces only acaulosporoid spores, and Arc. leptoticha and Arc. gerdemannii, both dimorphic - i.e., producing acaulosporoid and glomoid spores. Later, combining the morphology of the spore wall and the type of spore development (entrophosporoid), Sieverding and Oehl (2006) established the genus Intraspora within Archaeosporaceae to accommodate I. schenckii, previously classified as Entrophospora schenckii within the family Entrophosporaceae. Spain et al. (2006) also combined unique spore wall morphology and type spore development (acaulosporoid) to accommodate Acaulospora appendicula, Appendicispora jimgerdemannii and Arc. gerdemannii in a new genus Appendicispora in Archaeosporaceae. Walker et al. (2007a), using a molecular approach, described the genus Ambispora, which is the type genus for the family Ambisporaceae (formerly described as Appendicisporaceae) (Walker et al. 2007b) in Archaeosporales, accommodating Amb. leptoticha, Amb. gerdemannii, Amb. fennica and Amb. callosa, while only Arc. trappei remained in Archaeospora as previously suggested by Schüßler et al. (2001). Since Appendicispora represented a homonym of Appendicospora described previously by Hyde (1995) in Ascomycota, the names Ambispora and Ambisporaceae became legitimate (Walker 2008). Following SSU rDNA sequence analysis, Schüßler and Walker (2010) transferred I. schenckii to Archaeospora, thereby synonymizing the genus Intraspora. Based on spore morphology and phylogeny of the ITS/LSU rDNA regions, Oehl et al. (2015) described the genus Palaeospora, with the type species P. spainii, forming a clade sister to Arc. trappei and I. schenckii. Subsequent analyses by Schüßler and Walker (2019) led to the reclassification of P. spainii into the genus Archaeospora, rendering Palaeospora a synonym of Archaeospora. Morphologically, Archaeosporaceae is characterized by small, hyaline spores with distinctive developmental modes. While most species produce monomorphic acaulosporoid spores, some species are dimorphic by producing both acaulosporoid and glomoid spores, or, more rarely, also producing entrophosporoid spores (Sieverding and Oehl 2006). Oehl et al. (2011) transferred Acaulospora myriocarpa and A. undulata to Archaeospora considering their bi-walled spores, with both outer and inner walls composed of two-three layers each. 251 MycoKeys 124: 249–273 (2025), DOI: 10.3897/mycokeys.124.166449 Keyvan Esmaeilzadeh-Salestani et al.: New genera and recombinations in Archaeosporales Archaeospora spainii (formerly Palaeospora) is an exception in Archaeosporaceae, producing spores with three distinct two-layered walls (Oehl et al. 2015). The mycorrhizal structures consist of arbuscules and both intraand extraradical hyphae, which generally stain weakly in Trypan blue; vesicles are rarely observed. Currently, Archaeosporaceae is a monogeneric family represented by Archaeospora comprising seven species, of which Arc. myriocarpa and Arc. undulata (Oehl et al. 2011) lack phylogenetic characterization. However, the phylogenetic placement of Arc. trappei, the type species of the genus, remains uncertain, as sequences obtained from multiple isolates with similar morphology are found in separate clades (Schüßler and Walker 2019). Recent advances in environmental DNA (eDNA) sequencing have revolutionized our understanding of microbial diversity, enabling the detection and identification of previously unknown fungal taxa directly from soil and root samples (Hug et al. 2016; Tedersoo et al. 2024b). Accordingly, Tedersoo et al. (2024a) highlighted two possible undescribed genera in Archaeosporaceae. While analyzing trap cultures intended for the maintenance of AMF isolates, we discovered an unexpected fungus that proliferated as a contaminant. Preliminary molecular analyses suggested that this fungus might be accommodated in one of the suggested new genera. Additionally, our investigations revealed the need for new combinations within the family. Therefore, the aims of this study are (i) to describe and characterize the morphology of the new fungus; (ii) to determine its position within Glomeromycota using both traditional and eDNA sequence-based approaches; (iii) to uncover candidate new species based on eDNA within the Archaeosporaceae; (iv) to revisit the genus Palaeospora with P. spainii based on morphological and molecular evidence; and (v) to accommodate Arc. ecuadoriana in a new genus within Archaeosporaceae. Materials and methods Origin of study material Antiquispora disseminans pot culture was established in 2019 from spores collected in an old accession of Diversispora epigaea (formerly labeled as Glomus versiforme) available at the University of Turin (Italy). The substrate contained old spores (whose affiliation to D. epigaea was confirmed by rDNA sequencing) that had lost the ability to germinate, and smaller hyaline spores of unknown origin. About 100 spores were collected by F. Magurno and used to set up a pot culture with autoclaved sand and 5% bentonite as substrate, and Plantago lanceolata as host plant. After propagation, twelve single-spore pot cultures were also prepared under the same conditions. Pot cultures were maintained in the plant growth chamber at the Institute of Biology, Biotechnology and Environmental Protection (Katowice, Poland) and re-established approximately every six months upon verification of the presence of spores. Morphological analysis Spores were isolated from pure culture pots by suspending 20–30 ml of substrate in water and passing the decanted supernatant through a 45-μm sieve. Spores were mounted on microscope slides using water, polyvinyl alcohol– 252 MycoKeys 124: 249–273 (2025), DOI: 10.3897/mycokeys.124.166449 Keyvan Esmaeilzadeh-Salestani et al.: New genera and recombinations in Archaeosporales lactic acid–glycerol (PVLG), and a mixture of PVLG and Melzer’s reagent (1:1, v/v). Morphological characteristics of spores were described from at least 100 spores, examined and photographed using dissecting and compound microscopes. The terminology of the spore structures is that presented in Błaszkowski (2012). Color descriptions were based on Kornerup and Wanscher (1983). The fungal nomenclature and the authors of fungal names were verified in the Index Fungorum database at www.indexfungorum.org (accessed on 24 February 2025). Voucher specimens were deposited in the UFRN–Fungos herbarium, Brazil (holotype) and in the Herbário Parque das Dunas RN, Brazil (isotype). Molecular analysis Spores from both multiand single-spore cultures were collected by wet sieving of the pot substrate. Genomic DNA was extracted with DNeasy PowerSoil Pro Kit (Qiagen, Hilden, Germany), according to the manufacturer’s instructions with modifications as in Magurno et al. (2024). Amplicons of SSU-ITS-LSU nrDNA partial genes were obtained by PCR with newly designed primer FULlongF (CCT AGT AAG CGT GAG TCA TCA) and FULR (Malicka et al. 2022) using the Phusion Plus DNA Polymerase (Thermo Fisher Scientific, Waltham, MA, USA) with a universal annealing temperature of 60 °C, according to the producer’s instructions. The annealing sites of the two primers are slightly internal to the positions of SSUmCfx and LSUmBrx (Krüger et al. 2009). Since these primers are not Glomeromycota-specific, clones positive for the insert were also checked with the Archaeosporaceae-specific reverse primer SpAll_Archaeo_R (CAT TAY GTC AGC ATC CTT G), in combination with the vector sequencing primers. Additional shorter sequences were obtained with the FULF/FULR primer pair as in Malicka et al. (2022). PCR amplicons were purified and cloned with GeneJET PCR Purification and CloneJET PCR Cloning Kits (Thermo Fisher Scientific, Waltham, MA, USA) and sequenced at Genomed S.A. (Warsaw, Poland). Sequences were deposited in GenBank with accession numbers PV873150– PV873156, PV938315–PV938337. Bioinformatic analysis To infer the phylogenetic placement of the new species, a dataset was created including representatives of members of Archaeosporales, and Paraglomerales as the outgroup. More in detail, for Archaeosporaceae, the dataset comprised sequences from the new species and from all Archaeospora species (sensu Schüßler and Walker 2019) in possession of partial SSU-ITS-LSU nrDNA sequences or part of it, including sequences from twelve Archaeospora trappei isolates. The dataset was aligned with the online version of MAFFT v.7 (Katoh and Standley 2013) using the E-INS-i iterative refinement method (http://mafft. cbrc.jp/alignment/server/). Bayesian and maximum likelihood phylogenetic inference were performed via CIPRES Science Gateway 3.1 (Miller et al. 2010), using MrBayes v3.2.7 (Ronquist et al. 2012) and RAxML-NG (Kozlov et al. 2019) with partitions and nucleotide substitution models as described in Magurno et al. (2024). For Bayesian analysis, the number of generations was increased up to 5 million with a stop rule at split frequency standard deviation = 0.01. Phylogenetic trees from the two analyses were visualized, merged and edited in 253 MycoKeys 124: 249–273 (2025), DOI: 10.3897/mycokeys.124.166449 Keyvan Esmaeilzadeh-Salestani et al.: New genera and recombinations in Archaeosporales TreeGraph 2 (Stöver and Müller 2010). Clades were considered supported with Bayesian posterior probabilities ≥ 0.95 and ML bootstrap values ≥ 70%. Due to the uncertainty of phylogenetic delimitation of Arc. trappei, we included a phylogenetic placement based on a comprehensive eDNA dataset (see description below). LSU_D2 sequences from 8 pure cultures of Antiquispora disseminans and sequences of Arc. europaea, Arc. schenckii and Arc. ecuadoriana were also added to the eDNA dataset alignment with the online version of MAFFT 7 using the “mafft --add”. The GTR+G+I model parameters of the reference tree were derived in RAxML using the function “--evaluate”. Sequences’ placement was achieved using EPA-ng (Barbera et al. 2019) and the resulting jplace file was converted to Newick format using Gappa. A broader phylogeny, including a vast number of eDNA sequences, was conducted to confirm the phylogenetic placement of the new genus Antiquispora and detect candidate new species in Archaeosporaceae. Sequence data assigned to Glomeromycota were downloaded from three nucleotide sequence databases – EUKARYOME v.1.7 (Tedersoo et al. 2024b), NCBI (Sayers et al. 2024) and UNITE v.9.1 (Abarenkov et al. 2024). Unidentified fungi obtained from NCBI and UNITE were first assigned to rough taxonomic groups based on BLASTn queries against reference sequences in EUKARYOME v.1.7. Sequences affiliated with Archaeosporales were selected to assemble a SSU-ITS-LSU sequence dataset that was aligned using MAFFT v.7. The alignment was edited by manual trimming of overarching and misaligned ends and manual correction in case of obvious misalignments using AliView v.1.26 (Larsson 2014). In the alignment, at least one read from each described species was included to delimit clades and assign taxonomy. The alignments were further filtered to exclude unalignable regions and processed in ClipKIT v.1.4.0 (Steenwyk et al. 2020) to remove phylogenetically uninformative positions. Finally, five partitions (SSU, ITS1, 5.8S, ITS2, LSU) were defined and employed in phylogenetic analyses. Maximum-likelihood tree reconstruction was performed using IQ-TREE v.2.2.5 (Minh et al. 2020), with a partitioned dataset under the GTR+I+G substitution model, including 1000 ultrafast bootstrap replicates and 1000 SH-aLRT tests. The trees were visualized and used for taxonomic re-annotation in FigTree v.1.4.4 (Rambaut 2018). The first three rounds of alignments and analyses were primarily used to detect and remove low-quality reads and chimeric sequences. From the fourth round onwards, high-quality reads were retained and used to generate final phylogenies for species and genus delimitation. To detect possible novel species clades, we used the following criteria: (i) monophyly; (ii) bootstrap support > 95; (iii) phylogenetic breadth and divergence roughly comparable to previously described taxa; and (iv) minimizing the number of novel taxa (i.e. preferably retaining larger groups if there were multiple alternative splitting possibilities). Intraspecies and interspecies divergences were calculated in Mothur 1.48 using the ‘calc=eachgap’ parameter. To produce the distance matrix, only species described in Archaeosporaceae with sequences overlapping the same SSU-ITS-LSU region were considered. The maximum intraspecific distance that did not exceed the minimum interspecies distance was selected as a threshold for OTU clustering. Similarly, sequences of the eDNA dataset, covering the same target region, were used to produce a distance matrix, and then OTUs, according to the cutoff selected. For each OTU, sequences were mapped into the eDNA 254 MycoKeys 124: 249–273 (2025), DOI: 10.3897/mycokeys.124.166449 Keyvan Esmaeilzadeh-Salestani et al.: New genera and recombinations in Archaeosporales tree to detect possible species clades. Thereafter, the clades were evaluated based on the criteria described above. Diagnoses of candidate novel species were prepared based on sequence motifs in the ITS and LSU regions by visually selecting the most distinguishing oligonucleotide barcodes of typically 20 bases using multiple sequence alignments. The barcodes were selected as not having ambiguous base calls (e.g., N or other IUPAC codes) for the suggested novel species and had at least two differences from closely related taxa. We also limited the number of permitted alignment mismatches (typically 0 or 1) for the candidate novel species to ensure species-level resolution. Finally, metadata obtained from the EUKARYOME database were used to map the occurrences of members of Archaeosporaceae and their distribution across distinct biomes. Results Molecular data and phylogenetic analysis Overall, seven partial SSU-ITS-LSU nrDNA and 23 partial LSU nrDNA clones were successfully sequenced from all the cultures obtained from multiple and single spores of Antiquispora disseminans. The highest dissimilarity between partial SSU-ITS-LSU nrDNA sequences was ca. 1%. Phylogenies inferred using both Bayesian and Maximum Likelihood analysis recovered a similar topology (Fig. 1). The sequences of Ant. disseminans clustered in an autonomous, highly supported clade, shared with sequences from isolate SF119B (identified as Archaeospora trappei). Other sequences from this isolate and from the isolates FL327C, KE120, and NC104B (all from the INVAM collection) formed a sister clade with moderate support (0.99/73). Together, these two clades received full support (1.00/100) and were positioned as a sister group to the clade comprising all the other taxa in Archaeosporaceae. The analysis revealed fully or highly supported clades for Arc. ecuadoriana, Arc. spainii, and Archaeospora (sensu Oehl et al. 2015). The first two are here proposed as the basis for establishing the new genus Andinospora and for resurrecting the genus Palaeospora, respectively (Fig. 1). Archaeospora trappei sequences from eight isolates did not form a monophyletic clade but split into several clades with varying support, with each isolate’s sequences confined to a single clade. The EPA analysis returned a slightly different topology, with most Arc. trappei sequences placed in two supported clades, or in their vicinity, corresponding to lineages called Arc. sp5 and Arc. trappei (named after the Arc. trappei isolate Att1783, with SSU-ITS-LSU sequences available) in the eDNA-based phylogeny (Suppl. material 1). Sequences of Arc. schenckii and And. ecuadoriana were distributed across two distinct clades, whereas sequences of Arc. europaea clustered within the Arc. trappei. Finally, all 23 partial LSU rDNA sequences from 8 pure cultures of Antiquispora disseminans were placed in the corresponding species clade. The phylogenetic analysis based on the eDNA database confirmed the support and autonomy of Antiquispora, Andinospora, Palaeospora and Archaeospora as genera in the Archaeosporaceae (Fig. 2, Suppl. material 2), with a topology coherent with the one in Fig. 1. The clade of Archaeospora was the only one to 255 MycoKeys 124: 249–273 (2025), DOI: 10.3897/mycokeys.124.166449 Keyvan Esmaeilzadeh-Salestani et al.: New genera and recombinations in Archaeosporales 1.00/100 GeosiphonpyriformisFM876840 G. pyriformis AM183920 Polonospora polonica MZ359654 P. polonica MZ359657 P. polonica MZ359656 P. polonica MZ359655 P. polonica MZ359659 P. polonica MZ359658 1.00/98 1.00/93 Antiquisporadisseminans PV873150 Ant. disseminans PV873154 Ant. disseminans PV873152 Ant. disseminans PV873156 Ant. disseminans PV873151 Archaeosporatrappei SF119B KT250767 Arc. trappei SF119B MT832160 Arc. trappei SF119B KT250768 0.99/73 Arc. trappei SF119B KT250787 Arc. trappei SF119B KT250786 Arc. trappei SF119B KT250785 Arc. trappei NC104B MT832161 Arc. trappeiFL327C KT250781 Arc. trappei FL327C KT250784 Arc. trappei NC104B KT250780 Arc. trappei SF119B KT250783 Arc. trappei SF119B KT250778 Arc. trappei SF119B KT250777 Arc. trappei NC104BKT250779 Arc. trappei SF119B KT250774 Arc. trappei SF119B KT250770 Arc. trappei SF119B KT250772 Arc. trappei SF119B KT250771 Arc. trappei KE120KT250791 Arc. trappei KE120KT250790 Arc. trappei KE120 KT250789 Arc. trappei KE120KT250788 1.00/93 1.00/95 Andinosporaecuadoriana LR596344 And. ecuadoriana LR596346 And. ecuadoriana LR596347 And. ecuadorianaLR596348 And. ecuadoriana LR596345 And. ecuadoriana LR596351 And. ecuadoriana LR596352 And. ecuadoriana LR596354 And. ecuadoriana LR596353 And. ecuadorianaLR596349 And. ecuadoriana LR596350 1.00/98 Palaeosporaspainii HG977198 Pal. spainii HG977200 Pal. spainii HG977201 Pal. spainii HG977202 Pal. spainii HG977203 Pal. spainii HG977199 1.00/95 Archaeosporaschenckii FR750020 Arc. schenckiiFR750021 Arc. schenckiiMZ359660 -/90 Arc. trappei AU219KT250815 Arc. trappei AU219KT250816 Arc. trappei AU219KT250819 Arc. trappei ON201D MT832164 Arc. trappei IL203B KT250823 Arc. trappei IL203B MT832163 Arc. trappei PE102KT250814 -/70 1.00/71 Arc. trappeiAtt178_3 FR750034 Arc. trappei Att178_3 FR750037 Arc. trappei Att178_3 FR750035 Arc. trappei DSU87 ON113869 Arc. trappei Att178_3 FR750036 Arc. trappei Att178_3 FR750038 -/80 Arc. trappei AZ119KT250821 Arc. trappei AZ119KT250820 Arc. trappei AZ119KT250822 -/96 Arc. europaea MK940274 Arc. europaea MK940275 Arc. trappei SF113 KC166259 Arc. trappei SF113 KC166260 Arc. trappei SF113 MT832162 Ambispora appendicula JQ231205 Amb. appendicula FN547524 Amb. appendicula FN547529 Amb. leptoticha KC166263 Amb. leptoticha KC166276 Amb. leptoticha FJ461886 Amb. fennica FN547545 Amb. fennica FR750157 Amb. fennica FN547543 Amb. gerdemanniiJF439210 Amb. gerdemanniiKC166283 Amb. gerdemanniiKC166279 Pervetustussimplex KY630235 Per. simplexKY630238 Per. simplexKY630251 Paraglomus laccatum KY630227 Par. laccatum KY630228 Par. laccatum OP378060 Innosporamajewskii KY630229 I. majewskiiKY630232 I. majewskiiKY630233 1.00/100 1.00/100 1.00/100 1.00/100 0.98/- 1.00/100 1.00/100 1.00/100 1.00/100 1.00/100 1.00/100 1.00/100 1.00/100 1.00/100 1.00/100 1.00/100 1.00/99 1.00/79 Antiquispora Andinospora Palaeospora Archaeospora Archaeosporaceae pp 8 8 5 6 6 9 9 5 5 3 Z Z M Z M M M a a c c i polon P . P P P P 0.1 Figure 1. Phylogram generated from Maximum Likelihood (ML) and Bayesian Inference (BI) analyses displaying the phylogenetic relationships of Antiquispora disseminans among taxa in Archaeosporales. In the Antiquispora clade, sequences from isolates previously identified as Arc. trappei, are indicated with their original taxonomic affiliation. Colors in Archaeospora trappei (in Archaeospora clade) represent the different isolates, indicated before the accession numbers, from which sequences originated. Members of Paraglomerales were used to root the tree. Posterior probabilities and support values ≥ 0.98 and 70%, respectively, are indicated above or below the branches. Bar indicates 0.1 expected change per site per branch. 256 MycoKeys 124: 249–273 (2025), DOI: 10.3897/mycokeys.124.166449 Keyvan Esmaeilzadeh-Salestani et al.: New genera and recombinations in Archaeosporales sp.1(99.6/100) sp.3(96.7/100) sp.4(100/100) sp.6(99.7/99) sp.7(99.8/100) sp.8(100/100) sp.9(99.9/99) sp.10(100/100) sp.13(95.8/100) sp.14(97.1/100) Ant. disseminans(95.9/100) sp.16(99.7/100) sp.17(99.8/99) sp.18(99.4/100) sp.19(99.9/100) sp.20(100/100) sp.21(90/100)Archaeosporaceae gen02 (99.9/100) sp.1(98.8/100) And. ecuadoriana(100/100) sp.1(100/100) sp. 2(100/100) sp. 3(100/100) Arc. schenckii(100/100) sp. 4(85.1/99) sp. 5(99.8/100) Arc. trappei; Arc. europaea (100/100) sp.7(96.3/100) 0.2 Ambisporaceae(100/100) Polonosporaceae (100/100) Geosiphonaceae (100/100) Palaeospora(100/100) Archaeospora (65.6/91) Antiquispora (100/100) Andinospora (99.7/100) Pal. spainii(100/100) sp.2(97/100) sp.5(99.5/100) sp.11(99.8/99) sp.12(97.2/100) sp.15(99.9/100) sp.2(100/100) sp.6(96.3/99) Figure 2. Phylogram generated from Maximum Likelihood analysis based on the eDNA dataset of Archaeosporales. The analysis conducted in IQ-TREE2 involved 918 sequences, 563 of which overlapped the SSU-ITS-LSU region, representative of four families and seven genera (plus one putative without representative isolates) in Archaeosporales. In Archaeosporaceae, candidate lineages at species rank are highlighted by colored boxes. Support values (SH-aLRT and Ultrafast Bootstrap support) are shown beside the respective lineage labels. Bar indicates 0.1 expected change per site per branch. 257 MycoKeys 124: 249–273 (2025), DOI: 10.3897/mycokeys.124.166449 Keyvan Esmaeilzadeh-Salestani et al.: New genera and recombinations in Archaeosporales receive moderately strong support (91) as ultrafast bootstrap, and low support as SH-aLRT value (65.6). Additionally, a clade at genus rank (Archaeosporaceae_gen02), represented by a few sequences, was shown in a sister position to the clade containing the other genera. Intraand interspecies divergence analyses identified a 6% dissimilarity threshold, which was subsequently applied to delineate putative species-level lineages. Antiquispora comprised, besides Ant. disseminans, 21 well-supported lineages, which we suggest as candidate species pending further morphological and ecological characterization. The motifs TGGTCTCGGCCTA and TTCGTTGGCTGGTT based on the ITS2 and LSU regions, respectively, as indicated in Fig. 3, were selected as diagnostic barcodes for the genus Antiquispora. Similarly, unique LSU barcodes were determined for all putative species in Antiquispora and ITS barcodes for 19 lineages (Fig. 4 for Antiquispora disseminans, Suppl. material 3 for the others). Additionally, our analysis detected nine well-supported lineages in Archaeospora, including one represented by Arc. schenckii and one hosting the sequences of both Arc. trappei (isolate Att-178) and Arc. europaea. In Andinospora, two putative species lineages, distinct from And. ecuadoriana, were identified. The phylogenetic distinctiveness of Ant. disseminans, And. ecuadoriana, and P. spainii as autonomous clades was also supported by blastn comparisons of their sequences with sequences of similar length to Archaeospora. The sequence divergences of Ant. disseminans from those of Archaeospora, Andinospora, and Palaeospora, were significantly greater (ca. 19–21%) than sequence divergences between other genera in Archaeosporaceae. The average sequence divergences between the genera Antiquispora vs. Andinospora, Antiquispora vs. Archaeospora, Antiquispora vs. Palaeospora, Andinospora vs. Palaeospora, Andinospora vs. Archaeospora, and Palaeospora vs. Archaeospora amounted to 19.5%, 21%, 21%, 13%, 14%, and 11.5%, respectively. A ntiquispora sp.1 EUK1635791 A ntiquisporasp.2 EUK1672043 A ntiquispora sp.3 EUK1702220 A ntiquispora sp.4 EUK1672047 A ntiquispora sp.5 EUK1672028 A ntiquispora sp.6 EUK1702111 A ntiquispora sp.7 EUK1702041 A ntiquispora sp.8 EUK1631017 A ntiquispora sp.9 EUK1701894 A ntiquispora sp.10 EUK1701933 A ntiquispora sp.11 EUK1672034 A ntiquispora sp.12 EUK1671994 A ntiquispora sp.13 EUK1702226 A ntiquispora sp.14 EUK1672027 A ntiquispora sp.15 EUK1672001 A ntiquispora disseminans PV873151 A ntiquispora sp.16 EUK1672059 A ntiquispora sp.17 EUK1702150 A ntiquispora sp.18 EUK1671998 A ntiquispora sp.19 EUK1702214 A ntiquispora sp.20 EUK1671990 A ntiquispora sp.21 EUK1672044 A mbispora EUK1671785 A mbispora appendicula FN547529 A rchaeospora EUK1631756 A rchaeospora schenckii FR750020 A rchaeosporaceae_gen02 MT765709 Polonospora polonic aMZ359659 Polonospor a EUK1672148 Polonospora EUK1672597 101 ITS2 subregion 112 492 LSU 504 Antiquispora sp.1 EUK1635791 Antiquispora sp.2 EUK1672043 Antiquispora sp.3 EUK1702220 Antiquispora sp.4 EUK1672047 Antiquispora sp.5 EUK1672028 Antiquispora sp.6 EUK1702111 Antiquispora sp.7 EUK1702041 Antiquispora sp.8 EUK1631017 Antiquispora sp.9 EUK1701894 Antiquispora sp.10 EUK1701933 Antiquispora sp.11 EUK1672034 Antiquispora sp.12 EUK1671994 Antiquispora sp.13 EUK1702226 Antiquispora sp.14 EUK1672027 Antiquispora sp.15 EUK1672001 Antiquispora disseminans PV873151 Antiquispora sp.16 EUK1672059 Antiquispora sp.17 EUK1702150 Antiquispora sp.18 EUK1671998 Antiquispora sp.19 EUK1702214 Antiquispora sp.20 EUK1671990 Antiquispora sp.21 EUK1672044 Ambispora EUK1671785 Ambispora appendicula FN547529 Archaeospora EUK1631756 Archaeospora schenckii FR750020 Archaeosporaceae_gen02 MT765709 Polonospora polonica MZ359659 Polonospora EUK1672148 Geosiphon pyriformis AM183920 Figure 3. Separation of Antiquispora from other genera of Archaeosporaceae based on the ITS region (ITS2 positions 101–112 tggtctcggcct; one mismatch allowed) and LSU (positions 492–504 ttcgttggctggtt; one mismatch allowed). 264 MycoKeys 124: 249–273 (2025), DOI: 10.3897/mycokeys.124.166449 Keyvan Esmaeilzadeh-Salestani et al.: New genera and recombinations in Archaeosporales wall producing a delicate germinal shield. However, Palaeospora present a distinctive spore development, characterized by (i) small (<100 μm), hyaline, spores produced laterally to sporiferous saccule, and (ii) spore wall with three walls, an outer, middle, and inner (germinal) wall with eight layers, resembling Acaulospora species but without a beaded layer (Sieverding and Oehl 2006). Also considering spore wall organization and phylogenetic analysis Błaszkowski et al. (2021) describe a new family, Polonosporaceae in Archaeosporales with a new genus Polonospora based on species previously described as Acaulospora polonica. A comparative overview of spore type, number of walls and wall layers, and the mycorrhizal structures, including their staining patterns in Trypan blue, observed in all species of Archaeosporaceae is summarized in Table 1. Traits such as spore type and the number of inner wall layers proved inconclusive for distinguishing species, particularly within Archaeospora. Moreover, numerous inconsistencies in the number of inner wall layers are common in the Archaeospora literature, often arising from descriptions based on different isolates. These discrepancies may indicate either (i) that these isolates correspond to different species, or (ii) that a single species exhibits high morphological variation. Based on the current evidence, we consider the first explanation to be more plausible. Therefore, for morphological comparisons, we prioritize the original descriptions or redescriptions that include examination of type specimens. The re-establishment of Palaeospora with P. spainii is supported by morphological features of the spore wall, as originally suggested by Oehl et al. (2015). Palaeospora spainii remains the only species in Archaeosporaceae known to possess eight layers in three spore walls (a feature observed in other Archaeosporales families such as Ambisporaceae and Polonosporaceae), while other members of Archaeospora typically exhibit two spore walls (Spain et al. 2006; Błaszkowski et al. 2021). The new genera Antiquispora and Andinospora within Archaeosporaceae also exhibit spores with two walls. Although And. ecuadoriana was described by Schüßler and Walker (2019) as having “three wall Table 1. Comparison of spore type, number of walls and wall layers and mycorrhizal structures among species of the family Archaeosporaceae. Species Spore type Number of spore walls Number of layers (sw) Number of layers (iw) Mycorrhizal structures/ Trypan blue staining Palaeospora spainii Acaulosporoid/ Glomoid 3 (sw, mw, iw) 3 5 Arbuscules, intraand extraradical hyphae do not stain or stain weakly. Vesicles absent. Andinospora ecuadoriana Acaulosporoid/ Entrophosporoid/ Glomoid 2 (sw, iw) 1 2 Spores and intraradical hyphae staining weakly to moderately. Antiquispora disseminans Acaulosporoid 2 (sw, iw) 2 2 Spores, intraand extraradical hyphae, and vesicles stain darkly. Archaeospora trappei Acaulosporoid 2 (sw, iw) 2 1 Arbuscules, intraand extraradical hyphae stain weakly. Archaeospora schenckii Entrophosporoid 2 (sw, iw) 2 1 or 3 Arbuscules vesicles and intraradical hyphae stain weakly, whereas the inner spore wall stains darkly. Archaeospora europaea Acaulosporoid/ Glomoid 2 (sw, iw) 2 3 Arbuscules, intraand extraradical hyphae not or stain weakly. Vesicles absent. Archaeospora myriocarpa Acaulosporoid 2 (sw, iw) 2 1 Intraradical hyphae stain weakly. Typical arbuscules and vesicles absent. Archaeospora undulata Acaulosporoid 2 (sw, iw) 2 1 Unknown *Abbreviation of spore walls and spore wall layers: spore wall (sw), middle wall (mw) and inner wall (iw) with 1–3 layers. 265 MycoKeys 124: 249–273 (2025), DOI: 10.3897/mycokeys.124.166449 Keyvan Esmaeilzadeh-Salestani et al.: New genera and recombinations in Archaeosporales components (C1-C3)”, this terminology deviates from the standard wall-andlayer nomenclature (Błaszkowski 2012). Our reassessment of the published micrographs indicates that spores of And. ecuadoriana presents the typical biwalled structure (see fig. 2h, k, m, n in Schüßler and Walker 2019), where C1 represents a single-layered outer wall and C2-C3 correspond to the bi-layered inner (germinal) wall (see fig. 2i, n in Schüßler and Walker 2019). Andinospora ecuadoriana differs from Archaeospora species by having a single-layered spore wall, whereas species of Archaeospora present a two-layered spore wall. Additionally, And. ecuadoriana has a two-layered inner wall, whereas Archaeospora species typically have a single-layered inner wall. Archaeospora europaea is the only Archaeospora species described with five wall layers, including three of these in the inner wall. Although Arc. schenckii was initially described as having three wall layers, Sieverding and Oehl (2006), upon reexamining the type material, concluded that it also possesses five layers. Antiquispora disseminans differs from Andinospora by having a two-layered outer wall (vs. a single-layered), and from Archaeospora species by having a two-layered inner wall (vs. oneor three-layered). However, the most distinctive feature of Antiquispora is the staining of its mycorrhizal structures in Trypan blue. While Archaeospora and Palaeospora generally do not stain or stain only faintly, and Andinospora exhibits slight staining, Antiquispora shows consistently dark staining. In addition, the sporiferous saccule of Antiquispora stains similarly to the spores, whereas in Andinospora the saccule stains weakly and in Arc. schenckii shows no staining. For other species in the family, information on saccule staining is currently unavailable. Based on our analysis and results, we follow the recommendations to bi-morphic species proposed by Błaszkowski et al. (2022) for the upcoming works involving species in the Archaeosporaceae that (i) Arc. trappei-like culture collections should be carefully described to characterize the spore wall structure, germinal shield (or orb) and the possible presence of dimorphism, (ii) the morphology of Arc. trappei-like and/or glomoid-like spores should be described to present possible differences, (iii) descriptions of new species based on Arc. trappei-like cultures should be based on morphological specimens-based approaches with a strong phylogenetic dataset to clarify potential new species in the clade, (iv) spore wall description should apply standardized terminology to spore wall organization, avoiding confusion in spore wall interpretations, and finally (v) staining spores and mycorrhizal structures with Trypan blue must be included in species descriptions and characterization of isolates, since it often provides more informative results than Melzer’s reagent, which frequently gives weak or irrelevant reactions within this group. Finally, the large number of sequences retrieved from the Eukaryome database, along with the associated metadata, provided a powerful tool to map the occurrences of Archaeosporaceae members and to shed light on their ecology and distribution. Antiquispora and Archaeospora accounted for 250 and 221 occurrences (sample IDs in the database), respectively, while Rhizoglomus, one of the most widespread genera in Glomeromycota, was found in 889. Funneliformis, to provide another example, was recorded in “only” 203 samplings. When scaling up one taxonomic rank, the family Archaeosporaceae accounted for 429 occurrences, compared to 2727 for Glomeraceae sensu Schüßler and Walker (2010). Based on these data, Antiquispora appears to be not only quite common but also the most widespread genus within Archaeosporaceae. 266 MycoKeys 124: 249–273 (2025), DOI: 10.3897/mycokeys.124.166449 Keyvan Esmaeilzadeh-Salestani et al.: New genera and recombinations in Archaeosporales The reason it has been “hidden” until now remains unclear, but mismatches in Glomeromycota-specific primers commonly employed in environmental studies might be one of the culprits. Indeed, most sequences originated from studies using universal fungal primers, which could have helped overcome such primer bias. Similarly, non-Glomeromycota-specific primers were used to obtain the sequences from the Antiquispora disseminans isolate. Additional information Conflict of interest The authors have declared that no competing interests exist. Ethical statement No ethical statement was reported. Use of AI No use of AI was reported. Funding We thank (i) the Conselho Nacional de Desenvolvimento Cientfico e Tecnolgico (CNPq) for the research grant awarded to MB Queiroz (proc. 306632/2022-5) and to BT Goto (proc. 306632/2022-5); (ii) the University of Katowice for the visiting professor invitation to BT Goto as collaborative research between April and May of 2023; (iii) the Centre of Excellence AgroCropFuture funding to L Tedersoo and K Esmaeilzadeh-Salestani. Author contributions Conceptualization: FM. Data curation: BTG, FM, LT, MBQ, KES. Formal analysis: KES, VM, BTG, MBQ, FM. Funding acquisition: LT, FM. Investigation: FM, BTG, MBQ, KES, VM, LT, SU. Methodology: SU, FM, BTG, KES, MBQ. Software: FM, KES. Supervision: BTG, FM, LT. Writing - original draft: KES, MBQ, BTG, FM. Writing - review and editing: MBQ, BTG, KES, FM, LT. Author ORCIDs Keyvan Esmaeilzadeh-Salestani https://orcid.org/0000-0002-6882-7616 Mariana Bessa de Queiroz https://orcid.org/0000-0003-0197-7203 Vladimir Mikryukov https://orcid.org/0000-0003-2786-2690 Sylwia Uszok https://orcid.org/0000-0001-5961-7695 Bruno Tomio Goto https://orcid.org/0000-0001-6157-4954 Leho Tedersoo https://orcid.org/0000-0002-1635-1249 Franco Magurno https://orcid.org/0000-0002-3117-8149 Data availability All of the data that support the findings of this study are available in the main text or Supplementary Information. References Abarenkov K, Nilsson RH, Larsson KH, Taylor AFS, May TW, Frøslev TG, Pawlowska J, Lindahl B, Põldmaa K, Truong C, Vu D, Hosoya T, Niskanen T, Piirmann T, Ivanov F, Zirk A, Peterson M, Cheeke TE, Ishigami Y, Jansson AT, Jeppesen TS, Kristians- 267 MycoKeys 124: 249–273 (2025), DOI: 10.3897/mycokeys.124.166449 Keyvan Esmaeilzadeh-Salestani et al.: New genera and recombinations in Archaeosporales son E, Mikryukov V, Miller JT, Oono R, Ossandon FJ, Paupério J, Saar I, Schigel D, Suija A, Tedersoo L, Kõljalg U (2024) The UNITE database for molecular identification and taxonomic communication of fungi and other eukaryotes: Sequences, taxa and classifications reconsidered. Nucleic Acids Research 5: 791–797. https://doi. org/10.1093/nar/gkad1039 Ames RN, Linderman RG (1976) Acaulospora trappei sp. nov. Mycotaxon 3: 565–569. https://doi.org/10.5962/p.414006 Barbera P, Kozlov AM, Czech L, Morel B, Darriba D, Flouri T, Stamatakis A (2019) EPA-ng: Massively Parallel Evolutionary Placement of Genetic Sequences. Systematic Biology 68(2): 365–369. https://doi.org/10.1093/sysbio/syy054 Błaszkowski J (2012) Glomeromycota. W. Szafer Institute of Botany, Polish Academy of Sciences, Krakw, Poland. Błaszkowski J, Czerniawska B (2005) Entrophospora schenckii and Pacispora franciscana, arbuscular mycorrhizal fungi (Glomeromycota) new for Europe and Poland, respectively. Acta Mycologica 40(1): 11–18. https://doi.org/10.5586/ am.2005.002 Błaszkowski J, Niezgoda P, Meller E, Milczarski P, Zubek S, Malicka M, Uszok S, Casieri L, Goto BT, Magurno F (2021) New taxa in Glomeromycota: Polonosporaceae fam. nov., Polonospora gen. nov., and P. polonica comb. nov. Mycological Progress 20: 941–951. https://doi.org/10.1007/s11557-021-01726-4 Błaszkowski J, Sánchez-Garca M, Niezgoda P, Zubek S, Fernández F, Vila A, Al-Yahya’ei MN, Symanczik S, Milczarski P, Malinowski R, Cabello M, Goto BT, Casieri L, Malicka M, Bierza W, Magurno F (2022) A new order, Entrophosporales, and three new Entrophospora species in Glomeromycota. Frontiers in Microbiology 13: 962856. https:// doi.org/10.3389/fmicb.2022.962856 Błaszkowski J, Zubek S, Milczarski P, Malinowski R, Niezgoda P, Goto BT (2025a) New taxa and a combination in Glomerales (Glomeromycota, Glomeromycetes). MycoKeys 112: 253–276. https://doi.org/10.3897/mycokeys.112.136158 Błaszkowski J, Goto BT, Zubek S, Milczarski P, Malinowski R, Niezgoda P, Błaszkowski T (2025b) Paracorymbiglomus gen. nov., Diversispora conica sp. nov., and new combinations in Diversisporaceae (Glomeromycota). MycoKeys 117: 171–190. https://doi. org/10.3897/mycokeys.117.148052 Brundrett MC, Tedersoo L (2018) Evolutionary history of mycorrhizal symbioses and global host plant diversity. The New Phytologist 220: 1108–1115. https://doi. org/10.1111/nph.14976 Corradi N, Antunes PM, Magurno F (2025) A call for reform: Implementing genome-based approaches for species classification in Glomeromycotina. The New Phytologist 247(1): 50–54. https://doi.org/10.1111/nph.70148 Cowgill C, Gilbert JD, Convery I, Handley L (2025) Monitoring terrestrial rewilding with environmental DNA metabarcoding: A systematic review of current trends and recommendations. Frontiers in Conservation Science 5: 1473957. https://doi.org/10.3389/ fcosc.2024.1473957 da Silva GA, de Assis DMA, Sieverding E, Oehl F (2024) Four New Families of Arbuscular Mycorrhizal Fungi Within the Order Glomerales. Taxonomy 4: 761–779. https://doi. org/10.3390/taxonomy4040041 da Silva GA, Sieverding E, Assis DMA, Goto BT, Corazon-Guivin MA, Oehl F (2025) Revision of Entrophosporales, with Three Genera and an Identification Key for All Species Currently Attributed to This Order. Journal of Fungi 11: 97. https://doi.org/10.3390/ jof11020097 268 MycoKeys 124: 249–273 (2025), DOI: 10.3897/mycokeys.124.166449 Keyvan Esmaeilzadeh-Salestani et al.: New genera and recombinations in Archaeosporales Duarte S, Vieira PE, Lavrador AS, Costa FO (2021) Status and prospects of marine NIS detection and monitoring through (e) DNA metabarcoding. The Science of the Total Environment 751: 141729. https://doi.org/10.1016/j.scitotenv.2020.141729 Fall AF, Nakabonge G, Ssekandi J, Founoune-Mboup H, Apori SO, Ndiaye A, Badji A, Ngom K (2022) Roles of Arbuscular Mycorrhizal Fungi on Soil Fertility: Contribution in the Improvement of Physical, Chemical, and Biological Properties of the Soil. Frontiers in Fungal Biology 3: 723892. https://doi.org/10.3389/ffunb.2022.723892 Fediajevaite J, Priestley V, Arnold R, Savolainen V (2021) Meta-analysis shows that environmental DNA outperforms traditional surveys, but warrants better reporting standards. Ecology and Evolution 11: 4803–4815. https://doi.org/10.1002/ece3.7382 Goto BT, Queiroz MB, Magurno F, Souza FA, Błaszkowski J (2024) How far have we progressed in Glomeromycota taxonomy and systematic? IMS newsletter 5: 18–24. Hafeel KM (2004) Spore ontogeny of the arbuscular mycorrhizal fungus Archaeospora trappei (Ames & Schneider) Morton & Redecker (Archaeosporaceae). Mycorrhiza 14: 213–219. https://doi.org/10.1007/s00572-004-0300-y Hug LA, Baker BJ, Anantharaman K, Brown CT, Probst AJ, Castelle CJ, Butterfield CN, Hernsdorf AW, Amano Y, Ise K, Suzuki Y, Dudek N, Relman DA, Finstad KM, Amundson R, Thomas BC, Banfield JF (2016) A new view of the tree of life. Nature Microbiology 1: 16048. https://doi.org/10.1038/nmicrobiol.2016.48 Hyde KD (1995) Fungi from palms. XXVIII. Appendicospora coryphae, a new name for Apiosporella coryphae. Sydowia 47: 31–37. Katoh K, Standley DM (2013) MAFFT multiple sequence alignment software version 7: Improvements in performance and usability. Molecular Biology and Evolution 30: 772–780. https://doi.org/10.1093/molbev/mst010 Kestel JH, Field DL, Bateman PW, White NE, Allentoft ME, Hopkins AJM, Gibberd M, Nevill P (2022) Applications of environmental DNA (eDNA) in agricultural systems: Current uses, limitations and future prospects. The Science of the Total Environment 847: 157556. https://doi.org/10.1016/j.scitotenv.2022.157556 Kornerup A, Wanscher JH (1983) Methuen handbook of colour, 3rd edn. Eyre Methuen, London, 252 pp. Kozlov AM, Darriba D, Flouri T, Morel B, Stamatakis A (2019) RAxML-NG: A fast, scalable, and user-friendly tool for maximum likelihood phylogenetic inference. Bioinformatics 35(21): 4453–4455. https://doi.org/10.1093/bioinformatics/btz305 Krüger M, Stockinger H, Krüger C, Schüßler A (2009) DNA-based species level detection of Glomeromycota: One PCR primer set for all arbuscular mycorrhizal fungi. The New Phytologist 183(1): 212–223. https://doi.org/10.1111/j.1469-8137.2009.02835.x Larsson A (2014) AliView: A fast and lightweight alignment viewer and editor for large datasets. Bioinformatics 30: 3276–3278. https://doi.org/10.1093/bioinformatics/btu531 Magurno F, Uszok S, Bierza K, Bakr J, Kende Z, Queiroz MB, Casieri L (2024) Glomus mongioiense, a New Species of Arbuscular Mycorrhizal Fungi from Italian Alps and the Phylogeny-Spoiling Issue of Ribosomal Variants in the Glomus Genus. Agronomy 14: 1350. https://doi.org/10.3390/agronomy14071350 Malicka M, Magurno F, Piotrowska-Seget Z (2022) Phenol and polyaromatic hydrocarbons are stronger drivers than host plant species in shaping the arbuscular mycorrhizal fungal component of the mycorrhizosphere. International Journal of Molecular Sciences 23: 12585. https://doi.org/10.3390/ijms232012585 Miller MA, Pfeiffer W, Schwartz T (2010) Creating the CIPRES science gateway for inference of large phylogenetic trees. Proceedings of the Gateway Computing Envi- 269 MycoKeys 124: 249–273 (2025), DOI: 10.3897/mycokeys.124.166449 Keyvan Esmaeilzadeh-Salestani et al.: New genera and recombinations in Archaeosporales ronments Workshop, (USA), New Orleans, LA, 14: 1–8. https://doi.org/10.1109/ GCE.2010.5676129 Minh BQ, Schmidt HA, Chernomor O, Schrempf D, Woodhams MD, Von Haeseler A, Lanfear R (2020) IQ-TREE 2: New models and efficient methods for phylogenetic inference in the Genomic era. Molecular Biology and Evolution 37: 1530–1534. https:// doi.org/10.1093/molbev/msaa015 Morton JB, Redecker D (2001) Two new families of Glomales, Archaeosporaceae and Paraglomaceae, with two new genera Archaeospora and Paraglomus, based on concordant molecular and morphological characters. Mycologia 93(1): 181–195. https://doi.org/10.1080/00275514.2001.1206314 Oehl F, Silva G, Goto BT, Sieverding E (2011) New recombinations in Glomeromycota. Mycotaxon 117: 429–434. https://doi.org/10.5248/117.429 Oehl F, Sánchez-Castro I, Palenzuela J, da Silva GA (2015) Palaeospora spainii, a new arbuscular mycorrhizal fungus from Swiss agricultural soils. Nova Hedwigia 101(1): 89–102. https://doi.org/10.1127/nova_hedwigia/2014/02 Rambaut A (2018) FigTree v1.4.4. https://github.com/rambaut/figtree/releases Ronquist F, Teslenko M, van der Mark P, Ayres DL, Darling A, Höhna S, Larget B, Liu L, Suchard MA, Huelsenbeck JP (2012) MrBayes 3.2: Efficient Bayesian phylogenetic inference and model choice across a large model space. Systematic Biology 61(3): 539–542. https://doi.org/10.1093/sysbio/sys029 Sayers EW, Cavanaugh M, Clark K, Pruitt KD, Sherry ST, Yankie L, Karsch-Mizrachi I (2024) GenBank 2024 Update. Nucleic Acids Research 52: 134–137. https://doi. org/10.1093/nar/gkad903 Schüßler A, Walker C (2010) The Glomeromycota: A Species List with New Families and New Genera. Royal Botanic Garden Edinburgh. Schüßler A, Walker C (2019) Archaeospora ecuadoriana sp. nov. from a mountainous biodiversity hotspot area in Ecuador, and transfer of Palaeospora spainiae to Archaeospora, as A. spainiae comb. nov. Mycorrhiza 29: 435–443. https://doi.org/10.1007/ s00572-019-00913-2 Schüßler A, Schwarzott D, Walker C (2001) A new fungal phylum, the Glomeromycota: Evolution and phylogeny. Mycological Research 105: 1413–1421. https://doi. org/10.1017/S0953756201005196 Sieverding E, Oehl F (2006) Revision of Entrophospora and description of Kuklospora and Intraspora, two new genera in the arbuscular mycorrhizal Glomeromycetes. Journal of Applied Botany and Food Quality 80: 69–81. Spain JL (2003) Emendation of Archaeospora and of its type species, Archaeospora trappei. Mycotaxon 87: 109–112. Spain J, Oehl F, Sieverding E (2006) Appendicispora a new genus in the arbuscular mycorrhiza-forming Glomeromycetes, with a discussion of the genus Archaeospora. Mycotaxon 97: 163–182. Steenwyk JL, Buida TJ, Li Y, Shen X-X, Rokas A (2020) ClipKIT: A multiple sequence alignment trimming software for accurate phylogenomic inference. PLoS Biology 18: e3001007. https://doi.org/10.1371/journal.pbio.3001007 Stöver BC, Müller KF (2010) TreeGraph 2: Combining and visualizing evidence from different phylogenetic analyses. BMC Bioinformatics 11(7): 1–9. https://doi. org/10.1186/1471-2105-11-7 Tedersoo L, Magurno F, Alkahtani S, Mikryukov V (2024a) Phylogenetic classification of arbuscular mycorrhizal fungi: New species and higher-ranking taxa in Glomeromyco- 270 MycoKeys 124: 249–273 (2025), DOI: 10.3897/mycokeys.124.166449 Keyvan Esmaeilzadeh-Salestani et al.: New genera and recombinations in Archaeosporales ta and Mucoromycota (class Endogonomycetes). MycoKeys 107: 249–271. https:// doi.org/10.3897/mycokeys.107.125549 Tedersoo L, Hosseyni Moghaddam MS, Mikryukov V, Hakimzadeh A, Bahram M, Nilsson RH, Yatsiuk I, Geisen S, Schwelm A, Piwosz K, Prous M, Chmolowska D, Rueckert S, Skaloud P, Laas P, Thines M, Jung J-H, Alkahtani S, Anslan S (2024b) EUKARYOME: The rRNA gene reference database for identification of all eukaryotes. Database : The Journal of Biological Databases and Curation 2024: baae043. https://doi.org/10.1093/database/baae043 Walker C (2008) Ambispora and Ambisporaceae resurrected. Mycological Research 112: 297–298. https://doi.org/10.1016/j.mycres.2008.02.001 Walker C, Vestberg M, Demircik F, Stockinger H, Saito M, Sawaki H, Nishmura I, Schüssler A (2007a) Molecular phylogeny and new taxa in the Archaeosporales (Glomeromycota): Ambispora fennica gen. sp. nov., Ambisporaceae fam. nov., and emendation of Archaeospora and Archaeosporaceae. Mycological Research 111(2): 137–153. https://doi.org/10.1016/j.mycres.2006.11.008 Walker C, Vestberg M, Schüssler A (2007b) Nomenclatural clarifications in Glomeromycota. Mycological Research 111: 253–255. https://doi.org/10.1016/j.mycres.2007.02.009 Supplementary material 1 Snapshot of EPA analysis to infer the position of Archaeospora trappei, Arc. schenckii, Arc. europaea, Andinospora ecuadoriana and Antiquispora disseminans isolates in the reference eDNA tree Authors: Keyvan Esmaeilzadeh-Salestani, Mariana Bessa de Queiroz, Vladimir Mikryukov, Sylwia Uszok, Bruno Tomio Goto, Leho Tedersoo, Franco Magurno Data type: pdf Explanation note: Names of isolates are labeled in color. Accession numbers: Archaeospora trappei Att178_3 (FR750034, FR750035, FR750036, FR750038, FR750037), AU219 (KT250815, KT250816, KT250819), SF113 (KC166259, KC166260, MT832162), AZ119 (KT250821, KT250820, KT250822), ON201D (MT832164), PE102 (KT250814), IL203B (KT250823, MT832163), DSU87 (ON113869). Archaeospora schenckii Att2124 (FR750020, FR750021), CL401 (KT250807, KT250792, KT250802, KT250812, KT250797, KT250811, KC166258, KT250813). Archaeospora europaea SAF1151 (MK940274, MK940275). Andinospora ecuadoriana N2 (LR596344, LR596345, LR596349, LR596350, LR596351, LR596352, LR596353, LR596354), N6 (LR596346, LR596347, LR596348). Antiquispora disseminans 1 (PV938315, PV938316, PV938317), 3 (PV938327, PV938328), 4 (PV938318, PV938319, PV938320), 5 (PV938329, PV938330, PV938331), 6 (PV938321, PV938322, PV938323), 8 (PV938324, PV938325, PV938326), 9 (PV938332, PV938333, PV938334), 12 (PV938335, PV938336, PV938337). Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/mycokeys.124.166449.suppl1 271 MycoKeys 124: 249–273 (2025), DOI: 10.3897/mycokeys.124.166449 Keyvan Esmaeilzadeh-Salestani et al.: New genera and recombinations in Archaeosporales Supplementary material 2 Phylogram generated from Maximum Likelihood analysis based on the eDNA dataset of Archaeosporales Authors: Keyvan Esmaeilzadeh-Salestani, Mariana Bessa de Queiroz, Vladimir Mikryukov, Sylwia Uszok, Bruno Tomio Goto, Leho Tedersoo, Franco Magurno Data type: pdf Explanation note: The analysis conducted in IQ-TREE 2 involved 918 sequences, 563 of which overlapping the SSU-ITS-LSU region, representative of four families and seven genera in Archaeosporales. In Archaeosporaceae, candidate lineages at species rank are highlighted by colored boxes. Support values (SH-aLRT and Ultrafast Bootstrap support) are shown beside the respective lineage labels. Bar indicates 0.1 expected change per site per branch. Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/mycokeys.124.166449.suppl2 Supplementary material 3 ITS and LSU barcodes selected for the lineages identified in the study for each genus in Archaeosporaceae Authors: Keyvan Esmaeilzadeh-Salestani, Mariana Bessa de Queiroz, Vladimir Mikryukov, Sylwia Uszok, Bruno Tomio Goto, Leho Tedersoo, Franco Magurno Data type: pdf Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/mycokeys.124.166449.suppl3 272 MycoKeys 124: 249–273 (2025), DOI: 10.3897/mycokeys.124.166449 Keyvan Esmaeilzadeh-Salestani et al.: New genera and recombinations in Archaeosporales Supplementary material 4 Global distribution of the genus Antiquispora based on EUKARYOME database records Authors: Keyvan Esmaeilzadeh-Salestani, Mariana Bessa de Queiroz, Vladimir Mikryukov, Sylwia Uszok, Bruno Tomio Goto, Leho Tedersoo, Franco Magurno Data type: pdf Explanation note: The map shows the geographic locations (colored dots) where the genus has been recorded. A total of 596 sequences were considered from the reference database, spanning 65 countries (Poland for the type species, not included). Map generated with MapCustomizer (https://www.mapcustomizer.com/). Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/mycokeys.124.166449.suppl4 Supplementary material 5 Occurrence of fungal genera in the family Archaeosporaceae across different biomes, based on EUKARYOME database records Authors: Keyvan Esmaeilzadeh-Salestani, Mariana Bessa de Queiroz, Vladimir Mikryukov, Sylwia Uszok, Bruno Tomio Goto, Leho Tedersoo, Franco Magurno Data type: pdf Explanation note: The table summarizes the presence of each genus in natural and anthropogenic biomes, including forests, grasslands, shrublands, woodlands, deserts, and aquatic environments. Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/mycokeys.124.166449.suppl5 273 MycoKeys 124: 249–273 (2025), DOI: 10.3897/mycokeys.124.166449 Keyvan Esmaeilzadeh-Salestani et al.: New genera and recombinations in Archaeosporales Supplementary material 6 Global distribution of the genus Andinospora based on EUKARYOME database records Authors: Keyvan Esmaeilzadeh-Salestani, Mariana Bessa de Queiroz, Vladimir Mikryukov, Sylwia Uszok, Bruno Tomio Goto, Leho Tedersoo, Franco Magurno Data type: pdf Explanation note: The map shows the geographic locations (colored dots) where the genus has been recorded. A total of 52 sequences from the reference database, spanning 14 countries (Ecuador for the type species, not included). Map generated with MapCustomizer (https://www.mapcustomizer.com/). Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/mycokeys.124.166449.suppl6 Supplementary material 7 Global distribution of the genus Archaeospora based on EUKARYOME database records Authors: Keyvan Esmaeilzadeh-Salestani, Mariana Bessa de Queiroz, Vladimir Mikryukov, Sylwia Uszok, Bruno Tomio Goto, Leho Tedersoo, Franco Magurno Data type: pdf Explanation note: The map shows the geographic locations (colored dots) where the genus has been recorded. A total of 251 sequences were considered from the reference database, spanning 38 countries. Map generated with MapCustomizer (https://www. mapcustomizer.com/). Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/mycokeys.124.166449.suppl7