Additions to Macgarvieomyces in Iran: Morphological and Phylogenetic Analyses Reveal Six New Species

Abstract

The genus Macgarvieomyces (Magnaporthales, Sordariomycetes, Ascomycota) currently includes three species, which are associated with leaf spots on plants belonging to the Cyperaceae and Juncaceae families and are known only in Europe and New Zealand. During a comprehensive survey conducted between 2020 and 2022 targeting host plants from these families across various regions of Iran, six novel species of Macgarvieomyces—M. caspica, M. cyperi, M. junci-acuti, M. juncigenus, M. salkadehensis, and M. schoeni—were uncovered. These species were identified based on detailed morphological characterizations and multi-locus phylogenetic analyses using ITS-rDNA, RPB1, ACT, and CAL gene regions. This study provides thorough descriptions and illustrations of the new taxa, including information on their morphology, ecological preferences, and geographic distribution. The phylogenetic relationships among the species are also discussed. This work significantly enhances the known diversity of Macgarvieomyces associated with Cyperaceae and Juncaceae, expands their geographic distribution, and underscores the value of integrating morphological and molecular data in fungal taxonomy; accordingly, the findings of this study lay the groundwork for future ecological and evolutionary studies of this genus.

1. Introduction

The order Magnaporthales (Sordariomycetes, Ascomycota), which was originally established to accommodate the Magnaporthaceae family according to the combined phylogenetic data of the small and large subunits of ribosomal DNA (SSU and LSU) and morphological features [1], comprises a diverse group of fungi that includes important and destructive plant pathogens, mainly on cereals and grasses—endophytes and saprophytes—associated with submerged wood and dead grasses [2,3,4,5]. Historically, the taxonomy of Magnaporthales was based on morphological characteristics, such as the structure of perithecia, asci, and ascospores, as well as the morphological characteristics of the asexual morphs [2,6]. However, morphological traits exhibit high levels of convergence and plasticity among different lineages and have been proven to be insufficient for taxonomic resolution. Consequently, an integrative approach combining morphology, ecology, and multi-locus phylogeny has been adopted for stable classification [2,5]. At present, multi-locus phylogenetic analyses utilizing the large subunit of ribosomal DNA (LSU), the internal transcribed spacer (ITS) region, parts of the largest subunit of RNA polymerase II (RPB1), actin (ACT), calmodulin (CAL), translation elongation factor 1-alpha (TEF1), and minichromosome maintenance complex component 7 (MCM7) gene sequences have been used to delimit the ordinal boundaries and confirm the monophyly of the order Magnaporthales. These studies have resolved the order into six families: Ceratosphaeriaceae, Magnaporthaceae, Ophioceraceae, Plagiosphaeraceae, Pseudohalonectriaceae, and Pyriculariaceae. These comprise 52 genera and 317 species [2,5,7,8,9,10,11].

The family Pyriculariaceae was established by Klaubauf et al. [2], with Pyricularia as the type of genus, according to the combination of morphological features and phylogenetic data derived from LSU, ITS, CAL, ACT, and RPB1 sequences. At present, 13 genera—Bambusicularia, Barretomyces, Bipyricularia, Macgarvieomyces, Neocordana, Neopyricularia, Nothopyricularia, Proxipyricularia, Pseudopyricularia, Pyricularia, Pyriculariomyces, Utrechtiana, and Xenopyricularia—are recognized in Pyriculariaceae. These genera are primarily characterized by the production of denticulate conidiogenous cells and ellipsoid, obclavate, or pyriform, septate conidia (pyricularia-like conidia) [2,5,7,8,9,12]. Most species within the Pyriculariaceae are ecologically characterized by their pathogenicity on cereals and other monocotyledonous plants. A prominent example is Pyricularia oryzae, the pathogen responsible for rice blast disease, which is associated with yield losses reaching up to 30% on a global scale [13,14]. The genus Macgarvieomyces was introduced to accommodate two species, Macgarvieomyces borealis and M. juncicola, which were previously classified under Pyricularia and were isolated from Juncus effusus [2]. The third species, M. luzulae (syn. Pyricularia luzulae), isolated from Luzula spp. (Juncaceae), was later included in the genus, bringing the total to three species [9]. Species of Macgarvieomyces have been reported from leaf spots on plants in the Cyperaceae and Juncaceae families in Europe and New Zealand [2,9,15].

Iran hosts remarkably diverse ecosystems, including temperate forests, montane grasslands, arid steppe, and semi-desert biomes with diverse vegetation. In our ongoing studies of fungi from plants in the families Cyperaceae and Juncaceae, we focused our survey on Macgarvieomyces isolates collected in Iran between 2020 and 2022, gathering specimens from a wide array of host plants exhibiting leaf and stem spots and blights to explore the species diversity of this genus. This study aimed to identify and describe Macgarvieomyces species based on combined morphological characteristics and multigene phylogenetic analyses using ITS, RPB1, ACT, and CAL sequences.

2. Materials and Methods

2.1. Sample Collection and Isolation of Fungi

Leaf and culm samples exhibiting brown lesions and blight symptoms were collected from various wetland plants belonging to the families of Cyperaceae and Juncaceae across six Provinces in Iran: Ardebil, Golestan, Guilan, Mazandaran, Tehran, and West Azarbaijan, between 2020 and 2022 (Figure 1). The samples were labelled [16], stored at low temperatures, and transported to the laboratory. Fungal isolation, purification, and preservation followed the methods described by Ahmadpour et al. [17,18]. The pure cultures of all identified isolates were deposited in the fungal culture collections at the Iranian Research Institute of Plant Protection (IRAN) and Urmia University (FCCUU).

Figure 1.

Map of Iran indicating the sampling locations across the provinces of Ardebil, Golestan, Guilan, Mazandaran, Tehran, and West in the present study.

2.2. Morphological Examination

Mycelial discs (5 mm in diameter) were excised from the actively growing edges of seven-day-old cultures and transferred to fresh culture media, including Potato Dextrose Agar (PDA; 39 g/L, Merck, Darmstadt, Germany), Potato Carrot Agar (PCA; composed of 20 g white potato, 20 g carrot, 20 g agar, and 1000 mL distilled water), and Malt Extract Agar (MEA; 50 g/L, Quelab, Montreal, QC, Canada). The culture plates were incubated in darkness at 25 °C for periods of 7 and 14 days. Colony characteristics, including colour, growth pattern, and diameter, were observed and documented. Colony colours were determined using Rayner’s [19] colour charts. The morphological characteristics were examined and recorded from 10–14-day-old cultures on synthetic nutrient-poor agar medium (SNA) (KH₂PO₄ 1.0 g, KNO₃ 1.0 g, MgSO₄ + 7H₂O 0.5 g, KCl 0.5 g, glucose 0.2 g, sucrose 0.2 g, agar 20 g, distilled water 1000 mL) [20] supplemented with autoclaved barley seeds and incubated at 23–25 °C under 12 h dark/12 h near-ultraviolet light for 10–14 days [2]. Microscopic examination and measurements of hyphae, conidiophores, conidia, hyphopodia, and chlamydospores were performed from fungal slides mounted in clear lactic acid or lactophenol cotton blue solutions. For each morphological structure, 30 to 50 measurements were obtained, and images were captured using an Olympus AX70 microscope equipped with differential interference contrast (DIC) illumination (Olympus Optical Co., Ltd., Tokyo, Japan). Images were processed using Adobe Photoshop 2020 version 2.10.8 (Adobe Inc., San Jose, CA, USA). Newly described taxa were registered in the MycoBank database (www.mycobank.org; accessed 20 May 2025) [21].

2.3. DNA Extraction, PCR Amplification, and Sequencing

Total genomic DNA was isolated from the mycelial biomass of each isolate, obtained from 10-day-old cultures grown on PDA, using the protocol outlined by Ahmadpour et al. [18]. The ITS region, RPB1, ACT, and CAL genes were amplified using the primer pairs ITS1/ITS4 [22], RPB1F/RPB1R [2], ACT-512F/ACT-783R [23], and CAL-228F/CAL-737R [23], respectively. PCR amplification was conducted using a SimpliAmp™ Thermal Cycler (Applied Biosystems™, Thermo Fisher Scientific Corp., Waltham, MA, USA) in a final reaction volume of 30 μL. Each reaction mixture comprised 0.4 μM of each primer, 10 μL of 2X Master Mix containing Taq DNA polymerase and 2 mM MgCl₂ (Ampliqon, Odense, Denmark), and approximately 10 ng of genomic DNA. PCR amplification was carried out under the following thermal cycling conditions: an initial denaturation at 95 °C for 5 min, followed by 35 cycles consisting of denaturation at 95 °C for 45 s, annealing at 62–57 °C for ITS, ACT, and CAL regions, and 57–52 °C for RPB1 (with a gradual decrease of 0.5 °C per cycle during the first 10 cycles), for 45 s, and extension at 72 °C for 45 s. A final extension step was performed at 72 °C for 7 min. PCR amplicons were visualized on a 1% agarose gel stained with FluoroVue™ Nucleic Acid Gel Stain (SMOBIO Technology Inc., Hsinchu, China), and fragment sizes were estimated using the FluoroBand™ 100 bp + 3K Fluorescent DNA Ladder (SMOBIO Technology Inc., China). Macrogen Corp. (Seoul, Republic of Korea) cleaned and sequenced the amplified products using the same primer sets employed for PCR amplification. The new sequences were submitted to GenBank (Table 1).

Table 1.

Information of taxa and corresponding GenBank accession numbers used for phylogenetic analyses. The sequences generated in this study are in bold, ^T indicates ex-type strains, and “-“ indicates that data were unavailable.

Species	Culture Collection Number	Host/Substrate	Location	GenBank Accession Numbers
				ITS	RPB1	ACT	CAL
Bambusicularia brunnea	CBS 133599 T	Sasa sp.	Japan	KM484830	KM485043	AB274449	AB274482
Bambusicularia brunnea	CBS 133600	Phyllostachys bambusoides	Japan	AB274436	KM485044	AB274450	AB274483
Barretomyces calatheae	CBS 129274	Calathea longifolia	Brazil	KM484831	KM485045	KM485162	KM485231
Bipyricularia graminis	YNE01013 T	Poaceae sp.	China	MW479090	MW482852	OQ918100	-
Bipyricularia graminis	YNE01016	Poaceae sp.	China	MW479091	MW482853	OQ918101	-
Macgarvieomyces borealis	CBS 461.65 T	Juncus effusus	Scotland	KM484854	KM485070	KM485170	KM485239
Macgarvieomyces caspica	IRAN 5071C T	Juncus acutus	Iran	PQ453843	PQ450660	PQ450624	PQ450642
Macgarvieomyces caspica	FCCUU 1956	Juncus acutus	Iran	PQ453844	PQ450661	PQ450625	PQ450643
Macgarvieomyces cyperi	IRAN 5070C T	Cyperus sp.	Iran	PQ453841	PQ450658	PQ450622	PQ450640
Macgarvieomyces cyperi	FCCUU 1955	Cyperus sp.	Iran	PQ453842	PQ450659	PQ450623	PQ450641
Macgarvieomyces junci-acuti	IRAN 4233C T	Juncus acutus	Iran	PQ453836	PQ450653	PQ450617	PQ450635
Macgarvieomyces junci-acuti	FCCUU 1951	Juncus sp.	Iran	PQ453837	PQ450654	PQ450618	PQ450636
Macgarvieomyces junci-acuti	FCCUU 1952	Juncus sp.	Iran	PQ453838	PQ450655	PQ450619	PQ450637
Macgarvieomyces junci-acuti	FCCUU 1953	Juncus sp.	Iran	PQ453839	PQ450656	PQ450620	PQ450638
Macgarvieomyces junci-acuti	FCCUU 1954	Schoenus sp.	Iran	PQ453840	PQ450657	PQ450621	PQ450639
Macgarvieomyces juncicola	CBS 610.82	Juncus effusus	Netherlands	KM484855	KM485071	KM485171	KM485240
Macgarvieomyces juncigenus	IRAN 5073C T	Juncus sp.	Iran	PQ453850	PQ450667	PQ450631	PQ450649
Macgarvieomyces juncigenus	FCCUU 1961	Juncus sp.	Iran	PQ453851	PQ450668	PQ450632	PQ450650
Macgarvieomyces luzulae	CBS 143401 T	Luzula sylvatica	Ukraine	MG934440	MG934469	MG934462	MG934519
Macgarvieomyces luzulae	CPC 31555	Luzula sylvatica	Ukraine	MG934441	MG934470	MG934463	MG934520
Macgarvieomyces salkadehensis	IRAN 5072C T	Juncus inflexus	Iran	PQ453845	PQ450662	PQ450626	PQ450644
Macgarvieomyces salkadehensis	FCCUU 1957	Juncus sp.	Iran	PQ453846	PQ450663	PQ450627	PQ450645
Macgarvieomyces salkadehensis	FCCUU 1958	Scirpoides sp.	Iran	PQ453847	PQ450664	PQ450628	PQ450646
Macgarvieomyces salkadehensis	FCCUU 1959	Juncus sp.	Iran	PQ453848	PQ450665	PQ450629	PQ450647
Macgarvieomyces salkadehensis	FCCUU 1960	Juncus sp.	Iran	PQ453849	PQ450666	PQ450630	PQ450648
Macgarvieomyces schoeni	IRAN 5074C T	Schoenus sp.	Iran	PQ453852	PQ450669	PQ450633	PQ450651
Macgarvieomyces schoeni	FCCUU 1962	Schoenus sp.	Iran	PQ453853	PQ450670	PQ450634	PQ450652
Magnaporthiopsis incrustans	M35	-	-	JF414843	Genome	Genome	Genome
Magnaporthiopsis poae	ATCC 64411	Triticum sp.	USA	Genome	Genome	AF395973	AF396032
Neocordana musarum	CBS 142116 T	Musa sp.	France	KY173425	KY173577	KY173568	-
Neocordana musigena	CBS 142624 T	Musa sp.	Morocco	KY979749	KY979886	KY979855	-
Neopyricularia commelinicola	CBS 128307	Commelina communis	South Korea	FJ850125	KM485086	KM485174	KM485243
Neopyricularia commelinicola	CBS 128308 T	Commelina communis	South Korea	FJ850122	KM485087	KM485175	-
Nothopyricularia junci	CBS 148308 T	Juncus effusus	Netherlands	OK664720	OK651152	OK651127	OK651142
Proxipyricularia zingiberis	CBS 132195	Zingiber mioga	Japan	KM484869	KM485088	AB274448	KM485244
Proxipyricularia zingiberis	CBS 303.39	Zingiber officinale	Japan	KM484871	KM485092	KM485177	KM485247
Pseudopyricularia bothriochloae	CBS 136427 T	Bothriochloa bladhii	Thailand	KF777186	KY905701	KY905700	-
Pseudopyricularia caricicola	CBS 149674 T	Carex disticha	Netherlands	OQ628482	-	OQ627932	-
Pseudopyricularia cyperi	CBS 133595 T	Cyperus iria	Japan	KM484872	AB818013	AB274453	AB274485
Pseudopyricularia cyperi	Cr88383	Cyperus rotundus	Philippines	KM484874	KM485094	KM485179	KM485249
Pseudopyricularia festucae	CBS 146629 T	Festuca californica	USA	MW883447	MW890057	-	MW890044
Pseudopyricularia hagahagae	CPC 25635 T	Unidentified Cyperaceae	South Africa	KT950851	KT950877	KT950873	-
Pseudopyricularia higginsii	CBS 121934	Typha orientalis	New Zealand	KM484875	KM485095	KM485180	KM485250
Pseudopyricularia kyllingae	CBS 133597 T	Kyllinga brevifolia	Japan	KM484876	KM485096	AB274451	AB274484
Pseudopyricularia kyllingae	PH0054 = Cb8959	Cyperus brevifolius	Philippines	KM484877	KM485097	KM485181	KM485251
Pyricularia grisea	CBS 138707 T	Digitaria sp.	USA	KM484885	KM485105	KM485187	KM485258
Pyricularia oryzae	CBS 255.38	-	Romania	KM484889	KM485109	KM485190	KM485261
Pyricularia oryzae	CBS 657.66	Oryza sativa	Egypt	KM484893	KM485113	KM485194	KM485265
Pyricularia penniseticola	BF0017	Pennisetum typhoides	Burkina Faso	KM484925	KM485144	DQ240878	DQ240894
Pyricularia urashimae	CBS 142117 T	Urochloa brizantha	Brazil	KY173437	KY173578	KY173571	KX524100
Pyricularia zingibericola	RN0001 T	Zingiber officinale	Réunion	KM484941	KM485157	KM485157	KM485297
Pyriculariomyces asari	CPC 27442	Asarum sp.	Malaysia	KX228290	MG934472	KX228360	-
Pyriculariomyces asari	CPC 27444 T	Asarum sp.	Malaysia	KX228291	KX228368	KX228361	MG934541
Utrechtiana arundinacea	CPC 33994 T	Phragmites sp.	Netherlands	MG934461	MG934473	MG934468	MG934542
Utrechtiana roumeguerei	CBS 128780 T	Phragmites australis	Netherlands	JF951153	KM485047	KM485163	KM485232
Xenopyricularia zizaniicola	CBS 133593 T	Zizania latifolia	Japan	KM484947	KM485161	KM485230	AB274479
Xenopyricularia zizaniicola	CBS 132356	Zizania latifolia	Japan	KM484946	KM485160	AB274444	AB274480

2.4. Molecular Phylogeny

Preliminary identification of the isolates was conducted by comparing newly generated ITS, RPB1, ACT, and CAL sequences using the NCBI Basic Local Alignment Search Tool (BLAST) (www.ncbi.nlm.nih.gov/blast/; accessed 30 April 2025). Subsequently, pairwise sequence comparisons were carried out between the putative novel species and their closest relatives using the same platform. Reference sequences from type or representative species were retrieved from GenBank (Table 1), based on recent studies by Klaubauf et al. [2], Feng et al. [5], and Marin-Felix et al. [9], and incorporated into the analyses. A multi-locus phylogenetic analysis was performed using a concatenated dataset consisting of four genetic loci (ITS, RPB1, ACT, and CAL). Sequence alignments were generated with the online platform MAFFT version 7 (https://mafft.cbrc.jp/alignment/server/; accessed 20 April 2025) [24]. The most appropriate nucleotide substitution models were selected based on the Akaike Information Criterion (AIC) implemented in MrModeltest version 2.3 [25]. Maximum likelihood (ML), Bayesian phylogenetic inference (BI), and maximum parsimony (MP) analyses were performed using the CIPRES Science Gateway portal version 3.3 (https://www.phylo.org/; accessed 20 April 2025) [26]. ML analysis was conducted with RAxML-HPC BlackBox v. 8.2.12 employing the GTR + GAMMA model and 1000 bootstrap replicates [27]. Bayesian inference was carried out using MrBayes on ACCESS v. 3.2.7a, applying the Markov Chain Monte Carlo (MCMC) method with four chains run for 1,000,000 generations, sampling every 1000 generations, and discarding the first 25% as burn-in [28]. Parsimony analysis was performed in PAUP on ACCESS v. 4.a168 using a heuristic search strategy and tree bisection and reconnection (TBR) branch swapping, with 1000 bootstrap replicates [29]. Descriptive statistics for the parsimony trees, including Tree Length (TL), Consistency Index (CI), Retention Index (RI), and Homoplasy Index (HI), were also calculated. In all phylogenetic analyses, Magnaporthiopsis incrustans (M35) and Ma. poae (ATCC 64411) were employed as outgroup taxa [5]. Phylogenetic trees were visualized using FigTree version 1.4.4 [30] and subsequently refined with Adobe Illustrator^® CC 2021 (Adobe Inc., San Jose, CA, USA) for graphical presentation.

2.5. Genealogical Concordance Phylogenetic Species Recognition Analysis

Genealogical Concordance Phylogenetic Species Recognition (GCPSR) was applied to detect notable recombination events among phylogenetically close species [31]. A concatenated dataset comprising four loci (ITS, RPB1, ACT, and CAL) was analysed using SplitsTree version 5 software, applying the pairwise Homoplasy Index (PHI or Φw) test [32,33]. The phylogenetic relationships between the newly identified species and their closely related taxa were visualized by generating split graphs from the concatenated dataset, employing the LogDet transformation and split decomposition methods. A PHI test value below 0.05 (Φw < 0.05) signifies the occurrence of significant recombination within the dataset.

3. Results

3.1. Phylogenetic Analyses

In this study, 95 isolates were obtained from various plants in the Cyperaceae and Juncaceae families. All isolates were examined based on their morphology, and 18 representative isolates were then selected according to different plant hosts for phylogenetic study. A total of 57 ITS, 56 RPB1, 56 ACT, and 48 CAL sequences were subjected to multiple sequence alignment (nucleotides + gaps), resulting in 517-, 735-, 513-, and 670-character datasets, respectively. The four-gene sequence combination for a total of 57 strains consisted of 2435 characters; of these, 1185 were constant, 129 were variable and parsimony-uninformative, and 1121 were parsimony-informative (Table 2). The most parsimonious tree yielded the following values: TL = 4826, CI = 0.474, RI = 0.760, and HI = 0.526. The MrModeltest findings suggested the GTR+I+G, GTR+I+G, HKY+I+G, and HKY+I+G models for ITS, RPB1, ACT, and CAL datasets, respectively (Table 2). Phylogenetic information and substitution models for each dataset is summarized in Table 2. The ML, MP, and BI phylogenetic analyses yielded congruent tree topologies with no significant conflicts observed. The combined dataset analysis conducted using RAxML produced the best-scoring tree (Figure 2), with a final ML optimization likelihood value of –23,503.286101. The estimated nucleotide base frequencies were A = 0.251726, C = 0.283076, G = 0.255946, and T = 0.209252. The substitution rates were calculated as follows: AC = 1.085584, AG = 2.836763, AT = 1.235728, CG = 0.790339, CT = 4.384725, and GT = 1.000000. The gamma distribution shape parameter (α) was estimated at 1.489603. All 18 studied isolates were clustered into the genus Macgarvieomyces (Figure 2). Based on morphological characteristics, multi-locus phylogenetic analyses (ITS, RPB1, ACT, and CAL), and the pairwise Homoplasy Index (PHI or Φw) test, six new Macgarvieomyces species viz. Macgarvieomyces caspica, M. cyperi, M. junci-acuti, M. juncigenus, M. salkadehensis, and M. schoeni were identified. Comprehensive morphological descriptions and illustrations were provided for all species, alongside detailed discussions of their habitat, distribution, and phylogenetic relationships with other Macgarvieomyces taxa.

Table 2.

Phylogenetic details of individual and concatenated sequence datasets utilized in phylogenetic analyses.

Parameter	Gene
	ITS	RPB1	ACT	CAL	Combined
Number of taxa	57	56	56	48	57
Total characters	517	735	513	670	2435
Constant sites	327	382	224	252	1185
Variable sites	190	353	289	418	1250
Parsimony informative sites	162	329	258	372	1121
Parsimony uninformative sites	28	24	31	46	129
AIC substitution model *	GTR+I+G	GTR+I+G	HKY+I+G	HKY+I+G	GTR+I+G
Lset nst, Rates	6, invgamma	6, invgamma	2, invgamma	2, invgamma	6, invgamma
−lnL	4564.091206	6165.249261	5332.845681	7571.390091	23503.286101

* Substitution models selected based on the Akaike Information Criterion (AIC) and applied in Bayesian inference analyses.

Figure 2.

Phylogenetic tree constructed using maximum likelihood (ML) analysis based on the combined ITS, RPB1, ACT, and CAL sequence data from Macgarvieomyces species and related genera within the Pyriculariaceae family. Support values at each node include bootstrap percentages from both maximum likelihood and maximum parsimony (MLBS/MPBS) analyses equal to or greater than 70%, as well as Bayesian posterior probabilities (BIPP) of 0.90 or higher. The newly identified strains are highlighted in bold blue, with the phylogenetic tree rooted using Magnaporthiopsis incrustans (M35) and Ma. poae (ATCC 64411). The number of substitutions for nucleotides is displayed on the scale bar and ex-type strains are denoted by ^T.

3.2. Taxonomy

3.2.1. Macgarvieomyces caspica A. Ahmadpour, Y. Ghosta, F. Alavi, Z. Alavi, and E. Hashemlou sp. nov. (Figure 3)

MycoBank No. 859375

Etymology: The name refers to the Caspian Sea, where the holotype was collected.

Typification: Iran, Golestan Province, Torkaman County, Qareh Su Village, isolated from the culms of Juncus sp. (Juncaceae, Poales), 7 August 2021, A. Ahmadpour (holotype IRAN 18495F, ex-type culture IRAN 5071C).

Asexual morph on SNA medium with sterile barley seed: Mycelium consisting of smooth, hyaline, branched, septate hyphae, 1–2 μm diam. Conidiophores semi–macronematous, solitary, erect, straight–flexuous, unbranched, thick-walled, pale–medium brown, 1–2-septate, lacking a swollen base, 45–125 × 4–5 µm ($\overline{x}$ = 77 × 4.5 μm, n = 50). Conidiogenous cells integrated, terminal, rarely intercalary, pale brown, smooth-walled, forming a rachis with several sympodially protruding flat-tipped denticles, 1–2 × 1–1.5 μm. Conidia solitary, obclavate–pyriform–fusoid, hyaline at first and becoming pale brown with age, smooth, granular, guttulate, 1-septate, not or slightly constricted at septum, apex obtusely rounded, base tapering towards a protruding hilum, 1–1.5 μm diam., not thickened, not darkened, 22–27 × 6–7 µm ($\overline{x}$ = 24.5 × 6.5 μm, n = 50). Hyphopodia, sexual morph, microconidiation, and chlamydospores were not observed.

Culture characteristics: Colony on PDA reaching up to 35 and 55 mm diam. after 7 and 14 days at 25 °C in the dark, respectively; flat, circular, margin regular, white with buff centre, reverse white with buff centre. Colony on PCA reaching up to 35 and 53 mm diam. after 7 and 14 days at 25 °C, respectively; flat, circular, margin regular, white with buff centre and white aerial mycelium, reverse white with buff centre. Colony on MEA reaching up to 40 and 54 mm diam. after 7 and 14 days at 25 °C, respectively; flat, circular, margin entire, transparent, velvety, isabelline, or pale luteous with white aerial mycelium at the centre, reverse ochreous–pale luteous towards the edge.

Additional specimen examined. Iran, Golestan Province, Torkaman County, Qareh Su Village, isolated from the culms of Juncus sp. (Juncaceae, Poales), 7 August 2021, A. Ahmadpour (culture FCCUU 1956).

Notes: Phylogenetic analyses indicate that Macgarvieomyces caspica is closely affiliated with M. junci-acuti and M. schoeni, supported by high confidence values (MLBS/MPBS/BIPP = 100/100/1.0) (Figure 2). A comparison of nucleotide differences in ITS, RPB1, ACT, and CAL indicates that M. caspica (IRAN 5071C) differs from M. junci-acuti (IRAN 4233C) by 2/434 bp (0.69%) in ITS, 10/693 bp (1.44%) in RPB1, 15/300 bp (5%, with five gaps (1%)) in ACT, and 23/460 bp (5%) in CAL and from M. schoeni (IRAN 5074C) by 3/479 bp (0.62%) in ITS, 3/688 bp (0.43%) in RPB1, 5/303 bp (1.65%, with one gap (0%)) in ACT, and 3/473 bp (0.63%, with two gaps (0%)) in CAL. The PHI analysis results indicated that M. caspica does not exhibit significant genetic recombination with its closely related species (Φw > 0.05, Figure 4). Macgarvieomyces caspica can be differentiated from M. junci-acuti by producing shorter conidiophores (45–125 µm vs. 80–150 µm in M. junci-acuti), shorter and slightly wider conidia (22–27 × 6–7 µm vs. 25–33 × 5–6 μm in M. junci-acuti), and conidial shape (obclavate to pyriform vs. narrowly obclavate in M. junci-acuti), and from M. schoeni by shorter and slightly narrower conidia (22–27 × 6–7 µm vs. 26–32 × 4–5 μm in M. schoeni), and absence of hyphopodia.

Figure 3.

Macgarvieomyces caspica (IRAN 5071C, ex-type). (a,b) Symptoms on the culms of Juncus sp.; (c–e) colony on PDA (c), PCA (d), and MEA (e) after 14 days; (f–i) sporulation pattern on SNA medium ((f) = 10×, (g) = 20×, (h,i) = 40×); (j–o) conidiophores and conidia. Scale bars: (j–o) = 10 μm.

Figure 4.

Split graph illustrating the pairwise Homoplasy Index (PHI) test results for the newly identified species and their closely related taxa, generated using LogDet transformation and split decomposition methods. A PHI test p-value (Φw) ≤ 0.05 signifies significant recombination among the aligned isolates. Newly described taxa are highlighted in bold blue, with the PHI test value and scale bars presented in the lower right corner.

3.2.2. Macgarvieomyces cyperi A. Ahmadpour, Y. Ghosta, F. Alavi, Z. Alavi, and E. Hashemlou, sp. nov. (Figure 5)

MycoBank No. 859376

Etymology: Named after the host genus, Cyperus, from which the holotype was collected.

Typification: Iran, Guilan Province, Fuman County, Masuleh City, isolated from the leaves and culms of Cyperus sp. (Cyperaceae, Poales), 6 August 2022, A. Ahmadpour (holotype IRAN 18494F, ex-type culture IRAN 5070C).

Description: Asexual morph on SNA medium with sterile barley seed: Mycelium consisting of smooth, hyaline, branched, septate hyphae, 1.2–2 μm diam. Conidiophores, semi–macronematous, solitary, erect, straight–flexuous, smooth, unbranched, medium brown–dark brown, 1–3-septate, tapering and paler towards the apex, mostly simple, occasionally swollen at the base, 50–125 × 4–5 µm ($\overline{x}$ = 85 × 4.5 μm, n = 50). Conidiogenous cells integrated, terminal, rarely intercalary, pale brown to brown, smooth, forming a rachis with several sympodially protruding denticles, 1–1.5 × 1–1.2 μm. Conidia solitary, narrowly obclavate, hyaline, becoming pale brown with age, smooth, granular, guttulate, 1-septate, not or slightly constricted at septum, apex obtusely rounded, base tapering to a protruding hilum, 1–1.2 μm diam., not thickened, not darkened, 19–25 × 4–6 µm ($\overline{x}$ = 22 × 5 μm, n = 50). Hyphopodia, sexual morph, microconidiation, and chlamydospores were not observed.

Culture characteristics: Colony on PDA reaching up to 34 and 50 mm diam. after 7 and 14 days at 25 °C in the dark, respectively; flat, circular, margin regular, white with buff centre and white aerial mycelium, reverse white with buff centre. Colony on PCA reaching up to 35 and 52 mm diam. after 7 and 14 days at 25 °C, respectively; flat, circular, margin regular, velvety or wool-like texture, white with pale olivaceous grey mycelium at the centre, reverse white with pale olivaceous grey centre. Colony on MEA reaching up to 35 and 52 mm diam. after 7 and 14 days at 25 °C, respectively; flat, circular, margin entire, velvety, isabelline, or pale luteous with white aerial mycelium at the centre, reverse ochreous–pale luteous towards the edge.

Additional specimen examined. Iran, Guilan Province, Asalem County, isolated from the leaves and culms of Cyperus sp. (Cyperaceae, Poales), 6 June 2022, A. Ahmadpour (culture FCCUU 1955).

Notes: Macgarvieomyces cyperi is phylogenetically closely allied with M. juncicola, supported by strong bootstrap and posterior probability values (MLBS/MPBS/BIPP = 100/100/1.0) (Figure 2). A comparison of nucleotide differences in ITS, RPB1, ACT, and CAL indicates that M. cyperi (IRAN 5070C) differs from M. juncicola (CBS 610.82) by 12/453 bp (2.64%, with three gaps (0%)) in ITS, 37/689 bp (5.37%, with one gap (0%)) in RPB1, 43/292 bp (14.72%, with 14 gaps (4%)) in ACT, and 52/455 bp (11.42%, with four gaps (0%)) in CAL. The PHI analysis results confirmed that M. cyperi does not exhibit significant genetic recombination with its closely related species (Φw > 0.05, Figure 4). Morphologically, M. cyperi can be differentiated from M. juncicola by shorter conidiophores (up to 125 vs. up to 200 μm in M. juncicola), shorter conidia (19–25 vs. 17–32 μm) in M. juncicola, and the formation of chlamydospores in intercalary chains in M. juncicola [2].

Figure 5.

Macgarvieomyces cyperi (IRAN 5070C, ex-type). (a) Symptoms on the leaves of Cyperus sp.; (b–d) colony on PDA (b), PCA (c), and MEA (d) after 14 days; (e,f) sporulation pattern on SNA medium (20×); (g–n) conidiophores and conidia. Scale bars: (g–n) = 10 μm.

3.2.3. Macgarvieomyces junci-acuti A. Ahmadpour, Y. Ghosta, F. Alavi, Z. Alavi, and E. Hashemlou, sp. nov. (Figure 6)

MycoBank No. 859377

Etymology: Named after the host genus, Juncus acutus, from which the holotype was collected.

Typification: Iran, West Azarbaijan Province, Urmia County, Baran Duz Village, isolated from the culms of Juncus acutus (Juncaceae, Poales), 31 August 2020, A. Ahmadpour (holotype IRAN 18105F, ex-type culture IRAN 4233C).

Description: Asexual morph on SNA medium with sterile barley seed: Mycelium consisting of smooth, hyaline, branched, septate hyphae, 1–2 μm diam. Conidiophores semi–macronematous, solitary, erect, straight–flexuous, smooth, mostly unbranched, occasionally branched, hyaline–pale brown, 1–4-septate, thick-walled near the base, 80–150 × 4–5 µm ($\overline{x}$ = 103 × 4.5 μm, n = 50). Conidiogenous cells integrated, terminal, rarely intercalary, hyaline–pale brown, smooth, forming a rachis with several sympodially protruding flat-tipped denticles, 1–2 × 1–1.5 μm diam. Conidia solitary, obclavate–narrowly obclavate, hyaline, becoming pale brown with age, smooth, granular, guttulate, 1-septate, not or slightly constricted at septum, apex obtusely rounded, base tapering to a protruding hilum, 1–1.5 μm diam., not thickened, not darkened, 25–33 × 5–6 µm ($\overline{x}$ = 28.5 × 5.5 μm, n = 50). Hyphopodia commonly formed, elongated, dome-shaped–lobulate, brown–dark brown, smooth, 10–15 × 5–6 µm. Sexual morph, microconidiation, and chlamydospores were not observed.

Culture characteristics: Colony on PDA reaching up to 25 and 49 mm diam. after 7 and 14 days at 25 °C in the dark, respectively; flat, circular, margin entire, transparent, velvety, isabelline, or pale luteous, reverse ochreous–pale luteous towards the edge. Colony on PCA reaching up to 22 and 45 mm diam. after 7 and 14 days at 25 °C, respectively; flat, circular, margin regular, white–grey with white aerial mycelium, reverse grey at the centre and hyaline at the margin. Colony on MEA reaching up to 20 and 35 mm diam. after 7 and 14 days at 25 °C, respectively; flat, circular, margin entire, cottony appearance, white with white aerial mycelium, reverse white–hyaline towards the edge.

Additional specimens examined: Iran, Golestan Province, Torkaman County, Qareh Su Village, isolated from the culms of Juncus sp. (Juncaceae, Poales), 3 November 2021, A. Ahmadpour (culture FCCUU 1951); Iran, Mazandaran Province, Sari County, Qajar Kheyl Village, isolated from the culms of Juncus sp. (Juncaceae, Poales), 2 November 2021, A. Ahmadpour (culture FCCUU 1952); Iran, Mazandaran Province, Sari County, Farahabad City, isolated from the culms of Juncus sp. (Juncaceae, Poales), 2 November 2021, A. Ahmadpour (culture FCCUU 1953); Iran, West Azarbaijan Province, Khoy County, Salkadeh Village, isolated from the culms of Schoenus sp. (Cyperaceae, Poales), 5 October 2020, A. Ahmadpour (culture FCCUU 1954).

Notes: Isolates of Macgarvieomyces junci-acuti formed a distinct clade with strong support values, including 100% maximum likelihood (ML) bootstrap, 100% maximum parsimony (MP) bootstrap, and a Bayesian posterior probability (BI) of 1.0, and were resolved as sister taxa to M. schoeni and M. caspica (Figure 2). A comparison of nucleotide differences in ITS, RPB1, ACT, and CAL indicates that M. junci-acuti (IRAN 4233C) differs from M. caspica (IRAN 5071C) by 2/434 bp (0.69%) in ITS, 10/693 bp (1.44%) in RPB1, 15/300 bp (5%, with five gaps (1%)) in ACT, and 23/460 bp (5%) in CAL and from M. schoeni (IRAN 5074C) by 7/682 bp (1.02%) in RPB1, 14/294 bp (4.76%, with six gaps (2%)) in ACT, and 26/460 bp (5.65%, with two gaps (0%)) in CAL. The PHI analysis further confirmed the absence of significant genetic recombination between M. junci-acuti and its closely related taxa (Φw > 0.05, Figure 4). Macgarvieomyces junci-acuti can be differentiated from M. capsica by having longer conidiophores (80–150 vs. 45–125 µm in M. capsica), longer conidia (25–33 vs. 22–27 µm in M. capsica), and the presence of hyphopodia, and from M. schoeni by having longer conidiophores (80–150 vs. 37–50 µm in M. schoeni).

Figure 6.

Macgarvieomyces junci-acuti (IRAN 4233C, ex-type). (a) Host (Juncus sp.); (b–d) colony on PDA (b), PCA (c), and MEA (d) after 14 days; (e,f) hyphopodia formed on SNA medium; (g–i) sporulation pattern on SNA medium ((g,h) = 10×, (i) = 40×); (j–p) conidiophores and conidia. Scale bars: (e,f,j–p) = 10 μm.

3.2.4. Macgarvieomyces juncigenus A. Ahmadpour, Y. Ghosta, F. Alavi, Z. Alavi, and E. Hashemlou, sp. nov. (Figure 7)

MycoBank No. 859378

Etymology: Named after the host, Juncus sp., from which the holotype was collected.

Typification: Iran, Ardebil Province, Ardebil County, Fandoqlu Forest, isolated from the culms of Juncus sp. (Juncaceae, Poales), 10 June 2021, A. Ahmadpour (holotype IRAN 18497F, ex-type culture IRAN 5073C).

Description: Asexual morph on SNA medium with sterile barley seed: Mycelium consisting of smooth, hyaline, branched, septate hyphae, 1–2 μm diam. Conidiophores semi–macronematous, solitary, erect, straight–flexuous, smooth, mostly unbranched, occasionally branched, hyaline–pale brown, 1–3-septate, thick-walled near the base, occasionally swollen at the base, 35–75 × 5–7 µm ($\overline{x}$ = 49 × 6 μm, n = 50). Conidiogenous cells integrated, terminal, rarely intercalary, hyaline–pale brown, smooth, forming a rachis with several sympodially protruding flat-tipped denticles, 1–1.5 × 1–1.2 μm diam. Conidia solitary, narrowly obclavate–narrowly pyriform, hyaline, becoming pale brown with age, smooth, granular, guttulate, 1-septate, not or slightly constricted at septum, apex obtusely rounded, base tapering to a protruding hilum, 1–1.5 μm diam., not thickened, not darkened, 19–26 × 4–6 µm ($\overline{x}$ = 24.5 × 5.3 μm, n = 50). Chlamydospores 8–10 μm diam., formed in intercalary chains, spherical–ellipsoid, hyaline–pale brown, smooth, frequently giving rise to conidiophores. Hyphopodia, sexual morph, and microconidiation were not observed.

Culture characteristics: Colony on PDA reaching up to 30 and 49 mm diam. after 7 and 14 days at 25 °C in the dark, respectively; flat, circular, margin regular, velvety, white with buff centre and white aerial mycelium, reverse white with buff centre. Colony on PCA reaching up to 35 and 53 mm diam. after 7 and 14 days at 25 °C, respectively; flat, circular, margin regular, white–grey with white aerial mycelium, reverse grey at the centre and hyaline at the margin. Colony on MEA reaching up to 32 and 52 mm diam. after 7 and 14 days at 25 °C, respectively; flat, circular, margin entire, velvety, isabelline, or pale luteous with sparse white aerial mycelium, reverse ochreous–pale luteous towards the edge.

Additional specimen examined. Iran, Ardebil Province, Ardebil County, Fandoqlu Forest, isolated from the culms of Juncus sp. (Juncaceae, Poales), 10 June 2021, A. Ahmadpour (culture FCCUU 1961).

Notes: Phylogenetic analysis indicates a close relationship between Macgarvieomyces juncigenus and M. salkadehensis. A comparison of nucleotide differences in ITS, RPB1, ACT, and CAL indicates that M. juncigenus (IRAN 5073C) differs from M. salkadehensis (IRAN 5072C) by 1/481 bp (0.20%) in ITS, 2/653 bp (0.30%) in RPB1, 4/270 bp (1.48%) in ACT, and 4/462 bp (0.86%) in CAL. The PHI analysis results additionally verified that M. juncigenus does not exhibit significant genetic recombination with its closely related species (Φw > 0.05, Figure 4). Morphologically, Macgarvieomyces juncigenus differs from M. salkadehensis by having slightly longer and wider conidia (19–26 × 4–6 µm ($\overline{x}$ = 24.5 × 5.3 μm) vs. 15–28 × 4–5 µm ($\overline{x}$ = 22.5 × 4.8 μm) in M. salkadehensis); however, the overlapping morphological features and shared host between these two species complicate their distinction based solely on morphology. Consequently, molecular phylogenetic analyses are essential for accurately distinguishing Macgarvieomyces species and identifying any cryptic species.

Figure 7.

Macgarvieomyces juncigenus (IRAN 5073C, ex-type). (a) Host (Juncus sp.); (b–d) colony on PDA (b), PCA (c), and MEA (d) after 14 days; (e) sporulation pattern on SNA medium (10×); (f,g) chlamydospores formed on SNA medium; (h–p) conidiophores and conidia. Scale bars: (f–p) = 10 μm.

3.2.5. Macgarvieomyces salkadehensis A. Ahmadpour, Y. Ghosta, F. Alavi, Z. Alavi, and E. Hashemlou, sp. nov. (Figure 8)

MycoBank No. 859379

Etymology: Named after the location, Salkadeh Village, Khoy County, from where the holotype was collected.

Typification: Iran, West Azarbaijan Province, Khoy County, Salkadeh Village, isolated from the culms of Juncus sp. (Juncaceae, Poales), 10 August 2021, A. Ahmadpour (holotype IRAN 18496F, ex-type culture IRAN 5072C).

Description: Asexual morph on SNA medium with sterile barley seed: Mycelium consisting of smooth, hyaline, branched, septate hyphae, 1–2 μm diam. Conidiophores semi–macronematous, solitary, erect, straight–flexuous, smooth, mostly unbranched, occasionally branched, hyaline–pale brown, 1–3-septate, thick-walled near the base, occasionally swollen at the base, 30–75 × 4–5 µm ($\overline{x}$ = 50 × 4.5 μm, n = 50). Conidiogenous cells integrated, terminal, rarely intercalary, hyaline–pale brown, smooth, forming a rachis with several sympodially protruding flat-tipped denticles, 1–2 × 1–1.5 μm. Conidia solitary, obclavate–narrowly obclavate, hyaline, becoming pale brown with age, smooth, granular, guttulate, 1-septate, not or slightly constricted at septum, apex obtusely rounded, base tapering to a protruding hilum, 1–1.5 μm diam., not thickened, not darkened, 15–28 × 4–5 µm ($\overline{x}$ = 22.5 × 4.8 μm, n = 50). Chlamydospores 6–10 μm diam, formed in intercalary chains, spherical–ellipsoid, hyaline–pale brown, smooth, frequently giving rise to conidiophores. Hyphopodia, sexual morph, and microconidiation were not observed.

Culture characteristics: Colony on PDA reaching up to 28 and 47 mm diam. after 7 and 14 days at 25 °C in the dark, respectively; flat, circular, margin entire, velvety, isabelline, or pale luteous with sparse white aerial mycelium, reverse ochreous–pale luteous towards the edge. Colony on PCA reaching up to 30 and 52 mm diam. after 7 and 14 days at 25 °C, respectively; flat, circular, margin regular, velvety, isabelline, or pale luteous with sparse white aerial mycelium, reverse ochreous–pale luteous towards the edge. Colony on MEA reaching up to 30 and 52 mm diam. after 7 and 14 days at 25 °C, respectively; flat, circular, margin entire, pale luteous without aerial mycelium, reverse pale luteous–hyaline towards the edge.

Additional specimens examined: Iran, Golestan Province, Gorgan County, Nahar Khoran Forest, Ziarat Village, isolated from the culms of Juncus sp. (Juncaceae, Poales), 4 November 2021, A. Ahmadpour (culture FCCUU 1957).—Iran, Ardebil Province, Ardebil County, Meshgin Shahr County, Razey City, isolated from the leaves and culms of Scirpoides sp. (Cyperaceae, Poales), 2 June 2022, A. Ahmadpour (culture FCCUU 1958).—ibid. on the culms of Juncus sp. (Juncaceae, Poales), 2 June 2022, A. Ahmadpour (culture FCCUU 1959).—Iran, Tehran Province, Damavand County, Haraz Road, isolated from the culms of Juncus sp. (Juncaceae, Poales), 20 June 2022, E. Hashemlou (culture FCCUU 1960).

Notes: Phylogenetic analyses (Figure 2) revealed that the five examined isolates of Macgarvieomyces salkadehensis formed a distinct clade, supported by 100% maximum likelihood (ML) bootstrap, 98% maximum parsimony (MP) bootstrap, and a Bayesian posterior probability (BI) of 0.93, and were closely related as sister taxa to M. juncigenus. A comparison of morphological characteristics, nucleotide sequence variations, and PHI analysis results (Φw > 0.05, Figure 4) for these species is provided in the notes section for M. juncigenus.

Figure 8.

Macgarvieomyces salkadehensis (IRAN 5072C, ex-type). (a) Host (Juncus sp.); (b–d) colony on PDA (b), PCA (c), and MEA (d) after 14 days; (e–g) sporulation pattern on SNA medium ((e,g) = 10×, (f) = 20×); (h) chlamydospores formed on SNA medium; (i–s) conidiophores and conidia. Scale bars: (h–s) = 10 μm.

3.2.6. Macgarvieomyces schoeni A. Ahmadpour, Y. Ghosta, F. Alavi, Z. Alavi, and E. Hashemlou, sp. nov. (Figure 9)

MycoBank No. 859380

Etymology: Named after the host, Schoenus, from which the holotype was collected.

Typification: Iran, West Azarbaijan Province, Khoy County, Salkadeh Village, isolated from the culms of Schoenus sp. (Cyperaceae, Poales), 11 September 2021, A. Ahmadpour (holotype IRAN 18498F, ex-type culture IRAN 5074C).

Description: Asexual morph on SNA medium with sterile barley seed: Mycelium consisting of smooth, hyaline, branched, septate hyphae, 1–2 μm diam. Conidiophores semi–macronematous, solitary, erect, straight–flexuous, smooth, unbranched, hyaline–pale brown, 1–3-septate, thick-walled near the base, 37–50 × 4–5 µm ($\overline{x}$ = 45 × 4.5 μm, n = 50). Conidiogenous cells integrated, terminal, rarely intercalary, hyaline–pale brown, smooth, forming a rachis with several sympodially protruding flat-tipped denticles, 1–1.5 × 1–1.2 μm. Conidia solitary, obclavate–narrowly obclavate, hyaline, becoming pale brown with age, smooth, granular, guttulate, 1-septate, not or slightly constricted at septum, apex obtusely rounded, base tapering to a protruding hilum, 1–1.5 μm diam., not thickened, not darkened, 26–32 × 4–5 µm ($\overline{x}$ = 29.5 × 4.3 μm, n = 50). Hyphopodia commonly formed, elongated, dome-shaped to multilobulate, brown–dark brown, smooth, 10–13 × 5–6 µm. Sexual morph, microconidiation, and chlamydospores were not observed.

Culture characteristics: Colony on PDA reaching up to 32 and 50 mm diam. after 7 and 14 days at 25 °C in the dark, respectively; flat, circular, margin entire, cottony appearance, white with white–grey aerial mycelium, reverse white–olivaceous grey towards the edge. Colony on PCA reaching up to 35 and 53 mm diam. after 7 and 14 days at 25 °C, respectively; flat, circular, margin regular, pale olivaceous grey with white aerial mycelium, reverse pale olivaceous grey at the centre and grey at the margin. Colony on MEA reaching up to 31 and 52 mm diam. after 7 and 14 days at 25 °C, respectively; flat, circular, margin entire, velvety, pale luteous with sparse white aerial mycelium; reverse ochreous–pale luteous towards the edge.

Additional specimen examined. Iran, West Azarbaijan Province, Khoy County, Salkadeh Village, isolated from the culms of Schoenus sp. (Cyperaceae, Poales), 11 September 2021, A. Ahmadpour (culture FCCUU 1962).

Notes: Phylogenetic analysis indicates that Macgarvieomyces schoeni is closely related to M. caspica and M. junci-acuti (Figure 2). Detailed comparisons of their morphological features, nucleotide variations, and PHI analysis results (Φw > 0.05, Figure 4) are provided in the notes sections for M. caspica and M. junci-acuti.

Figure 9.

Macgarvieomyces schoeni (IRAN 5074C, ex-type). (a,b) Symptoms on the culms of Schoenus sp.; (c–e) colony on PDA (c), PCA (d), and MEA (e) after 14 days; (f,g) hyphopodia formed on SNA medium; (h–j) sporulation pattern on SNA medium ((h) = 10×, (i,j) = 20×); (k–n) conidiophores and conidia. Scale bars: (f,g,k–o) = 10 μm.

4. Discussion

This study assessed the species diversity of the genus Macgarvieomyces in Iran, focusing on host plants from the Cyperaceae and Juncaceae families. Our results reveal greater diversity within this genus than previously documented, leading to the identification and formal description of six new species. Until now, only three species had been described in this genus globally, all associated with hosts from these two plant families [2,5,9,15]. The newly discovered species in this study were also isolated exclusively from Cyperaceae and Juncaceae, suggesting a potential host-specific relationship. Interestingly, none of the previously described species, originally reported from Europe and New Zealand, were found in our samples. Among the newly described taxa, Macgarvieomyces junci-acuti and M. salkadehensis were isolated from multiple locations and host species within the same plant families, indicating a broader ecological distribution. In contrast, M. caspica, M. cyperi, M. juncigenus, and M. schoeni, were each isolated from a single host species and location. Despite these limitations in collection sites, the variety of habitats from which these fungi were obtained points to a wider ecological amplitude than previously recognized [2,9,15]. Further studies will be necessary to explore host specificity and geographical range within this genus.

Morphological features in Macgarvieomyces, as in other Pyricularia-like fungi, are often subtle and overlapping, making reliable identification based on morphology difficult, even at the generic level. Taxonomic features such as conidiophore structure, conidial shape and septation, pigmentation, and the presence of microconidia, hyphopodia, and chlamydospores show considerable variability and are often inconspicuous [2,5,9,34]. These challenges underscore the limitations of morphology-based classification and emphasize the necessity of molecular tools in fungal taxonomy. Recent studies using DNA-based phylogenetic analyses, particularly multi-locus sequence data, have greatly improved fungal taxonomy, resulting in the description of new families, genera, and species and have significantly helped in the understanding of evolutionary relationships within Macgarvieomyces and related genera [1,2,5,7,8,9,10]. The sequences of four genes, ITS, RPB1, ACT, and CAL, were used as DNA barcodes to differentiate Macgarvieomyces spp. Our study employed sequences from four genetic markers, ITS, RPB1, ACT, and CAL, as DNA barcodes to resolve species boundaries and infer phylogenetic relationships within Macgarvieomyces. Notably, M. salkadehensis and M. juncigenus shared overlapping morphological traits that made them difficult to distinguish based on morphology alone; however, phylogenetic analyses successfully differentiated them.

As part of our broader investigation into the fungal diversity of Iranian wetlands, we have recovered numerous fungal taxa from hosts in the Cyperaceae and Juncaceae families [17,18,35,36,37]. These taxa include fungi with varied ecological roles, ranging from pathogens and saprophytes to endophytes. The discovery of new Macgarvieomyces species in this study reinforces the ecological richness and taxonomic novelty of fungi associated with Iran’s wetland habitats. It also highlights the value of under-studied ecosystems and plant-fungal associations for uncovering previously unknown fungal taxa. Given Iran’s unique geographic position and environmental diversity, it will likely harbour additional, undescribed fungal species, especially among ecologically specialized genera like Macgarvieomyces. Continued exploration of these habitats, guided by integrative taxonomic approaches that combine morphological and molecular data, is essential for revealing hidden fungal diversity and informing future conservation and ecological efforts.

5. Conclusions

This study makes a substantial contribution to the understanding of Macgarvieomyces species diversity in Iran by identifying and describing six novel species, collected from wetland habitats and host plants belonging to the Cyperaceae and Juncaceae families. By integrating morphological features with multi-locus phylogenetic analyses of DNA barcodes, we present robust evidence supporting the delineation of distinct species. Our results not only broaden the taxonomic scope of the genus Macgarvieomyces but also highlight the ecological significance of Iranian wetlands as vital reservoirs of both known and previously undocumented fungal diversity. Continued research and conservation of these ecosystems are crucial for protecting fungal biodiversity and discovering species with potential applications in agriculture, biotechnology, and environmental monitoring. This study provides a foundation for future taxonomic, ecological, and functional research on fungi in these largely unexplored regions.

Author Contributions

A.A., E.H., and Y.G. designed and performed sampling, fungal isolation, experiments, writing, and editing; A.A., Z.A., and F.A. took the photographs; A.A. and E.H. carried out phylogenetic analyses; S.C.K., J.K., and N.S. reviewed the manuscript; J.K. and N.S. provided the funding. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest.

Funding Statement

This work was financially supported by the Research Deputy of Urmia University and Chiang Mai University, Thailand SCK thanks the National Natural Science Foundation of China (Number 32260004), the High-Level Talent Recruitment Plan of Yunnan Province (High-End Foreign Experts program), and the Key Laboratory of Yunnan Provincial Department of Education of the Deep-Time Evolution on Biodiversity from the Origin of the Pearl River for their support.