Description of Allocanariomyces and Parachaetomium, two new genera, and Achaetomium aegilopis sp. nov. in the Chaetomiaceae

We describe Allocanariomyces tritici gen et sp. nov. and Achaetomium aegilopis sp. nov. as seed endophytes of Triticum boeoticum and Aegilops triuncialis, respectively, in the west and northwestern Iran using morphological traits and sequences of the internal transcribed spacer regions 1 and 2 including the intervening 5.8S nuclear ribosomal DNA (ITS), partial nuclear 28S ribosomal DNA (LSU), β-tubulin (TUB2), and the second-largest subunit of DNA-directed RNA polymerase II (RPB2) gene. Chaetomium carinthiacum, C. iranianum, and C. truncatulum are also combined here under the new genus, Parachaetomium. Allocanariomyces is differentiated from Canariomyces, its closest relative, by having solitary and glabrous ascomata, cells of the perithecial wall forming a textura epidermoidea, stalked asci, densely granular ascospores with a distinct subapical germ pore, and producing only solitary conidia. Parachaetomium is characterized by distinctly ostiolate ascomata and equi- or inequilaterally fusiform, typically less than 13-μm-long ascospores with an oblique or subapical germ pore. Achaetomium aegilopis is mainly distinguished from A. strumarium, its closest relative, by possessing brown, ascomata, hyaline ascomatal hairs covered with hyaline crystals, hyaline chlamydospores, and lacking an anamorph.


Introduction
The family Chaetomiaceae was introduced by Winter (1885), as Chaetomiea, with Chaetomium Kunze as the type genus. Members of this family occur worldwide and live as saprobes on various substrates including dung, seeds, paper, plant debris, soil, air, and wood (Wang et al. 2016a, b). Endophytic, parasitic (Violi et al. 2007), and mycoparasitic (Marin-Felix et al. 2015) representatives have also been reported. Furthermore, some species have been found as human opportunistic pathogens (Abbott et al. 1995;de Hoog et al. 2013;Ahmed et al. 2016).

Section Editor: Hans-Josef Schroers
Electronic supplementary material The online version of this article (https://doi.org/10.1007/s11557-020-01636-x) contains supplementary material, which is available to authorized users.
Morphological characteristics of perithecia, hair/setae, asci, ascospores, anamorphs, and cultures and some physiological traits have been used to delimit genera and species in the Chaetomiaceae Asgari and Zare 2011;Wang et al. 2016aWang et al. , 2019a. Because of the plasticity of these characteristics, solid taxonomic concepts were established only with the help of phylogenetic analyses. Wang et al. (2016b) reevaluated generic and species concepts within Chaetomium globosum Kunze species complex based on phylogenetic inference from six loci and morphological characters. They resurrected six species that had been treated as synonyms of C. globosum by von Arx et al. (1986). Based on the phylogenetic analyses together with morphological studies, Wijayawardene et al. (2017) recognized 24 genera within the Chaetomiaceae. Wang et al. (2016aWang et al. ( , 2019a additionally expanded the Chaetomiaceae and proposed several new genera. They also restricted the genus Thielavia to its type species, T. basicola, and transferred it to the Ceratostomataceae (Melanosporales). More recently, one additional genus was added to the family, namely, Batnamyces Noumeur . Greif et al. (2009) investigated the taxonomy of the genus Chaetomidium using partial nuclear 28S ribosomal DNA (LSU), β-tubulin (TUB2) gene, and the second-largest subunit of the DNA-directed RNA polymerase II (RPB2) gene sequence data. The results of their analyses showed that Chaetomidium is polyphyletic. Furthermore, the genus Chaetomidium was rejected (Wang et al. 2016b).
In an investigation on fungal endophytes of wheat and its relatives (Poaceae) in the west and northwestern provinces of Iran, 2018-2019, three strains belonging to the Chaetomiaceae were isolated. Based on morphological characteristics and multilocus phylogeny, a new genus, Allocanariomyces, is established and a new species of Achaetomium is described. Two species of Chaetomium previously described by Asgari and Zare (2011), C. iranianum from leaves of Hordeum vulgare L. and C. truncatulum from cysts of Heterodera schachtii Schmidt, and C. carinthiacum (Sörgel 1961;von Arx et al. 1986) are combined here under the new genus, Parachaetomium.

Isolation and identification
Fungal isolates were obtained from seeds of wheat (Triticum aestivum L.) and its wild relatives (Triticum boeoticum Boiss. and Aegilops triuncialis L.) collected from the west and northwestern provinces of Iran, 2018-2019. The spikelets were detached from symptomless plants and immediately placed in paper bags, labeled, and transferred to the laboratory. Seeds were manually separated from each spikelet and surface-disinfected with 50% H 2 SO 4 for 30 min and 2% sodium hypochlorite for 20 min as described by Florea et al. (2015). Seeds were then rinsed three times with sterile water, drained on sterile filter paper, and placed on potato dextrose agar (PDA, Merck, Germany) plates containing 150 mg/L of penicillin G (Jiangxi Dongfeng Pharmaceutical Co., Ltd., China) and streptomycin sulfate (Sigma-Aldrich, Inc., USA). The plates were sealed, incubated for 2 months at 25°C, and examined periodically for the growth of fungal endophytes. Potato carrot agar (PCA; Domsch et al. 2007) was used to induce sporulation. Single-ascospore cultures were obtained by serial dilutions and transferring a single germinating ascospore to a new PDA plate.
Colony growth and characters were determined on PDA, OA (Sigma-Aldrich, Inc., USA), and PCA at 25°C. Colony colors were determined with the color charts of Rayner (1970). Microscopic characters were recorded from colonies grown on PCA. Microscopic features, such as the shape of ascomata, ascomatal hairs, and ascospores, were determined in lactic acid mounts. Ornamentations of ascomatal hairs, structures of the ascomatal wall, the shape of asci, and guttulation of ascospores were determined in water mounts (Asgari and Zare 2011). Photographs were taken using a DinoCapture 2.0 image software installed on an Olympus BH-2 microscope (Olympus, Tokyo, Japan). Macroscopic observations were carried out using an Olympus SZH stereo microscope.
Holotypes are preserved at the Fungus Reference Collection (IRAN…F) of Herbarium Ministerii Iranici Agriculturae "IRAN," Iranian Research Institute of Plant Protection (Tehran). Ex-type cultures are deposited at the Iranian Fungal Culture Collection (IRAN…C) of "IRAN" Herbarium.

DNA extraction, amplification, and sequencing
Fresh fungal mycelium (500 mg) was scraped from the margin of a PDA plate and transferred into a 1.5-mL centrifuge tube, followed by grounding in liquid nitrogen by a mini pestle. DNA extraction was performed according to Liu et al. (2000). The following primers were used for PCR amplification and sequencing: RPB2AM-1bf/RPB2AM-7R (Miller & Huhndorf 2005) for the RPB2 gene; ITS1/ITS4 (White et al. 1990) for the internal transcribed spacer regions 1 and 2 including the intervening 5.8S nuclear ribosomal DNA (ITS) and LROR/LR3 (Rehner and Samuels, 1995) for the D1/D2 domains of the LSU; and Bt2a/Bt2b (Glass and Donaldson 1995) for the partial TUB2 gene.

Sequence alignment and phylogenetic analyses
New sequences generated in this study were checked with FinchTV v. 1.4.0 (Geospiza Inc.). Separate ITS, LSU, TUB2, and RPB2 sequences were subjected to the BLAST search engine tool of NCBI for verification and selection of taxa for subsequent phylogenetic analyses. The sequences generated in this study were aligned against sequences of members of the Chaetomiaceae, mostly from Wang et al. (2016aWang et al. ( , 2019a and Asgari and Zare (2011) (see supplementary materials, Table 1). The alignments were obtained using MAFFT v. 7 (http://mafft.cbrc.jp/ alignment/server/index.html) (Katoh et al. 2019) and manually optimized with MEGA6 (Tamura et al. 2013). Sequences of the ITS region, partial LSU, TUB2, and RPB2 were analyzed individually and in combination. Maximum likelihood (ML), maximum parsimony (MP), and Bayesian inference (BI) analyses were used to estimate phylogenetic relationships.

Phylogeny
A partition homogeneity test in PAUP v. 4.0b10 (Swofford 2002) did not show any significant divergence (P = 0.1), indicating that the individual datasets were congruent and produced trees with a similar topology. Therefore, the four datasets were combined in a single analysis, with Podospora fimicola Ces. (CBS 482.64) and Triangularia bambusae (J.F.H. Beyma) Boedijn (CBS 352.33) used as outgroups ). The ML analysis of the combined dataset yielded a best-scoring tree ( Fig. 1) with a final ML optimization likelihood value of − 47,790.561115. The matrix had 1636 distinct alignment patterns, with 23.76% of undetermined characters or gaps. Parameters for the GTRGAMMA model of the combined ITS, LSU rDNA, TUB2, and RPB2 were as follows: estimated base frequencies A = 0.228173, C = 0.284233, G = 0.281294, and T = 0.206300; substitution rates AC = 1.254869, AG = 3.400447, AT = 1.496079, CG = 1.498467, CT = 5.257741, and GT = 1.000000; gamma distribution shape parameter α = 0. 297987. The phylogenetic tree obtained in this study showed similar results to previous studies (Wang et al. 2019a,b, Noumeur et al. 2020. The MP dataset contained 3057 positions, of which 1601 were constant, 240 parsimony uninformative, and 1216 parsimony informative. Parsimony analysis resulted in a single most parsimonious tree of 10,547 steps with a CI of 0.270, HI of 0.730, RI of 0.558, and RC of 0.150. For BI, the GTR+I+G model was selected as optimal for ITS, LSU, TUB2, and RPB2 based on the result of the jModelTest. The BI tree resulted in 20,002 trees after 1,000,000 generations. The first 5000 trees, representing the burn-in phase of the analysis, were discarded, while the remaining 15,002 trees were used for calculating posterior probabilities in the majority rule consensus tree. The BI and MP trees had the same topology as the ML tree. Therefore, the information obtained from the three analyses were combined, and the tree from ML analysis is illustrated (Fig. 1).
Members of the Chaetomiaceae, included in our phylogenetic analyses, were divided into 39 genera similar to Wang et al. (2019b) (Fig. 1). This analysis supports the position of Allocanariomyces and Parachaetomium as new genera and Achaetomium aegilopis as a new species within the family Chaetomiaceae, concordant with morphological traits. ascospores. Distinguished from other genera by cells of the perithecial wall arranged in a textura epidermoidea, densely granulated ascospores showing a distinct subapical germ pore, and humicola-like anamorph. Ascomata superficial, globose to subglobose, non-translucent, solitary, non-ostiolate, glabrous, connected to the agar by rhizoidal hyphae; tissue type in ascomatal wall textura epidermoidea in surface view. Asci evanescent, spherical, ovate or pyriform, stalked, eight-spored. Ascospores arranged irregularly in the ascus, one-celled, brown, fusiform, densely granulate, with a distinct, subapical germ pore. Anamorph humicola-like.
Anamorph not observed.
Colonies on PDA growing rapidly, attaining 60 mm diam. in 4 days at 25°C, circular, cottony, consisting of submerged and aerial mycelium, at first subhyaline, becoming buff (45); reverse of the same color. Colonies on OA at 25°C similar as those on PDA.

Discussion
Phylogenetic analyses based on ITS, LSU rDNA, tub2, and rpb2 sequence data showed that the new genera Allocanariomyces and Parachaetomium were placed in distinct lineages in the family Chaetomiaceae (Fig. 1). Allocanariomyces formed a strongly supported monophyly with a lineage of Batnamyces, Canariomyces, Madurella, and Stolonocarpus. Batnamyces, typified by B . globulariicola, is distinguished by the lack of reproductive structures ). Species of Madurella, a group of fungi causing human mycetoma, often do not sporulate, grow restrictedly in culture, and produce buff, cinnamon, sienna, or orange exudates diffusing into the agar (see Wang et al. 2019b). Stolonocarpus, typified by S. gigasporus , also produces non-ostiolate perithecia and fusiform ascospores, but it is distinct from Allocanariomyces in having perithecia covered by hyphae- Fig. 3 Achaetomium aegilopis (IRAN 3453C). a Colony on PDA after 1 week at 25°C. b Part of the colony on PCA, showing mature perithecia. c, d Perithecia. e, f Outer surface of the perithecial wall. g Perithecial hairs. h, i Asci. j Ascospores. k Chlamydospores. Scale bars: b = 500 μm; c, d = 100 μm; e-k = 10 μm