A New Genus of the Microascaceae (Ascomycota) Family from a Hypersaline Lagoon in Spain and the Delimitation of the Genus Wardomyces

The Saladas de Sástago-Bujaraloz is an endorheic and arheic complex of lagoons located in the Ebro Basin and protected by the Ramsar Convention on Wetlands. Due to the semi-arid climate of the region and the high salinity of their waters, these lagoons constitute an extreme environment. We surveyed the biodiversity of salt-tolerant and halophilic fungi residents of the Laguna de Pito, a lagoon belonging to this complex. Therefore, we collected several samples of water, sediments, and soil of the periphery. Throughout the study, we isolated 21 fungal species, including a strain morphologically related to the family Microascaceae. However, this strain did not morphologically match any of genera within this family. After an in-depth morphological characterization and phylogenetic analysis using a concatenated sequence dataset of four phylogenetically informative molecular markers (the internal transcribed spacer region (ITS) of the nuclear ribosomal DNA (nrDNA); the D1-D2 domains of the 28S gene of the nuclear ribosomal RNA (LSU); and a fragment of the translation elongation factor 1-alpha (EF-1α) and the β-tubulin (tub2) genes), we established the new genus Dactyliodendromyces, with Dactyliodendromyces holomorphus as its species. Additionally, as a result of our taxonomic study, we reclassified the paraphyletic genus Wardomyces into three different genera: Wardomyces sensu stricto, Parawardomyces gen. nov., and Pseudowardomyces gen. nov., with Parawardomyces ovalis (formerly Wardomyces ovalis) and Pseudowardomyces humicola (formerly Wardomyces humicola) as the type species of their respective genera. Furthermore, we propose new combinations, including Parawardomyces giganteus (formerly Wardomyces giganteus) and Pseudowardomyces pulvinatus (formerly Wardomyces pulvinatus).


Introduction
Extreme environments are habitats where physical and/or chemical conditions are exceptionally hostile to the survival and proliferation of most life forms [1].Typically, these harsh conditions are determined by remarkably high or low ("extreme") temperatures or pH, a high salt concentration, osmolarity, hydrostatic pressure, UV radiation, low water activity, or a high concentration of toxic compounds, such as organic solvents and heavy metals [1,2].Organisms that can survive and proliferate in such environmental conditions are known as "extremophiles", and their ability to thrive relies on their distinctive biochemical machinery and physiology [3] and on their cell ultrastructure and composition [4].
Endorheic basins and their associated lagoons and lakes are landlocked hydrogeological structures that do not drain into large waterbodies (rivers, oceans), only experiencing water loss through evapotranspiration and percolation to the underground [5].Commonly found in arid and semi-arid continental regions, these lagoons and lakes tend to be saline due to the gradual salt accumulation resulting from thousands of years of evaporative

Sampling and Fungal Isolation
We collected several samples of water, sediments, and soil from the surrounding areas of Laguna de Pito in January 2022.This lagoon covers approximately 50 ha., dries intermittently, and is surrounded by fields designated for the cultivation of cereals; its conservation status was reported as good [34].The salinity of the water samples, measured by an Aokuy refractometer (Shenzhenshi Jinshenghe Shangmao Youxiangongsi, Guangdong, China), was 50‰ w/v, and the pH measured with SRSE water test strips (Tepcom GmbH & Co., KG, Bendorf, Germany) was 7.8.The samples were transferred to 100 mL sterile plastic containers and were transported whilst being refrigerated (at 4-7 • C) to the laboratory.To maximize the diversity of isolated fungi, the following culture media were employed: 18% of glycerol agar (G18; 2.5 g peptone, 5 g dextrose, 0.5 g KH 2 PO 4 , 0.25 g MgSO 4 , 90 mL glycerol, 7.5 g agar-agar, 410 mL distilled water; [35]), potassium acetate agar (5 g potassium acetate, 1.25 g yeast extract, 0.5 g dextrose, 15 g agar-agar, 500 mL distilled water; [36]), potato dextrose agar (PDA; Laboratorios Conda S.A., Madrid, Spain; [37]) supplemented with 10% NaCl, and 2% malt extract agar (MEA; Difco Inc., Detroit, MI, USA; [38]) plus 30% glycerol.Moreover, sediment samples were activated with acetic acid following the modified protocol of Furuya and Naito [39,40].All culture media were supplemented with 250 mg/L of L-chloramphenicol to prevent the development of bacteria.Sediment samples were vigorously shaken in the same containers they were collected in and were settled for 1 min.Once settled, water was removed by decantation and the sediment was poured onto several layers of sterile filter paper placed over plastic trays until dry [41].Approximately, one gram of dried sediments and soil samples was sprinkled onto all the types of culture media in 90 mm Petri dishes.Different volumes of water for each of the samples (5, 15, and 30 mL) were filtered through a filter membrane of 0.45 µm diameter (Millipore SA, Molsheim, France) using a vacuum pump.Later, the filter membranes were placed onto the different culture media in 90 mm Petri dishes.Every sample was cultured by duplicate, being incubated in darkness at 15 • C and 37 • C, respectively.Plates were examined daily for up to two months by using a stereomicroscope.Each colony developed was transferred to 55 mm Petri dishes containing oatmeal agar (OA; 15 g filtered oat flakes, 7.5 g agar, 500 mL tap water; [38]) by using sterile disposable tuberculin-type needles, and these colonies were incubated at room temperature until axenic cultures of each isolate were obtained.Fungal strains suspected to be novel species or pertaining to uncommon taxa were deposited in the culture collection of the Faculty of Medicine of Reus (FMR; Reus, Tarragona Province, Spain), and the ex-type strains and the herborized specimens (as holotypes) were deposited at the Westerdijk Fungal Biodiversity Institute (CBS; Utrecht, The Netherlands).

Phenotypic Study
The macroscopic characterization of the colonies was performed on OA, MEA, PDA, and potato carrot agar (PCA; 10 g potato, 10 g carrot, 6.5 g agar, 500 mL distilled water) after incubation for 7-14 d at 25 • C in darkness [37,38].The color description of the colonies was made according to Kornerup and Wanscher [42].Cardinal growth temperatures were determined on PDA, ranging from 5 to 40 • C at 5 • C intervals, with an additional measurement at 37 • C.
The microscopic characterization of vegetative and reproductive structures was carried out by using fungal material from the colonies grown on OA under the same conditions as specified for macroscopic characterization.Measurements of at least 30 of the structures were taken from slide mountings using Shear's medium (3 g potassium acetate, 60 mL glycerol, 90 mL ethanol 95%, 150 mL distilled water; [43]) and using an Olympus BH-2 bright field microscope (Olympus Corporation, Tokyo, Japan).Micrographs were taken employing a Zeiss Axio-Imager M1 light microscope (Zeiss, Oberkochen, Germany) with a DeltaPix Infinity × digital camera using Nomarski differential interference contrast.

DNA Extraction, Amplification, and Sequencing
Total genomic DNA was extracted from colonies grown on PDA for 7 to 10 days at 25 ± 1 • C in darkness following the modified protocol of Müller et al. [44] and quantified using a Nanodrop 2000 (Thermo Scientific, Madrid, Spain).For each fungal strain, we amplified the molecular marker that allowed for the most accurate preliminary identification according to the bibliography.The internal transcribed spacers (ITS) region and the D1-D2 domains of the 28S nrRNA (LSU) were amplified using the primer pairs ITS5/ITS4 [45] and LR0R/LR5 [46], respectively.Fragments of the translation elongation factor 1α (EF-1α) and the β-tubulin (tub2) genes were amplified using the primer pairs 983F/2218R and EF-728F/EF-986R [47,48] and BT2a/BT2b [49].For our strain of interest, we amplified the following markers: ITS, LSU, tub2, and EF-1α (using the 983F/2218R set of primers).Singleband PCR products were stored at −20 • C and sequenced at Macrogen Europe (Macrogen Inc., Madrid, Spain) with the same amplification primers.Lastly, the software SeqMan v. 7.0.0(DNAStarLasergene, Madison, WI, USA) was employed to edit and assemble the consensus sequences.

Phylogenetic Analysis
The sequences obtained were compared with all the sequences available at the National Center for Biotechnology Information (NCBI) database using the Basic Local Alignment Search Tool (BLAST; https://blast.ncbi.nlm.nih.gov/Blast.cgi,accessed on 11 October 2023) to obtain a preliminary molecular identification of each isolate.A maximum level of identity (MLI) of ≥98% was considered to allow for species-level identification [50].Single and combined phylogenetic analyses of all the specific molecular markers mentioned above were initially conducted by performing a sequences alignment with the software MEGA (Molecular Evolutionary Genetics Analysis) v. 7.0.[51] using the ClustalW algorithm [52] and refining with MUSCLE [53] or/and manually, if needed.Subsequently, the phylogenetic reconstruction was made by maximum likelihood (ML) and the Bayesian Inference (BI) methods were made by two different software, RAxML-HPC2 on XSEDE v. 8.2.12 [54] software on the online CIPRES Science gateway portal [55] and MrBayes v.3.2.6 [56], respectively.The best substitution model for all the gene matrices was settled by the software from CIPRES Science gateway portal (ML) and by jModelTest v.2.1.3following the Akaike criterion (BI) [55,57].Regarding the ML analysis, phylogenetic support for internal branches was established by 1000 ML bootstrapped pseudoreplicates, being considered significant bootstrap support (bs) values ≥70 [58].Regarding the BI analysis, 5 million Markov Chain Monte Carlo (MCMC) generations were used, with four runs (three heated chains and one cold chain), and samples were stored every 1000 generations.To calculate the 50% majority rule consensus tree and posterior probability values (pp), the first 25% of samples were discharged, and pp values of ≥0.95 were considered significant [59].The resulting phylogenetic trees were plotted using FigTree v.1.3.1 (http://tree.bio.ed.ac.uk/software/figtree/, accessed on 11 October 2023).The DNA sequences and the sequence alignments generated in this study were deposited in GenBank (Table 1) and in TreeBASE (https://treebase.org,accessed on 11 October 2023), respectively.The novel taxa have been registered in My-coBank (https://www.mycobank.org/,accessed 23 March 2023 for Dactyliodendromyces holomorphus gen.et sp.nov.).ET , ex-epitype strain.IT , ex-isotype strain.NT , ex-neotype strain.T , ex-type strain.

Results
The fungi isolated from various substrates collected in Laguna de Pito are listed in Table 2, which also includes their extremophilic character (if previously reported and/or determined during the development of our study).
In the analysis involving species within the Microascaceae, the individual dataset for ITS, LSU, EF-1α, and tub2 showed no conflicts related to the tree topologies for the 70% reciprocal bootstrap trees; thus, a multi-gene analysis was performed.The final concatenated dataset included 43 ingroup strains belonging to the genera Acaulium, Cephalotrichum, Gamsia, Fairmania, Wardomyces and Wardomycopsis, and Microascus longirostris CBS 196.61 and Scopulariopsis brevicaulis MUCL 40726 as the outgroup.The alignment encompassed a total of 3019 characters, including gaps (661 for ITS, 843 for LSU, 965 for EF-1α, and 550 for tub2), 732 of them parsimony informative (203 for ITS, 73 for LSU, 190 for EF-1α, and 266 for tub2) and 960 of them being variable sites (293 for ITS, 91 for LSU, 264 for EF-1α, and 312 for tub2).The tree obtained through the BI analysis was both congruent and similar in topology to the one obtained by ML analysis.Regarding the BI analysis, GTR + G, GTR + G + I, GTR + G + I, and HKY + G + I were selected as the models that fitted the best for ITS, LSU, EF-1α, and tub2, respectively.The support values showed slight differences between the two analysis methods, making the ML bootstrap support values lower than the BI posterior probabilities.

Taxonomy
Microascaceae Luttrell et Malloch, Mycologia 62:734 (1970).Mycobank MB 81001.Ascomata globose, pyriform or irregular in shape, dark brown to black, hairy, rarely bare, arising from coiled ascocarp initials, with or without an ostiole; asci arising singly or in chains on the ascogenous hyphae, without croziers, ovoid to globose, soon evanescent; ascospores reddish brown to coppery-colored, one-celled, with a germ pore at one or both ends, dextrinoid when young, smooth-and thin-walled.

Taxonomy
Microascaceae Luttrell et Malloch, Mycologia 62:734 (1970).Mycobank MB 81001.Ascomata globose, pyriform or irregular in shape, dark brown to black, hairy, rarely bare, arising from coiled ascocarp initials, with or without an ostiole; asci arising singly or in chains on the ascogenous hyphae, without croziers, ovoid to globose, soon evanescent; ascospores reddish brown to coppery-colored, one-celled, with a germ pore at one or both ends, dextrinoid when young, smooth-and thin-walled.
Culture characteristics (after 14 d at 25 • C)-Colonies on PDA reaching 11 mm diam., convex, smooth texture, cerebriform, white (1A1), undulate, sporulation absent; reverse white, (1A1), and olive brown (4E6) at centre, white (1A1) towards periphery, soluble pigment absent.Colonies on PCA reaching 18 mm diam., slightly raised at centre, flattened at the edges, granulose, smooth, grey (30F1) at centre, white (1A1) at the edges, filamentous margins, sporulation (conidiophores) moderate; reverse white (1A1), soluble pigment absent.Colonies on OA reaching 24 mm diam., flattened, granulose, smooth, grey (30F1) at centre, white (1A1) towards periphery, filamentous margins, sporulation moderate to abundant (conidiophores); reverse olive (3F5) at centre, white (1A1) towards periphery, soluble pigment absent.Non-halophilic nor highly halotolerant (does not grow above 10% w/v NaCl).Cardinal temperatures of growth: minimum 5 • C, optimum 20 Diagnosis: The sexual morph of the genus Dactyliodendromyces resembles those species of the Microascaceae producing heart-shaped ascospores: Acaulium albonigrescens, Fairmania singularis, and Wardomyces giganteus, as well as several species of Microascus and Scopulariopsis.Nevertheless, Dactyliodendromyces produces ostiolate ascomata with a superficially areolate peridium and true setae, features not seen in the other taxa.On the other hand, the asexual morph of genus Dactyliodendromyces differs from Wardomyces, and the newly proposed genera Parawardomyces and Pseudowardomyces, in having annellidic conidiogenous cells, which are holoblastic in all of them.The genus Gamsia differs from Dactyliodendromyces by the formation of hyaline, mostly undifferentiated and unbranched conidiophores bearing polyblastic and anellidic conidiogenous cells, which are dematiaceous, well-developed, penicillated, and bear exclusively annellidic conidiogenous cells in Dactyliodendromyces.Consequently, the asexual morph of Dactyliodendromyces is morphologically more similar to Acaulium, Cephalosporium, Fairmania, and Wardomycopsis.However, Dactyliodendromyces is easily discriminated from Fairmania and Wardomycopsis because these genera produce conidia with longitudinal striations or germ slits (not seen in Dactyliodendromyces), and from Cephalosporium, because it produces conidiophores grouped in synnemata (which are absent in Dactyliodendromyces), and also lacks a sexual morph (present in Dactyliodendromyces). Acaulium, in comparison to the Dactyliodendromyces, produces more simple hyaline conidiophores, which are dematiaceous and penicillate in the new genus.
Description: Hyphae hyaline, septate, smooth-and thin-walled, branched, sometimes aggregated and frequently anastomosing.Conidiophores hyaline, macronematous, mostly branched, bi-to terverticillate, with a stipe of short to medium length.Conidiogenous cells holoblastic, terminal or subterminal, globose to barrel-shaped, producing one to three conidia per cell.Conidia two-celled, smooth-and thick-walled, navicular, slightly constricted at Diagnosis: Wardomyces differs from Parawardomyces and Pseudowardomyces by presenting semi-macronematous, short-stipitate, mostly biverticillate conidiophores.Moreover, it differs from Parawardomyces because does not present the scopulariopsis-like synanamorph and lacks a sexual morph, and from Pseudowardomyces in the conidial shape, the absence of a septum and its schizolytic secession.

Discussion
Although some fungi found during the course of this study, such as Alternaria alternata, Aspergillus amstelodami, and Aspergillus versicolor, have been previously reported in hypersaline lagoons and lakes [75], there are no reports of these identified species in endorheic lagoons in Spain.Notably, the extremophilic nature of several species belonging to globally distributed genera recovered in this study, such as Aspergillus, Cladosporium, and Penicillium, has been documented in earlier studies [76][77][78][79].Additionally, most of the identified species have been previously reported as extremophilic or extremotolerant.For instance, Aspergillus amstelodami, Aspergillus intermedius, and Penicillium egyptiacum exhibit osmophilic, xerophilic, or xerotolerant behavior [64,72], while Parachaetomium truncatulum, Aspergillus calidoustus, and Chaetomium grande display thermotolerance [64,70].
Particularly interesting are the findings of Chaetomium grande and Parachaetomium truncatulum, which represent two new reports for Spain and Europe, also being the first time that this species has been isolated from lake sediments [70,[80][81][82].Furthermore, Acrostalagmus luteoalbus can thrive in soils with high pH values (alkali-tolerant), and Aspergillus versicolor and Stachybotrys chartarum exhibit halotolerance [60,68,74].Some species have not been previously reported as extremophilic or extremotolerant, yet our study reveals their ability to grow in up to 10% w/v NaCl, such as Cladosporium europaeum and Malbranchea zuffiana.Surprisingly, we recovered Cephalotrichiella penicillata, Fusarium culmorum, and Ovatospora amygdalispora, taxa that do not exhibit any extremophilic/extremotolerant characteristics and, consequently, should be considered as non-specialized.
Based on both phylogenetic analysis and phenotypic features, we introduced the new monotypic genus Dactyliodendromyces, isolated from a sediment sample from Laguna de Pito.This fungus does not exhibit a strong halotolerant behavior and belongs to the Microascaceae family.While members of Microascaceae have a global distribution, only a few have been initially discovered in Spain, such as Wardomycopsis litoralis and Pseudoscopulariopsis schumacheri [20,83].Although most of the members of Microascaceae are not usually isolated from extreme environments [29,[84][85][86], some species of the genera Microascus and Scopulariopsis have been isolated from halophyte plants and salt marshes, respectively [87,88].Moreover, only two species of this family were first isolated from salty habitats: Wardomyces pulvinatus and Wardomycopsis litoralis [83,89].Dactyliodendromyces holomorphus differs from its closely related genera Gamsia, Parawardomyces, and Pseudowardomyces by the production of the holomorph in vitro.The only exception is Parawardomyces giganteus, which also produces a sexual morph, yet they do not morphologically resemble each other [19].The sexual morph of D. holomorphus consists of short-naked ostiolate ascomata with true setae and a tomentose, carbonaceous peridium, with these features being uncommon among the Microascaeae [24,85,90,91].
Despite previous attempts to separate the genus Wardomyces into different genera, nowadays, it is still considered a paraphyletic genus [92].However, based on our phylogenetic analysis using the ITS-LSU-EF-1α-tub2 markers, Wardomyces could be segregated into three different genera.Furthermore, based on the morphological features, Wardomyces produces complex conidiophores, bi-to terverticillate, and one-celled conidia, whereas Parawardomyces is characterized by the production of monoverticillate conidiophores, and

Table 1 .
Fungi and nucleotide sequences of the molecular markers used to build the phylogenetic trees.

Table 2 .
Fungal taxa recovered from Laguna de Pito and their extremophilic properties.