New Taxa of the Family Amniculicolaceae (Pleosporales, Dothideomycetes, Ascomycota) from Freshwater Habitats in Spain

With the exception of the so-called Ingoldian fungi, the diversity and distribution of the freshwater aero-aquatic or facultative fungi are not well known in Spain. In view of that, we collected and placed into wet chambers 105 samples of submerged and decomposing plant debris from various places in Spain, looking for individuals belonging to these latter two morpho-ecological groups of fungi. As a result, we found and isolated in pure culture several fungi, the morphology of some of them belonging to the family Amniculicolaceae (order Pleosporales, class Dothideomycetes). After a careful phenotypic characterization and a phylogenetic tree reconstruction using a concatenated sequence dataset of D1-D2 domains of the 28S nrRNA gene (LSU), the internal transcribed spacer region (ITS) of the nrDNA, and a fragment of the translation elongation factor 1-alpha (tef1) gene, we report the finding of three new species of the genus Murispora: Murispora navicularispora, which produces cinnamon-colored, broadly fusiform to navicular ascospores; Murispora fissilispora, which has as a remarkable characteristic the production of both sexual and asexual morphs in vitro; and Murispora asexualis, the unique species of the genus that lacks a sexual morph. As a consequence of the phylogenetic study, we introduce the new aero-aquatic genus Fouskomenomyces, with a new combination (Fouskomenomyces cupreorufescens, formerly Spirosphaera cupreorufescens as the type species of the genus) and a new species, Fouskomenomyces mimiticus; we propose the new combinations Murispora bromicola (formerly Pseudomassariosphaeria bromicola) and Murispora triseptata (formerly Pseudomassariosphaeria triseptata); and we resurrect Massariosphaeria grandispora, which is transferred to the family Lopiostomataceae.


Introduction
Fungi are a diverse group of ubiquitous organisms present in almost all ecosystems on Earth, including aquatic habitats [1]. Several fungal taxa have been isolated from freshwater environments, which offer a wide range of organic substrates for fungal colonization [2]. The most important role of fungi in freshwater is the recycling of dead organic matter, typically submerged plant debris [3]. Freshwater fungi complete (at least one part of) their life cycle into the water, and disperse their propagules through the water. Freshwater fungi are generally classified into different sorts of morphological and ecological groups: the "Ingoldian", producing submerged star-like (stauro-) or worm-like (scoleco-) asexual spores (or propagules) in lotic habitats (moving waters) [2,4]; the aero-aquatic, forming helical, net-like or globose conidia above the surface of lentic (standing) waters [5]; and members of the Ascomycota reproducing by the formation of conidia or sexual propagules (ascospores) into fertile bodies (conidiomata and ascomata, respectively). Members of

Phenotypic Study
Macroscopic characterization of the colonies was performed on OA, 2% malt extract agar (MEA; Difco, Detroit, MI, USA) [19] and potato dextrose agar (PDA; Pronadisa, Madrid, Spain) [20] into 90 mm diam. Petri dishes, after incubation for three weeks at 15 • C in the dark for species of the genus Murispora [16], and in similar conditions but at 20 • C for other taxa. Color notations were according to Kornerup and Wanscher (1978) [21]. The ability of the isolates to grow at cardinal temperatures was determined on PDA after 7 d in the dark, ranging from 5 to 35 • C, at 5 • C intervals, but also at 37 • C. Measurements and descriptions of microscopic structures were taken from specimens mounted in Shear's mounting medium (3 g potassium acetate, 60 mL glycerol, 90 mL ethanol 95% and 150 mL distilled water) [22], using an Olympus BH-2 bright field microscope (Olympus Corporation, Tokyo, Japan). Photomicrographs were taken using a Zeiss Axio-Imager M1 microscope (Oberkochen, Germany) with a DeltaPix Infinity × digital camera using Nomarski differential interference contrast.

DNA Extraction PCR Amplification and Sequencing
The strains were cultured on PDA for 7 days at 25 • C in the dark. Total DNA was extracted using the FastDNA kit protocol (Bio101, Vista, CA, USA), with a FastPrep FP120 instrument (Thermo Savant, Holbrook, NY, USA) according to the manufacturer's protocol. DNA was quantified by using Nanodrop 2000 (Thermo Scientific, Madrid, Spain). The following loci were amplified and sequenced: LSU (28S nrRNA gene), with the primer pair LR0R [23] and LR5 [24]; ITS (internal transcribed spacer region), with the primer pair ITS5 and ITS4 [25]; and tef1 with EF1-983F and EF1-2218R [26]. The PCR amplifications were performed in a total volume of 25 µL containing 5 µL 10 × PCR Buffer (Invitrogen, CA, USA), 0.2 mM dNTPs, 0.5 µL of each primer, 1 U Taq DNA polymerase and 1-10 ng genomic DNA. PCR conditions for LSU and ITS were set as follows: an initial denaturation at 95 • C for 5 min, followed by 35 cycles of denaturation, annealing and extension, and a final extension step at 72 • C for 10 min. For the LSU and ITS amplification, the 35 cycles consisted of 45 s at 95 • C, 45 s at 53 • C and 2 min at 72 • C; and for the tef1 an initial denaturation at 94 • C for 2 min, followed by 30 cycles consisting of 30 s at 94 • C, 1 min 20 s at 57 • C and 1 min 30 s at 72 • C. PCR products were purified and stored at −20 • C until sequencing. The same pairs of primers were used to obtain the sequences at Macrogen Spain (Macrogen Inc., Madrid, Spain). The consensus sequences were obtained using the SeqMan software v. 7 (DNAStar Lasergene, Madison, WI, USA).

Phylogenetic Analysis
The sequences generated in this study were compared with those of the National Center for Biotechnology Information (NCBI) using the Basic Local Alignment Search Tool (BLAST; https://blast.ncbi.nlm.nih.gov/Blast.cgi). Alignment for each locus was performed with the MEGA (Molecular Evolutionary Genetics Analysis) software v. 7.0. [27], using the ClustalW algorithm [28] and refined with MUSCLE [29] or manually, if necessary, on the same platform. The alignment included our sequences, together with those available at the NCBI databases, of all genera and species belonging to the family Amniculiculaceae, and representatives of the families Lindgomytaceae, Teratospharriaceae, Lophiostomataceae and Sporormiaceae ( Table 1). The phylogenetic analyses were carried out using Maximum-Likelihood (ML) and Bayesian Inference (BI) with RAxML v. 8.2.10 [30] using the Cipres Science gateway portal [31] and MrBayes v. 3.2.6 [32], respectively. For ML analyses, the best nucleotide substitution model was General Time Reversible with Gamma distribution. Support for internal branches was assessed by 1000 ML bootstrapped pseudoreplicates. For the BI phylogenetic analysis, the best nucleotide substitution model was determined using jModelTest [33]. For ITS, we used the symmetrical model with gamma distribution (SYM + G), for LSU, we used the symmetrical model with proportion of invariable sites and gamma distribution (SYM + I + G), and for tef1, we used the General Time Reverse with proportion of invariable sites and gamma distribution (GTR + I + G). The parameter settings were two simultaneous runs of 5M generations, four Markov chain Monte Carlo (MCMC), sampled every 1000 generations. The 50% majority-rule consensus tree and posterior probability values were calculated after discarding the first 25% of the samples. Leptosphaeria dolium (CBS 125,979 and CBS 505.75) served as outgroup taxa. Confident branch support was defined as Bayesian posterior probabilities (PP) ≥ 0.95 and ML bootstrap support (BS) ≥ 70%. Sequences generated in this study were deposited in European Nucleotide Archive (ENA).

Phylogenetic Analyses
The final concatenated ITS-LSU-tef1 sequence dataset using both ML and Bayesian analyses contained 37 ingroup strains from five families (Amniculicolaceae, Lindgomytaceae, Lophiostomataceae, Sporormiaceae and Teratospharriaceae). The alignment comprised a total of 1936 characters including gaps (815 for LSU, 399 for ITS and 722 for tef1), of which 435 were parsimony informative (125 for LSU, 173 for ITS and 137 for tef1). The individual sequence datasets did not show any conflicts in the tree topologies for the 70% reciprocal bootstrap trees, which allowed the three genes for the multi-locus analysis to be combined. The ML analysis showed similar tree topology and was congruent with that obtained in the Bayesian analysis. For the BI multi-locus analysis, a total of 2706 trees were sampled after the burn-in with a stop value of 0.01. The support values were slightly different with the two analysis methods: with BI, posterior probabilities being higher than the ML bootstrap support values. In our phylogenetic analysis, the family Amniculicolaceae formed a well-supported main clade (99% BS/1 PP) ( Figure 1). All taxa in this family were split into two well-supported clades. The first one (99% BS/1 PP) included two accepted genera (Amniculicola, 96% BS/1 PP and Vargamyces, 91% BS/1 PP), plus another genus (81% BS/0.98 PP). We propose this one as the new Fouskomenomyces, comprising Fouskomenomyces cupreorufescens (basionym Spirosphaera cupreorufescens) and two of our strains (FMR 17,151 and FMR 16,958). The second main clade, corresponding to the genus Murispora (94% BS/1 PP), was represented by all previously described species (including the type species of the genus, M. rubicunda). Our strains FMR 17,248, FMR 17,251 and FMR 17,838 were placed into independent terminal branches, each one representing a new species for the genus and two new combinations, M. bromicola (basionym Pseudomassariosphaeria bromicola) and M. triseptata (basionym P. triseptata). Surprisingly, Pseudomassariosphaeria grandispora (formerly included in the Amniculicolaceae) fell into the Lopiostomataceae (96% BS/1 PP). with that obtained in the Bayesian analysis. For the BI multi-locus analysis, a total of 2,706 trees were sampled after the burn-in with a stop value of 0.01. The support values were slightly different with the two analysis methods: with BI, posterior probabilities being higher than the ML bootstrap support values. In our phylogenetic analysis, the family Amniculicolaceae formed a well-supported main clade (99% BS/1 PP) ( Figure 1). All taxa in this family were split into two well-supported clades.    Etymology. From Greek µιµητικóς, mimetic, because the morphological resemblance to other genera such as Pseudoagerita.
Culture characteristics (after 3 weeks at 20 °C): Colonies on natural substratum not evident, appearing as scattered propagules. Colonies on MEA 2% reaching 25 mm diam., velvety, umbonate, margins regular, with abundant aerial mycelium, orange white to brownish-orange (6C4); reverse dark brown to brown (7F8/7E8), orange white (6C4) at the margins. Colonies on OA reaching 34-36 mm diam., flattened, slightly floccose, margins regular, with sparse aerial mycelium, dark purple to purplish grey (14F6/14B2); reverse violet to grey (15E8/14D1/15A3), margins white (1A1). Colonies on PDA reaching 28 mm diam., convex, cottony at the center, slightly floccose and velvety in the rest of the colony, margins regular, pinkish-white (9A2), margins orange grey (6B2); reverse brownish orange to reddish brown (6C3/8E7), margins orange white (5A2 Notes: Fouskomenomyces mimiticus produces brown to dark brown, globose to sub-globose propagules, composed of a compact branched system of globose to polyhedral cells, whereas the propagules of Fouskomenomyces cupreorufescens are formed by branched, loosely spiralled, interwoven filaments, which are coppery-brown in mass. Saprobic fungi living in freshwater habitats. Ascomata scattered or in small groups, immersed, erumpent, or nearly superficial, dark brown to black, ostiolate, globose to subglobose, neck periphysate with an apex weakly papillate, conical or nearly so. Peridium 3-7-layered, outer layer of textura angularis or textura intricata. Pseudoparaphyses trabeculate, embedded in mucilaginous material. Asci (4-)8-spored, bitunicate, fissitunicate, short pedicellate, cylindrical to clavate, with an ocular chamber. Ascospores transversally septate or muriform, hyaline when young, mostly becoming pale brown to reddish brown with age, less commonly remaining hyaline, constricted at the septa, navicular to broadly ellipsoidal, usually surrounded by an irregular, hyaline, gelatinous sheath. Staining the substrate in purple. Asexual morph coelomycetous. Notes: Morphologically differing from the other species of Murispora by its production of hyaline ascospores (brown in the rest of the species of the genus), fusiform to lunate and narrower towards the apex (mostly ellipsoidal with rounded ends in other species), and not strongly constricted at the septa (although strongly constricted at septa in all other species of the genus).
Pseudomassariosphaeria triseptata, of marine origin, is a species recently introduced to the genus Pseudomassariosphaeria by Jones et al., in 2020 [35]. However, in our phylogenetic analysis, this species, as well as P. bromicola, was placed into the Murispora clade. Therefore, we propose the next new combination for this fungus. Etymology. From Latin fissile-, splitting, and -sporarum, spore, because the ascospores split at the middle when old.
Notes: Murispora fissilispora, genetically distinct from its neighboring Murispora asexualis, is the only species of the genus that produces both sexual and asexual morphs in vitro. Notes: Murispora fissilispora, genetically distinct from its neighboring Murispora asexualis, is the only species of the genus that produces both sexual and asexual morphs in vitro.
Culture characteristics (after 3 weeks at 15 °C). Colonies on PDA reaching 25-30 mm diam., umbonate, velvety, slightly cottony center, surface orange white to reddish white (5A2/6A2), pale orange (5A3) at the regular margins; reverse violet brown to yellowish white (10E4/4A2), diffusible pigment orange white (5A3). Colonies on MEA 2% reaching 26-28 × 17-20 mm diam, ellipsoidal,   [17], to accommodate Pseudomassariosphaeria bromicola, found in a dead stem of Bromus sterilis L., transferring also Massariosphaeria grandispora to this genus (as Pseudomassariospaheria grandispora). However, in our phylogenetic study P. bromicola is clearly placed into the family Amniculicolaceae (transferred by us to the genus Murispora as M. bromicola earlier in this manuscript), whereas P. grandispora was located in the family Lophiostomataceae, phylogenetically close to Lophiostoma macrostomum and L. arundinis. The placement of P. grandispora into the Lophiostomataceae was previously suggested by Wang in 2007 [36], based on a molecular analysis using 28S rDNA, 18S rDNA and rpb2 gene. Consequently, we resurrected the name Massariosphaeria grandispora for this fungus.

Discussion
Of the three morpho-ecological groups of freshwater fungi (Ingoldian's, aero-aquatic and facultative) only the latter two were addressed in this study. In our phylogenetic analysis, all of the Amniculicolaceae species clustered in a distinct sister clade to Lindgomycetae, which is similar to previous studies [16][17][18]. Most Aminiculicolaceae species are reported from freshwater habitats and are widely distributed across Austria, Italy, France, Germany, Denmark, China, Hungary and Spain [15][16][17][18]34,37]. However, with exception of Murispora aquatica and M. triseptata (basionym Pseudomassariospaheria triseptata), all species of Murispora were isolated from terrestrial habitats such as dead terrestrial stems and dead and fallen twigs [14][15][16][17][18]35]. In this study, we have introduced three new species of Murispora collected from Spain in freshwater habitats. Thanks to the phenotypic characterization of several fungal isolates and to the subsequent phylogenetic analysis based on a concatenate database of the ITS-LSU-tef1 sequences, we have erected three new species of Murispora: M. asexualis, the unique species of the genus because it lacks a sexual morph; M. fissilispora, the first species of this genus to produce a holomorph in vitro, and M. navicularispora, which produces cinnamon-colored, broadly fusiform to navicular ascospores, features never seen in the genus before. In addition, we have proposed the new combinations M. bromicola (formerly P. bromicola) and M. triseptata (formerly P. triseptata), demonstrating that this genus is monophyletic. Consequently, we have enlarged the current concept of Murispora, including species with hyaline, navicular and transversally septate ascospores, or lacking a sexual morph. Our results also indicate that some morphological features, such as the size and shape of the ascospores, have less phylogenetic significance than previously proposed by other authors. Despite Spirosphaera cupreorufescens displaying features considered as typical of that genus, it was phylogenetically distant in our phylogeny (in the class Dothideomycetes) from the type species of the genus (Spirosphaera floriformis, in the class Leotiomycetes), and because S. cupreorufescens formed a strongly supported clade together with two of our strains, we have proposed the erection of the new genus Fouskomenomyces, to include Fouskomenomyces cupreorufescens (the type species of the genus) and the new species Fouskomenomyces mimiticus, both aero-aquatic conidial fungi. Finally, we have also resurrected Massariosphaeria grandispora, because in our phylogeny it was placed into the Lopiostomataceae instead of the Amniculicolaceae. To date, there have been few reports of fungi isolated from freshwater habitats in Spain, therefore this work represents an important contribution to the knowledge of the Spanish mycobiota in aquatic environments.
Author Contributions: V.M.-D. performed the experimental work, the phenotypic characterization of the isolates, as well as the DNA extraction and purification, gene sequencing and data processing for phylogenetic analysis, being one of the major contributors of this manuscript. A.M.S., because of their experience with fungal biology and taxonomy, supervised all steps of the experimental work by V.M.-D., collaborating in the description of the novel fungi and in the writing of chapters "Introduction" and "Discussion", reviewing the draft several times. J.F.C.-L. supervised the experimental work too, especially those steps related to nucleotide sequencing of the molecular markers employed in this study, but also the nucleotide sequence alignment and the phylogenetic reconstructions, took the pictures that appear in the figures, contributed actively in the identification and the taxonomy of the fungal strains, gave useful suggestions to write the manuscript and reviewed the draft several times. All authors have read and agreed to the published version of the manuscript.
Funding: This work was supported by the Spanish Ministerio de Economía y Competitividad, grant CGL2017-88094-P.