New Dothideomycetes from Freshwater Habitats in Spain

The Dothideomycetes are a class of cosmopolitan fungi that are present principally in terrestrial environments, but which have also been found in freshwater and marine habitats. In the present study, more than a hundred samples of plant debris were collected from various freshwater locations in Spain. Its incubation in wet chambers allowed us to detect and to isolate in pure culture numerous fungi producing asexual reproductive fruiting bodies (conidiomata). Thanks to a morphological comparison and to a phylogenetic analysis that combined the internal transcribed spacer (ITS) region of the nrDNA with fragments of the RNA polymerase II subunit 2 (rpb2), beta tubulin (tub2), and the translation elongation factor 1-alpha (tef-1) genes, six of those strains were identified as new species to science. Three belong to the family Didymellaceae: Didymella brevipilosa, Heterophoma polypusiformis and Paraboeremia clausa; and three belong to the family Phaeosphaeriaceae: Paraphoma aquatica, Phaeosphaeria fructigena and Xenophoma microspora. The finding of these new taxa significantly increases the number of the coelomycetous fungi that have been described from freshwater habitats.


Introduction
Freshwater fungi are a taxonomically heterogeneous ecological group of organisms of cosmopolitan distribution playing an important ecological role in the recycling of dead organic matter [1], where some of them are restricted to tropical or temperate areas, and others are present in cold-water habitats [2] Most freshwater fungi belong to the phylum Ascomycota (the ascomycetes), whose biodiversity depends on their geographical location and substrates [3]. Around 740 species of ascomycetes have been reported in freshwater habitats (http://fungi.life.illinois.edu/search/world_records, accessed on 10 November 2021), and approximately one-third of these belong to the class Dothideomycetes [4,5].
The Dothideomycetes is among the earliest fungi that were reported in freshwater environments [5]. They are a group of fungi characterized by the production of fissitunicate (bitunicate) asci in unilocular and polylocular ascomata [6,7], currently classified into two subclasses: the Dothideomycetidae, comprising the orders Capnodiales, Dothideales, and Myriangiales; and the Pleosporomycetidae, comprising the orders Gloniales, Hysteriales, Jahnulales, Mytilinidiales, and Pleosporales [8,9]. The majority of freshwater Dothideomycetes belong to the Pleosporales and to the Jahnulales [5].
The Pleosporales is the largest order of the Dothideomycetes, comprising a quarter of its species [6,10]. Taxa in this order have been found in diverse habitats, and can act as saprobes, endophytes, pathogens, or parasites. Most of the Pleosporales are plant pathogens with a wide range of hosts and mainly cause leaf and stem lesions [6,9,11,12].
The diverse hydrography, topology, and climatology of Spain led to the formation of several well-defined ecological regions that have a wide spectrum of scarcely explored habitats potentially rich in fungal populations. During this study, we isolated several fungi living on plant debris in freshwater habitats. The objective was to perform phylogenetic analyses using the nucleotide sequences of informative molecular markers that can clarify the taxonomy of these fungal isolates, and to describe any noteworthy taxa.

Samples Collection and Fungal Isolation
A total of 119 samples of plant debris submerged in freshwater in Spain were collected as follows: two in Parque de Doña Casilda Iturriza (Vizcaya province), three in Les Guilleries (Barcelona province), ten in Cúber (Escorca, Mallorca), 22 in Capafonts (Tarragona province), 15 in Pontons (Barcelona province), 17 in Riaza (Segovia province), and 50 in Cascadas del Huéznar (Cazalla de la Sierra, Sevilla province). Samples were placed into sterile self-sealing plastic bags for transport to the laboratory and stored at 5 • C. Samples were processed and examined following the method described previously by Magaña-Dueñas et al. [13]. All isolates were stored in the culture collection at the Faculty of Medicine and Health Sciences (FMR), Reus (Spain), and herborized materials and living cultures of novel fungi were deposited in the fungal collection at Westerdijk Fungal Biodiversity Institute (CBS; Utrecht, The Netherlands). The nomenclature and descriptions were registered in MycoBank (https://www.mycobank.org/page/Registration%20home, accessed on 4 October 2021).

Phenotypic Study
The phenotypic studies were carried out according to Magaña-Dueñas et al. [14], except for strain FMR 17808, in which the measurements of the structures were carried out after 30 days, due to the slow production of fertile fruiting bodies.

DNA Extraction, Amplification, and Sequencing
DNA extraction, amplification, and sequencing were carried out following the protocols outlined by Magaña-Dueñas et al. [13,14]. SeqMan software v. 7.0.0 (DNAStar Lasergene, Madison, WI, USA) was used to obtain and edit the consensus sequences. Sequences generated in this study were deposited at the European Nucleotide Archive (ENA) (Table S1).

Phylogenetic Analysis
The sequences obtained were compared with other fungal sequences deposited 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 20 July 2021). For the phylogenetic study, an alignment for each locus was made using the MEGA (Molecular Evolutionary Genetics analysis) program v. 7.0 [15], using the Clustal W algorithm [16] and refined with MUSCLE [17], or manually, when necessary, on the same platform. Phylogenetic analyses were made by maximum-likelihood (ML) and Bayesian interference (BI) with RAxML v. 8.2.12 [18] software on the online Cipres Science gateway portal [19] and MrBayes v.3.2.6 [20], respectively. The final matrix used for phylogenetic analyses were deposited in TreeBASE (http://purl.org/phylo/treebase/phylows/study/ TB2:S295050 (accessed on 25 November 2021)).
For the Didymellaceae, the phylogenetic reconstructions were performed using the concatenated nucleotide sequences of three phylogenetic markers (ITS, rpb2, and tub2), while for the Phaeosphaeriaceae four phylogenetic markers (ITS, rpb2, tub2, and tef-1) were used. The best nucleotide substitution model for the BI analysis of the family Didymellaceae was the Kimura 2-parametrer with proportion of Invariable sites and Gamma distribution (K80 + I + G) for rpb2, and the Symmetrical model with proportion of Invariable sites and Gamma distribution (SYM + I + G) for ITS and tub2. For the Phaeosphaeriaceae, the best model was General Time Reversible with proportion of Invariable sites and Gamma distribution (GTR + I + G) for ITS, K80 + I+G for rpb2, Hasegawa-Kishino-Yano with Gamma distribution (HKY + G) for tub2 and General Time Reversible with Gamma distribution (GTR + G) for tef-1, all estimated using the program jModelTest [21]. The parameter settings used in BI analyses were two simultaneous runs of 5,000,000 generations, and Markov chain Monte Carlo (MCMC), with samples taken every 1000 generations. The 50% majority rule consensus tree and posterior probability values (PP) were calculated after discarding the first 25% of the resulting trees. A PP value ≥ 0.95 was considered as significant [22]. For ML analysis, support for internal branches was assessed by 1000 ML bootstrapped pseudo replicates. Bootstrap support value (BS) ≥ 70% was considered significant.

Taxonomy
Diagnosis: Morphologically, Didymella brevipilosa differs from the rest of the species located in the same clade in having slightly sinuous, nodose and verrucose setae mainly on the neck and around the pycnidial ostiole, while the other species produce glabrous conidiomata [23,24]. In the submerged plant material, from which the fungus was isolated, only the asexual stage was observed.
Diagnosis: Heterophoma polypusiformis is easily distinguishable from the other species of the genus because it produces a sexual stage, which is morphologically related to the genus Ascochyta, a member of the Didymellaceae [25]. Moreover, H. polypusiformis develops into OA pycnidia with numerous ostiolar necks that branch out giving them a cephalopod appearance, a feature never seen in the other species of the genus. Unlike H. polypusiformis, its phylogenetically closer species, H. verbasci-densiflori, produces in the same culture conditions pycnidia bearing 1-6 papillated to short (of less than 60 µm long, whereas these can reach up to 200 µm in H. polypusiformis) unbranched necks (branched in H. polypusiformis), with a 'potato-like' appearance. The sexual stage of H. polypusiformis was found on the submerged plant material, whereas its asexual stage was observed once the fungus was grown in pure culture.
Diagnosis: Paraboeremia clausa is phylogenetic close to P. putaminum, but differs from the latter because the pycnidia lack ostioles, and the conidia are hyaline in mass, while those of P. putaminum are greenish [26]. Both strains of P. clausa displayed similar phenotypic features in pure culture, and the asexual stage of P. clausa was originally detected on the freshwater submerged plant debris.
Diagnosis: Paraphoma aquatica differs from the phylogenetically closest species, P. radicina, because its pycnidia lack of a neck and are non-ostiolate. The asexual stage of P. aquatica was also observed in the submerged substrate.
Notes: Paraphoma aquatica is located in the clade with a weak support; however, it forms a fully supported clade with Paraphoma radicina. The concatenated ITS-rpb2-tub2-tef-1 nucleotide sequences of both species differs in 59 bp.
Diagnosis: Morphologically, Phaeosphaeria fructigena is characterized by the production in vitro of ascomata with up to three necks, and of fusiform ascospores. Only the sexual stage of P. fructigena has been observed in both original material and pure culture.
Notes: In our phylogenetic analysis P. fructigena is located at an independent branch, thus revealing itself as a new species. Diagnosis: Morphologically, Xenophoma microspora is distinguished from Xenophoma puncteliae by the production of up to three ostiolar necks, and also by producing smaller conidia than X. puncteliae (1.5-2.5 × 1-1.2 µm vs. 2.5-3 × 2-2.5 µm). The asexual stage of X. microspora was observed in plant debris submerged in freshwater.

Discussion
Recently, molecular biology helped to clarify the phylogenetic relationships between the members of the Dothideomycetes, especially among several phoma-like fungal taxa. Multilocus analyses based on LSU, ITS, rpb2, tef-1, and tub2 sequences have been widely used to define the species boundaries for the Didymellaceae, the Phaeosphaeriaceae and other families of the Dothideomycetes [23,24,[27][28][29][30]. However, rpb2 alone provides a phylogenetic tree with a similar topology to those obtained with more phylogenetic markers [24,30,31].
During the development of the present study, we isolated several fungi from submerged wood in certain freshwater habitats of Spain. We carried out phylogenetic analyses with concatenated of three loci (ITS-rpb2-tub2) for the Didymellaceae members, and of four loci (ITS-rpb2-tub2-tef-1) for those taxa in the Phaeosphaeriaceae. Thus, we report six new species to science: Didymella brevipilosa, Heterophoma polypusiformis, Paraboeremia clausa, Paraphoma aquatica, Phaeosphaeria fructigena, and Xenophoma microspora.
The species of Didymella-genus established by Saccardo [32] to accommodate D. exigua -are saprobes commonly found in living and dead parts of herbaceous and woody plants but are also important phytopathogens. Several species have also been isolated from inorganic substrates, such as asbestos, cement, and paint [23,28,33]. In 2015, Chen et al. carried out a multilocus phylogenetic analysis and the genus was defined as monophyletic and encompassing 37 species [28]. Approximately 30 new species have recently been included in the genus [23,24,29,30,[34][35][36]. In our phylogenetic analysis, Didymella brevipilosa was placed in an independent branch separated from the rest of Didymella spp. In addition, this species is characterized by having short, sinuous and asperulate setae mainly located around the ostioles, unlike most of the species of the genus, which lack these structures.
The genus Heterophoma was introduced by Chen et al. [28]  In our study, we report the finding of Heterophoma polypusiformis, the first species of the genus isolated from wood submerged in freshwater. Moreover, H. polypusiformis produces both asexual and sexual stages, being the first species of the genus reported to have sexual reproduction. The main features of the ascospores (smooth-walled, muriform, brown, and surrounded by a gelatinous sheath) correspond to those reported for other genera of the family Didymellaceae, such as Ascochyta and Neomicrosphaeropsis [25]. Heterophoma polypusiformis is easily distinguishable from the other species of the genus because the pycnidia submerged in the culture medium have an 'octopus' appearance.
Chen et al. [28] introduced the genus Paraboeremia into the Didymellaceae to accommodate P. adianticola, P. putaminum, and P. selaginellae. Nine species are currently accepted in the genus (http://www.indexfungorum.org/Names/Names.asp, accessed on 25 October 2021). The majority of the species of the genus are plant parasites, causing leaf or stem spots [28,31]. Moreover, Paraboeremia spp. have been isolated from the rhizosphere, soil, and healthy and dead plants [23,28,30,31]. Paraboeremia clausa is the first species reported in plant material submerged in freshwater and is characterized by the production of barrel-shaped to pyriform, translucent, very pale colored pycnidia covered by dark brown anastomosing hyphae and lacking ostioles.
The genus Phaeosphaeria was introduced by Miyake [37] to accommodate its type species, P. oryzae. Later, the lectotype was designated by Eriksson [38], and due to the morphological similarities with the Leptosphaeria, both genera were for a long time considered synonyms. Barr [39] subsequently established a new family Phaeosphaeriaceae, designating Phaeosphaeria as its type genus. Phaeosphaeria species have a cosmopolitan distribution, they are saprobic but also pathogenic stems, flowers, and leaves of monocotyledons, and hyperparasites of other fungi [27]. There are 219 species currently listed in the Index Fungorum (http://www.indexfungorum.org/Names/Names.asp, accessed on 25 October 2021). Phaeosphaeria fructigena was isolated from plant debris submerged in freshwater, and its sexual stage shares several features with other species of the genus (e.g., P. musae, P. oryzae, and P. thysanolaenicola), such as the fissitunicate asci and 3-septate, hyaline to pale yellowish ascospores [27,40]. In our phylogenetic analysis P. fructigena was placed in and independent branch in the clade.
In 1983, Morgan-Jones introduced the new genus Paraphoma in order to accommodate phoma-like species with setose conidiomata [41]. However, the genus was later treated at section level within Phoma by Boerema [42]. De Gruyter [43] reinstated the genus, and placed it into the family Phaeosphaeriaceae based on a phylogenetic analysis. Twelve species are currently accepted in the genus (http://www.indexfungorum.org/names/ Names.asp, accessed on 25 October 2021). Paraphoma spp. have been reported mainly as soilborne phytopathogens, causing root and crown rot diseases [27,43,44]. Paraphoma aquatica differs from the other species of the genus because the ascomata lacks of ostiolar necks.
In 2012, Lawrey and Driederich introduced the new species Phoma puncteliae, isolated from the parasitized thalli of Punctelia rudecta [45]. Based on a phylogenetic analysis, Trakunyingcharoen and Crous [46] erected the new genus Xenophoma and placed it in the Phaeosphaeriaceae, designating X. puncteliae as its type species. The morphology of Xenophoma is similar to that of Phoma, differing by the production of cauliflower-shaped, uni-to multilocular conidiomata, and of the subspherical to ellipsoid conidia. Our new species, X. microspora differs from X. puncteliae (the phylogenetically nearest species) by the production of more than one ostiole per conidiomata and by the smaller bacilliform conidia.
The sexual stages of the freshwater ascomycetes have undergone a series of morphological adaptations to survive in aquatic environments. Many of them produce ascospores with appendages and/or mucilaginous sheaths, which facilitate their attachment to substrates into the water [47][48][49]. In this study, the sexual stage of H. polypusiformis, found on plant debris submerged in freshwater, produces ascospores with a mucilaginous sheath, a feature also found in other genera living in similar environments, such as Murispora and Lolia [48,50]. On the other hand, some coelomycetous fungi exclusively reported in freshwater habitats, such as Aquasubmersa mircensis, Coelomyces aquaticus, and Lolia aquatica, are characterized by the production of conidia with mucilaginous appendages [50,51], a feature not observed in our fungal strains, nor in the terricolous counterparts.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/jof7121102/s1, Figure S1: ML phylogenetics tree of Phaeosphaeriaceae inferred from the LSU sequences of 59 strains. Support in nodes is indicated above by bootstrap values of 70% or higher. The tree was rooted with Neophaeosphaeria agaves CBS 136429 and Neophaeosphaeria filamentosa CBS 102202. Alignment length 713 b.p. Newly proposed taxa are given in blue. Type strains are indicated by a superscript T. Table S1: Sequences used in this study.