New Coelomycetous Fungi from Freshwater in Spain

Coelomycetous fungi are ubiquitous in soil, sewage, and sea- and freshwater environments. However, freshwater coelomycetous fungi have been very rarely reported in the literature. Knowledge of coelomycetous fungi in freshwater habitats in Spain is poor. The incubation of plant debris, from freshwater in various places in Spain into wet chambers, allowed us to detect and isolate in pure culture several pycnidia-producing fungi. Fungal strains were phenotypically characterized, and a phylogenetic study was carried out based on the analysis of concatenated nucleotide sequences of the D1–D2 domains of the 28S nrRNA gene (LSU), the internal transcribed spacer region (ITS) of the nrDNA, and fragments of the RNA polymerase II subunit 2 (rpb2) and beta tubulin (tub2) genes. As a result of these, we report the finding of two novel species of Neocucurbitaria, three of Neopyrenochaeta, and one of Pyrenochaetopsis. Based on the phylogenetic study, we also transferred Neocucurbitaria prunicola to the genus Allocucurbitaria. This work makes an important contribution to the knowledge of the mycobiota of plant debris in freshwater habitats.


Introduction
Coelomycetous fungi are characterized by the production of conidia within a cavity lined by fungal or fungal-host tissue called conidiomata [1]. Conidiomata can be acervular (open, cup-shaped asexual fruiting bodies developing below the epidermis of the plant host tissue and bearing a series of adpressed conidiophores), pycnidial (globose, pyriform to flask-shaped asexual reproductive structures whose conidia are liberated through an usually apical opening [ostiolum]), or stromatic (consisting of undifferentiated sclerotic tissues, ostiolate or not, in which one or more lysigenic cavities develops, upholstered inside by conidiophores/conidiogenous cells forming conidia). Coelomycetous fungi are mostly parasites of terrestrial vascular plants but are also saprobic, growing at the expense of dead organic matter on the ground, especially on plant debris. These are ubiquitous on soil, sewage, and in salt-and freshwater environments. [2]. Freshwater coelomycetous fungi occur on stream-side plants or on submerged wood litter, and their conidia can also be recovered from foam and water samples [3]. Usually, they produce brown to blackish pycnidial fruiting bodies on submerged woody debris and stems of herbaceous plants, and produce several conidia from the conidiogenous cells [4]. Identification of coelomycetous fungi has gone through dramatic changes over the last decade, and currently involves DNA sequencing of several (four to six) genetic markers and the building of phylogenetic trees [5]. In Spain, there have been a few reports of coelomycetous fungi recovered from freshwater habitats. In 1990, Roldán and Honrubia reported Bartalinia robillardoides and Truncatella angustata [6], and Giralt described Diplolaviopsis ranula [7]. Up to 2014, only 16 coelomycetous fungi had been reported from freshwater habitats [4,[8][9][10][11][12][13][14][15][16].
The main objective of this work was to characterize phenotypically and to identify molecularly those coelomycetous fungi found in different freshwater habitats in Spain.

Phenotypic Study
Macroscopic characterization of the colonies was performed on OA and on malt extract agar (MEA; Difco, Detroit, MI, USA) incubated for 14 d in the dark at 25 ± 1 • C [17]. Colony colour was determined according to Kornerup and Wanscher [18]. The ability of the isolates to grow at cardinal temperatures was determined on potato dextrose agar (PDA; Pronadisa, Madrid, Spain) after 7 d in the dark, ranging from 5 to 35 ± 1 • C at 5 • C intervals, plus 37 ± 1 • C [19]. Morphological characterization of vegetative and reproductive structures was performed growing the fungal strains on OA in the same conditions as for colony characterization, and examining at least 30 individuals of each structure [20,21] on Shear's mounting medium (3 g potassium acetate, 60 mL glycerol, 90 mL ethanol 95%, and 150 mL distilled water; [22]) using a 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 X digital camera using Nomarski differential interference contrast.

DNA Extraction, Amplification and Sequencing
Fungal strains were cultured on PDA for 7 days at 25 ± 1 • 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, with the primer pair LR0R [23] and LR5 [24]; ITS, with the primer pair ITS5 and ITS4 [25]; a fragment of the beta-tubulin gene (tub2) with the primers TUB2Fw and TUB4Rd [26]; and a fragment of the RNA polymerase II subunit 2 gene (rpb2) with RPB2-5F2 [27] and fRPB2-7cR primers [28]. 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, ITS, and tub2 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 tub2 region 30 s at 94 • C, 45 s at 56 • C, and 1 min at 72 • C. The PCR program for rpb2 amplification consisted of 5 cycles of 45 s at 94 • C, 45 s at 60 • C, and 2 min at 72 • C; then 5 cycles with 58 • C annealing temperature; and 30 cycles with a 54 • C annealing temperature. 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
We made a preliminary molecular identification by comparing the LSU, ITS, tub2, and rpb2 sequences of our isolates 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 (accessed on 16 March 2021)). For tub2 sequences, a maximum level of identity (MLI) of <98% provides identification only at genus level, and a value >98% was considered to allow for species-level identification. Alignment for each locus was performed with the MEGA (Molecular Evolutionary Genetics Analysis) software v. 7.0. (Tamura et al. 2013), using the ClustalW algorithm [29] and refined with MUSCLE [30] or manually, if necessary, on the same platform. Individual and concatenated phylogenetic trees were built after a maximum likelihood (ML) analysis carried out using the RAxML v. 8.2.10 [31] software on the online Cipres Science gateway portal [32], and a Bayesian Inference (BI) analysis using MrBayes v. 3.2.6 [33]. 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 [34]. For ITS we used the symmetrical model with gamma distribution (SYM + G), for LSU and tub2 the symmetrical model with proportion of invariable sites and gamma distribution (SYM + I + G), and for rpb2 the symmetrical model with gamma distribution (SYM + G). The parameter settings used were two simultaneous runs of 5 M generations and four Markov chain Monte Carlo (MCMC), sampled every 1000 generations. The 50% majority-rule consensus tree and posterior probability values (PP) were calculated after discarding the first 25% of the samples. Pleospora herbarum CBS 191.86 and P. typhicola CBS 132.69 served as outgroup taxa. Confident branch support is defined as Bayesian posterior probabilities (PP) >0.95 and maximum likelihood bootstrap support (BS) >70%. Sequences generated in this study were deposited in European Nucleotide Archive (ENA), the final matrix used for phylogenetic analyses in TreeBASE (http://purl.org/phylo/treebase/phylows/study/TB2:S28077 (accessed on 16 March 2021)) and the novel taxonomic descriptions and nomenclature in MycoBank (www.mycobank.org (accessed on 16 March 2021)).

Blast Search
Blast search results are shown in Table S2 (Supplementary Material).
Diagnosis: Neopyrenochaeta asexualis is grouped in the same terminal clade as N. thailandica, but as a distinct taxon. Morphological comparison between N. asexualis and N. thailandica is not possible because only the former produces the asexual morph and only the latter one forms ascomata [37]. However, it is noteworthy that N. asexualis produces conidiomata with doliiform phialides with up two conidiogenous loci.
Diagnosis: In our phylogenetic analysis, N. submersa is located in the same terminal clade as N. acicola, N. fragariae, N. inflorescentiae, and N. glabra. With the exception of N. glabra, all these species are morphologically very similar. However, N. submersa grows faster than N. fragariae (reaching 14 mm and 11 mm diameter after 7 days at 25 • C on OA and MEA, respectively). Also, N. submersa does not produce exopigment on OA, which is lilac-rose in N. acicola [38] and orange in N. fragariae.
Diagnosis: Pyrenochaetopsis aquatica differs morphologically from the phylogenetically nearest species P. leptospora and P. poae, because it is mostly glabrous or covered with few short setae, while the pycnidia of P. leptospora and P. poae are abundantly covered with long setae [39].
Diagnosis: Pyrenochaetopsis aquatica differs morphologically from the phylogenetically nearest species P. leptospora and P. poae, because it is mostly glabrous or covered with few short setae, while the pycnidia of P. leptospora and P. poae are abundantly covered with long setae [39].
Notes: Differences in nucleotide sequences (ITS-LSU-tub2-rpb2 concatenated dataset) between P. aquatica and the other species of the same terminal clade are: P. leptospora, 57 bp; and P. poae, 67 bp.

Discussion
The genus Neocucurbitaria was introduced by Wanasinghe et al. [40] to accommodate N. acerina, N. quercina and N. unguis-hominis (the type species of the genus). Twenty-two species are currently accepted (Index of Fungi; http://www.indexfungorum.org/names/ Names.asp (accessed on 16 March 2021)). Neocucurbitaria spp. has been isolated from human corneal and skin lesions, seawater, and trees and shrubs [5,40,41]. We described two new species for the genus, N. aquadulcis and N. variabilis, from submerged plant debris in freshwaters, the first report for this sort of habitat. It is remarkable that N. variabilis produces two sorts of conidiogenous cells (flask-shaped and long cylindrical) and that N. aquadulcis only produces ampulliform phialides, whereas the other species in the same subclade (N. acerina, N. aquatica, N. irregularis, N. keratinophila and N. unguis-hominis) produce doliiform phialides or well-developed conidiophores. In 2019, Crous & Akulov introduced N. prunicola to that genus [36]. However, in our phylogenic analysis, N. prunicola was located far from the type species of Neocucurbitaria (N. unguis-hominis), being located within the genus Allocucurbitaria. Consequently, we propose the new combination Allocucurbitaria prunicola.
A molecular study by Valenzuela-López et al. [5] allowed recognition of four new families of coelomycetous fungi included previously in the family Cucurbitariaceae: Neopyrenochaetaceae, Parapyrenochaetaceae, Pseudopyrenochaetaceae, and Pyrenochaetopsidaceae. In the latter family, the authors recognized four species belonging to the genus Neopyrenochaeta: N. acicola (basionym: Vermicularia acicola; originally described on decaying leaves of Pinus sylvestris, Vosges, France), N. fragariae (originally identified as Pyrenochaeta acicola; isolated from Fragaria (×) ananassa, The Netherlands), N. inflorescentiae (basionym: Pyrenochaeta in-florescentiae; from style of senescent flowerhead of Protea neriifolia, Western Cape Province, South Africa), and N. thelephonii (basionym: Pyrenochaeta telephonii; from surface of cell phone, Maharashtra, India) [40,42,43]. During 2019 and 2020, eight more species were described [37,44], three of them (Neopyrenochaeta annellidica, Neopyrenochaeta chiangraiensis and Neopyrenochaeta maesuayensis) from submerged decaying wood in Thailand. Interestingly, we also identified two of these latter three species in Spain (Figure 1). This implies that the geographical distribution of N. annellidica and N. maesuayensis is much broader than would be expected, since their original report was from tropical areas of Southeastern Asia. In the present study, we report the finding of three novel additional species from submerged plant debris in Spain: Neopyrenochaeta glabra, N. asexualis, and N. submersa. Neopyrenochaeta glabra is easily recognized by the absence of setae and the darker conidiomata wall around the ostiole. Neopyrenochaeta asexualis is distinguished from other species of the genus because it produces doliiform phialides with one or two conidiogenous loci. Otherwise, N. submersa is difficult to discriminate morphologically from N. acicola, N. fragariae, and N. inflorescentiae species phylogenetically related but differing molecularly.
The fungal genus Pyrenochaetopsis was introduced by De Gruyter et al. [45] to accommodate: P. decipens, P. indica, P. leptospora (type species of the genus), P. microspore, and P. pratorum. Currently 16 species are accepted (Index Fungorum 2020). The members of this genus have been found in terrestrial and marine environments, human dermatitis, sputum, and blood human samples, [5,37,[46][47][48][49]. In our phylogenetic analysis, the strain FMR 17337, named here as P. aquatica, clustered within the Pyrenochaetopsis clade, is distant from other species of this genus, with the exception of P. leptospora and P. poae, which forms a sister clade. Both species differ phylogenetically and morphologically from P. aquatica in having more abundant and longer setae.

Conclusions
In the present study, we have isolated several coelomycetous fungi from submerged plant debris collected in different freshwater habitats in Spain by incubation of the samples in wet chambers. After a phenotypic characterization and a phylogenic study based on the analysis of nucleotide sequences of the ITS, LSU, tub2, and rpb2 loci, six new species have been described: Neocucurbitaria aquadulcis and N. variabilis; Neopyrenochaeta glabra, N. asexualis and N. submersa; and Pyrenochaetopsis aquatica. Also, thanks to the phylogenetic analysis, Neocucurbitaria prunicola was transferred to the genus Allocucurbitaria. In our opinion, the present study makes an important contribution to the knowledge of the coelomycetous fungi growing on decomposing plant material in aquatic habitats.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/jof7050368/s1, Table S1: coelomycetous fungi sequences used in this study; Table S2: Results of blast search of the new proposed species. Figure S1. ML phylogenetic tree of Cucurbitariaceae, Neopyrenochaetaceae, Pseudopyrenochaetaceae, and Pyrenochaetopsidaceae inferred from the ITS sequences (455 bp). Support in nodes is indicated above by bootstrap values of 70% or higher. T = ex-type strains. New species are indicated in blue. New strains isolated during this study are indicated in bold; Figure S2. ML phylogenetic tree of Cucurbitariaceae, Neopyrenochaetaceae, Pseudopyrenochaetaceae, and Pyrenochaetopsidaceae inferred from the LSU sequences (791 bp). Support in nodes is indicated above branches by bootstrap values of 70% or higher. T = ex-type strains. New species are indicated in blue. Strains isolated during this study are indicated in bold; Figure  S3. ML phylogenetic tree of Cucurbitariaceae, Neopyrenochaetaceae, Pseudopyrenochaetaceae, and Pyrenochaetopsidaceae inferred from rpb2 sequences (734 bp). Support in nodes is indicated above branches by bootstrap values of 70% or higher. T = ex-type strains. New species are indicated in blue. New strains isolated during this study are indicated in bold; Figure S4. ML phylogenetic tree of Cucurbitariaceae, Neopyrenochaetaceae, Pseudopyrenochaetaceae, and Pyrenochaetopsidaceae inferred from tub2 sequences (272 bp). Support in nodes is indicated above branches by bootstrap values of 70% and higher. T = ex-type strains. New species are indicated in blue. Strains isolated during this study are indicated in bold. Alignment length.