1. Introduction
Hypersaline endorheic lagoons are located within endorheic basins—closed systems with no direct connection to seas, oceans, or rivers—that are predominantly found in salt flats or depressions within arid or semi-arid continental regions [1,2]. These lagoons represent fragile ecosystems that are particularly sensitive to fluctuations in air temperature and precipitation patterns. Consequently, they are highly vulnerable to the impacts of global climate change, as well as to direct anthropogenic pressures [3,4]. Furthermore, these lagoons support a rich biodiversity that differs from that of permanent lagoons and lakes, often contributing more significantly to regional biodiversity than larger water bodies and providing a refuge for uncommon and rare species [5,6].
The Saladas de Chiprana and the Saladas de Sástago-Bujaraloz are endorheic lagoon complexes located in the Ebro Basin (Zaragoza Province, Northeast Spain). Since 1994, the Saladas de Chiprana have been protected under the Ramsar convention and have been regularly monitored to preserve their unique characteristics [4,7]. The Salada Grande de Chiprana (41°14′26.8″ N 0°10′59.1″ W) is the main and deepest lagoon in the complex, and it is the only known permanent hypersaline lagoon in Western Europe [8]. The local climate is semi-arid Mediterranean, characterized by intense summer droughts and increased winter precipitations [9]. This hypersaline lagoon has a surface area of approximately 31.5 ha and a variable depth between 3.5 m and 5.6 m, and it is situated at 137 m a.s.l. The mean annual temperature is approximately 15.5 °C, with minimal and maximal temperatures of 3 °C and 34 °C, respectively [10].
The other lagoon complex, the Saladas de Sástago-Bujaraloz, is located on a platform of ~400 m a.s.l. and comprises more than a hundred basins. It is one of the best-preserved and the most representative lagoon systems associated with these basins and, thus, is protected by the Ramsar Convention on Wetlands [7,11]. The largest of these lagoons is the Laguna de La Playa, situated approximately 20 km south of the Salada Grande de Chiprana. It is the largest salt flat in the complex, with an area of 239.9 ha and a length of 2.7 km. Laguna de La Playa has one of the highest occurrences of water presence, following La Salineta, with water present on 77% of the 52 dates studied between 1984 and 2004. The largest water surface area recorded was 187 ha in January 1987, and the maximum depth of 51 cm was measured in December 1994 [7,12]. This lagoon complex shares its local climate with the Saladas de Chiprana [11,13]. However, unlike the Salada Grande de Chiprana, La Playa lagoon suffers dramatic changes in surface area and salt concentration throughout the year and over time, as can be observed by the variation of different physical–chemical parameters in the comparative Table 1.
Despite the archaeal and bacterial communities of some hypersaline lagoons being well-characterized [14,15,16,17,18,19], the contribution of halophilic and salt-tolerant fungi to these microbiotas has only recently been acknowledged. Species of “black yeasts”, such as Hortaea werneckii and Trimmatostroma salinum, are present in marine-origin solar salterns [20,21,22,23,24], where they are well-adapted to proliferate in water and sediments with high salt concentrations [25,26,27]. The most halophilic of these fungi, Wallemia ichthyophaga, can grow in NaCl concentrations up to 250 g/L [28]. Many other genera of filamentous fungi and yeasts have been found in salterns and natural salt lakes, such as the Dead Sea [20,29]. Recently, we described the new fungus Dactyliodendromyces holomorphus, from the waters of Laguna de Pito, a hypersaline endorheic lagoon in the Saladas de Sástago-Bujaraloz complex [30]. However, the fungus recovered in this place proved to be only moderately salt tolerant to non-salt tolerant.
Although extreme environments are recognized as reservoirs of a diverse array of fungi with biotechnological potential [31,32,33], research on fungi in hypersaline lagoons remains limited due to the underexplored nature of these ecosystems. Nevertheless, Gomoiu et al. [34] reported the production of hydrolytic enzymes by fungi isolated from brackish and hypersaline lakes, while Georgieva et al. [35] evaluated the antimicrobial potential of halo-alkali-tolerant fungi from the Big Tambukan Saline Lake (Northern Caucasus). Additionally, several halophilic fungal species from different environments have been reported to exhibit antibacterial activity [32,36,37,38].
Given the absence of prior studies on fungal diversity in the Salada Grande de Chiprana and La Playa lagoons, our study aimed to improve the understanding of the fungal biodiversity inhabiting these endorheic hypersaline lagoons. To achieve this, we employed culture-dependent techniques combined with a polyphasic taxonomic approach to identify fungal species and determine their phylogenetic position and relationships with other fungi.
Table 1Main physical-chemical parameters of the Salada Grande and La Playa lagoons.
| Parameter | Salada Grande (Saladas de Chiprana Lagoon Complex) [14] | La Playa (Saladas de Sástago-Bujaraloz Lagoon Complex) |
|---|---|---|
| pH | 9.2, 9.6 | 8.0 [15], 6.75–8.3 [39], 8.2 [40] |
| Temperature (°C; at surface) | 8.85, 11.4 | 23.8 [15], 16.4 [40] |
| Salinity (g/L) | 70–80 | 18.38 [40] |
| Conductivity (mS/cm) | 46, 40 | >200 [15], 28.8 [40] |
| Dissolved O2 (mg/L) | 8.65, 7.86 | 9.4 [15], 9.1 [40] |
| Phosphates (ppb) | ND | <100 [15] |
| Nitrates (ppm) | ND | <1 [15], <2 [40] |
| Ammonia (ppm) | 2.5 | 0.6 [15] |
| Cl− (ppm) | 8214 | 27,050–166,371 [39] |
| SO42− (ppm) | 25,679 | 8309–94,955 [39] |
| Na+ (ppm) | 9619 | 19,237–120,657 [39] |
| K+ (ppm) | 192 | 605–16,750 [39] |
| Ca2+ (ppm) | 586 | 84–968 [39] |
| Mg2+ (ppm) | 8233 | 3037–25,235 [39] |
2. Materials and Methods
2.1. Characterization of the Studied Area
Different physical–chemical parameters of the Salada Grande and La Playa lagoons, available in the literature, were compiled in Table 1.
2.2. Sampling and Fungal Isolation
We collected several samples of the hypersaline waters (from superficial and intermediate [from 10 to 50 cm depth depending of the lagoons] layers), lagoon sediments (of up to 60 mm depth), and soils (A horizon, up to 50 mm deep) surrounding the La Playa (Figure 1, Site 1) and Salada Grande de Chiprana lagoons (Figure 1, Site 2) in May 2021 and July 2022, respectively. To collect water and sediment samples, sterile plastic containers for biological samples, held with an extendable and retractable narrow telescopic grabber arm rod, were introduced into the aquatic medium and subsequently sealed with their respective screw caps.
The salinity and pH of the water samples were measured by an Aokuy refractometer (Shenzhenshi Jinshenghe Shangmao Youxiangongsi, Guangdong, China) and SRSE water test strips (Tepcom GmbH & Co. KG, Bendorf, Germany). The samples were collected using 100 mL sterile plastic containers and transported to the laboratory to be refrigerated at 4–7 °C. In order to isolate the largest possible number of fungal taxa, the following culture media were used: 2% malt extract agar (MEA; Difco Inc., Detroit, MI, USA; [41], supplemented with 30% glycerol); potato–dextrose agar (PDA; Laboratorios Conda S.A., Madrid, Spain; [42] plus 10% NaCl); ascospore agar (AA, 5 g potassium acetate, 1.25 g yeast extract, 0.5 g dextrose, 15 g agar-agar, 500 mL distilled water; [43]), and 18% of glycerol agar (G18; 2.5 g peptone, 5 g dextrose, 0.5 g KH2PO4, 0.25 g MgSO4, 90 mL glycerol, 7.5 g agar-agar, 410 mL distilled water; [44]). Bacterial growth was prevented by adding 250 mg/L of L-chloramphenicol to each culture media previously sterilized in an autoclave. For each sample of water, 5, 15, and 30 mL were filtered through a filter membrane with a 0.45 µm pore diam. (Millipore SA, Molsheim, France) using a vacuum pump. Then, the filter membranes were aseptically placed (using sterilizable metal tweezers) onto the culture media mentioned previously and deposited into 90 mm diam. sterile disposable Petri dishes. Samples of sediment were vigorously shaken inside their original containers and then settled for one minute for sedimentation. Then, the upper water layer was removed, and the solid sediment was dried over several layers of sterile filter paper placed on plastic trays [45]. One gram of dried sediment and an equal amount of soil sample were evenly sprinkled onto each culture media in 90 mm diam. sterile disposable Petri dishes. Additionally, to break the dormancy of the fungal spores, sediment samples were treated with 5% acetic acid [46,47]. Every sample was cultured in duplicate and incubated in darkness at 15 °C and 37 °C. Petri dishes were examined daily using a stereomicroscope for up to two months to detect fungal development. After visualization, every single colony was transferred to a 55 mm Petri dish containing oatmeal agar (OA; 15 g of filtered oat flakes, 7.5 g agar, 500 mL tap water; [41]) using sterile disposable tuberculin-type needles and syringes and then incubated at room temperature until obtaining pure cultures. Fungal strains were deposited in the culture collection of the Faculty of Medicine of Reus (FMR; Tarragona Province, Spain), and uncommon taxa, as well as the ex-type strains and the herborized specimens (holotypes), were deposited at the Westerdijk Fungal Biodiversity Institute (CBS; Utrecht, The Netherlands) for their preservation.
2.3. Phenotypic Study
The macroscopic characterization of the colonies was carried out 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 14 days at 25 °C in darkness [41,42]). The color description of the colonies was made according to Kornerup and Wanscher [48]. Cardinal temperatures of growing were determined on PDA, ranging from 5 to 40 °C at 5 °C intervals, with an additional measurement at 37 °C. Microscopic characterization of vegetative and reproductive structures was carried out by visualizing the vegetative and reproductive fungal structures from the colonies grown on OA in the previously stated conditions. At least 30 measurements of each structure were taken from slide mountings using Shear’s medium (3 g potassium acetate, 60 mL glycerol, 90 mL ethanol 95%, 150 mL distilled water; [49]) using an Olympus BH-2 bright-field microscope (Olympus Corporation, Tokyo, Japan). Micrographs were taken using a Zeiss Axio-Imager M1 light microscope (Zeiss, Ober-kochen, Germany) with a DeltaPix Infinity × digital camera using Nomarski differential interference contrast.
2.4. DNA Extraction, Amplification, and Sequencing
Total genomic DNA was extracted from colonies grown on PDA for 7 to 10 days at 25 °C in darkness following the modified protocol of Müller et al. [50] and quantified by a Nanodrop 2000 (Thermo Scientific, Madrid, Spain). The molecular markers used for each fungal strain were selected and amplified according to the bibliography. The primers used to amplify the internal transcribed spacers (ITS) region and the D1-D2 domains of the 28S nrRNA (LSU) were ITS5/ITS4 [51] and LR0R/LR5 [52], respectively. The primers used to amplify fragments of the beta-tubulin gene (tub2) were BT2a/BT2b [53]. For the fragments of the translation elongation factor 1α (TEF-1α), the primer pair EF-728F/2218R [54,55] was used. Single-band PCR products were stored at −20 °C and sequenced at Macrogen Europe (Macrogen Inc., Madrid, Spain) with the same primers employed for amplification. The software SeqMan v. 7.0.0 (DNAStarLasergene, Madison, WI, USA) was to used edit and assemble the consensus sequences.
2.5. Phylogenetic Analysis
The consensus sequences were compared with all of the sequences available at the National Center for Biotechnology Information (NCBI) database using the Basic Local Alignment Search Tool (BLAST;
3. Results
3.1. Salinity and pH of the Water Samples
Water samples displayed a pH of 8.2 and a salinity of 34‰ w/v for the La Playa and 8.4 and 62 ‰ w/v for the Salada Grande de Chiprana lagoons.
3.2. Phylogeny
Among all the strains isolated during the development of this study, the strains FMR 19580, FMR 20550, and FMR 20333 grabbed our attention due to their unique morphology, leading to an in-depth morphological and phylogenetic analysis. The results of the BLAST search using ITS, LSU, tub2, and TEF-1α sequences for these three strains are summarized in Table 3.
Based on the BLAST results, we conducted two phylogenetic analyses: one including species from the family Didymosphaeriaceae, given the preliminary placement of our strains FMR 19580 and FMR 20550 within the genus Montagnula, which belongs to this family, and another including species from the genus Monosporascus, based on the preliminary placement of our strain FMR 20333 within this genus.
For the individual analysis containing species belonging to the Didymosphaeriaceae family, the individual dataset for ITS, LSU, and TEF-1α did not display any conflicts related to the tree topologies for the 70% reciprocal bootstrap trees. Therefore, a multi-gene analysis was performed. The final concatenated dataset included 69 ingroup strains belonging to the genera Alloconiothyrium, Austropleospora, Chromolaenicola, Cylindroaseptospora, Didymosphaeria, Kalmusia, Kalmusibambusa, Karstenula, Montagnula, Neokalmusia, Neptunomyces, Paracamarosporium, Paraconiothyrium, Paramassariosphaeria, Paraphaeosphaeria, Pseudocamarosporium, Septofusispora, Verrucoconiothyrium, and Xenocamarosporium, plus two outgroup strains of the genus Periconia. The alignment encompassed a total of 2179 characters including gaps (484 for ITS, 814 for LSU, and 881 for TEF-1α), 466 of them parsimony informative (193 for ITS, 106 for LSU, and 167 for TEF-1α) and 626 of them variable sites (226 for ITS, 175 for LSU, and 225 for TEF-1α). The tree obtained by the BI analysis was congruent and similar in topology to the one obtained through the ML analysis (Figure 2). Regarding the ML analysis, K2 + G + I was the best-fitting model for ITS and LSU and TN93 + G + I was the best-fitting model for TEF-1α. Regarding the BI analysis, SYM + I + G was the best-fitting model for ITS, and GTR + I + G was the best-fitting model for both LSU and TEF-1α. The support values showed slight differences between the two analysis methods. However, no incongruences were shown.
The phylogenetic analyses revealed seven fully supported clades formed by different family members of Didymosphaeriaceae. Most of the species of the Montagnula genus clustered in a main clade (99% BS, 1 PP), whereas Montagnula jonesii was placed alone in a distant, independent, and fully supported terminal clade. Consequently, Montagnula jonesii was considered a novel genus. Within the Montagnula clade, our strains were placed in a well-supported terminal clade (95% BS, 0.99 PP), the strain FMR 19580 clustered in the same terminal branch (99% BS, 1 PP) with CBS 100254, CBS 100256, and CBS 100394 and FMR 20550 were placed in an independent well-supported branch. The strains CBS 100254, CBS 100256, and CBS 100394 were deposited in the Westerdijk Fungal Biodiversity Institute collection under the non-validated name of ‘Aporospora terricola’. Our phylogenetic analysis demonstrates that these strains are co-specific to our strain FMR 19580, representing a novel species of the genus Montagnula. On the other hand, the strain CBS 385.65, previously classified within the genus Herpotrichia as the type strain of H. striatispora, was included in our phylogenetic analysis because the BLAST searches using all of the phylogenetic markers placed it within the genus Montagnula. Since CBS 385.65 was positioned as an independent species in the same terminal clade as the ‘Aporospora terricola’ strains (including FMR 19580 and FMR 20550), it represents a novel combination, Montagnula striatispora.
Regarding the individual analysis containing species belonging to the genus Monosporascus (Figure 3), to carry out a multi-gene analysis, we first corroborated that the tree topologies for the 70% reciprocal bootstrap trees of the individual molecular markers, ITS, LSU, tub2, and TEF-1α did not show contradictions. The final multi-gene analysis dataset included 21 ingroup strains of the genus Monosporacus, and, as outgroups, Eutypa petrakii CBS 285.87 and Eutypa camelliea HKAS 107022. The alignment comprised 2425 characters, including gaps (535 for ITS, 688 for LSU, 553 for tub2, and 649 for TEF-1α). Among the total characters, 422 of them were parsimony informative (122 for ITS, 39 for LSU, 90 for tub2, and 171 for TEF-1α) and 506 of them were variable sites (133 for ITS, 54 for LSU, 120 for tub2, and 199 for TEF-1α). For both the ML and the BI analyses, the support values obtained displayed little to no differences. Thus, both analyses were consistent. In the ML analysis, the model that fitted the best for all of the molecular markers was K2 + G. In the BI analysis, the model that fitted the best for ITS, tub2, and TEF-1α was K80 + G, and the model that fitted the best for LSU was K80 + I.
The phylogenetic analysis revealed a fully supported main clade encompassing all species of Monosporascus, which was further divided into three subclades. The first of these subclades is well-supported (92 BS, 0.99 PP) and includes most species within the genus. The second subclade comprises two fully supported terminal branches corresponding to the species Monosporascus caatinguensis and Monosporascus bulgaricus. The third subclade contains two terminal branches, one with Monosporascus solitarius and the other with Monosporascus ibericus and our strain FMR 20333, with enough phylogenetic distance between them to be considered separate species.
3.3. Taxonomy
Pleosporales Luttr. ex M.E. Barr, Prodr. Cl. Loculoasc. (Amherst): 67 (1987). Mycobank MB 90563.
Didymosphaeriaceae Munk, Dansk bot. Ark. 15 (no. 2): 128 (1953). Mycobank MB 80702.
Montagnula Berl., Icon. fung. (Abellini) 2 (2–3): 68 (1896). Mycobank MB 3265.
Type species—Montagnula infernalis (Niessl) Berl. 1896 (designated by Clements and Shear, Gen. fung., Edn 2 (Minneapolis): 276. 1931).
Montagnula striatispora (Papendorf and Arx, 1967) Barnés-Guirado, Stchigel and Cano, comb. nov. MycoBank MB854493 Figure 4.
Basionym—Herpotrichia striatispora Papendorf and Arx, Nova Hedwigia 12 (3 + 4): 395 (1967).
Description: Papendorf and Arx (1967).
Notes: The distinctive features of M. striatispora are related to the nature of their ascospores, which are bicellular, asymmetrically biconical with acute ends, and longitudinally striate. Also, the hamathecium is composed of paraphyses, not as in the most of species of the genus (pseudoparaphysate), having a peryphysate ostiolate ascomata.
Montagnula terricola Barnés-Guirado, Cano and Stchigel, sp. nov. MycoBank MB850890 Figure 5.
Etymology. From Latin terra-, soil, and -colere, living in, because of the (mostly) geophilic habit of the fungus.
Description: Mycelium superficial to immersed, composed of hyaline to brown, septate, smooth-walled to rugose (due to the external deposition of melanin-like pigments), thin- to thick-walled hyphae 2–9 µm wide, often anastomosing and grouped to form rope-like dark-brown structures. Sexual state — Ascomata erumpent, dark brown, non-ostiolate, setose, globose to subglobose, 130–260 µm diam.; peridial wall brown to dark brown, translucent, 2–3-layered, 5–8 µm thick, of textura angularis in upper view, composed by flattened, brown polygonal cells of 4–12 µm diam., covered by an irregular net of dark brown, septate, smooth-walled to tuberculate anastomosing hyphae; setae scarce, distributed irregularly on the surface of the ascomata, septate, brown at the base but becoming subhyaline to hyaline at the rounded apex, sinuous, up to 7 µm wide at the base, up to 100 µm long, and covered by a gelatinous sheath then becoming tuberculate with the age. Asci 8-spored, bitunicate, cylindric-clavate, 38–48 × 4.5–7.5 µm, non-stipitate, without apical apparatuses. Paraphyses hyaline, septate, unbranched, 40–55 × 2 µm. Ascospores are two-celled, non-constricted to slightly constricted at the broad median septum, smooth- and thick-walled, chocolate brown when mature, broadly fusiform to biconical or bicampanulate, 9.5–12 × 5 µm, without appendages nor gelatinous sheath; aberrant ascospores seen in culture, one-celled, brown, smooth- to rough-walled, globose to ellipsoidal, and 5–7 µm diam. when globose, similar in size to the “normal” ones when ellipsoidal. Asexual state—not observed.
Culture characteristics (14 days at 25 °C)—Colonies on PDA reach 83 mm diam., slightly raised at center flattened at the edges, cottony, smooth, white (1A1) at center, grey (4B1) towards periphery, filamentous margins, sporulation absent; reverse yellowish orange (4A6) to pale yellow (4A3) at center, greyish yellow (4B3) to pale yellow (4A3) at periphery, and diffusible pigment absent. Colonies on PCA reach 82 mm diam., slightly raised at center flattened at the edges, cottony to velvety, smooth, white (1A1) to greyish brown (5E3) at center, olive brown (4F8) to grey (4B1) towards periphery, filamentous margins, sporulation moderate to abundant, reverse, yellowish-brown (5F8) at center, olive brown (4F3) to grey (4B1) at towards periphery, and diffusible pigment absent. Colonies on OA reach 80 mm diam., flattened and immersed hyphae towards periphery, cottony to velvety, smooth, white (1A1) to olive brown (4F3) at center, olive brown (4E4) to grey (4B1) at the edges, entire, sporulation moderate; reverse brownish grey (4F2) at center, olive brown (4E4) to grey (4B1) at the edges, and diffusible pigment absent. Colonies on MEA reach 75 mm diam., slightly raised at center and flattened at the edges, cottony to velvety, smooth, white (1A1) at center, grey (4B1) to brownish grey (4D2) towards periphery, filamentous margins, sporulation absent; reverse yellowish orange (4A6) at center, olive brown (4D3) to grey (4B1) at the edges, and diffusible pigment absent. Cardinal growth temperatures: minimum of 15 °C, an optimum of 30 °C, and a maximum of 40 °C.
Specimen: MALAWI, Southern Region Mulanje Dist., Mulanje Mountain Chamble Basin, track to Knife Edge, 15°55′ S 35°34′ E, 2100 m.a.s.l., isolated from soil, 13/04/1991, collected and isolated by J.C. Krug, holotype TRTC 51988, and cultures ex-type CBS 100256 = ATCC 201452 = FMR 19807.
Other specimens: TRTC 51987 = CBS 100254 = ATCC 201451 = FMR 19806, Egypt, Western Desert, Wadid Gedeed Governorate, Dakleh Oasis, Ein Birbiyeh, 25°32′ N 29°19′ E, isolated from soil near an irrigation canal, collected and isolated by J.C. Krug; TRTC 51989 = CBS 100394 = ATCC 201650 = FMR 19805, N-exposed cliff, Tunisia, 5 km NW of Ksar Haddada, NW from Tatahouine 470 m.a.s.l., soil attached to rhizoids of Grimmia orbicularis, collected and isolated by J.C. Krug; CBS 150901 = FMR 19580, Spain, Aragon Community, Zaragoza province, La Playa lagoon, 41°25′16.2″ N 0°11′18.4″ W, isolated from lagoon sediment, 14/05/2021, collected by María Barnés, Alan O. Granados and José F. Cano, and isolated by María Barnés.
Notes: All four strains of Montagnula terricola produce globose to subglobose (150–230 µm diam.), dark brown, setose, thin-walled, non-ostiolate ascomata, non-stipitate cylindric-clavate (43–50 × 6–10 µm) asci, and smooth-walled ascospores, whereas Montagnula striatispora produces a globose to pear-shaped (340–600 µm diam.), black, glabrous, very thick-walled, ostiolate ascomata, clavate (62–90 × 9–12 µm) asci, and ascospores ornamented with longitudinal ridges. However, it is remarkable that both species produce true paraphyses, not as was described for most of the species of the genus, which produce pseudoparaphyses.
Montagnula globospora Barnés-Guirado, Cano and Stchigel, sp. nov. MycoBank. MB 854553 Figure 6.
Etymology From the Latin globosus-, globose, and -sporae, spore, because of the nature of their ascospores.
Description: Mycelium superficial composed of hyphae hyaline to subhyaline, septate, thick-walled, branched, 1.0–2.5 µm wide. Sexual state—Ascomata cleistothecial, dark brown to black, subglobose, 220–385 × 230–395 µm; peridium 5–15 µm thick, outer wall 1–5-layered, composed of pale golden brown, smooth- and thick-walled, translucent prismatic cells, 5–15 µm in the main axis, inner wall 1–4-layered, composed of dark brown, smooth- and thick-walled, slightly translucent flattened prismatic cells, 5–15 µm in the main axis, cephalothecoid, composed of dark brown polygonal plates of 25–60 µm diam., separating by preformed paler sutures dark brown to black, and covered by subhyaline septate hyphae. Paraphyses hyaline is septate, unbranched, 45–55 × 1–1.5 µm. Asci bitunicate, 5 to 8-spored, ascospores uni- to biseriate, cylindric clavate to clavate, 105–135 × 24–34 µm, and without apical apparatuses. Ascospores one-celled, dark brown, very thick-walled, granulate, globose to subglobose, 9.5–19.5 × 9.5–17 µm, and guttulate. Asexual state—Conidiomata pycnidial, mostly unilocular, occasionally bilocular, clustering or more rarely solitary, pale brown to golden brown, becoming paler towards the apex, globose, broadly ellipsoidal, rarely lenticular, 20–40 µm diam, opening by a late dehiscence at the upper part of the peridium; peridium translucent, with a few digitiform projections, 1–2-layered, 4–7 µm thick, textura angularis, composed of smooth- and thin-walled, flattened polygonal cells up to 5 µm diam.; conidiogenous cells phialidic, globose, 1–1.5 × 2–3 µm, lacking a distinctive collarette or neck; conidia enteroblastic, unicellular, solitary, hyaline, cylindric, 2.5–4 × 1–2 µm, and produced in mass into a hyaline mucilaginous matrix.
Culture characteristics (after 14 d at 25 °C)—Colonies on PDA reaching 85 mm diam., raised at the center, flattened at the margins, cottony to velvety, radially sulcate at the center, smooth at the margins, white (1A1) to yellowish brown (5E8) at the center, white (1A1) to light brown (5D6) at the filamentous margins, sporulation abundant; reverse brown (7E8) at center, golden brown (5D7) at the margins; soluble pigment absent. Colonies on PCA reaching 85 mm diam., raised at the center, flattened at the entire margins, velvety, smooth, pale red (12A3), sporulation abundant; reverse reddish-white (12A2); and soluble pigment absent. Colonies on the OA reach 85 mm diam., flattened velvety, smooth, hyaline, margins filamentous, sporulation abundant; reverse hyaline; and soluble pigment absent. Colonies on MEA reach 82 mm diam., flat, velvety, smooth, hyaline, margins filamentous, sporulation abundant; reverse hyaline; and soluble pigment absent. Cardinal temperatures of growth: minimum of 15 °C, an optimum of 30 °C, and a maximum of 37 °C.
Specimen: FMR 20550 = CBS 152803. SPAIN, Aragon community, Zaragoza province, Salada Grande de Chiprana, 41°14′19.2″ N 0°10′49.6″ W, isolated from lagoon sediment, 11 July 2022, collected by María Barnés, Alan O. Granados and José F. Cano, and isolated by María Barnés, holotype CBS H-25643.
Notes, Montagnula globospora differs from all previously known species by the production of cleistothecioid ascomata with a cephalothecoid peridium, one-celled, granulate ascospores, and by the presence of a pycnidial asexual state. Also, in addition to M. terricola and M. stratispora, M globospora forms true paraphyses.
Because the description of the genus Montagnula does not include species with cleistothecial ascomata nor unicellular ascospores, thus, we emended the generic description as follows:
Montagnula Berl. emended by Barnés-Guirado, Cano, and Stchigel.
It is saprobic on dead wood, branches, stems, bark, leaves, freshwater, soil, and sediments of hypersaline lagoons. Sexual state is—Ascomata usually ostiolate (perithecioid), occasionally non-ostiolate (cleistothecioid), scattered singly or beneath a blackened clypeus, or within a stromatic development of hyphae, peridium mostly thick-walled and composed several layers of flattened cells, occasionally thin-walled (when cleisthotheciod), and very rarely cephalothecoid. Hamathecium mostly composed of cellular, branched, septate, numerous pseudoparaphyses, and is occasionally paraphysate. Asci mostly 8-spored, uni- to biseriate, bitunicate, clavate to elongate-clavate or cylindrical, sometimes with a minute ocular chamber at the top, wall somewhat thickened, and tapered below or with a somewhat elongated stalk. Ascospores rarely unicellular, then globose, mostly multicellular, then ellipsoidal, bi-cupulate, fusoid or fusiform and with one or few transverse septa, medial septum mostly constricted, or muriform, then with one or more longitudinal septa, outer wall smooth-walled, papillate–tuberculate, verruculose, verrucose, or with longitudinal striations, surrounded or not by a mucilaginous sheath, and very rarely with polar appendages. Asexual state is — coelomycetous and pycnidial.
A dichotomous key to the accepted species of Montagnula is provided as Supplementary Material (S1).
Given that Montagnula jonesii was positioned in our phylogenetic analysis distantly from the Montagnula spp. main clade and exhibits a phylogenetic distance comparable to one of the coelomycetous genus Neptunomyces, we propose reclassifying this fungus into the new genus Neomontagnula, as described below.
Neomontagnula Barnés-Guirado, Stchigel and Cano, gen. nov. MycoBank MB 854535.
Etymology: From Latin neo-, new, because of the phylogenetic and phenotypic relationship with the genus Montagnula.
Description: Sexual state—Ascomata solitary, scattered to clustered, immersed or erumpent on the host tissue, globose to subglobose, glabrous, uniloculate, and ostiolate. Peridium composed of two layers of pseudoparenchymatous cells, and the outer layer is arranged in a textura angularis. Pseudoparaphyses distinctly septate, not constricted at the septa, and anastomosing at the apex. Asci 8-spored, bitunicate, fissitunicate, clavate, long pedicellate, and apically rounded, with an ocular chamber. Ascospores are overlapping, 1–2-seriate, brown to reddish brown at maturity, transversally 3-septate when mature, fusiform, with rounded ends, constricted at the septa, straight to curved, enlarged at the second cell from the apex, smooth-walled, and guttulated. Asexual state—unknown.
Type species: Neomontagnula jonesii (Tennakoon, Hyde, Wanasinghe, Bahkali, Camporesi, Khan and Phookamsak 2016) Barnés-Guirado, Stchigel and Cano, comb. nov. MycoBank MB 854536.
Description: Tennakoon, Hyde, Wanasinghe, Bahkali, Camporesi, Khan, and Phookamsak (2016).
Notes: Neomontagnula jonesii produces reddish-brown, transversally 3-septate ascospores, such as in M. aloes, M. camporesii, M. cirsii, M. scabiosae, and M. shangrilana. However, the ascospores of N. jonesii are smaller (14–16 × 5–6 μm) than in the previously consigned species of Montagnula (33–36 × 13–14 μm in M. aloes; 18–25 × 5–8 μm in M. camporesii; 18–23.5 × 6.5–9.5 μm in M. cirsii; 20–23 × 7–9 μm in M. scabiosae; and 48–60 × 17–22 µm in M. shangrilana) and have an enlarged second cell from the apex [66,67,68]. Montagnula aquatica also produces four-celled ascospores. But, these are dark brown when mature, and their ascomata are globose (lenticular in N. johnesii) and smaller (140–210 μm diam. vs. 250–340 × 250–430 μm). Neomontagnula jonesii differs from the closest genus Neptunomyces by having a sexual state and lacking an asexual coelomycetous state, exactly the opposite of Neptunomyces [69].
Xylariales Nannf., Nova Acta R. Soc. Scient. upsal., Ser. 4 8 (no. 2): 66 (1932). Mycobank MB 90505.
Diatrypaceae Nitschke [as ’Diatrypeae’], Verh. naturh. Ver. preuss. Rheinl. 26: 73 (1869). Mycobank MB 80692.
Monosporascus Pollack and Uecker, Mycologia 66(2): 348 (1974). Mycobank MB 3260.
Monosporascus auratispora Barnés-Guirado, Cano and Stchigel, sp. nov. MycoBank. MB 84809 Figure 7.
Etymology: From Latin auratus-, golden, -sporae, spores, because of the color of the ascospores under the microscope.
Description: Mycelium superficial to immersed and composed of hyphae hyaline to pale brown, septate, smooth- and thick-walled, branched, and 1.0 µm wide. Sexual state — Ascomata immersed to semi-immersed, scattered, pale to dark brown, translucent, non-ostiolate, globose, and 350–550 µm diam. Peridial wall subhyaline to brown, translucent, tomentose, 8 to 14-layered, texture epidermoid to angularis, 25–35 µm thick, and consisting of pale yellow to pale brown, thin-walled cells. Asci one-to-5-spored (mostly 2–3 spored), fasciculate, short stipitate, clavate to subcylindrical, 35–90 × 15–30 µm, and rounded at the apex, with no apical apparatuses. Ascospores are one-celled, hyaline when young, becoming brown at maturity, very thick-walled, three-layered, smooth-walled to slightly granulose, globose, and 20–30 µm diam., without germ pores. Asexual state — not observed.
Culture characteristics (14 days at 25 °C) Colonies on PDA reach 85 mm diam., flattened, cottony, round, hyaline, and white (1A1) at the center to white (1A1) and hyaline at the margins, filamentous margins, sporulation absent; reverse white (1A1), and soluble pigment absent. Colonies on PCA and OA reach 85 mm diam., flattened, velvety, filamentous, hyaline, sporulation absent; reverse hyaline, and soluble pigment absent. Colonies on MEA reach 85 mm diam., flattened, velvety, filamentous, hyaline at the center, white (1A1) at the margins, sporulation absent; reverse hyaline at the center, white (1A1) at the margins, and soluble pigment absent. The cardinal temperatures of growth are a minimum of 15 °C, an optimum of 30 °C, and a maximum of 37 °C.
Specimen: SPAIN, Aragon Community, Zaragoza province, Salada Grande de Chiprana, 41°14′19.2″ N 0°10′49.6″ W, isolated from lagoon sediment, 11/07/2022, collected by María Barnés, Alan O. Granados and José F. Cano, isolated by María Barnés, holotype CBS H-25251, and culture ex-type FMR 20333 = CBS 149967.
Notes: Monosporascus auratispora can be distinguished from its phylogenetically closest species, Monosporascus ibericus, by producing smaller ascomata (350–550 µm diam. vs. 400–700 µm diam) and asci (35–90 × 15–30 µm compared to 100–240 × 32–72 µm). Additionally, the asci of Mo. auratispora lacks apical structures, whereas those of Mo. ibericus exhibit a broad distinct apical ring.
A dichotomous key to the accepted species of the genus Montagnula is provided in the Supplementary Material (S2).
4. Discussion
The family Didymosphaeriaceae was established by Munk in 1953 to accommodate the genus Didymosphaeria [70]. Nowadays, there are 33 accepted genera in this family, with most of these reported as saprobes, phytopathogens, and endophytes of a broad diversity of living plants. But, some of them are also pathogens for animals and even humans [67,71]. The genus Montagnula is typified by M. infernalis (no molecular data available) and was established in 1896 by Berlese [72]. This genus has 47 species according to the Index Fungorum [
The family Diatrypaceae was established by Nitschke in 1869, with Diatrype designated as the type of genus [77]. Members of Diatrypaceae are globally distributed and encompass a diverse range of ecological roles, including saprobic species, endophytes, and significant plant pathogens in both terrestrial and marine biomes [78]. Nowadays, 26 genera are accepted in this family [79]. The genus Monosporascus has twelve species accepted according to the Index Fungorum [
5. Conclusions
Hypersaline endorheic lagoons, such as those examined in this study, represent rare, small, and fragile ecological niches of considerable intrinsic value. Due to their unique characteristics, these ecosystems are highly vulnerable to the effects of global warming and anthropogenic pressures. They have a diverse array of endemic organisms that have evolved to thrive under the extreme physicochemical conditions of these habitats. Thus, the need for their conservation is both capital and urgent. Despite their ecological importance, the fungal biota of hypersaline endorheic lagoons and lakes in Europe remains largely uncharted, primarily due to limited research in these specialized environments. This study addresses this gap by contributing critical insights into the mycobiota of these threatened habitats, including the identification and description of three novel ascomycete species: Montagnula terricola, Montagnula globospora, and Monosporascus auratispora. These species, all of them characterized by the production of cleistothecial ascomata, are specifically adapted to the extreme environmental conditions of these lagoons.
Conceptualization, A.M.S. and J.F.C.-L.; methodology, M.B.-G., A.M.S. and J.F.C.-L.; software, M.B.-G. and J.F.C.-L.; validation, A.M.S. and J.F.C.-L.; formal analysis, M.B.-G., A.M.S. and J.F.C.-L.; investigation, M.B.-G.; resources, J.F.C.-L.; data curation, M.B.-G.; writing—original draft preparation, M.B.-G.; writing—review and editing, M.B.-G., A.M.S., A.N.M. and J.F.C.-L.; visualization, M.B.-G., A.M.S. and J.F.C.-L.; supervision, A.M.S. and J.F.C.-L.; project administration, J.F.C.-L.; funding acquisition, J.F.C.-L. All authors have read and agreed to the published version of the manuscript.
Not applicable.
Not applicable.
Data are contained within the article.
To Alan Omar Granados, University Rovira i Virgili, Medical School, Mycology Unit, C/Sant Llorenç 21, 43201 Reus, Tarragona, Spain, for his participation in the different samplings carried out. To Simona Margaritescu, TRTC Collection, and Jean-Marc Moncalvo, Curator of Fungi and Deputy Head, Department of Natural History, Royal Ontario Museum (CA), for their kind help locating the herbarium specimens of ‘Aporospora terricola’ and some clarifications about its history. To Trix Merkx, technician, and Gerard Verkleij, Curator of the Fungal and Yeast Collection (CBS), Westerdijk Fungal Biodiversity Institute (The Netherlands), for providing several of the strains employed in the present work. To Margarita I. Hernández-Restrepo, Post-Doctoral Researcher, Westerdijk Fungal Biodiversity Institute, for providing relevant information for this study. And to Konstanze Bensch, Mycobank Curator, Westerdijk Fungal Biodiversity Institute, for your inestimable assistance in fungal nomenclature. M.B.-G. is grateful to the University Rovira i Virgili and Diputació de Tarragona for a Martí-Franqués doctoral grant.
The authors declare no conflicts of interest.
The following abbreviations are used in this manuscript:
| AA | Ascospore Agar |
| BI | Bayesian Inference |
| BLAST | Basic Local Alignment Search Tool |
| BRIP | The Plant Pathology Herbarium |
| BS | Bootstrap Support |
| CBS | Westerdijk Fungal Biodiversity Institute |
| CGMCC | China General Microbiological Culture Collection Center |
| CMG | Culture collection of Mark Gleason |
| CMM | Culture Collection of Phytopathogenic Fungi Prof. Maria Menezes |
| CPC | Culture Collection of P.W. Crous |
| FMR | Faculty of Medicine of Reus |
| G18 | 18% of Glycerol Agar |
| GUCC | Culture Collection at Department of Plant Pathology, Agriculture College, Guizhou University |
| HKAS | Herbarium of the Kunming Institute of Botany |
| HVVV | Personal collection of Wayne Pitt from Vitis vinifera |
| ITS | Internal Transcribed Spacers |
| KUMCC/KUNCC | Kunming Institute of Botany Culture Collection Kunming |
| LSU | D1-D2 domains of the 28S nrRNA |
| M. | Montagnula |
| MEA | 2% Malt Extract Agar |
| MEGA | Molecular Evolutionary Genetics Analysis |
| MFLU | Herbarium of the Mae Fah Luang University |
| MFLUCC | Culture Collection of the Mae Fah Luang University |
| ML | Maximum Likelihood |
| MLI | Maximum Level of Identity |
| Mo. | Monosporascus |
| NCY-UCC | National Chiayi University Culture Collection |
| NTUCC | National Taiwan University Culture Collection |
| OA | Oatmeal Agar |
| PCA | Potato–Carrot Agar |
| PDA | Potato–Dextrose Agar |
| PP | Posterior Probability |
| TEF-1α | Fragments of the Translation Elongation Factor 1α |
| tub2 | Beta-tubulin gene |
| UESTCC | University of Electronic Science and Technology Culture Collection |
Footnotes
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Enlarge this image.Figure 1. Sampled locations (https://www.ign.es/iberpix/visor; accessed on 19 March 2024).
Enlarge this image.Figure 2. Phylogenetic analysis of members of the family Didymosphaeriaceae was conducted using ITS, LSU, and TEF-1α nucleotide sequences. RA × ML bootstrap support (BS) values ≥70% and Bayesian posterior probabilities (PP) ≥0.95 are shown above the branches. Branches with 100% BS/1 PP are indicated as broad lines. Novel genus, species, and combinations are indicated in bold type. The tree is rooted to Periconia didymosporum MFLU 15-0057 and Periconia alishanica MFLUCC 19-0145. T = Ex-type strains.
Enlarge this image.Figure 3. Phylogenetic analysis of members of Monosporascus using the molecular markers ITS, LSU, tub2, and TEF-1α. RA × ML bootstrap support (BS) values ≥70% and Bayesian posterior probabilities (PP) ≥0.95 are displayed above the branches. Branches with 100% BS/1 PP are indicated as broad lines. Novel species is indicated in bold; type species in red. Tree rooted to Eutypa camelliae HKAS 107022 and Eutypa petrakii CBS 285.87. T = Ex-type strain.
Enlarge this image.Figure 4. Montagnula striatispora ILL00000264 (isotype of Papendorf 83). (A). Ascoma, cross-section. (B,C). Hymenium, showing young and mature asci within ascospores and paraphyses. (D–G). Mature, superficially striated ascospores. Scale bars: (A) = 50 µm; (B,C) = 20 µm; (D–G) = 10 µm.
Enlarge this image.Figure 5. Montagnula terricola CBS 100256T. (A–D). Colonies on PCA, OA, MEA, and PDA, respectively, after two weeks at 25 ± 1 °C (left, surface; right, reverse). (E). Ascoma. (F). Bitunicate asci (young, black arrows) and paraphyses (white arrows). (G). Mature ascospores. Scale bars: (E–G) = 10 µm.
Enlarge this image.Figure 6. Montagnula globospora CBS 152803T. (A–D). Colonies on PCA, OA, MEA, and PDA, respectively, after two weeks at 25 ± 1 °C (left, surface; right, reverse). (E). Broken ascoma. (F). Detail of the cephalothecoid peridium. (G,H). Young asci and paraphysis (white arrows). (I). Mature asci within ascospores. (J). Mature ascospores, most in cross-section. (K). Pycnidium. (L). Mature pycnidia (Cotton Blue stained). Scale bars: (E) = 50 µm; (F–L) = 10 µm.
Enlarge this image.Figure 7. Monosporascus auratispora CBS 149967T. (A–D). Colonies on PCA, OA, MEA, and PDA, respectively, after two weeks at 25 ± 1 °C (left, surface; right, reverse). (E). Broken ascoma. (F). Hymenium (paraphyses, white arrows). (G). Mature, 3-spored ascus. (H). Asci and free ascospores. Scale bars: (E) = 100 µm; (F–H) = 10 µm.
Taxa and GenBank accession numbers of the molecular markers used in the phylogenetic analysis.
| Taxon | Strain Number | GenBank Accession Number | |||
|---|---|---|---|---|---|
| ITS | LSU | TEF-1α | tub2 | ||
| Alloconiothyrium camelliae | NTUCC 17-032-1T* | MT112294 | MT071270 | MT232967 | |
| Alloconiothyrium aptrootii | CBS 980.95T | JX496121 | JX496234 | ||
| Austropleospora osteospermi | BRIP51628T | FJ481946 | |||
| Chromolaenicola nanensis | MFLUCC 17-1473T | MN325015 | MN325003 | ||
| Chromolaenicola thailandensis | MFLUCC 17-1510T | MN325018 | MN325006 | MN335651 | |
| Cylindroaseptospora leucaenae | MFLUCC 17-2424T | NR_163333 | NG_066310 | MK360047 | |
| Didymosphaeria rubi-ulmifolii | MFLUCC 14-0023T | KJ436586 | |||
| Eutypa camelliae | HKAS 107022T | NR_175674 | NG_081500 | ||
| Eutypa petrakii | CBS 285.87 | MH862077 | MH873766 | ||
| Kalmusia ebuli | CBS 123120T | KF796674 | NG_070920 | ||
| Kalmusia longispora | CBS 582.83T | MH861658 | NG_070449 | ||
| Kalmusibambusa triseptata | MFLUCC 13-0232 | KY682697 | KY682695 | ||
| Karstenula lancangensis | KUMCC 21-0670T | OP058969 | OP059060 | ||
| Karstenula rhodostoma | CBS 691.94 | LC014559 | AB807531 | AB808506 | |
| Monosporascus auratispora sp. nov. | FMR 20333T | PP973385 | PP973719 | PP973931 | PP973383 |
| Monosporascus brasiliensis | CMM-4839T | MG735234 | MG748803 | MG720040 | MG725317 |
| CMM-4840 | MG735235 | MG748804 | MG720041 | MG725318 | |
| Monosporascus bulgaricus | CBS 151406T | KT269184 | PP454707 | PP460994 | |
| P1811 | KT269083 | ||||
| Monosporascus caatinguensis | CMM-4833T | MG735228 | MG748797 | MG720034 | MG725311 |
| CMM-4834 | MG735229 | MG748798 | MG720035 | MG725312 | |
| Monosporascus cannonballus | CBS 609.92T | NR_111370 | |||
| MC0603 | JQ762364 | MG748824 | JQ907314 | JQ907307 | |
| Monosporascus europaeus | CBS 150022T | KT269082 | PP454705 | PP481183 | |
| P1889 | KT269158 | PP454706 | |||
| Monosporascus eutypoides | MT45T | JQ958963 | MG748827 | JQ958959 | JQ973834 |
| MT47 | JQ958964 | JQ958956 | |||
| Monosporascus ibericus | CBS 110550T | JQ973832 | JQ958958 | JQ958958 | JQ973833 |
| Monosporascus mossoroensis | CMM-4857T | MG735252 | MG720058 | MG720058 | MG725335 |
| CMM-4858 | MG735253 | MG720059 | MG720059 | MG725336 | |
| Monosporascus nordestinus | CMM-4846T | MG735241 | MG720047 | MG720047 | MG725324 |
| CMM-4847 | MG735242 | MG720048 | MG720048 | MG725325 | |
| Monosporascus solitarius | CBS 150023T | KT269777 | PP454708 | ||
| Monosporascus semiaridus | CMM-4830T | MG735222 | MG748791 | MG720028 | MG725305 |
| CMM-4831 | MG735223 | MG748792 | MG720029 | MG725306 | |
| Montagnula acaciae | MFLUCC 18-1636 | ON117280 | ON117298 | ||
| NCYUCC 19-0087T | ON117281 | ON117299 | |||
| Montagnula aloes | CBS 132531T | NR_111757 | NG_042676 | ||
| Montagnula appendiculata | CBS 109027T | DQ435529 | AY772016 | ||
| Montagnula aquatica | MFLU 22-0171T | OP605992 | OP605986 | ||
| KUNCC 23-14425 | OR583097 | OR583116 | OR588088 | ||
| Montagnula aquilariae | KUNCC 22-10815T | OP452927 | OP482265 | OP426318 | |
| KUNCC 22-10816 | OP554219 | OP482266 | OP426319 | ||
| Montagnula bellevaliae | MFLUCC 14-0924T | KT443906 | KT443902 | ||
| Montagnula camporesii | MFLUCC 16-1369T | MN401746 | NG_070946 | MN397908 | |
| Montagnula chiangraiensis | MFLUCC 17-1420T | NR_168864 | NG_068707 | ||
| Montagnula chromolaenae | MFLUCC 17-1435T | NR_168865 | NG_068708 | ||
| Montagnula cirsii | MFLUCC 13-0680 | KX274242 | KX274249 | KX284707 | |
| Montagnula cylindrospora | CBS 146572T | LT796834 | LN907351 | LT797074 | |
| Montagnula donacina | HVVV01 | KJ628375 | KJ628377 | ||
| MFLUCC 17-1469 | NR_168866 | NG_070948 | MT235773 | ||
| Montagnula graminícola | MFLUCC 13-0352T | KM658314 | KM658315 | ||
| Montagnula guiyangensis | HKAS 124556T | OP605989 | OP600484 | ||
| GUCC 22–0817 | OP605990 | OP600485 | |||
| Montagnula krabiensis | MFLUCC 16-0250T | NR168179 | NG068826 | MH412776 | |
| Montagnula lijiangensis | HKAS 126540 | OR583107 | OR583126 | OR588098 | |
| HKAS 126541T | OR583108 | OR583127 | OR588099 | ||
| Montagnula menglaensis | KUNCC 23-14424T | OR583111 | OR583130 | OR588102 | |
| KUNCC 23-14423 | OR583110 | OR583129 | OR588101 | ||
| Montagnula opulenta | CBS 168.34 | DQ678086 | DQ678086 | ||
| Montagnula scabiosae | MFLUCC 14-0954T | NR_155378 | NG_059602 | ||
| Montagnula shangrilana | KUNCC 23-14433 | OR583112 | OR583131 | OR588103 | |
| KUNCC 23-14434T | OR583113 | OR583132 | OR588104 | ||
| Montagnula striatispora | CBS 385.65T | MH858624 | NG_064048 | ||
| Montagnula terricola sp.nov. | FMR 19580 | OQ708962 | PP973381 | PP973930 | |
| CBS 100254 | OQ708960 | PP973379 | PP973928 | ||
| CBS 100256T | OQ708961 | PP973380 | PP973929 | ||
| CBS 100394 | OQ708959 | PP973378 | PP973927 | ||
| Montagnula thevetiae | HKAS 126963 | OR583114 | OR583133 | OR588105 | |
| HKAS 126964T | OR583115 | OR583134 | OR588106 | ||
| Montagnula globospora sp.nov. | FMR 20550T | PP973384 | PP973382 | PP973931 | |
| Montagnula verniciae | CGMCC 3.24435T | OR269139 | OR253273 | OR251166 | |
| UESTCC 23.0029 | OR269140 | OR253274 | OR251167 | ||
| Neokalmusia brevispora | CBS 120248 | MH863078 | MH874633 | ||
| Neokalmusia jonahhulmei | KUMCC 21-0818T | ON007043 | ON007039 | ON009133 | |
| Neomontagnula jonesii gen. nov. | MFLUCC 16-1448T | KY313619 | KY273276 | KY313620 | |
| MFLU 18-0084 | ON117282 | ON117300 | ON158095 | ||
| Neptunomyces aureus | CMG10AT | MK912119 | MK947998 | ||
| CMG13 | MK912122 | MK948001 | |||
| Neptunomyces litoralis | BRIP 75555aT | OR271911 | NG_242141 | ||
| Paracamarosporium psoraleae | CPC 21632T | KF777143 | KF777199 | ||
| Paracamarosporium hawaiiense | CBS 120025T | NR_154287 | NG_070608 | ||
| Paraconiothyrium estuarinum | CBS 109850T | NR_166007 | MH874432 | ||
| Paramassariosphaeria anthostomoides | CBS 615.86 | MH862005 | MH873693 | ||
| Paramassariosphaeria clematidicola | MFLUCC 16-0172 | KU743206 | KU743207 | ||
| Paraphaeosphaeria michotii | CBS 591.73 | MH860778 | GU456326 | GU456267 | |
| Paraphaeosphaeria rosae | MFLUCC 17-2547T | MG828935 | MG829044 | MG829222 | |
| Periconia didymosporum | MFLU 15-0057T | NR_176693 | NG_081448 | KP761727 | |
| Periconia alishanica | MFLUCC 19-0145T | MW063165 | MW063229 | MW183790 | |
| Pseudocamarosporium piceae | MFLUCC 14-0192T | KJ747046 | KJ803030 | ||
| Pseudocamarosporium propinquum | MFLUCC 13-0544T | NR_154309 | NG_069202 | ||
| Septofusispora thailandica | MFLU 22-0043T | OP058971 | OP059062 | OP135945 | |
| Verrucoconiothyrium nitidae | CBS 119209T | EU552112 | EU552112 | ||
| Xenocamarosporium acaciae | CBS 139895T | NR_137982 | NG_058163 | ||
T* = Ex-type strains. FMR: Facultad de Medicina y Ciencias de la Salud, Reus, Spain; CBS: culture collection of the Westerdijk Fungal Biodiversity Institute, Utrecht, The Netherlands; MFLUCC: culture collection of the Mae Fah Luang University, Chiang Rai, Thailand; MFLU: herbarium of the Mae Fah Luang University, Chiang Rai, Thailand; NTUCC: National Taiwan University Culture Collection, Taipei. En Taiwan; BRIP: The Plant Pathology Herbarium, Queensland, Australia; HKAS: herbarium of the Kunming Institute of Botany, Kunming, China; KUMCC/KUNCC: Kunming Institute of Botany Culture Collection Kunming, China; CMM: culture collection of phytopathogenic fungi Prof. Maria Menezes, Dois Irmaos, Brazil; NCYUCC: National Chiayi University Culture Collection, Chiayi City, Taiwan; HVVV: personal collection of Wayne Pitt from Vitis vinifera, Bathurst, Australia; GUCC: culture collection at Department of Plant Pathology, Agriculture College, Guizhou University, Guizhou, China; CGMCC: China General Microbiological Culture Collection Center, Beijing, China; UESTCC: University of Electronic Science and Technology Culture Collection, Chengdu, China; CMG: culture collection of Mark Gleason, Ames, USA; CPC: culture collection of P.W. Crous, Utrecht, The Netherlands. In bold, sequences generated in this study.
BLAST results for our strains FMR 19580, FMR 20550, and FMR 20333.
| Strain | Molecular Marker | Closest Species | Identity | GenBank | Identities |
|---|---|---|---|---|---|
| FMR 19580 | ITS | Aporospora terricola * | 99.78% | AF049088.1 | 446/447 (no gaps) |
| Herpotrichia striatispora CBS 385.65 | 98.43% | MH858624.1 | 440/447 (no gaps) | ||
| LSU | Herpotrichia striatispora CBS 385.65 | 99.50% | NG_064048.1 | 796/800 (no gaps) | |
| Montagnula cylindrospora UTHSC DI16-208 | 98.75% | LN907351.1 | 790/800 (4 gaps) | ||
| Montagnula bellevaliae MFLUCC 14-0924 | 98.50% | NG_059601.1 | 788/800 (2 gaps) | ||
| TEF-1α | Montagnula donacina MFLU 22-0046 | 95.91% | OP135938.1 | 820/855 (no gaps) | |
| Montagnula puerensis KUMCC:20-0225 | 95.49% | MW573959.1 | 763/799 (no gaps) | ||
| FMR 20550 | ITS | Herpotrichia striatispora CBS 385.65 | 96.42% | MH858624.1 | 431/447 (7 gaps) |
| Montagnula terricola FMR 19580 | 96.42% | OQ708962.1 | 431/447 (7 gaps) | ||
| LSU | Herpotrichia striatispora CBS 385.65 | 98.26% | NG_064048.1 | 791/805 (6 gaps) | |
| Montagnula cirsii MFLUCC:13-0680 | 97.86% | KX274249.1 | 787/804 (6 gaps) | ||
| TEF-1α | Montagnula donacina MFLUCC:22-0046 | 96.23% | OP135938.1 | 842/875 (no gaps) | |
| Montagnula chromolaenicola MFLUCC:17-1469 | 96.03% | MT235773 | 846/881 (no gaps) | ||
| FMR 20333 | ITS | Monosporascus ibericus CBS 110550 | 96.60% | JQ973832 | 454/470 (5 gaps) |
| LSU | Monosporascus caatinguensis CMM-4832 | 97.37% | MG748796 | 667/685 (5 gaps) | |
| Monosporascus eutypoides CBS:132472 | 96.93% | MH877468 | 664/685 (8 gaps) | ||
| tub2 | Monosporascus ibericus CBS 110550 | 98.62% | JQ973833 | 502/509 (1 gap) | |
| Monosporascus ibericus CBS 110550 | 93.27% | JQ958958 | 554/594 (6 gaps) |
* non-legitimate name; author’s note.
Supplementary Materials
The following supporting information can be downloaded at
References
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; Miller, Andrew N 2
; Stchigel, Alberto M 1
1 Mycology Unit, Medical School, University Rovira i Virgili, C/Sant Llorenç 21, 43201 Tarragona, Spain;
2 Illinois Natural History Survey, University of Illinois Urbana-Champaign, 1816 South Oak Street, Champaign, IL 61820-6970, USA;