Diversity of Ascomycota in Jilin: Introducing Novel Woody Litter Taxa in Cucurbitariaceae

Abstract

Cucurbitariaceae has a high biodiversity worldwide on various hosts and is distributed in tropical and temperate regions. Woody litters collected in Changchun, Jilin Province, China, revealed a distinct collection of fungi in the family Cucurbitariaceae based on morphological and molecular data. Phylogenetic analyses of the concatenated matrix of the internal transcribed spacer (ITS) region, the large subunit (LSU) of ribosomal DNA, the RNA polymerase II subunit (rpb2), the translation elongation factor 1-alpha (tef1-α) and β-tubulin (β-tub) genes indicated that the isolates represent Allocucurbitaria and Parafenestella species based on maximum likelihood (ML), maximum parsimony (MP) and Bayesian analysis (BPP). We report four novel species: Allocucurbitaria mori, Parafenestella changchunensis, P. ulmi and P. ulmicola. The importance of five DNA markers for species-level identification in Cucurbitariaceae was determined by Assemble Species by Automatic Partitioning (ASAP) analyses. The protein-coding gene β-tub is determined to be the best marker for species level identification in Cucurbitariaceae.

1. Introduction

Fungi are known to have a high diversity; however, the number of named and classified fungi is still lower than the estimated number of species [1,2,3,4]. This could be because several regions are yet to be explored. China is the third largest country in the world by area, with several different climatic conditions [5,6,7,8]. Jilin is a province located in northeast (NE) China where the temperature is hot and dry in summers and has a harsh winter with temperatures down to −20 °C [9]. The vegetation in the eastern mountains includes tree genera such as the Betula, Fraxinus, Juglans, Larix, Pinus, Quercus, Salix, Sorbus and Ulmus [10]. These trees are common in the northern hemisphere and in temperate climates [11].

The family Cucurbitariaceae was established by Winter [12], and it is characterized by clustered ascomata and scattered, black, and shiny ostioles, surrounded with olivaceous-to-brown hyphae and having yellow-to-dark olivaceous, brown and muriform ascospores [13,14,15]. Asexual morphs are known to occur as pycnidia with hyaline conidia [14]. Cucurbitariaceae has received much attention in recent years, and it includes 13 genera: Allocucurbitaria Valenz.-Lopez, Stchigel, Guarro & Cano, Astragalicola Jaklitsch & Voglmayr, Cucitella Jaklitsch & Voglmayr, Cucurbitaria Gray (=Pleurostromella Petr.), Fenestella Tul. & Tul C., Neocucurbitaria Wanas., E.B.G. Jones & K.D. Hyde, Paracucurbitaria Valenz.-Lopez Stchigel, Guarro & Cano, Parafenestella Jaklitsch & Voglmayr, Protofenestella Jaklitsch & Voglmayr, Rhytidiella Zalasky, Seltsamia Jaklitsch & Voglmayr, Syncarpella Theiss. & Syd. and Synfenestella Jaklitsch & Voglmayr [13]. Jaklitsch et al. [15] provided a comprehensive study of fenestelloid clades of Cucurbitariaceae using fresh collections. Various type specimens were verified, and all the genera of Cucurbitariaceae formed a well-supported clade in a multi-locus phylogeny [15]. However, the phylogenetic placement of Rhytidiella and Syncarpella remain to be confirmed as they lack molecular data [15]. Fenestella, Neocucurbitaria and Parafenestella have a wide distribution mainly in temperate regions and can be found on various hosts [14,16,17,18,19]. For example, Parafenestella salicum was found on the twigs of Salix alba and Fenestella parafenestrata on the branches of Quercus robur in Austria, while Neocucurbitaria subcaespitosa was isolated from the twigs of Sorbus aria in Switzerland [14,15].

This study mainly focuses on ascomycetous fungi from the northern part of China. The novel taxa are introduced based on morphology and molecular data. In this study, Allocucurbitaria was used to demonstrate important characteristics for distinguishing the asexual morph at the generic level. This study also determines the best barcode out of five DNA markers for species delineation in Cucurbitariaceae by applying assemble species by automatic partitioning (ASAP) analyses.

2. Materials and Methods

2.1. Collection and Isolation

Dried branches of Morus alba, Populus species and Ulmus pumila were collected from Jilin Agricultural University in Changchun, Jilin Province, China (longitude: 125.410385; latitude: 43.810433). Specimens were kept in sealed paper bags indicating the location, time and host details. The specimens were processed following Senanayake et al. [20] for isolation. Single-spore isolation was performed using potato dextrose agar (PDA) and incubated at 25 °C in the dark [16]. Germinated ascospores were transferred aseptically to PDA and grown at 25 °C for 2 weeks. Pure cultures were deposited at the Engineering Research Center of the Chinese Ministry of Education for Edible and Medicinal Fungi at the Jilin Agricultural University (CCMJ), Changchun, China, and type specimens were deposited in the Herbarium of Mycology, Jilin Agricultural University (HMJAU). The new taxa were registered with Mycobank [17,18].

2.2. Morphological Observation

The specimens were examined using a Zeiss Stemi 2000C stereomicroscope equipped with a Leica DFC450C (Leica, Heidelberg, Germany) digital camera. A thin section of partial ascoma was prepared and placed on glass slides with a drop of sterile water. The structure and size of microcharacters were observed and photographed using a digital Axiocam 506 color camera equipped with Zeiss Image A2 (Zeiss, Oberkochen, Germany). Fructification of asexual morph in the sterile culture was observed after four weeks of incubation in the dark.

2.3. DNA Extraction, PCR Amplification and Sequencing

Genomic DNA was extracted using NuClean PlantGen DNA Kit (CWBIO, Taizhou, China) according to the manufacturer’s protocol. The internal transcribed spacer region of ribosomal DNA (ITS) [21], the large subunit (LSU) of ribosomal DNA [22], the RNA polymerase II second-largest subunit (rpb2) [23], the translation elongation factor 1-alpha (tef1-α) and beta-tubulin (β-tub) were amplified as described in

Table 1. The PCR primers and amplifying conditions used in this study.

Amplification Loci (Primer Pair Forward/Reverse)	PCR Conditions	References
ITS (ITS5/ITS4)	An initial denaturation step of 5 min at 94 °C, followed by 35 cycles of 30 s at 94 °C, 30 s at 56 °C and 90 s at 72 °C, and a final extension step of 10 min at 72 °C, and 10 °C for holding temperature	White et al. [21]
rpb2 (fRPB2-5F/fRPB2-7cR)		Vilgalys et al. [23]
tef1-α (2218F/983R)		Carbone and Kohn [24] Rehner and Buckley [25]
LSU (LROR/LR5)	An initial denaturation step of 5 min at 94 °C, followed by 35 cycles of 30 s at 94 °C, 45 s at 53 °C and 90 s at 72 °C, and a final extension step of 10 min at 72 °C, and 10 °C for holding temperature	Vilgalys and Hester [22]
Β-tub (T1/Bt2b)		O’Donnell and Cigelnik [26]

. The amplification reactions were performed using 20 μL PCR mixtures containing 9 μL of ddH₂O, 10 μL of 2× EsTaq MasterMix (Dye), 0.4 μL of DNA template and 2 μL of 2 μmol/μL of each forward and reverse primer. All PCR products were visualized with electrophoresis using a 1% agarose gel. The PCR products were sequenced by Sangon Biotech (Shanghai) Co., Ltd., China.

2.4. Phylogenetic Analysis

The sequence data were assembled using Geneious Prime 2021 (Biomatters Ltd., Auckland, New Zealand). The closest matches for the new strains were obtained using BLASTn searches (http://www.blast.ncbi.nlm.nih.gov/, accessed on 17 December 2021), and reference sequence data were downloaded from recent publications [14,15]. The sequences were aligned with MAFFT version 7 (https://mafft.cbrc.jp/alignment/server/, accessed on 8 July 2022) [27], and ambiguous nucleotides were manually adjusted following visual examination in AliView version 1.26 [28]. Leading or trailing gaps exceeding the primer binding site were trimmed from the alignments, and the alignment gaps were treated as missing data. The concatenation of the multilocus data was created using Sequence Matrix version 1.8 [29].

Phylogenetic analyses were conducted using maximum likelihood, maximum parsimony and Bayesian inference methods. Maximum likelihood analysis was performed using RAxML-HPC2 on XSEDE on the CIPRES web portal (http://www.phylo.org/portal2/, accessed on 8 July 2022) [30,31,32]. The GTR+I+G model of nucleotide evolution was used for the datasets, and RAxML rapid bootstrapping of 1000 pseudo-replicates was performed [33]. The best-fit evolutionary models for individual and combined datasets were estimated under the Akaike information criterion (AIC) using jModeltest 2.1.10 on the CIPRES web portal for posterior probability [34]. The GTR+I+G model was the best model for the datasets. Maximum parsimony analysis of the combined matrices was performed using a parsimony ratchet approach. Descriptive tree statistics for parsimony (Consistency Index [CI], Homoplasy Index [HI] Tree Length [TL], Retention Index [RI] and Relative Consistency Index [RC]) were calculated for the trees generated under the different optimality criteria. The resulting best trees were then analyzed using PAUP and subjected to a heuristic search with TBR branch swapping (MulTrees option in effect, steepest descent option not in effect) [35]. Bayesian inference analyses were conducted using MrBayes v. 3.2.6 on the CIPRES web portal. Simultaneous Markov chains were run for seven million generations, and trees were sampled every 100th generation [36]. The phylogenetic trees were visualized in FigTree 1.4.3 [37] and edited in Adobe Illustrator CS v. 6 (Adobe, San Jose, CA, USA).

2.5. Analysis of Matrix Partitions by Assemble Species by Automatic Partitioning

Puillandre et al. [38] introduced the assemble species by automatic partitioning (ASAP) method to build species partitions. The ASAP method circumscribes species partitions using an implementation of a hierarchal clustering algorithm based on pairwise genetic distances (Kimura 2-Parameter). The pairwise genetic distances are used to build a list of partitions ranked by a score that is computed using the probabilities of groups to define panmictic species. The ASAP delimitations were run on the online version (https://bioinfo.mnhn.fr/abi/public/asap/ (accessed on 13 January 2022)) using single-locus datasets that included 107 strains of Cucurbitariaceae. The partition with the lowest ASAP score is known to represent the best partitions [38,39], and thus partitions with the lowest ASAP score were considered for each dataset [39,40].

3. Results

3.1. Phylogenetic Analyses

The final concatenated dataset comprised 110 ingroup taxa and two outgroup taxa, with 4607 characters including gaps (651 bases for ITS, 911 bases for LSU, 1063 bases for rpb2, 1281 bases for tef1-α, and 701 bases for β-tub). The RAxML analysis yielded a best-scoring tree with a final ML optimization likelihood value of −39123.587750. The matrix consisted of 1740 distinct alignment patterns, with 25.90% undetermined characters or gaps. Estimated base frequencies were as follows: A = 0.234707, C = 0.269983, G = 0.265086, T = 0.230223; substitution rates AC = 1.287870, AG = 4.563896, AT = 1.434736, CG = 1.144629, CT = 6.919700, GT = 1.000000; proportion of invariable sites I = 0.606319; gamma distribution shape parameter α = 0.967784. The maximum parsimony dataset consisted of 1230 parsimony-informative characters and 246 variable characters. The parsimony analysis yielded 256 most parsimonious trees out of 1000 (TL = 6467, CI = 0.368, RI = 0.806, RC = 0.296, HI = 0.632). In the BPP analysis, 2437 trees were sampled after the 20% burn-in with a stop value of 0.009904. The maximum parsimony dataset consisted of 3132 parsimony-informative characters and 241 variable characters. The parsimony analysis yielded 512 most parsimonious trees out of 1000 (TL = 6468, CI = 0.368, RI = 0.806, RC = 0.297, HI = 0.632). In the BPP analysis, 1461 trees were sampled after the 20% burn-in with a stop value of 0.009955. The phylogenetic trees generated from the ML, MP and BPP had similar topologies (Figures S6 and S7).

In the ML analysis of the ITS region, Parafenestella ulmi (CCMJ 5001 and CCMJ 5002) and P. ulmicola (CCMJ 5003 and CCMJ 5004) clustered together with high support (ML = 95%), while P. changchunensis (CCMJ 5007) formed a clade with P. vindobonensis (CBS 145256) with relatively low support (ML = 63%) in Parafenestella (Figure S1). Parafenestella ostryae (MFLU 16-0184) and P. pittospori (CPC 34462) resided in the Neocucurbitaria clade (Figure 1) similar to the combined dataset. Allocucurbitaria mori (CCMJ 5005 and CCMJ 5006) formed a clade with A. botulispora (CBS 142452), Seltsamia galinsogisoli (CBS 140956), S. ulmi (CBS 143002) and two unidentified Seltsamia species (EAB-67-11b and SGSF207) (ML = 100%). The LSU locus could not accurately distinguish taxa at the genus and species level in Cucurbitariaceae (Figure S2). In the ML analysis of β-tub gene, P. ulmi (CCMJ 5001 and CCMJ 5002) and P. ulmicola (CCMJ 5003 and CCMJ 5004) formed a clade with high support (ML = 94%), while P. changchunensis (CCMJ 5007) clustered with P. pseudosalicis (CBS 145264) with moderate support (ML = 71%). Allocucurbitaria mori (CCMJ 5005 and CCMJ 5006) and A. botulispora (CBS 142452) formed a clade with moderate support (ML = 54%, Figure S5). In the tef1-α analysis, Parafenestella ulmi (CCMJ 5001 and CCMJ 5002) and P. ulmicola (CCMJ 5003 and CCMJ 5004) formed a clade with relatively high support (ML = 89%) (Figure S4). Parafenestella changchunensis (HMJAU 60182) formed a clade with P. salicis (CBS 145270 and C303), P. pseudosalicis (CBS 145264), P. vindobonensis (CBS 145265) and P. alpina (CBS 145263 and C249) with relatively high support (ML = 79%). Allocucurbitaria mori (CCMJ 5005 and CCMJ 5006) clustered with Synfenestella pyri (CBS 144855) with low support (ML = 41%).

In the multi-locus phylogenetic analysis, Parafenestella ulmi (CCMJ 5001 and CCMJ 5002) and P. ulmicola (CCMJ 5003 and CCMJ 5004) formed a clade with high support (ML = 100%; MP = 100%; BPP = 1.00). Parafenestella changchunensis (CCMJ 5007) clustered with P. pseudosalicis (CBS 145264) and P. salicis (CBS 145270 and C303) with high support (ML = 99%; MP = 96%; BPP = 1.00). Parafenestella changchunensis (CCMJ 5007) is closely related to P. pseudosalicis (ML = 75%; MP = 96%). The fresh collections from Morus alba revealed a new species Allocucurbitaria mori (CCMJ 5005 and CCMJ 5006). The two isolates (CCMJ 5005 and CCMJ 5006) formed a close relationship to an unidentified Seltsamia species (SGSF207) with strong statistical support (ML = 100%; MP = 100%; BPP = 1.00).

Figure 1. The Bayesian 50% majority-rule consensus phylogram based on a concatenated ITS, LSU, rpb2, tef1-α and β-tub dataset of Cucurbitariaceae. The tree is rooted with Pyrenochaetopsis americana (UTHSC DI16225) and P. confluens (CBS 142459). Bootstrap support values for maximum likelihood and maximum parsimony analysis greater than 70% (ML = left; MP = middle) and Bayesian posterior probabilities ≥ 0.90 (BPP, right) are shown at the nodes. The new species are indicated in blue. The type-derived strains are indicated in bold and marked with ^T.

Figure 1. The Bayesian 50% majority-rule consensus phylogram based on a concatenated ITS, LSU, rpb2, tef1-α and β-tub dataset of Cucurbitariaceae. The tree is rooted with Pyrenochaetopsis americana (UTHSC DI16225) and P. confluens (CBS 142459). Bootstrap support values for maximum likelihood and maximum parsimony analysis greater than 70% (ML = left; MP = middle) and Bayesian posterior probabilities ≥ 0.90 (BPP, right) are shown at the nodes. The new species are indicated in blue. The type-derived strains are indicated in bold and marked with ^T.

3.2. ASAP: Assemble Species by Automatic Partitioning

Five single-locus datasets were used that comprised 110 sequences of ITS, 109 sequences of LSU, 101 sequences of rpb2, 96 sequences of β-tub and 88 sequences of tef1-α. The ASAP analysis of the ITS region assigned all members of Cucurbitariaceae into 45 groups (Figure 2); β-tub gene into 65 groups (Figure 2); LSU into 43 groups (Figure S8); rpb2 gene into 65 groups (Figure S9); tef1-α gene into 45 groups (Figure S10).

The ASAP analysis recovered P. ulmi (CCMJ 5001 and CCMJ 5002), P. ulmicola (CCMJ 5003 and CCMJ 5004) and twelve other strains including P. pseudoplatani (CBS 142392), P. austriaca (CBS 145262), P. rosacearum (C203, FM1, C269, C283, CBS 145268, C315, CBS145272, C320), P. germanica (CBS 145267) and P. tetratrupha (CBS 145266) as one group in the LSU data. Parafenestella changchunensis (CCMJ 5007) and P. pseudosalicis (CBS 145264) were recovered as one group in the LSU data. The ASAP analysis of the ITS region recovered P. ulmi and P. ulmicola as one group (Figure 2). The ASAP result of the β-tub gene was similar to the combined dataset (Figure 2). Parafenestella ulmi and P. ulmicola were not delineated by the tef1-α and rpb2 genes (Figures S9 and S10). Parafenestella changchunensis, P. pseudosalicis (CBS 145264) and P. salicis (CBS 145270 and C303) were recovered as one group in the tef1-α data. Allocucurbitaria mori (CCMJ 5005 and CCMJ 5006) grouped with Synfenestella pyri (CBS 144855) in the ASAP analysis of the tef1-α gene, but both were recovered as individual groups in the ITS, LSU, rpb2, and β-tub datasets.

In the ASAP analysis, the β-tub gene was the best marker for identifying Parafenestella and Allocucurbitaria taxa. Parafenestella ulmi and P. ulmicola were recovered as a group in ASAP analysis of the ITS and other markers but were recovered as separate groups in the β-tub dataset (similar to the combined dataset). Parafenestella changchunensis and P. vindobonensis (CBS 145265) were recovered as a group in the ITS region but were recovered as distinct species in the β-tub dataset. Allocucurbitaria mori was recovered as an individual group in all single-marker analyses (except tef1-α gene). Based on the current results, the β-tub gene is the best marker for the identification of Cucurbitariaceae taxa at the species level.

3.3. Taxonomy

Allocucurbitaria mori W.X. Su, Phukhams. & Y. Li, sp. nov. (Figure 3).

MycoBank Number: MB844413.

Etymology: Named after the host genus Morus.

Holotype: HMJAU 60183.

Description: Saprobic on dead twigs of Morus alba.

Sexual morph: Undetermined.

Asexual morph: Stromata poorly developed, multiloculate, with 5–8 locules forming groups in stromata, immersed. Conidiomata 108–180 × 103–201 μm ($\overline{x}$ = 142 × 143 μm, n = 6), pycnidia, solitary or aggregated, sometimes confluent, semi-immerged, visible as black protrusions, globose to ellipsoid, coriaceous, black, without distinguishable ostioles. Pycnidial wall 5–9 μm wide, thick-walled, composed of 7–10 layers of thin-walled cells of textura angularis, dark brown on the outside to gradually lighter on the inside, inner layer subhyaline, lining layer bearing conidiogenous cells. Conidiophores reduced to conidiogenous cells. Conidiogenous cells 6–14 × 1–5 μm ($\overline{x}$ = 10 × 2 μm, n = 30), enteroblastic, solitary, long cylindrical, arising from the inner layer of conidioma, smooth-walled, hyaline. Conidia 3–5 × 1–2 μm ($\overline{x}$ = 4 × 1.5 μm, n = 50), oblong, hyaline, aseptate, with a minute guttule, smooth.

Cultural characters: Colonies on MEA reaching 32–38 mm diam after 4 weeks at 25 °C. Cultures from above, gray at the center, dense in the middle, sparse at the edge, circular, papillate, black lumps produced on the surface of cultures, white at the edge.

Material examined: CHINA, Jilin Province, Changchun, Jilin Agricultural University, from Morus alba (Moraceae) twigs, 20 May 2021, Wenxin Su and C. Phukhamsakda, S057 (HMJAU 60183, holotype); ex-type living culture, CCMJ5005; isotype = HMJAU 60184; ex-isotype living culture, CCMJ5006.

GenBank accession numbers: CCMJ5005: LSU = OL897171, ITS = OL996120, tef1-α = OL944601, rpb2 = OL944505, and β-tub = OL898725. CCMJ5006: LSU = OL897172, ITS = OL996121, tef1-α = OL944602, rpb2 = OL944506 and β-tub = OL898720.

Notes: Allocucurbitaria mori (CCMJ5005 and CCMJ5006) formed a separate clade in Allocucurbitaria/Seltsamia with high support (ML = 98%; MP = 97%; BPP = 1.00). Morphologically, A. mori (HMJAU 60183) is similar to A. botulispora (CBS 142452) and S. galinsogisoli (CBS 140956) in having cylindrical, enteroblastic, solitary conidiogenous cells and aseptate conidia [41,42] (Figure 4). However, S. galinsogisoli (CBS 140956) has longer conidia, while A. botulispora (CBS 142452) has distinct guttulate at the conidia ends [41,42].

A BLASTn search of the ITS region of A. mori strain CCMJ 5005 showed a high query cover and similarity (99.80%) to an unidentified Seltsamia sp. (SGSF207) from soil. However, there are no other loci available in public databases for comparison. Hence, we introduce Allocucurbitaria mori as a novel species, and this is the first report of Allocucurbitaria on Morus tree [41,42,43].

Figure 3. Allocucurbitaria mori (HMJAU 60183, holotype) The red arrow indicates the conidiomata in face view. (a,b) Appearance of conidiomata on host substrate. (c,d) Vertical section of partial conidiomata. (e) Section of partial conidioma wall. (f–h) Conidiogenous cells and conidia. (i) Conidia. (j) Culture characteristics on PDA. Scale bars: (a) = 500 µm; (b) = 200 µm; (c,f) = 100 µm; (d) = 50; (e,g,h) = 10 µm; (i) = 5 µm.

Figure 3. Allocucurbitaria mori (HMJAU 60183, holotype) The red arrow indicates the conidiomata in face view. (a,b) Appearance of conidiomata on host substrate. (c,d) Vertical section of partial conidiomata. (e) Section of partial conidioma wall. (f–h) Conidiogenous cells and conidia. (i) Conidia. (j) Culture characteristics on PDA. Scale bars: (a) = 500 µm; (b) = 200 µm; (c,f) = 100 µm; (d) = 50; (e,g,h) = 10 µm; (i) = 5 µm.

Figure 4. Morphology of related taxa in the Allocucurbitaria clade. (a–c) Characters of Allocucurbitaria botulispora were redrawn from Valenzuela-Lopez et al. [41]: (a) Pycnidia. (b,c) Conidiogenous cells and conidia. (d–f) Characters of Seltsamia galinsogisoli redrawn from Zhang et al. [42]: (d) Pycnidia. (e,f) Conidiogenous cells and conidia. (g–j) Characters of Allocucurbitaria mori (CCMJ 5005): (g) Appearance of conidiomata of Allocucurbitaria mori on host substrate. (h–j) Conidiogenous cells and conidia. Scale bars: (b,c,e,f,h,i) = 10 µm; (d) = 20 µm; (g) = 200 µm; (j) = 5 µm.

Figure 4. Morphology of related taxa in the Allocucurbitaria clade. (a–c) Characters of Allocucurbitaria botulispora were redrawn from Valenzuela-Lopez et al. [41]: (a) Pycnidia. (b,c) Conidiogenous cells and conidia. (d–f) Characters of Seltsamia galinsogisoli redrawn from Zhang et al. [42]: (d) Pycnidia. (e,f) Conidiogenous cells and conidia. (g–j) Characters of Allocucurbitaria mori (CCMJ 5005): (g) Appearance of conidiomata of Allocucurbitaria mori on host substrate. (h–j) Conidiogenous cells and conidia. Scale bars: (b,c,e,f,h,i) = 10 µm; (d) = 20 µm; (g) = 200 µm; (j) = 5 µm.

Parafenestella changchunensis W. X. Su, Phukhams. & Y. Li, sp. nov. (Figure 5).

MycoBank Number: MB844412.

Etymology: referring to Changchun City where the sample was collected.

Holotype: HMJAU 60182.

Description: Saprobic on dead stems of Populus L.

Sexual morph: Ascomata 174–416 × 226–486 μm ($\overline{x}$ = 280 × 353 μm, n = 5), single or gregarious, scattered, globose to depressed globose, submerged, visible as black dots and protruding host surface, solitary or aggregated. Ostioles 61 × 100 μm, center, protruding filled with periphyses. Peridium 12–27 μm wide, thick-walled, composed of 6–10 wall layers, outer part comprising dark brown cells of textura angularis, inner layer thin-walled, dark brown from the outside radiating light brown cells to hyaline towards the inside. Hamathecium of dense, 1.6–2.0 μm ($\overline{x}$ = 1.7 μm, n = 10) wide, filamentous, septate, cellular pseudoparaphyses surrounding asci. Asci 95–138 × 16–21 μm ($\overline{x}$ = 121 × 18 μm, n = 10), 6–8 ascospores, bitunicate, fissitunicate, broad cylindrical, some curved, short-pedicellate, apically rounded with an ocular chamber. Ascospores 18–25 × 8–13 μm ($\overline{x}$ = 21 × 10 μm, n = 30), uniseriate, partially overlapping, fusiform to oval, slightly asymmetrical, the middle of ascospores is slightly contracted, with 4–6 transverse septa, 2–3 vertical septa, the upper part is slightly larger than the lower part, light yellow to dark brown.

Asexual morph: Pycnidia produced in PDA after 2 weeks of incubation in the dark, mycelium white. Conidiomata confluent or scattered, superficial, covered with dense vegetative hyphae, with turbid whitish drops, globose, black. Conidia 5–8 × 2.5–4.5 μm ($\overline{x}$ = 6.5 × 3.7 μm, n = 30), oblong to allantoid, hyaline, aseptate, with 1–2 guttules.

Culture characteristics: Colonies on PDA, reaching 26–31 mm diam after 2 weeks at 25 °C. Culture from above, mycelium dense and producing hyphal coil structures; from the center to the outer edge, the color changes from grey to greyish-green to white, with obvious concentric wheel patterns, a clear radiation pattern at the back, round.

Material examined: CHINA, Jilin Province, Changchun, Jilin Agricultural University, from dead stems of Populus L. (Salicaceae), 18 April 2021, Wenxin Su, S12-16 (HMJAU 60182, holotype); ex-type living culture, CCMJ5007.

GenBank accession numbers: CCMJ5007: LSU = OL897170, SSU = OL891808, ITS = OL996119, tef1-α = OL944600, and β-tub = OL898719.

Notes: In our phylogenetic analysis, P. changchunensis (CCMJ5007) is closely related to P. pseudosalicis (CBS 145264) with moderate support (ML = 75%; MP = 96 %; Figure 1). Parafenestella changchunensis is morphologically similar to P. pseudosalicis in having immersed, concave apex ascomata, with the upper part of young ascospores often wider, ends concolorous and smooth walled [14]. The immature spores of P. changchunensis have four horizontal septa and form 2–3 vertical septa during the maturation process. However, the immature spores of P. pseudosalicis have 2 transverse septa turning into 2–4 longitudinal septa during the maturation process [15]. Parafenestella changchunensis mycelium nodules gradually form fruiting bodies on the medium, while there are no reports of the asexual morph of P. pseudosalicis [15].

A BLASTn search of the ITS region of P. changchunensis (CCMJ 5007) showed a high similarity and query cover (98.81%) to P. vindobonensis (CBS 145265). The β-tub sequence of P. changchunensis (CCMJ 5007) showed a high query cover and similarity (96.82%) to P. pseudosalicis (C301). There were 0.96% (6/627 bases), 0.34% (3/885), 1.78% (13/730) and 7.99% (43/538 bases) base differences in the ITS, LSU, tef1-α and β-tub genes between P. changchunensis (CCMJ 5007) and P. vindobonensis (CBS 145265), excluding gaps. There were 1.75% (11/627 bases), 0.11% (1/885), 1.10% (8/730) and 3.16% (17/538 bases) base differences in the ITS, LSU, tef1-α and β-tub genes of P. changchunensis (CCMJ 5007) and P. pseudosalicis strain C301, excluding gaps. Therefore, we introduce P. changchunensis as a novel species, and this is the first report of Parafenestella on the Populus tree [14,15].

Figure 5. Parafenestella changchunensis (HMJAU 60182, holotype). (a) Ascomata on host surface. (b) Vertical section through partial ascoma. (c) Ostioles. (d) Partial peridium. (e) Pseudoparaphyses. (f–i) Asci. (j–v) Development stages of ascospores. (w) Germinating ascospore (x) Culture characteristics on PDA. (y) Pycnidia. (z) Hyphal coil structures formed by mycelia. (a1) Conidia. Scale bars: (a) = 500 µm; (b,c) = 100 µm; (d,e) = 20 µm; (f–i) = 50 µm; (j–v,a1) = 10 µm.

Figure 5. Parafenestella changchunensis (HMJAU 60182, holotype). (a) Ascomata on host surface. (b) Vertical section through partial ascoma. (c) Ostioles. (d) Partial peridium. (e) Pseudoparaphyses. (f–i) Asci. (j–v) Development stages of ascospores. (w) Germinating ascospore (x) Culture characteristics on PDA. (y) Pycnidia. (z) Hyphal coil structures formed by mycelia. (a1) Conidia. Scale bars: (a) = 500 µm; (b,c) = 100 µm; (d,e) = 20 µm; (f–i) = 50 µm; (j–v,a1) = 10 µm.

Parafenestella ulmi W.X. Su, Phukhams., & Y. Li, sp. nov. (Figure 6).

MycoBank Number: MB844410.

Etymology: Named after the host genus Ulmus.

Holotype: HMJAU 60178.

Description: Saprobic on dead stems of Ulmus pumila.

Sexual morph: Ascomata 170–225 × 194–260 μm ($\overline{x}$ = 201 × 229 μm, n = 5), immersed, visible as black spots or having a convex surface, solitary, scattered, globose to ellipsoid, flat at the base, coriaceous, black. Peridium 19–39 μm wide, composed of 6–10 layers, outer part comprising dark brown cells of textura angularis, inner layer comprising thin-walled, light brown cells of textura angularis. Hamathecium of dense, 1.5–4.5 μm wide ($\overline{x}$ = 2.2 μm, n = 20), filamentous, septate, pseudoparaphyses surrounding asci. Asci 115–181 × 11–15 μm ($\overline{x}$ = 132 × 13 μm, n = 20), 8 ascospores, bitunicate, cylindrical, mostly curved, short-pedicellate, apically rounded with an ocular chamber, clearly visible when immature. Ascospores 18–24 × 8–12 μm ($\overline{x}$ = 22 × 10 μm, n = 30), uniseriate to partially overlapping, broadly ellipsoid, slightly pointed at both ends, 5–8 transversely septate, 1–2 vertically septate, mature spores constricted at the middle septum, slightly curved, initially hyaline, becoming yellowish to brown at maturity, the cell above median septum slightly wider, smooth-walled.

Asexual morph: Pycnidia produced in PDA after 2 weeks of incubation in the dark, mycelium greenish, 1–3 μm ($\overline{x}$ = 2.2 μm, n = 20), uniloculate, confluent or scattered, superficial, covered with dense vegetative hyphae, globose, dark brown to black. Conidiogenous cells 18–24 × 8–12 μm ($\overline{x}$ = 22 × 10 μm, n = 30), enteroblastic, phialidic, determinate, discrete, solitary, short cylindrical or conical, straight, with broad base, hyaline. Conidia 3–5 × 1–2 μm ($\overline{x}$ = 4.3 × 1.5 μm, n = 30), long ellipsoid to cylindrical, aseptate, with two small guttulate at the polar ends, hyaline, smooth-walled.

Culture characteristics: Colonies on PDA, reaching 45–48 mm diam after two weeks at 25 °C. Culture from above the center to the outer edge, the color radiating from black to dark green to yellow and white edges, with obvious concentric wheel patterns, dense intermediate hyphae and sparse white mycelium at the outer circle; reverse greenish-black, round.

Material examined: CHINA, Jilin Province, Changchun, Jilin Agricultural University, from Ulmus pumila (Ulmaceae) stem litter, 15 March 2021, Wenxin Su and C. Phukhamsakda, S12 (HMJAU 60178, holotype); ex-type living culture, CCMJ 5001, isotype = HMJAU 60179; ex-isotype living culture, CCMJ 5002.

GenBank accession numbers: CCMJ5001: LSU = OL897166, SSU = OL891806, ITS = OL996115, tef1-α = OL944596, rpb2 = OL944501, and β-tub = OL898723. CCMJ5002: LSU = OL897167, ITS = OL996116, tef1-α = OL944597, rpb2 = OL944502, and β-tub = OL898717.

Notes: In our phylogenetic analysis, P. ulmi (CCMJ 5001 and CCMJ 5002) and P. ulmicola (CCMJ 5003 and CCMJ 5004) formed a clade in Parafenestella with high statistical support (ML = 100%; MP = 100%; BPP = 1.00; Figure 1). Both P. ulmi and P. ulmicola were found on dead branches of Ulmus pumila in Jilin Province, China, which lies in the temperate zone. Parafenestella taxa are mainly recorded in Austria, followed by England, Germany and Ukraine, which are all temperate countries [15]. Morphologically, the ascomata of P. ulmi and P. ulmicola are semi-immersed, visible as black spots or convex surfaces. The asci of P. ulmi are longer than P. ulmicola but similar in width (132 × 13 vs. 119 × 13 μm). The immature ascospores of P. ulmi present 2–3 transverse septa without longitudinal septate, but the spores have 4–8 transverse septa with 1–3 longitudinal septate at mature stages. The ascospores of P. ulmicola showed indentation when immature that disappeared during maturation. The ascospores of P. ulmicola showed 5–8 transverse septa and 1–2 vertically septate after maturity with less constriction at the septum. The ascospores of P. ulmi are yellowish to brown, while P. ulmicola have dark brown ascospores at maturity. In PDA, the colonies of P. ulmicola have wavy and aggregated colony edges. The colonies of P. ulmi are blue-black (reverse view) with black-green edges, while P. ulmicola is gray-brown with white edges.

A BLASTn search of the ITS region of P. ulmi strain CCMJ 5001 showed a high query cover and similarity (96.45%) to P. tetratrupha (CBS 145266) while the β-tub sequence of P. ulmi strain CCMJ 5001 showed a high similarity and query cover (97.07%) to P. germanica strain C307. Therefore, we introduce P. ulmi as a novel species.

Figure 6. Parafenestella ulmi (HMJAU 60178, holotype). (a) Ascomata on host surface. (b) Vertical section through ascoma. (c) Partial peridium in vertical section. (d,e) Asci arrangement along with pseudoparaphyses. (f–h) Development stages of asci. (i–s) Development stages of ascospores. (t) Germinating ascospore. (u) Four-week-old culture characteristics on PDA. (v) Pycnidia formed in sterile culture after two weeks of incubation on PDA. (w,x) Conidiogenous cells and conidia. (y) Conidia. Scale bars: (a) = 500 µm; (b) = 100 µm; (c–h) = 50 µm; (i–s) = 20 µm; (v) = 200 µm; (w–y) = 5 µm.

Figure 6. Parafenestella ulmi (HMJAU 60178, holotype). (a) Ascomata on host surface. (b) Vertical section through ascoma. (c) Partial peridium in vertical section. (d,e) Asci arrangement along with pseudoparaphyses. (f–h) Development stages of asci. (i–s) Development stages of ascospores. (t) Germinating ascospore. (u) Four-week-old culture characteristics on PDA. (v) Pycnidia formed in sterile culture after two weeks of incubation on PDA. (w,x) Conidiogenous cells and conidia. (y) Conidia. Scale bars: (a) = 500 µm; (b) = 100 µm; (c–h) = 50 µm; (i–s) = 20 µm; (v) = 200 µm; (w–y) = 5 µm.

Parafenestella ulmicola W.X. Su, Phukhams., & Y. Li, sp. nov. (Figure 7).

MycoBank Number: MB844411.

Etymology: Named after the host genus Ulmus.

Holotype: HMJAU 60180.

Description: Saprobic on twigs debris of Ulmus pumila L.

Sexual morph: Ascomata 242–434 × 310–462 μm ($\overline{x}$ = 306 × 359 μm, n = 5) μm wide, semi-immersed, visible as a convex hemisphere, globose to subglobose, solitary or mostly aggregated, scattered, coarse-walled, coriaceous, black, with a papilla. Ostiole 21 × 24 μm, centrally located. Peridium 21–68 μm wide, composed of 11–20 wall layers, with dark brown cells of textura angularis. Asci 105–153 × 11–14 μm ($\overline{x}$ = 119 × 13 μm, n = 20), 8 ascospores, bitunicate, fissitunicate, broadly cylindrical, apically rounded, some curved, short-pedicellate, ocular chamber is not visible at maturity. Ascospores 17–22 × 8–12 μm ($\overline{x}$ = 19 × 9 μm, n = 30), uniseriate, rarely overlapping, broadly oval, blunt at both ends, narrow towards the ends, with 4–8 transversely septate, 1–3 vertically septate, constricted at the middle septum, initially hyaline, becoming yellowish to brown at maturity, smooth-walled.

Asexual morph: Pycnidia produced in cultures on PDA after four weeks of incubation in the dark, mycelium greenish, 41–158 μm diam, covered with white mycelium, ellipsoid, semi-immersed, scattered or aggregated, black, ostiole central. Peridium with brown cells of textura angularis. Conidia 1.4–2.5 × 0.6–0.9 μm ($\overline{x}$ = 1.9 × 0.7 μm, n = 30), cylindrical to allantoid, hyaline, smooth, aseptate, with a minute guttulate.

Culture characteristics: Colonies on PDA reaching 35–41 mm diam after 2 weeks at 25 °C. Culture from above the center to the outer edge, the color changes from grey to taupe to white, with obvious concentric wheel patterns; a few weeks later, the outer circle hyphae grow into round dark green hyphae with a thin surface.

Material examined: CHINA, Jilin Province, Changchun, Jilin Agricultural University, from Ulmus pumila (Ulmaceae) twigs debris, 15 March 2021, Wenxin Su and C. Phukhamsakda, S16 (HMJAU 60180, holotype); ex-type living culture, CCMJ 5003, isotype = HMJAU 60181; ex-isotype living culture, CCMJ 5004.

GenBank accession numbers: CCMJ5003: LSU = OL897168, SSU = OL891807, ITS = OL946117, tef1-α = OL944598, rpb2 = OL944503 and β-tub = OL898724. CCMJ5004: LSU = OL897169, ITS = OL996118, tef1-α = OL944599, rpb2 = OL944504 and β-tub = OL898719.

Notes: Sixteen Parafenestella species are listed in Species Fungorum [44], of which six species were reported on Rosaceae, four on Salicaceae and three on Betulaceae, while one species was reported on Pittosporaceae, Salicaceae and Sapindaceae [14,15,45,46]. Parafenestella ulmicola (CCMJ 5003 and CCMJ 5004) is closely related to P. ulmi (CCMJ 5001 and CCMJ 5002) within Parafenestella (ML = 100%; MP = 100%; BPP = 1.00, Figure 1). There were 2.31% (12/518) base differences in the β-tub, 0.14% (1/733) base differences in the tef1-α and 0.27% (2/736) base differences in the rpb2 gene between P. ulmicola (CCMJ 5003 and CCMJ 5004) and P. ulmi (CCMJ 5001 and CCMJ 5002), excluding gaps. There were no base differences in the ITS and LSU sequences.

Figure 7. Parafenestella ulmicola (HMJAU 60180, holotype). (a) Ascomata on host surface. (b) Vertical section through ascoma. (c) Ostiole. (d) Partial peridium wall. (e) Pseudoparaphyses. (f–h) Asci. (i–s) Developmental stages of ascospores. (t) Germinating ascospore. (u) Pycnidia produced in four weeks old cultures on PDA. (v) Conidiomata. (w) Conidia. (x) Four weeks old culture on PDA. Scale bars: (b) = 100 µm; (c) = 50 µm; (d,e) = 20 µm; (f–h) = 50 µm; (i–s) = 10 µm; (u) = 200 µm; (v) = 100 µm; (w) = 5 µm.

Figure 7. Parafenestella ulmicola (HMJAU 60180, holotype). (a) Ascomata on host surface. (b) Vertical section through ascoma. (c) Ostiole. (d) Partial peridium wall. (e) Pseudoparaphyses. (f–h) Asci. (i–s) Developmental stages of ascospores. (t) Germinating ascospore. (u) Pycnidia produced in four weeks old cultures on PDA. (v) Conidiomata. (w) Conidia. (x) Four weeks old culture on PDA. Scale bars: (b) = 100 µm; (c) = 50 µm; (d,e) = 20 µm; (f–h) = 50 µm; (i–s) = 10 µm; (u) = 200 µm; (v) = 100 µm; (w) = 5 µm.

Parafenestella ulmi and P. ulmicola are phylogenetically close to P. tetratrupha but differ from P. tetratrupha by having a less longitudinal septa being visible at the surface [20]. Parafenestella tetratrupha ascospores are ellipsoid, yellow-brown to reddish-brown to dark brown, with 1–3 main septa, 8–17 distinct transverse and 2–4 longitudinal septa; they are darker and longer than P. ulmi and P. ulmicola (26.5–33.5 × 13–16.5 vs. 18–24 × 8–12 vs. 17–22 × 8–12 µm) and have more transverse septa than P. ulmi and P. ulmicola (

Table 2. The dataset used for phylogenetic analysis. The type-derived sequences are in bold.

Taxon	Strain	Host/Substrate	Typification Status	GenBank Accession Numbers
				ITS	LSU	rpb2	tef1-α	β-tub
Allocucurbitaria botulispora	CBS 142452	human scab on leg	Holotype	LT592932	LN907416	LT593070	–	LT593001
Allocucurbitaria mori	HMJAU 60183	Morus alba	Holotype	OL996120	OL897171	OL944505	OL944601	OL898725
Allocucurbitaria mori	HMJAU 60184	Morus alba	Isotype	OL996121	OL897172	OL944506	OL944602	OL898720
Astragalicola amorpha	CBS 142999	Astragalus angustifolius	Holotype	MF795753	MF795753	MF795795	MF795842	MF795883
Cucitella opali	CBS 142405	Acer opalus	Holotype	MF795754	MF795754	MF795796	MF795843	MF795884
Cucurbitaria berberidis	C39	Berberis vulgaris subsp. atropurpurea	–	MF795755	MF795755	MF795797	MF795844	MF795885
Cucurbitaria berberidis	CB	Berberis vulgaris	–	MF795757	MF795757	MF795799	MF795846	MF795887
Cucurbitaria berberidis	CBS 130007 = CB1	Berberis vulgaris	Epitype	MF795758	MF795758	MF795800	–	–
Cucurbitaria berberidis	CBS 142401 = C241	Berberis sp.	–	MF795756	MF795756	MF795798	MF795845	MF795886
Cucurbitaria oromediterranea	C265	Berberis aetnensis	–	MF795762	MF795762	MF795804	MF795850	MF795891
Cucurbitaria oromediterranea	C29	Berberis hispanica	–	MF795759	MF795759	MF795801	MF795847	MF795888
Cucurbitaria oromediterranea	C86	Berberis hispanica	–	MF795760	MF795760	MF795802	MF795848	MF795889
Cucurbitaria oromediterranea	CB2	Berberis cretica	–	MF795763	MF795763	MF795805	MF795851	MF795892
Cucurbitaria oromediterranea	CB3	Berberis hispanica	–	MF795764	MF795764	MF795806	MF795852	–
Cucurbitaria oromediterranea	CBS 142399 = C229	Berberis cretica	Holotype	MF795761	MF795761	MF795803	MF795849	MF795890
Fenestella crataegi	C287	Crataegus monogyna	–	MK356281	MK356281	–	MK357554	MK357598
Fenestella crataegi	CBS 144857 = C314	Crataegus monogyna	Epitype	MK356282	MK356282	MK357512	MK357555	MK357599
Fenestella fenestrata	CBS 143001 = FP9	Alnus glutinosa	Epitype	MF795765	MF795765	MF795807	MF795853	MF795893
Fenestella gardiennetii	CBS 144859 = FM	Acer saccharum	Holotype	MK356283	MK356283	MK357513	MK357556	MK357600
Fenestella granatensis	CBS 144854 = C279	Acer granatense	Holotype	MK356284	MK356284	MK357514	MK357557	MK357601
Fenestella media	CBS 144860 = FP	Corylus avellana	Epitype	MK356285	MK356285	MK357515	MK357558	MK357602
Fenestella media	FCO	Carpinus orientalis	–	MK356286	MK356286	MK357516	MK357559	–
Fenestella media	FP1	Corylus avellana	–	MK356287	MK356287	MK357517	MK357560	MK357603
Fenestella media	FP3	Acer pseudoplatanus	–	MK356288	MK356288	MK357518	MK357561	MK357604
Fenestella media	FP7	Castanea sativa	–	MK356289	MK356289	MK357519	MK357562	MK357605
Fenestella media	FP10	Tilia cordata	–	MK356290	MK356290	MK357520	MK357563	MK357606
Fenestella parafenestrata	CBS 144856 = C306	Quercus robur	Holotype	MK356291	MK356291	MK357521	MK357564	MK357607
Fenestella parafenestrata	C317	Salix sp.	–	MK356292	MK356292	MK357522	MK357565	MK357608
Fenestella subsymmetrica	CBS 144861 = FP6	Acer campestre	Holotype	MK356297	MK356297	MK357525	MK357569	MK357610
Fenestella subsymmetrica	C285	Juglans regia	–	MK356293	MK356293	MK357523	MK357566	–
Fenestella subsymmetrica	C286	Juglans regia	–	MK356294	MK356294	–	MK357567	–
Fenestella subsymmetrica	C286x	Juglans regia	–	MK356295	MK356295	–	–	–
Fenestella subsymmetrica	FP4	Corylus avellana	–	MK356296	MK356296	MK357524	MK357568	MK357609
Fenestella subsymmetrica	FP8	Salix caprea	–	MK356298	MK356298	MK357526	MK357570	MK357611
Fenestella viburni	CBS 144863 = FVL	Viburnum lantana	Holotype	MK356300	MK356300	MK357528	MK357572	MK357613
Fenestella viburni	FP2	Viburnum lantana	–	MK356299	MK356299	MK357527	MK357571	MK357612
Neocucurbitaria acanthocladae	CBS 142398 = C225	Genista acanthoclada	Holotype	MF795766	MF795766	MF795808	MF795854	MF795894
Neocucurbitaria acerina	C26a	Acer pseudoplatanus	–	MF795767	MF795767	MF795809	MF795855	MF795895
Neocucurbitaria acerina	CBS 142403 = C255	Acer pseudoplatanus	–	MF795768	MF795768	MF795810	MF795856	MF795896
Neocucurbitaria aetnensis	CBS 142404 = C261	Genista aetnensis	Holotype	MF795769	MF795769	MF795811	MF795857	MF795897
Neocucurbitaria aetnensis	C270	Genista aetnensis	–	MF795770	MF795770	MF795812	MF795858	MF795898
Neocucurbitaria aquatica	CBS 297.74	Sea water	Holotype	LT623221	EU754177	LT623278	–	LT623238
Neocucurbitaria cava	CBS 115979	–	–	AY853248	EU754198	LT623273	–	LT623234
Neocucurbitaria cava	CBS 257.68	Wheat-field soil	Epitype	JF740260	EU754199	LT717681	–	KT389844
Neocucurbitaria cinereae	CBS 142406 = KU9	Genista cinerea	Holotype	MF795771	MF795771	MF795813	MF795859	MF795899
Neocucurbitaria cisticola	CBS 142402 = C244	Cistus monspeliensis	Holotype	MF795772	MF795772	MF795814	MF795860	MF795900
Neocucurbitaria hakeae	CBS 142109 = CPC 28920	Hakeasp.	Holotype	KY173436	KY173526	KY173593	–	KY173613
Neocucurbitaria irregularis	CBS 142791	Subcutaneous tissue from injured human arm	Holotype	LT592916	LN907372	LT593054	–	LT592985
Neocucurbitaria juglandicola	C316	Quercus rubra	–	MK356301	MK356301	MK357529	MK357573	MK357614
Neocucurbitaria juglandicola	CBS 142390 = BW6	Juglans regia	Holotype	MF795773	MF795773	MF795815	MF795861	MF795901
Neocucurbitaria keratinophila	CBS 121759	From human corneal scrapings (keratitis)	Holotype	EU885415	LT623215	LT623275	–	LT623236
Neocucurbitaria populi	CBS 142393 = C28	Populussp.	Holotype	MF795774	MF795774	MF795816	MF795862	MF795902
Neocucurbitaria prunicola	CBS 145033	Prunus padus	–	MK442594	MK442534	MK442668	–	MK442737
Neocucurbitaria quercina	CBS 115095	Quercus robur	Neotype	LT623220	GQ387619	LT623277	–	LT623237
Neocucurbitaria rhamni	CBS 142391 = C1	Rhamnus frangula	Epitype	MF795775	MF795775	MF795817	MF795863	–
Neocucurbitaria rhamni	C112	Rhamnus frangula	–	MF795776	MF795776	MF795818	MF795864	MF795903
Neocucurbitaria rhamni	C133	Rhamnus frangula	–	MF795777	MF795777	MF795819	MF795865	MF795904
Neocucurbitaria rhamni	C190	Rhamnus frangula	–	MF795778	MF795778	MF795820	MF795866	–
Neocucurbitaria rhamni	C277	Rhamnus saxatilis	–	MF795779	MF795779	MF795821	MF795867	MF795905
Neocucurbitaria rhamnicola	CBS 142396 = C185	Rhamnus lycioides	Holotype	MF795780	MF795780	MF795822	MF795868	MF795906
Neocucurbitaria rhamnicola	KRx	Rhamnus alaternus	–	MF795781	MF795781	MF795823	MF795869	MF795907
Neocucurbitaria rhamnioides	C222	Rhamnus saxatilis subsp. prunifolius	–	MF795783	MF795783	MF795825	MF795871	MF795909
Neocucurbitaria rhamnioides	C223	Rhamnus saxatilis subsp. prunifolius	–	MF795784	MF795784	MF795826	MF795872	MF795910
Neocucurbitaria rhamnioides	CBS 142395 = C118	Rhamnus myrtifolius	Holotype	MF795782	MF795782	MF795824	MF795870	MF795908
Neocucurbitaria ribicola	CBS 142394 = C55	Ribes rubrum	Holotype	MF795785	MF795785	MF795827	MF795873	MF795911
Neocucurbitaria ribicola	C155	Ribes rubrum	–	MF795786	MF795786	MF795828	MF795874	MF795912
Neocucurbitaria unguis-hominis	CBS 111112	Agapornis sp.	–	LT623222	GQ387623	LT623279	–	LT623239
Neocucurbitaria vachelliae	CBS 142397 = C192	Vachellia gummifera	Holotype	MF795787	MF795787	MF795829	MF795875	MF795913
Paracucurbitaria italica	CBS 234.92	Olea europaea	Holotype	LT623219	EU754176	LT623274	–	LT623235
Paracucurbitaria riggenbachii	CBS 248.79	Fraxinus excelsiorwith bacterial canker	Holotype	LT903672	GQ387608	LT903673	–	LT900365
Parafenestella alpina	CBS 145263 = C198	Cotoneaster integerrimus	Holotype	MK356302	MK356302	MK357530	MK357574	MK357615
Parafenestella alpina	C249	Salix appendiculata	–	MK356303	MK356303	MK357531	MK357575	MK357616
Parafenestella austriaca	CBS 145262 = C152	Rosa canina	Holotype	MK356304	MK356304	MK357532	MK357576	MK357617
Parafenestella changchunensis	HMJAU 60182	PopulusL.	Holotype	OL996119	OL897170	–	OL944600	OL898719
Parafenestella faberi	MFLUCC 16-1451	Rosa canina	Holotype	KY563071	KY563074	–	–	–
Parafenestella germanica	CBS 145267 = C307	Corylus avellana	Holotype	MK356305	MK356305	MK357533	MK357577	MK357618
Parafenestella ostryae	MFLU 16-0184	Ostrya carpinifolia	–	KY563072	KY563075	–	–	–
Parafenestella pittospori	CPC 34462	Pittosporum tenuifolium	Holotype	MN562098	MN567606	–	–	–
Parafenestella pseudoplatani	CBS 142392 = C26	Acer pseudoplatanus	Holotype	MF795788	MF795788	MF795830	MF795876	MF795914
Parafenestella pseudosalicis	CBS 145264 = C301	Salixcf. alba	Holotype	MK356307	MK356307	MK357535	MK357579	MK357620
Parafenestella rosacearum	CBS 145268 = C309	Pyracantha coccinea	Holotype	MK356311	MK356311	MK357539	MK357583	MK357624
Parafenestella rosacearum	C203	Pyrus communis	–	MK356308	MK356308	MK357536	MK357580	MK357621
Parafenestella rosacearum	C269	Crataegus monogyna	–	MK356309	MK356309	MK357537	MK357581	MK357622
Parafenestella rosacearum	C283	Pyrus communis	–	MK356310	MK356310	MK357538	MK357582	MK357623
Parafenestella rosacearum	C315	Rosa canina	–	MK356312	MK356312	MK357540	MK357584	MK357625
Parafenestella rosacearum	C320	Sorbus aria	–	MK356315	MK356315	MK357543	MK357587	–
Parafenestella rosacearum	CBS 145272 = FP11	Prunus domestica	–	MK356314	MK356314	MK357542	MK357586	MK357627
Parafenestella rosacearum	FM1	Rosa canina	–	MK356313	MK356313	MK357541	MK357585	MK357626
Parafenestella salicis	CBS 145270 = C313	Salix alba	Neotype	MK356317	MK356317	MK357545	MK357589	MK357629
Parafenestella salicis	C303	Salix alba	–	MK356316	MK356316	MK357544	MK357588	MK357628
Parafenestella salicum	CBS 145269 = C311	Salix alba	Holotype	MK356318	MK356318	MK357546	MK357590	MK357630
Parafenestella tetratrupha	CBS 145266 = C304	Alnus glutinosa	Epitype	MK356319	MK356319	MK357547	MK357591	MK357631
Parafenestella ulmi	HMJAU 60178	Ulmus pumilaL.	Holotype	OL996115	OL897166	OL944501	OL944596	OL898723
Parafenestella ulmi	HMJAU 60179	Ulmus pumilaL.	Isotype	OL996116	OL897167	OL944502	OL944597	OL898717
Parafenestella ulmicola	HMJAU 60180	Ulmus pumilaL.	Holotype	OL996117	OL897168	OL944503	OL944598	OL898724
Parafenestella ulmicola	HMJAU 60181	Ulmus pumilaL.	Isotype	OL996118	OL897169	OL944504	OL944599	OL898718
Parafenestella vindobonensis	CBS 145265 = C302	Salix babylonica	Holotype	MK356320	MK356320	MK357548	MK357592	MK357632
Protofenestella ulmi	CBS 143000 = FP5	Ulmus minor	Holotype	MF795791	MF795791	MF795833	MF795879	MF795915
Pyrenochaeta nobilis	CBS 407.76 = AFTOL-ID 1856	Laurus nobilis leaves	Neotype	MF795792	MF795792	MF795834	MF795880	MF795916
Pyrenochaetopsis americana	UTHSC DI16-225	–	Holotype	LT592912	LN907368	LT593050	–	LT592981
Pyrenochaetopsis confluens	CBS 142459	Deep tissue/ fluids from human blood sample	Holotype	LT592950	LN907446	LT593089	–	LT593019
Seltsamia galinsogisoli	CBS 140956 = CGMCC 3.17981 =SYPF 7336	Soil of a Galinsoga parviflora	Epitype	KU759584	KU759581	–	–	–
Seltsamia sp.	EAB-67-11b	Emerald ash borer	–	MT777389	–	–	–	–
Seltsamia sp.	SGSF207	–	–	MK192899	–	–	–	–
Seltsamia ulmi	CBS 143002 = L150	Ulmus glabra	Holotype	MF795794	MF795794	MF795836	MF795882	MF795918
Synfenestella pyri	CBS 144855 = C297	Pyrus communis	Holotype	MK356321	MK356321	MK357549	MK357593	MK357633
Synfenestella sorbi	C298	Sorbus aucuparia	–	MK356325	MK356325	MK357553	MK357597	MK357636
Synfenestella sorbi	CBS 144858 = C196	Sorbus aucuparia	Holotype	MK356324	MK356324	MK357552	MK357596	MK357635
Synfenestella sorbi	CBS 144862 = FR	Sorbus aucuparia	Epitype	MK356322	MK356322	MK357550	MK357594	MK357634
Synfenestella sorbi	FRa	Sorbus aucuparia	–	MK356323	MK356323	MK357551	MK357595	–

). In the multi-locus phylogenetic analysis, although P. rosacearum was divided into six groups (Figure 1), it was still identified as one species because the tef1-α sequences of C203, C283 and C309 are almost the same. The rpb2 sequences of strains C203, C315, FM1 and FP11 are identical, while C269 and C283 differ from C203, C283 and C309 by 20 nucleotides [15]. In the phylogenetic analysis, P. germanica and P. pseudoplatani clustered in the same clade as P. parasalicum and P. salicum. These strains were identified as different species due to morphological distinctiveness [15]. The ascospores of P. germanica were larger than P. pseudoplatani (29–39.5 × 13–16.5 vs. 25–29 × 12–14 µm). The ascospores of P. parasalicum were larger than P. salicum (36–44 × 15.8–19.3 vs. 27–33 × 12.5–16 µm) (

Table 3. Synopsis of sexual morph characteristics of eleven Parafenestella species with the newly introduced species in bold.

Taxon	Sexual Morph
	Ascomata	Asci	Ascospores
P. alpina	240–375 μm diam, globose, subglobose or pyriform, usually tightly aggregated in bark on a perithecial host fungus in small numbers, with brown to black, subicular hyphae.	170–208 × 18.5–21.5 μm, cylindrical to oblong, a short stipe and simple or knob-like base, containing 6–8 ascospores in uniseriate arrangement.	24–30.5 × 12–14 μm, typically ellipsoid to fusoid often inequilateral, pale or yellowish-brown, eventually dark brown, with 7–15 transverse and 2–4 longitudinal septa.
P. austriaca	283–431 μm diam, subglobose to pyriform, scattered or aggregated, basally and laterally surrounded by subhyaline to dark brown subicular hyphae.	159–205 × 16–19.5 μm, cylindrical, with a short stipe and simple or knob-like base, containing 4–8 ascospores in uniseriate arrangement.	27–32.5 × 13–15 µm, broadly ellipsoid, symmetric, dark brown or dark reddish-brown, with 9–14 distantly spaced transverse and 3–5 longitudinal septa.
P. changchunensis	280 × 353 μm, globose to depressed globose, solitary or aggregated forming visible black bumps submerged under bark.	95–138 × 16–21 μm, broad cylindrical, short-pedicellate, curved, some curved, 6–8 spores ocular chamber is not visible at maturity, uniseriate arrangement.	18–25 × 8–13 μm, fusiform to oval, light yellow to dark brown, developing 2 main septa, 4–6 transverse septa, 2–3 longitudinal septa.
P. faberi	300–500 μm diam, tightly or loosely aggregated in small numbers, with ostiolar, partly erumpent through bark fissures, maxing with Cytospora species.	135–180 × 18.5–23.5 μm, cylindrical to oblong or narrowly clavate, a short stipe and simple or knob-like base, 4–8 ascospores in uniseriate to partly biseriate arrangement.	28.5–36 × 12.5–16 µm, variable in shape, pale or yellowish-brown to dark brown, with 1–4 main septa, 7–14 transverse and 1–5 longitudinal septa.
P. germanica	230–450 μm diam, black, solitary or in small groups on inner bark or on the ostiolar level of old Diaporthe decedens.	140–173 × 17.5–22 μm, cylindrical to oblong, with a short stipe and simple or knob-like base, containing 2–8 ascospores (obliquely or overlapping), uniseriate arrangement.	29–39.5 × 13–16.5 μm, ellipsoid to broadly fusoid, turning yellow to yellow-brown to dark brown, with 1–3 main septa, 8–15 transverse and 3–6 longitudinal septa.
P. parasalicum	270–400 μm diam, immersed in bark, globose, subglobose or pyriform, forming groups, maxing with Cytospora species.	185–219 × 22–27 μm, cylindrical to oblong, with a short stipe and simple or knob-like base, containing 4–8 ascospores (overlapping, obliquely), uniseriate to partly biseriate arrangement.	36–44 × 15.8–19.3 μm, fusoid or ellipsoid, yellow-brown to dark brown, with 2 main septa, 11–16 distinct transverse septa and 3–5 longitudinal septa.
P. pseudosalicis	300–400 diam, subglobose to subpyriform, immersed in bark or on ascomata of an effete perithecial fungus, often with concave apex, covered with subicular hyphae.	186–215 × 17.5–19 μm, cylindrical to oblong, with a short stipe and simple or knob-like base, containing 4–8 ascospores in uniseriate arrangement.	25–29 × 12–14 μm, ellipsoid, yellow-brown to dark brown, with 1–3 main septa, 7–11 transverse and 2–4 longitudinal septa, with minute guttules.
P. rosacearum	285–432 μm diam, globose, subglobose to subpyriform, immersed on often blackened inner bark, scattered or in small groups, erumpent through bark fissures.	181–240 × 19–22 μm, cylindrical to oblong, with a short-contorted stipe and simple or knob-like base, containing 2–8 ascospores in uniseriate, rarely partly biseriate arrangement.	28–35 × 13.5–16.5 μm, ellipsoid, symmetric to inequilateral, yellow-brown to dark brown, with 1–3 main septa, 7–15 transverse and 2–5 longitudinal septa.
P. salicis	275–442 μm diam, globose, subglobose to pyriform or subconical, immersed below the epidermis on inner bark, partly erumpent through bark fissures.	141–188 × 16–19 μm, cylindrical to oblong, with a short stipe and simple or knob-like base, containing 1–8 ascospores in (obliquely) uniseriate to partly biseriate arrangement.	23–29 × 11–13.5 μm, ellipsoid to fusoid, symmetric, golden yellow-brown (when fresh) to dark brown, with 1–3 main septa, 5–11 transverse and 1–3 longitudinal septa.
P. salicum	270–420 diam, globose, subglobose or pyriform, immersed in bark, the inner bark layers connected to the host, scattered or aggregate, cover with subicular hyphae.	181–228 × 19.5–24 μm, cylindrical, with a short stipe and simple or knob-like base, containing 6–8 ascospores in (overlapping) uniseriate arrangement.	27–33 × 12.5–16 μm, broadly ellipsoid to broadly fusoid, first 2-celled and hyaline, turning golden yellow to dark brown or dark reddish-brown, with 9 –14 transverse and 3–4 longitudinal septa.
P. tetratrupha	300–500 μm diam, globose, subglobose or pyriform, immersed, tightly or loosely aggregated in whitish to dark brown subiculum, erumpent through fissures.	154–229 × 18.5–22.2 μm, cylindrical to oblong, with a short stipe and simple or knob-like base, containing 2–8 ascospores in uniseriate arrangement.	26.5–33.5 × 13–16.5 μm, ellipsoid, yellow-brown to reddish-brown to dark brown, with 1–3 main septa, 8–17 distinct transverse and 2–4 longitudinal septa.
P. ulmi	170–225 × 194–260 μm, globose to ellipsoid, immersed under the host epidermis, visible as black spots or having a convex surface.	115–181 × 11–15 μm, cylindrical, mostly curved, short-pedicellate, containing 8 ascospores, uniseriate to partially overlapping.	18–24 × 8–12 μm, broadly ellipsoid, yellowish to brown, with 5–8 transversely septate, 1–2 longitudinal septa.
P. ulmicola	242–434 × 310–462 μm, globose to subglobose, on the surface, semi-immersed, visible as a convex hemisphere, with a papilla.	105–153 × 11–14 μm, broad cylindrical, some curved, short-pedicellate, containing 8 ascospores, short-pedicellate, uniseriate, rarely overlapping.	17–22 × 8–12 μm, broadly oval, yellowish to brown, with 4–8 transversely septate and 1–3 vertical septate.
P. vindobonensis	308–425 μm diam, globose, subglobose or pyriform, immersed in bark, partially erumpent, tightly aggregated in small groups on inner bark mixing with pseudostromata of a Cytospora sp.	179–214 × 13.5–15.5 μm, cylindrical, with a short stipe and simple or knob-like base, containing 4–8 ascospores in uniseriate arrangement.	24.5–30.5 × 9.5–11 μm, oblong, fusoid or narrowly ellipsoid, turning yellowish to medium brown, 1–6 main septa, when mature with 7–11 thick transverse and 1–3 septa, containing minute droplets.

). There were 0.40% (2/494) base differences in the ITS, 2.28% (16/701) base differences in β-tub, 0.51% (4/789) base differences in tef1-α and 1.41% (15/1063) base differences in rpb2 between P. germanica and P. pseudoplatani. There were 3.42% (24/701) base differences in β-tub, 1.90% (15/789) base differences in tef1-α and 1.32% (14/1063) base differences in rpb2 between P. parasalicum and P. salicum. Thus, the species boundaries of P.ulmi and P. ulmicola were justified based on their distinct morphological traits and nucleotides differences. Therefore, we introduce P. ulmicola as a novel species, and this is first report of Parafenestella on Ulmus trees.

4. Discussion

The family Cucurbitariaceae was introduced by Winter [12] and typified by Cucurbitaria berberidis (Pers.) Gray [46]. Members of this family occur worldwide and are commonly recorded in Austria, Germany, England and Ukraine as saprobic or necrotrophic on various substrates including plant debris, soil and wood [14,15,47]. Although ribosomal markers and the ITS region are important for phylogenetic analyses, other loci are often needed for better resolution at the species level [48,49,50,51]. The ITS region can have low support values on key evolutionary nodes and cannot be used to accurately classify species in most genera [52,53]. Housekeeping genes and protein-coding genes such as act, β-tub, cal, gapdh, rpb2 and tef1-α are thus usually recommended for a stable and reliable topology in phylogenetic analyses [54,55,56].

In this study, ASAP [38] was used to determine the most informative loci for Parafenestella. The β-tub gene provided the best species level identification of Parafenestella, followed by rpb2, tef1-α, ITS and LSU based on ASAP analyses (Figure 2, Figures S8–S11). ASAP analyses based on the β-tub gene provided the best resolution of P. ulmi and P. ulmicola, in addition to P. changchunensis, P. pseudosalicis and P. salicis (Figure 2). The ITS region is an important marker; however, it could not delineate between P. pseudoplatani (CBS 142392), P. parasalicum (CBS 145271), P. salicum (CBS 145269), P. austriaca (CBS 145262), P. germanicola (CBS 145267) and P. rosacearum (C203, C269, C283, C315, C320, CBS 145272, CBS 145268, FM1) as they were recovered as a group in ASAP analysis. In the ASAP analysis of the β-tub gene, this clade was divided into seven groups: (1) P. austriaca (CBS 145262), (2) P. germanicola (CBS 145267), (3) P. rosacearum (C269, C283, C315, FM1), (4) P. rosacearum (CBS 145272, CBS 145268) and P. rosacearum (C203), (5) P. pseudoplatani (CBS 142392), (6) P. parasalicum (CBS 145271) and (7) P. salicum (CBS 145269) (Figure 2). The β-tub gene exists in all eukaryotes and is involved in the formation of the spindle during cell division [57]. β-tubulin plays an important role in defining the characteristics of species [58]. The ASAP analysis of the β-tub gene likely reflects the interspecific relationship within Parafenestella. Thus, we encourage the inclusion of β-tub in the phylogenetic studies of Parafenestella species. This result is also supported by the phylogeny of single genes, two loci datasets (ITS + β-tub, Figure S14); ITS + rpb2, Figure S12); ITS + tef1-α, Figure S13) and multi-loci dataset (Figures S7 and S11).

Valenzuela-Lopez et al. [58] established Allocucurbitaria in Cucurbitariaceae based on morphological and phylogenetic analysis. Allocucurbitaria botulispora (CBS 142452) was classified as Pyrenochaeta species [43]. Valenzuela-Lopez et al. [41] examined the morphology of Pyrenochaeta and suggested that A. botulispora was more similar to phoma-like taxa. As it clustered in Cucurbitariaceae, the authors classified the species under the genus Allocucurbitaria within Cucurbitariaceae [41]. Seltsamia was introduced with the unique characteristics of pleomassaria-like fungus [14]. There is no confirmed report of the holomorph character of the type species (S. ulmi), and thus the generic status is constrained. Three species of Allocucurbitaria are listed in Species Fungorum [44], with one species reported on Ulmus glabra in Norway, one species from soil in China and one species reported from diseased human scab in the USA [41,59]. Notably, the Allocucurbitaria strains can be saprophyte and can harbor soil and/or opportunistic fungal disease in humans [41,42,43]. We provide the first report of Allocucurbitaria on dead twigs of Populus morus.

Parafenestella is the fourth most speciose genera in Cucurbitariaceae (Cucurbitaria 94 species; Fenestella 28 species; Neocucurbitaria 21 species; Parafenestella 14 species; Syncarpella 7 species; Rhytidiella 4 species; Allocucurbitaria 2 species; Astragalicola 2 species; Paracucurbitaria 2 species; Synfenestella 2 species; Cucitella 1 species; Protofenestella 1 species; Seltsamia 1 species) [44]. Parafenestella species are commonly distributed over temperate areas including northeast China but are rarely found in the tropical regions [11,13]. All three novel species in this study were collected during early spring in Changchun, Jilin Province, China. Jilin Province (40°52′~46°18′ N) belongs to a temperate continental climate, and the study of similar vegetation from similar climates is likely to result in many Parafenestella taxa [60]. We speculate that extensive investigations in the temperate regions would result in numerous Parafenestella members. Climate conditions also affect the infection degree of Cucurbitariaceae fungi to hosts, as temperatures below 0 °C may stop fungal development [15]. The age of the host including branch size and thickness may also affect the development of Cucurbitariaceae [15].

Parafenestella is characterized by immersed to erumpent and aggregated or clusters of ascomata [15]. The number of ascomata in Parafenestella (as a cluster) is often less than 10, which is higher than in Fenestella and Synfenestella [14,15]. Parafenestella does not form distinct pseudostromata, while Fenestella forms a pustular pseudostroma appearing as bumps, and Synfenestella forms conspicuous pseudostromatic pustules on pseudostromata [15]. The ascospores of Parafenestella are irregularly arranged and partially overlapping, while the ascospores of Fenestella and Synfenestella are borne in a uniseriate arrangement [14,15]. The sexual morph of Cucurbitariaceae is usually found on the wood and bark of trees and shrubs (Corylus avellana, Prunus domestica, Rosa canina, Sorbus aucuparia) [15]. The asexual morph of Parafenestella has not been reported from the natural host and is successfully produced only in culture [14,15]. However, pycnidia in artificial culture often lack conidiophores, which could be due to environmental conditions [61].