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

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.


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  [13]. Jaklitsch et al. [15] provided a comprehensive study of fenestelloid Table 1. The PCR primers and amplifying conditions used in this study.

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 ( [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).

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]. 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 tef 1-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 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 tef 1-α gene). Based on the current results, the βtub gene is the best marker for the identification of Cucurbitariaceae taxa at the species level.
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.
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].
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 greenishblack, round.
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.
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.

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 tef 1-α are thus usually recommended for a stable and reliable topology in phylogenetic analyses [54][55][56].
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].
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/jof8090905/s1, Figure S1: The best-scoring RAxML tree based on a concatenated ITS dataset. Figure S2: The best-scoring RAxML tree based on a concatenated LSU dataset. Figure S3: The best-scoring RAxML tree based on a concatenated rpb2 dataset. Figure S4: The best-scoring RAxML tree based on a concatenated tef 1-α dataset. Figure S5: The best-scoring RAxML tree based on a concatenated tub2 dataset. Figure S6. The best-scoring RAxML tree based on a concatenated ITS, LSU, rpb2, tef 1-α and tub2 dataset. Figure S7. Phylogram generated from maximum parsimony analysis based on combined ITS, LSU, rpb2, tef 1-α and tub2 dataset. Figure S8: Phylogram generated from ASAP analysis using LSU sequence data. Figure S9: Phylogram generated from ASAP analysis using rpb2 sequence data. Figure S10: Phylogram generated from ASAP analysis using tef 1-α sequence data. Figure S11: Phylogram generated from ASAP analysis using ITS, LSU, rpb2, tef 1-α and tub2 dataset. Figure S12: The best-scoring RAxML tree based on a concatenated ITS + rpb2 dataset. Figure S13: The best-scoring RAxML tree based on a concatenated ITS + tef 1-α dataset. Figure S14: The best-scoring RAxML tree based on a concatenated ITS + tub2 dataset.
Author Contributions: Conceptualization, Y.L. and C.P.; Writing-original draft and formal analysis, W.S.; Data curation, W.S., R.X., C.P. and C.S.B.; Investigation, W.S. and C.P.; Methodology, W.S., R.X., C.P., C.S.B., S.T. and Y.D.; Supervision, Y.L. and C.P.; Writing-review & editing, W.S., C.S.B. and C.P.; funding acquisition, Y.L. and C.P. All of the authors have read and approved the final draft. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement: Not applicable.

Informed Consent Statement: Not applicable.
Data Availability Statement: All sequences generated in this study were submitted to GenBank.