Description of Four Novel Species in Pleosporales Associated with Coffee in Yunnan, China

In Yunnan Province, the coffee-growing regions are mainly distributed in Pu’er and Xishuangbanna. During the surveys of microfungi associated with coffee in Yunnan Province, seven taxa were isolated from coffee samples. Based on molecular phylogenetic analyses of combined ITS, LSU, SSU, rpb2, and tef1-α sequence data and morphological characteristics, four new species viz. Deniquelata yunnanensis, Paraconiothyrium yunnanensis, Pseudocoleophoma puerensis, and Pse. yunnanensis, and three new records viz. Austropleospora keteleeriae, Montagnula thailandica, and Xenocamarosporium acaciae in Pleosporales are introduced. In addition, Paracamarosporium fungicola was transferred back to Paraconiothyrium based on taxonomy and DNA sequences. Full descriptions, illustrations, and phylogenetic trees to show the placement of new and known taxa are provided. In addition, the morphological comparisons of new taxa with closely related taxa are given.


Introduction
The coffee genus Coffea, belonging to the botanical family Rubiaceae, has about 170 varieties [1], and one of the varieties, Coffea arabica, is the most popular coffee variety around the world [2]. Coffee, as the world's second best-selling beverage and a food additive, sells all over the world, with many additional advantages such as refreshment, diuresis, invigorating the stomach, and stimulating appetite [3,4]. China's total production of coffee ranks 12th place in the world, and the annual export volume of coffee beans reaches 82.7 thousand tons [5], making it the fourth largest coffee exporter in Asia after Vietnam, Indonesia, and India [5,6]. In 2020, the export and import of coffee in China were worth $145 million and $310 million, respectively [7]. Yunnan Province is the largest coffee capital in China, and the planting area and total yield account for 95-98% of the total in China [5,8].
Coffee is susceptible to microfungi during pre-harvest and post-harvest processing, especially pathogenic fungi, which can affect coffee tree growth, fruit yield, and quality [9,10]. The most recent coffee fungi review in 2022 counted about 966 coffee-associated microfungi

Sequence Alignment and Phylogenetic Analyses
Raw sequences, both reverse and forward, generated in this study were assembled with the Geneious program (9.1.2) [38]. The newly generated assembled sequences in this study were used for BLAST searches in GenBank [39]. The BLAST search results and sequences from the latest publications were used to obtain sequence data for the phylogenetic analyses. Single gene sequence alignments were made with the online program MAFFT v.7.110 [40]. They moved the uninformative gaps and ambiguous regions by trimAL v1. 2 [41] and combined multigene sequencing by Sequence Matrix program (1.7.8) [42]. The fasta files have transferred the format in the AliView program [43], PHYLIP for maximum likelihood analysis (ML), and NEXUS for Bayesian analysis (BYPP).
Dissanayake et al. [44] was referenced for the phylogenetic analyses, considering both maximum likelihood and Bayesian methods. Maximum likelihood analysis was performed by RAxML-HPC v.8 on the online program CIPRES Science Gateway [45] with rapid bootstrap analysis, followed by 1000 bootstrap replicates, with GTRGAMMA substitution model. Bayesian analysis was performed by MrBayes v3.1.2, and the best models of evolution were estimated by MrModeltest 2.2 [46] and PAUP v. 4.0b10 [47]. The best-fit model was the GTR + I + G substitution model for each locus under the Akaike Information Criterion (AIC). Six simultaneous Markov Chains were run for 2 million generations, and trees were sampled at every 200th generation (resulting in 10,000 trees). Phylogenetic trees were visualized by FigTree v. 1.4.2 [48], the trees were edited in Microsoft Office PowerPoint 2020, and reliable bootstrap support values from ML and BYPP were inserted. All the obtained alignments and phylogenetic trees were deposited in Figshare
In 2022, eight epithets (seven species) isolated as saprotrophic on different hosts and substrates from both terrestrial and freshwater habitats are listed in Index Fungorum (2022) [55][56][57][58][59][60]. Pseudocoleophoma clematidis was transferred to Pseudocyclothyriella by phylogenetic status and morphological distinctiveness [55]. Herein, we introduce two new species in Pseudocoleophoma, and this is the first report of Pseudocoleophoma from coffee.

Montagnula thailandica
Material Notes: Paraconiothyrium (Paraco.) was proposed to accommodate four new species, Paraco. estuarinum, Paraco. brasiliense, Paraco. cyclothyrioides, and Paraco. fungicola by Verkley et al. [75]. The genus is reported as a phytopathogen, saprophyte, and endophyte in a wide range of hosts and substrates worldwide [69,75,76]. The asexual morph characteristics of Paraconiothyrium are eustromatic conidiomata, phialidic conidiogenous cells and aseptate, sometimes 1-septate, thin-walled, smooth or minutely warted, and hyaline to brown conidia [75]. Sexual morph characteristics are globose or subglobose ascomata, clavate or cylindrical asci, and fusiform to ellipsoidal ascospores [76]. The genus comprises 29 epithets (21 species) in the Index Fungorum (2022), but some species have already been moved to other genera. The taxonomic affiliation of the Paraconiothyrium species is still confusing, with contrasting differences at the phylogenetic and morphological levels [77]. In our study, we introduce a new species of Paraconiothyrium from coffee.  Notes: Xenocamarosporium (X.) was first proposed to includethe Camarosporium complex by Crous et al. [78], and the type species X. acaciae was an asexual morph isolated from leaf spots of Acacia mangium in Malaysia [78]. Later, the sexual morph of X. acaciae was introduced by Jayasiri et al. [57] from a decaying pod of Leucaena sp. as a saprotrophic fungus in Thailand. The asexual characteristics of this genus are brown and globose Notes: Phylogenetic analyses show that Paraconiothyrium yunnanensis is well-separated from Paraco. fungicola, with 100% ML, 1.00 BYPP statistical support (Figure 1). Based on BLAST search results of sequence data, ITS and LSU are closely related to Paraco. fungicola, with similarity rates of 98.9% (MK619287) and 99.8% (JX496133). SSU is 99.8% similar to Paraco. variabile (KM096136), rpb2 is 88% (MT473955) similar to Paraconiothyrium sp., and tef 1-α is 97% (LT797134) similar to Paraco. cyclothyrioides. In terms of morphological characteristics, our new species is similar to Paraco. magnoliae in that it has ellipsoidal, yellowish to light brown, and septate ascospores [68]. However, the difference between our new species and Paraco. magnoliae is that the ascospores have 2 or 4 septa, without a sheath, and the penultimate cell is enlarged, while Paraco. magnoliae ascospores have three septa, a sheath, and a distinct guttule. Therefore, our isolate is described as a new species from Coffea sp. in China. Notes: Xenocamarosporium (X.) was first proposed to includethe Camarosporium complex by Crous et al. [78], and the type species X. acaciae was an asexual morph isolated from leaf spots of Acacia mangium in Malaysia [78]. Later, the sexual morph of X. acaciae was introduced by Jayasiri et al. [57] from a decaying pod of Leucaena sp. as a saprotrophic fungus in Thailand. The asexual characteristics of this genus are brown and globose conidiomata, brown textura angularis cells of the peridium, hyaline and smooth conidiogenous cells lining the inner conidiomatal cavity, and ellipsoidal to subcylindrical, hyaline to golden-brown, verruculose, septate conidia [78]. The sexual characteristics of this genus are brown and immersed ascomata, textura angularis cells of the peridium, filiform and septate hamathecium, bitunicate, cylindrical to cylindric-clavate asci, and hyaline to brown, cylindrical, septate ascospores, which are often enlarged at the fourth cell [57]. To date, this genus consists of only one species [22,79]. Here, we introduce one new host and country record in Xenocamarosporium for coffee in China.  Notes: In phylogeny, our strains form a clade with Xenocamarosporium acaciae, and the BLAST results of ITS, LSU, SSU, and tef 1-α of our strain give 100% (MK347766), 99% (MK347983), 99.9% (MK360093), and 99.9% (MK347873) similarities with X. acaciae, respectively. Furthermore, the comparison of morphological characteristics of asci and ascospores shows that our isolate is highly consistent with the sexual morph of X. acaciae morphologically [57]. Therefore, in this study, X. acaciae (ZHKU 22-0117) is reported as a new host record and a country record from coffee in China.  [76]. Based on nucleotide comparisons, Paraco. fungicola (CBS 113269) is different from Paraca. psoraleae (CPC 21632) by 38/552 bp (6.8%) of the ITS and 3/896 bp (0.3%) of the LSU, while it is different from Paraco. magnoliae (MFLUCC 10-0278) in 33/524 bp (6.2%) of the ITS, and 0/899 bp (0%) of the LSU. In addition, the morphology fits with the characteristics of Paraconiothyrium [71,73]. Therefore, we recommend that Paraco. fungicola should be transferred back to the genus Paraconiothyrium, as first reported by Verkley et al. [75].

Discussion
In this study, four new taxa and three new records isolated from coffee are introduced based on morphological and phylogenetic analyses. In addition, a known species was transferred to Paraconiothyrium based on our phylogenetic analyses and the study of Wang et al. [76].
Dictyosporiaceae contains 17 genera and 125 species [22], but none belong to coffeeassociated fungi. Herein, we introduce two new species in the genus of Pseudocoleophoma viz. Pse. puerensis and Pse. yunnanensis, from Yunnan Province, China. This is the first report of coffee-associated fungi in Dictyosporiaceae [10,74].
In our multigene phylogeny, Paraconiothyrium is polyphyletic and paraphyletic within Didymosphaeriaceae, which repeats the results of previous studies [74,76,77,81]. Most species of Paraconiothyrium that were published earlier were mainly based on morphological taxonomy, especially asexual morphology [76,82]. In subsequent studies, some species were classified into other genera. In our phylogenetic tree, we also found the same taxonomic problems in this genus as Phukhamsakda et al. [83]. Paraconiothyrium nelloi and Paraco. fuscomaculans clustered in the genus Kalmusia, while Paraco. nelloi clustered with K. italica with high statistical support (ML/BYPP = 96/1.00). Paraconiothyrium nelloi (MFLU 14-0813) shows few nucleotide differences compared with K. italica (MFLUCC 13-0066), by 1/502 bp (0.2%) of the ITS, 3/857 bp (0.35%) of the LSU, and 2/995 bp (0.2%) of the SSU. The asexual morph of Paraco. nelloi was isolated from a dead twig, while K. italica was observed from the culture of PDA. Conidia of both species differ in size but are similar in shape and color [84]. Paraconiothyrium fuscomaculans (CBS 116.16), grouped with K. longispora, showed good statistical support (ML/BYPP = 82/0.90). Based on nucleotide comparisons, Paraco. fuscomaculans (CBS116.16) is slightly different from K. longispora (CBS 582.83) by 0/532 bp (0%) of the ITS and 1/901 bp (0.1%) of the LSU. Morphologically, Paraco. fuscomaculans and K. longispora have septate conidiogenous cells and almost the same size conidia [58,85]. In 2020, Gonçalves et al. [77] also reported Paraco. nelloi (MFLU 14-0813) and Paraco. fuscomaculans (CBS 116.16) that fit in the genus Kalmusia, and mentioned that morphological analyses of Didymosphaeriaceae are needed to evaluate the redisposition of Paraconiothyrium-like species. Therefore, in future research, we strongly recommend that these two species should be re-collected and re-identified with more collections in order to demonstrate their taxonomic positions. When studying the species of Paraconiothyrium, both morphology and multigene phylogeny are important, and all the Paraconiothyrium taxa should be included in the phylogenetic analyses. In our study, we introduce a new species of Paraconiothyrium based on multigene phylogeny and morphology.
We excluded the type species Austropleospora osteospermi in our phylogenetic analyses due to the lack of multi-gene sequence data [61]. In ITS phylogenetic analysis (not shown), A. osteospermi was not grouped within Austropleospora, thus, we believe that only the ITS gene is insufficient to distinguish intra-species in the phylogeny. The sexual morph of A. osteospermi is similar to A. ochracea [61,63]; therefore, we suggest adding multi-gene sequence data for A. osteospermi in future studies.
In this research, we mainly focused on coffee-associated saprotrophic fungi in Yunnan Province, China, which is one of the biodiversity hotspots in the Greater Mekong Subregion [86,87]. Saprotrophic fungi are considered one of the most active plant litter decomposers that play an important role in the cycling of carbon, nitrogen, and soil nutrients [88]. Some saprotrophic fungi can also release various chemical compounds to increase plant resistance in harsh environments [89]. In addition, some saprotrophic fungi have been investigated for plant disease control [14]. Including the fungi that are reported in this study, to date, about 40 saprotrophic fungi have been recorded from coffee [3,8,10]. Even though coffee is one of the important economic crops worldwide, very few saprotrophic fungi studies on coffee have been carried out in China, and thus, it is important to study coffee saprophytic fungi and their contribution to coffee plants. Our research increases the knowledge of coffee saprophytic fungi and provides a basis for future research on the applied aspects of coffee saprotrophic fungi.

Conclusions
In conclusion, four novel taxa belonging to Pleosporales that are associated with coffee were discovered. Pseudocoleophoma puerensis and Pse. yunnanensis are introduced as new species in Dictyosporiaceae, while Deniquelata yunnanensis and Paraconiothyrium yunnanensis are introduced as new species in Didymosphaeriaceae. The phylogenetic relationships among species of these three genera were also updated in this study. In addition, Austropleospora keteleeriae as a new host record for coffee, as well as Montagnula thailandica, and Xenocamarosporium acaciae as the new host and country records for coffee in China were reported. Furthermore, based on taxonomy and phylogenetic analyses, Paracamarosporium fungicola was transferred back to Paraconiothyrium.