Morphological and Phylogenetic Analyses Reveal Four New Species of Gnomoniopsis (Gnomoniaceae, Diaporthales) from China

The fungal genus Gnomoniopsis (Gnomoniaceae, Diaporthales) has been reported all around the world and isolated from multiple plant hosts. Based on multilocus phylogenies from a combined dataset of internal transcribed spacer (ITS) region, the ribosomal RNA gene cluster, and partial regions of translation elongation factor 1 alpha (tef1) and partial beta-tubulin (tub2), in conjunction with morphological characteristics, we describe and illustrate herein four new species, including Gnomoniopsis diaoluoshanensis sp. Nov., G. lithocarpi sp. Nov., G. mengyinensis sp. Nov. and G. yunnanensis sp. Nov. Alongside this, their similarity and dissimilarity to morphologically-allied and phylogenetically-related species are annotated and discussed. For facilitating future identification, we update the key to all species currently recognized in this genus.


Introduction
Diaporthales Nannf. is an important order in the perithecial ascomycetes Sordariomycetes Erikss. & Winka, accommodating not only saprophytes but also endophytes or phytopathogens on various hosts [1][2][3][4][5]. Gnomoniaceae Winter, which contains 60 genera and 919 species, the second largest family in this order, occurs on growing and overwintering leaves and twigs of hardwood trees, shrubs, and herbaceous plants [6,7]. This family was first established in 1886 [8] and conserved by Hawksworth and Eriksson in 1988 [9,10]. Gnomoniaceae was circumscribed by Sogonov et al. in 2008 [11], and since then, their concept has been followed by others. At the present time, besides morphology and molecular data, host specificity has become a key characteristic for species identification and a single species in the Gnomoniaceae is often associated with a single host genus or species [6,[12][13][14][15][16][17].
Gnomoniopsis Berl. was initially described as a subgenus within Gnomonia Ces. & De Not. for species with multi-septate ascospores [11]. Subsequently, multiple septa were found not to be a stable characteristic; thus, the Gnomoniopsis was synonymized with Gnomonia [17]. Currently, Gnomoniopsis is accepted as a separate genus in the Gnomoniaceae and typified by Gnomoniopsis chamaemori (Fr.) Berl. This genus is characterized by having small, black perithecia immersed in the host tissue and one-septate, oval to fusiform ascospores [4]. Species in this genus are delimitated by a combination of morphological and molecular data, and are known to inhabit three plant families only, viz. Fagaceae, Onagraceae and Rosaceae [4,5,11,15,18]. A total of 36 names are documented for Gnomoniopsis in the Index Fungorum (accessed on 20 June 2022) and 26 species possess sequence data.
Fungi associated with leaf spots were collected from Castanea mollissima Bl. (Fagaceae), Castanopsis chinensis Hance (Fagaceae), and Lithocarpus fohaiensis (Hu) A. Camus (Fagaceae). We obtained their respective morphological characteristics by separation and purification,   Notes: New species established in this study are in bold. Ex-type or ex-epitype strains are marked with "*".

Phylogenetic Analyses
The generated sequences for each gene were subjected to BLAST searches for identifying closely related sequences in the NCBI's GenBank nucleotide database [27]. For the ITS-tef1-tub2 analysis, subsets of sequences from the alignments of Jiang et al. [4] were used as backbones. Newly generated sequences in this study were aligned with additional related sequences downloaded from GenBank (Table 1), using MAFFT 7 online service with the auto strategy (http://mafft.cbrc.jp/alignment/server/, accessed on 20 June 2022) [28]. To establish the identity of the isolates at species level, phylogenetic analyses were conducted first individually for each marker and then combinedly (ITS-tef1-tub2) (Supplementary File S1).
Phylogenetic analyses were conducted for the multi-marker data based on maximum likelihood (ML) and Bayesian inference (BI) algorithms. For BI, the best evolutionary model for each partition was determined using MrModeltest v. 2.3 [29] and incorporated into the analyses. ML and BI run on the CIPRES Science Gateway portal (https://www.phylo. org/, accessed on 20 June 2022) [30]. ML was performed in RaxML-HPC2 on XSEDE (8.2.12) [31] and 1000 rapid bootstrap replicates were run with the GTRGAMMA model of nucleotide evolution. BI was performed in MrBayes on XSEDE (3.2.7a) [32][33][34]. For ML analyses, the default parameters were used and BI was carried out using the rapid bootstrapping algorithm with the automatic halt option. Bayesian analyses included 4 parallel runs of 5,000,000 generations, with the stop rule option and a sampling frequency of 100 generations. The burn-in fraction was set to 0.25 and posterior probabilities (PP) were determined from the remaining trees. All resulted trees were plotted using FigTree v. 1.4.4 (http://tree.bio.ed.ac.uk/software/figtree, accessed on 20 June 2022) and the layout of the trees was carried out in Adobe Illustrator CC 2019.

Phylogenetic Analyses
The alignment contained 50 isolates representing Gnomoniopsis and allied taxa, and the strain CBS 109778 of Melanconis stilbostoma was used as the outgroup. A total of 1751 characters were used for phylogenetic analyses, viz. 1-550 (ITS), 551-1222 (tef1), 1223-1751 (tub2). Of these characters, 979 were constant, 69 were variable and parsimonyuninformative and 703 were parsimony-informative. MrModelTest recommended that the Bayesian inference should use the Dirichlet base frequencies and the GTR+I+G evolutionary mode for all the three partitions. The topology of the Bayesian tree was consistent with that of the ML tree, and therefore is shown as a representative for recapitulating evolutionary history within the genus Gnomoniopsis (Figure 1). The final ML optimization likelihood was -13036.518679. The 50 strains were assigned to 28 species clades on the phylogram (Figure 1).

Taxonomy
Culture characteristics-Colonies on PDA entirely occupy a 90 mm petri dish in 14 days at 25 • C in dark, with a growth rate of 6.0-6.5 mm/day, are grey-white to creamy white with an irregular margin, spreading out in circles in a similar way to petals and the reverse is similar in color.
Notes-Phylogenetic analyses of a combined three genes (ITS, tef1 and tub2) showed that Gnomoniopsis diaoluoshanensis sp. nov. formed an independent clade and is phylogenetically closely related to G. daii, G. mengyinensis sp. nov. and G. yunnanensis sp. nov. (Figure 1). In detail, G. diaoluoshanensis is distinguished from G. daii by 14/496, 25/314 and 32/445 characters in ITS, tef1 and tub2 sequences, respectively. It is distinguished from G. mengyinensis   Culture characteristics-Colonies on PDA entirely occupy a 90 mm petri dish in 14 days at 25 °C in dark, with a growth rate of 6.0-6.5 mm/day, are grey-white to creamy white with an irregular margin, spreading out in circles in a similar way to petals and the reverse is similar in color.
Culture characteristics-Cultures incubated on PDA at 25 • C in dark attain 82.0-86.0 mm in diameter after 14 days, with a growth rate of 5.8-6.2 mm diam/day and the colonies are flat, spreading with moderate aerial mycelia and lobate to undulate margins, grey-white to creamy, spreading out in a similar way to petals and the reverse is similar in color.

Key to the Species of Gnomoniopsis
Together with the 4 new species proposed in this study, we have currently accepted a worldwide total of 30 species in the genus Gnomoniopsis. In order to facilitate identification in the future, a key to the species of Gnomoniopsis is provided herein. Characteristics Culture characteristics-Cultures incubated on PDA at 25 • C for 14 days in dark attain 69.0-72.0 mm in diameter, with a growth rate of 4.9-5.2 mm diam/day, with moderate aerial mycelia and a lobate to undulate margin, grey-white to creamy, spreading out in a similar way to petals and the reverse is similar in color.
Notes-Strains SAUCC YN1659, SAUCC YN1657 and SAUCC YN1641 are identified to the same species Gnomoniopsis yunnanensis sp. nov. on the basis of similar morphology and molecular monophyly. For details, one can refer to the notes for G. diaoluoshanensis.

Key to the Species of Gnomoniopsis
Together with the 4 new species proposed in this study, we have currently accepted a worldwide total of 30 species in the genus Gnomoniopsis. In order to facilitate identification in the future, a key to the species of Gnomoniopsis is provided herein. Characteristics adopted in the key include perithecia, septa, asci, ascospores, conidiogenous cells, conidia, and chlamydospores.

Discussion
In the present study, four new species (Gnomoniopsis diaoluoshanensis, G. lithocarpi, G. mengyinensis, and G. yunnanensis) from three hosts (Castanea mollissima, Castanopsis chinensis, and Lithocarpus fohaiensis) in three provinces of China were described and illustrated (Figures 2-5), and all these three hosts belong to the family Fagaceae. Currently, Gnomoniopsis species were found from hosts that belong to three plant families (Fagaceae, Onagraceae and Rosaceae). Sixteen Gnomoniopsis species (including the four new species herein) were described from fagaceous hosts. Only one species (G. racemula) was described from the Onagraceae family [11,15,36]. The remaining 11 species were from the family Rosaceae. The Fagaceae, Onagraceae and Rosaceae plants are widely distributed in China, suggesting abundant potentially new Gnomoniopsis species.
The Gnomoniopsis species were reported with 200 records in Fungal Databases (https://nt.ars-grin.gov/fungaldatabases/index.cfm, accessed on 20 June 2022). Among these, G. daii and G. chinensis were determined to be phytopathogenic, causing fruit rot and leaf spot diseases and branch canker of Chinese chestnut, respectively [40,41]. Gnomoniopsis smithogilvyi were illustrated and described in 12 countries (Australia, New Zealand, Chile, France, India, Ireland, Italy, Portugal, Spain, Switzerland, United Kingdom and USA) with 30 records in Fungal Databases, causing sweet chestnut branch canker and fruit rot in Australia, Europe and USA [42][43][44]. Apart from this, Linaldeddu et. al. revealed some fungi associated with branch diseases of hazelnut in Sardinia (Italy), including Dothiorella iberica, Do. omnivora, Do. symphoricarposicola and G. smithogilvyi. Gnomoniopsis smithogilvyi was isolated from rotting chestnut kernels as an endophyte from asymptomatic flowers, leaves and stems of the genus Chestnut [45]. The descriptions, pathogenicity testing and molecular data for species of Gnomoniopsis by taxonomists represent an important resource for plant pathologists and plant quarantine officials.