Fresh specimens of Gnomoniaceae-related fungi were collected from branches and leaves of hosts in Beijing, Jiangxi and Shaanxi provinces (Tables 1–3). Isolates from host material were obtained by removing a mucoid spores mass from perithecia and pycnidia-like conidiomata, spreading the suspension on the surface of 1.8% potato dextrose agar (PDA), and incubating at 25 °C for up to 24 h. Single germinating conidia/ascospore was removed and plated on to fresh PDA plates. Specimens are deposited in the Museum of the Beijing Forestry University (BJFC). Axenic cultures are maintained in the China Forestry Culture Collection Centre (CFCC).

Morphological observations of the asexual/sexual morph in the natural environment were based on features of the conidiomata or ascomata on infected plant tissues and micromorphology, supplemented by cultural characteristics. Ascomata and conidiomata from tree barks were sectioned by hand, using a double-edged blade and structures were observed under a dissecting microscope. The gross morphology of conidiomata or ascomata was recorded using a Leica stereomicroscope (M205 FA). Fungal structures were mounted in clear lactic acid and micromorphological characteristics were examined using a Leica compound microscope (DM 2500) with differential interference contrast (DIC) optics. Thirty measurements of each structure were determined for each collection. Colony characters and pigment production on PDA were noted after 10 d. Colony colors were described according to Rayner (1970).

Total genomic DNA was extracted from fresh mycelium grown on PDA using a cetyltrimethylammonium bromide (CTAB) method (Doyle and Doyle 1990). PCR amplifications were performed in a DNA Engine Peltier Thermal Cycler (PTC-200; Bio-Rad Laboratories, Hercules, CA, USA). The primer sets ITS1 and ITS4 (White et al. 1990) were used to amplify the ITS region. The primer sets LR0R and LR7 (Vilgalys and Hester 1990; Vilgalys and Sun 1994) were used to amplify the nuclear ribosomal large subunit (LSU) region. The primer sets EF1-728F (Carbone and Kohn 1999) and EF1-1567R (Rehner 2001) were used to amplify a partial fragment of the translation elongation factor 1-α gene (tef1-α). The primer sets RPB2-5F and fRPB2-7cR (Liu et al. 1999) were used to amplify the partial RNA polymerase II subunit (rpb2) region. The primer sets T1 (O’Donnell and Cigelnik 1997) and Bt2b (Glass and Donaldson 1995) were used to amplify the beta-tubulin gene (tub2). The PCR conditions were: an initial denaturation step of 5 min at 94 °C followed by 35 cycles of 30 sec at 94 °C, 50 sec at 48 °C (ITS, LSU) or 54 °C (tef-1α) or 55 °C (rpb2, tub2) and 1 min at 72 °C, and a final elongation step of 7 min at 72 °C. PCR amplification products were assayed via electrophoresis in 2% agarose gels. DNA sequencing was performed using an ABI PRISM 3730XL DNA Analyser with a BigDye Terminater Kit v.3.1 (Inv-itrogen, USA) at the Shanghai Invitrogen Biological Technology Company Limited (Beijing, China).

The quality of our amplified nucleotide sequences was checked and combined by SeqMan v.7.1.0 and reference sequences were retrieved from the National Center for Biotechnology Information (NCBI), based on Mejía et al. (2011a), Senanayake et al. (2018), Jiang and Tian (2019), and Jiang et al. (2019), supplemented by sequences of Tenuignomonia styracis and Neognomoniopsis quercina from Crous et al. (2019) and Minoshima et al. (2019). Sequences were aligned using MAFFT v. 6 (Katoh and Toh 2010) and manually corrected using Bioedit 7.0.9.0 (Hall 1999).

The phylogenetical analyses were conducted using Maximum Parsimony (MP), Maximum Likelihood (ML) and Bayesian inference (BI). MP was performed with PAUP v. 4.0b10 (Swofford 2003) using tree-bisection-reconnection (TBR) as the branch-swapping algorithm. Other calculated parsimony scores were tree length (TL), consistency index (CI), retention index (RI), and rescaled consistency (RC). ML was performed with RAxML (Stamatakis 2006) as implemented in raxmlGUI 1.3 (Silvestro and Michalak 2012), using the ML + rapid bootstrap setting and the GTRGAMMA substitution model with 1000 bootstrap replicates. BI was performed using a Markov Chain Monte Carlo (MCMC) algorithm in MrBayes v. 3.0b4 (Ronquist and Huelsenbeck 2003). Two MCMC chains, started from random trees for 1,000,000 generations and trees, were sampled every 100^th generation, resulting in a total of 10,000 trees. The first 25% of trees were discarded as burn-in of each analysis. Branches with significant Bayesian Posterior Probabilities (BPP) were estimated in the remaining 7500 trees. Phylogenetic trees were viewed with FigTree v.1.4.3 (Rambaut 2016) and processed by Adobe Illustrator CS5. Alignment and trees were deposited in TreeBASE (submission ID: S26271). The nucleotide sequence data of the new taxa have been deposited in GenBank (Tables 1–3).

The first sequences dataset for the ITS, LSU, tef1, and rpb2 was analyzed to focus on Gnomoniaceae. The alignment included 45 taxa, including the outgroup sequences of Melanconis marginalis (Table 1). The aligned four-locus datasets included 3388 characters. Of these, 2180 characters were constant, 198 variable characters were par-simony-uninformative and 1010 characters were parsimony informative. The heuristic search using maximum parsimony (MP) generated 4 parsimonious trees (TL = 3241, CI = 0.539, RI = 0.672, RC = 0.362), from which one was selected (Fig. 1). In the phylogenetic tree, two strains form a well-supported clade (MP/ML/BI=100/100/1) sister to the species Flavignomonia rhoigena from Rhus chinensis.

Figure 1. 

Maximum parsimony phylogram of Gnomoniaceae based on a combined matrix of ITS, LSU, tef1 and rpb2 genes. The MP and ML bootstrap support values above 50% are shown at the first and second position, respectively. Thickened branches represent posterior probabilities above 0.90 from BI. Scale bar: 80 nucleotide substitutions. Strains in this study are in blue and ex-type strains are in blod.

The second dataset with ITS, tef1 and tub2 sequences were analyzed in combination to infer the interspecific relationships within Gnomoniopsis. The alignment included 36 taxa, including the outgroup sequences of Apiognomonia veneta and Plagiostoma euphorbiae (Table 2). The aligned three-locus datasets included 2481 characters. Of these, 1443 characters were constant, 186 variable characters were par-simony-uninformative and 852 characters were parsimony informative. The heuristic search using maximum parsimony (MP) generated one parsimonious tree (TL = 2644, CI = 0.620, RI = 0.781, RC = 0.485), which is shown in Fig. 2. In the phylogenetic tree, three strains form a well-supported clade (MP/ML/BI=100/100/1) that does not include any previously described species.

Figure 2. 

Maximum parsimony phylogram of Gnomoniosis based on a combined matrix of ITS, tef1-α and tub2 genes. The MP and ML bootstrap support values above 50% are shown at the first and second position, respectively. Thickened branches represent posterior probabilities above 0.90 from BI. Scale bar: 80 nucleotide substitutions. Strains in this study are in blue and ex-type strains are in blod.

The third dataset with ITS, tef1 and tub2 sequences were analyzed in combination to infer the interspecific relationships within Plagiostoma. The alignment included 48 taxa, including the outgroup sequences of Apiognomonia errabunda (Table 3). The aligned three-locus datasets included 2311 characters. Of these, 1556 characters were constant, 204 variable characters were parsimony-uninformative and 551 characters were parsimony informative. The heuristic search using maximum parsimony (MP) generated 6 parsimonious trees (TL = 1462, CI = 0.685, RI = 0.779, RC = 0.534), from which one was selected (Fig. 3). In the phylogenetic tree, four strains from this study group in a well-supported clade with Plagiostoma populinum. The topologies resulting from MP, ML and BI analyses of the concatenated dataset were congruent.

Figure 3. 

Maximum parsimony phylogram of Plagiostoma based on a combined matrix of ITS, tef1-α and tub2 genes. The MP and ML bootstrap support values above 50% are shown at the first and second position, respectively. Thickened branches represent posterior probabilities above 0.90 from BI. Scale bar: 30 nucleotide substitutions. Strains in this study are in blue.