Fallen leaves of E. ulmoides were collected in a forest plantation of Guizhou University, Guiyang, Guizhou Province, China, in January 2021. The samples were stored in envelopes and several topsoil samples from the forest were stored in self-sealing bags, then taken back to the laboratory and photographed. Before isolation, collected leaves samples were sprayed two to three times with 75% ethanol to disinfect the leaf surface. Pure cultures of the fungi were obtained by single spore isolation (Chomnunti et al. 2014). Fungi in the soil samples were isolated by the dilution plate method (Zhang et al. 2015). A small amount of soil (1 g) per sample was collected and added to 9 mL of sterile water in a 15 mL sterile glass test tube. It was manually mixed and then the suspension was diluted to a series of concentrations (10^–1, 10^–2, 10^–3, 10^–4, 10^–5 and 10^–6), and 100 µL from each concentration was spread onto 90-mm-diam Petri dishes containing Synthesis of low nutrient Agar (SNA), Potato Dextrose Agar (PDA), Malt Extract Agar (MEA) and Oatmeal Agar (OA) (Zhang et al. 2017). These SNA, PDA, MEA and OA plates were incubated at constant temperature (25 °C) in a controlled temperature light incubator. Holotype specimens of the new species were conserved in the Herbarium of the Department of Plant Pathology, Agricultural College, Guizhou University (HGUP). Ex-type cultures were conserved in the Culture Collection at the Department of Plant Pathology, Agriculture College, Guizhou University, P.R. China (GUCC).

Pure cultures were grown on SNA, PDA, MEA and OA media in a constant temperature incubator (25 °C). Culture characteristics were recorded and examined using a dissecting microscope (LEICA S9i, Germany). The morphological observations and measurements on SNA were made using a Zeiss Scope 5 compound microscope (Axioscope 5, China) with an attached camera AxioCam 208 color (ZEN 3.0) and measurements were made using ZEN 3.0. Taxonomic information for the two new taxa were deposited in MycoBank (www.mycobank.org).

Fresh mycelia were scraped from the PDA plates with a sterilized scalpel. Genomic DNA was extracted using Fungal gDNA Kit (Biomiga #GD2416, San Diego, California, USA) in accordance with the manufacturer’s instructions. PCR amplification was performed in a 25 μL reaction volume following Liang et al. (2018). Primer pairs ITS4/ITS5 (White et al. 1990), EF1-728F/EF1-986R (Carbone and Kohn 1999) and ACT-512F/ACT-783R (Carbone and Kohn 1999) were used for ITS, tef1 and act, respectively. The amplification procedures were performed using the method described by Halo et al. (2019). Purification and sequencing of these three gene loci were carried out by the SinoGenoMax company (Beijing, China).

Sequences used in this study (Table 1) were assembled based on the closest matches from the BLASTn search results (https://blast.ncbi.nlm.nih.gov/Blast.cgi) and previous publications (Sandoval-Denis et al. 2016; Bensch et al. 2018; Halo et al. 2019). Alignments were conducted with the online version of MAFFT v. 7.307 (Katoh and Standley 2016), checked visually and improved manually where necessary using BioEdit v. 7.1.3.0 (Hall 1999). SequenceMatrix v. 1.7.8 (Vaidya et al. 2011) was used to concatenate the aligned sequences of the different loci. Ambiguous areas were excluded from the analysis using AliView (Larsson 2014) and gaps were coded as missing data.

The Maximum Likelihood (ML) analyses were carried out at the CIPRES web portal (Miller et al. 2010) using RAxML (Stamatakis 2006). The tree search included 1,000 non-parametric bootstrap replicates and the best scoring tree was selected from suboptimal trees under the GTRGAMMA substitution model. The resulting replicates were plotted on to the best scoring tree obtained previously. Non-parametric bootstrap analysis was implemented with 1,000 duplicates. Maximum Parsimony (MP) analyses were performed with PAUP v. 4.0a (Swofford 2003), using the heuristic search option with 1,000 random sequence addition replicates and tree bisection-reconnection (TBR) with reconnection limit (=8) as the branch swapping algorithm. Maxtrees was set at 5,000. Branches collapsed (creating polytomies) if maximum branch length is zero. The Tree Length (TL), Consistency Indices (CI), Retention Indices (RI), Rescaled Consistency Indices (RC) and Homoplasy Index (HI) were calculated for each tree generated. Bayesian Inference (BI) analyses were performed in MrBayes v. 3.2.7a (Ronquist et al. 2012). Six Markov chain Monte Carlo runs were started, and the random start trees were calculated for 50,000,000 generations and sampled every 1,000 generations. 25% of the trees initially produced were discarded as burn-in. ML bootstrap support (MLBS) and MP bootstrap support (PBS) equal or greater than 70% (Hillis and Bull 1993) and Bayesian posterior probabilities (PP) equal or greater than 0.95 (Hespanhol et al. 2019) are displayed on the edited phylogenetic tree. The phylogenetic tree was drawn with FigTree v. 1.4.4 (Rambaut 2009).

Morphological and phylogenetically related species were analyzed using the genealogical consistency phylogenetic species identification (GCPSR) model as described by Taylor et al. (2000) by pin-pair homogeneity index test (PHI) (Bruen et al. 2006). The PHI tests were performed in SplitsTree v. 4.17.1 (Huson 1998; Huson and Bryant 2006) as described by Quaedvlieg et al. (2014) to determine the level of recombination within phylogenetically closely related species. The results can be visualized by constructing a split graph using LogDet conversion and the Splits options. The hypothesis of this analysis is if the PHI value is below 0.05 (Фw < 0.05), there is significant evidence for the presence of recombination.

DNA sequences used in this study (Table 1) were selected to obtain phylogenetic trees based on the closest matches by the BLASTn search with strain GUCC 401.6 and eight strains (GUCC 401.1–401.5 to GUCC 401.7–401.9), respectively, with outgroup C. neolangeronii (CBS 797.97) and C. langeronii (CBS 189.54). The final alignment (GUCC 401.6) of ITS, tef1 and act comprised 1,033 characters, viz. ITS: 1–543, act: 544–770 and tef1: 771–1033, which included 843 constant characters, 38 variable characters and 152 parsimony-informative characters, and the alignment (GUCC 401.1–GUCC 401.9 except for GUCC 401.6) comprised 1,040 characters, viz., ITS: 1–544, act: 545–780 and tef1: 781–1040; which included 813 constant characters, 46 variable characters and 181 parsimony-informative characters. The RAxML results were selected to show the topology (Fig. 1 for GUCC 401.6 and Fig. 2 for GUCC 401.1–GUCC 401.9 except for GUCC 401.6), because the MP and Bayesian analyses resulted in similar topologies. The parameter settings that were used are shown in Table 3.

Figure 1. 

Maximum Likelihood (RAxML) tree from the combined analysis of ITS, tef1 and act sequences of Cladosporium, which includes our strain GUCC 401.6. The tree was rooted with C. neolangeronii (CBS 797.97) and C. langeronii (CBS 189.54). ML and MP bootstrap values (≥ 70%) and Bayesian posterior probability (≥ 0.95) are indicated along branches (ML/MP/PP). Our species is highlighted with a yellow background. T = ex-holotype strain.

GUCC 401.6 clustered very close to C. perangustum (CBS 125996 = ex-holotype strain) with relatively high statistical support (79% MLBS/1 PP) (Fig. 1). Strains GUCC 401.2, GUCC 401.3, GUCC 401.4 and GUCC 401.5 had a very close relationship to C. tenuissimum (CBS 125995), variedly supported by MLBS (93%), PBS (70%) and PP (1) (Fig. 2). The comparison of DNA bases (Table 2) showed that our strains cluster with the ex-type strain of C. tenuissimum (CBS 125995, ex-epitype strain) with only one base pair difference in the ITS, two to fifteen base pair difference in the tef1, and one to five base pair difference in the act. Cladosporium eucommiae (GUCC 401.1) is a sister to C. magnoliigena (MFLUCC 18-1559) and C. cladosporioides (CBS 101367) with high statistical support (75% MLBS / 87% MPBS)/(99 MLBS / 87% MPBS / 1 PP) (Fig. 2). The comparison of DNA bases composition (Table 2) indicated that, between C. eucommiae (GUCC 401.1) and C. magnoliigena, there were identical sequences in the ITS region, but 29 bases different in the tef1 region. Unfortunately, Cladosporium magnoliigena did not have act sequence data for comparison. The comparison of DNA bases composition (Table 2) indicated that, between C. eucommiae (GUCC 401.1) and C. cladosporioides (CBS 112388, ex-neotype strain), there were 18 bp differences in the tef1 region, and 13 in the act region, but without difference in the ITS sequences. GUCC 401.7 was closer to C. cladosporioides (CBS 112388, ex-neotype strain) with high support in their respective branches (100% MLBS / 99% MPBS / 1 PP)/(100% MLBS / 100% MPBS / 1 PP) (Fig. 2). The comparison of DNA bases (Table 2) reveals 29 bp difference on tef1 and 14 bp difference in act between C. guizhouense and C. cladosporioides (CBS 112388, ex-neotype strain), but only 1 bp difference in ITS sequences.

Figure 2. 

Maximum Likelihood (RAxML) tree from the combined analysis of ITS, tef1 and act sequences of Cladosporium, which includes our strains GUCC 401.1–GUCC 401.9 (except for GUCC 401.6). The tree was rooted with C. neolangeronii (CBS 797.97) and C. langeronii (CBS 189.54). ML and MP bootstrap values (≥ 70%) and Bayesian posterior probability (≥ 0.95) are indicated along branches (ML/MP/PP). Our species are emphasized with a yellow background. T = ex-holotype strain, ET = ex-epitype strain, NT = ex-neotype strain.

The pairwise homoplasy index (PHI) test revealed that there was no significant recombination (Фw = 0.4589) between C. eucommiae (GUCC 401.1 and GUCC 401.9) and the related taxa C. magnoliigena, C. cladosporioides, C. guizhouense (GUCC 401.7 and GUCC 401.8). The PHI test did not find any statistically significant evidence for recombination (Фw = 0.02487) between our four strains (GUCC 401.2, GUCC 401.3, GUCC 401.4 and GUCC 401.5) and the related taxon C. tenuissimum (CBS 126359, ex-epitype strain, CPC 12795, CPC 10882, CPC 10538, DTO 323-C5, CBS 125995, CPC 13732, CPC 14196 and CPC 13252). Based on the PHI test, there was a statistically significant recombination (Фw = 0.0104) between GUCC 401.6 and the related taxon C. perangustum (CBS 125996, = ex-holotype strain, CPC 13870, DTO 323-E4, CPC 12216, CPC 14247 and CPC 22297).

In this section, we introduced two new species and report two new substrate records.

 Cladosporium eucommiae S.Y. Wang, Yong Wang bis & Y. Li, sp. nov.

MycoBank No: 842406

Fig. 3a–h

Etymology

eucommiae, in reference to the genus name of the host plant (Eucommia ulmoides), from which the fungus was isolated.

Figure 3. 

Cladosporium eucommiae (GUCC 401.1, ex holotype strain). a–d colonies on SNA, PDA, MEA and OA (left: above, right: reverse) e branching conidiophore, secondary ramoconidia and conidia on SNA f–h conidiogenous cells, secondary ramoconidia and conidia on SNA. Scale bars: 10 µm (e–h).

Type

China, Guizhou Province, Guiyang, Huaxi district, plantation forest of E. ulmoides, Guizhou University (26°24'16"N, 106°40'29"E), on fallen leaves of E. ulmoides, S.Y. Wang, Y. Wang & Y. Li, 13 January 2021 (HGUP 401.1, holotype; ex-type living culture GUCC 401.1; additional living culture GUCC 401.9).

Description

Saprobic on fallen leaves of Eucommia ulmoides. Sexual morph: Not developed. Asexual morph: Hyphomycetous. Mycelium abundant, superficially and submerged, overgrowing whole culture dishes, thin to dense, hyphae straight to slightly sinuous, branched, light olive-green to olive-brown, 1.5–5 µm wide, thin-walled, smooth. Conidiophores (7–)22–198 × 2.5–4.5 µm (x̄= 77.2 × 3.3 µm; n = 20), erect, branching, slightly attenuated towards the apex, light olive-green, smooth and thick-walled. Conidia 3–9 × 2.5–4.5 µm (x̄= 5.6 × 3.3 µm; n = 30), in simple and branched acropetal chains, mostly light olive, aseptate, smooth-walled and thin-walled, variable in size and shape, subglobose, ellipsoid-ovoid, obovoid, fusiform, subcylindrical. Secondary ramoconidia 5–25 × 2.5–4.0 µm (x̄= 11.9 × 3.4 µm; n = 30), olive-green, ellipsoid-ovoid, obovoid, fusiform, subcylindrical, aseptate, smooth-walled and thin-walled, rarely thick-walled.

Culture characteristics

Colonies on SNA 35–45 mm diam, after 2 weeks at 25 °C, pale olive, flat, velvety, with a regular edge, reverse light olive. Colonies on PDA 30–45 mm diam, after 2 weeks at 25 °C, olive-brown to gray-olive to iron-gray, with a regular white edge, irregularly folded, slightly depressed at the center, thatched, and often forming a bulge in the colony kernel, reverse olive to dark olive, with a whitish final edge. Colonies on MEA 35–45 mm diam, after 2 weeks at 25 °C, gray-green to olive, less radially furrowed, velvety, with an even gray white edge, reverse olive to dark olive, with an even gray-green final edge. Colonies on OA 35–40 mm diam, after 2 weeks at 25 °C, olive to gray-green, white at the final edge, flat, velvety, margin regular; reverse dark green to black, with a whitish final edge.

GenBank numbers

ITS: OL587465, tef1: OL504966, act: OL519775 (GUCC 401.1); ITS: ON334729, act: ON383337 (GUCC 401.9).

 Cladosporium guizhouense S.Y. Wang, Yong Wang bis & Y. Li, sp. nov.

MycoBank No: 842407

Fig. 4a–h

Etymology

guizhouense , in reference to the type location (Guizhou Province), where the fungus was isolated.

Type

China, Guizhou Province, Guiyang, Huaxi district, plantation forest of Eucommia ulmoides, Guizhou University (26°24'16"N, 106°40'29"E), on fallen leaves of E. ulmoides, S.Y. Wang, Y. Wang & Y. Li, 13 January 2021 (HGUP 401.6, holotype; living culture GUCC 401.7; additional living culture GUCC 401.8).

Figure 4. 

Cladosporium guizhouense (GUCC 401.7). a–d colony on SNA, PDA, MEA and OA (left: above, right: reverse) e–h conidiogenous cells, secondary ramoconidia and conidia on SNA. Scale bars: 10 µm (e–h).

Description

Saprobic on fallen leaves of Eucommia ulmoides. Sexual morph: Not developed. Asexual morph: Hyphomycetous. Mycelium abundant, submerged, overgrowing whole culture dishes, hyphae straight to slightly sinuous, septate, branching, light olive-green to olive-brown, mostly smooth- and thin-walled, 1.5–6 µm wide. Conidiophores 13–100 × 3–4.5 µm (x̄= 60.8 × 3.5 µm; n = 10), erect, branching, light olive-green, smooth- and thin-walled. Conidia 3–7.5 × 2.5–4 µm (x̄= 4.8 × 3.1 µm; n = 30), in simple and branched acropetal chains, mostly light olive, aseptate, mostly smooth- and thin-walled, variable in size and shape, ellipsoid-ovoid, obovoid, fusiform. Secondary ramoconidia 6.5–23 × 3–5.5 µm (x̄= 11.3 × 4.1 µm; n = 30), pale olive-green, narrowly ellipsoid to cylindrical-oblong, subcylindrical, aseptate, smooth- and thin-walled.

Culture characteristics

Colonies on SNA 45–55 mm diam, after 2 weeks at 25 °C, pale olive, flat, velvety, margin regularly, reverse light olive. Colonies on PDA 40–50 mm diam, after 2 weeks at 25 °C, smoke-gray to light olive-gray, reverse leaden-gray, gray-olive at edge both surface and reverse, woolly or felty, broad edge, regular, growth low convex, without protuberant exudates, reverse formed cracks in the middle small circle. Colonies on MEA 30–40 mm diam, after 2 weeks at 25 °C, smoke-gray to light olive-gray, woolly or felty, fluffy, with a whitish narrow final edge; reverse olive-yellow or olive-brown, radially furrowed, irregularly folded, with a whitish narrow final edge. Colonies on OA 30–45 mm diam, after 2 weeks at 25 °C, gray-green or olive, granular and fluffy mycelium, woolly and felty edge, with an irregularly folded whitish and olive final edge; reverse olive-yellow or olive-brown, with a whitish narrow final edge.

GenBank numbers

ITS: OL579741, tef1: OL504965, act: OL519780 (GUCC 401.7); ITS: ON334728, tef1: ON383470, act: ON383338 (GUCC 401.8).

 Cladosporium perangustum Bensch, Crous & U. Braun, Studies in Mycology 67: 65 (2010)

MycoBank No: 517085

Fig. 5a–h

Material examined

China, Guizhou Province, Guiyang, Huaxi district, plantation forest of E. ulmoides, Guizhou University (26°24'16"N, 106°40'29"E), on fallen leaves of E. ulmoides, S.Y. Wang, Y. Wang & Y. Li, 13 January 2021 (HGUP 401.6, living culture GUCC 401.6) (new substrate record).

Figure 5. 

Cladosporium perangustum (GUCC 401.6, new substrate record from Guizhou Province). a–d colonies on SNA, PDA, MEA and OA (left: above, right: reverse) e branching conidiophore, secondary ramoconidia and conidia on SNA f–h conidiogenous cells, secondary ramoconidia and conidia on SNA. Scale bars: 50 µm (e); 10 µm (f–h).

Description

Saprobic on fallen leaves of Eucommia ulmoides. Sexual morph: Not developed. Asexual morph: Hyphomycetous. Mycelium superficial, hyphae branched, hyaline to subhyaline, 2.5–5 μm wide, usually slightly constricted at the septa and somewhat swollen, smooth to somewhat verruculose or irregularly rough-walled. Conidiophores macro- and micronematous, 14–167 × 2.5–4.5 µm (x̄= 65.4 × 3.4 µm; n = 20), erect, branched, slightly attenuated towards the apex, light olive-green, smooth and thick-walled. Conidia in acropetal chains, 2–9.5 × 2–4 µm (x̄= 5.6 × 3.3 µm; n = 30) mostly light olive-green, aseptate, mostly smooth-walled and thin-walled, variable in size and shape, subglobose, ellipsoid-ovoid, obovoid, fusiform. Secondary ramoconidia 6–24 × 2–5.5 µm (x̄= 11.2 × 3.3 µm; n = 30), olive-green, narrowly ellipsoid to cylindrical-oblong, subcylindrical, aseptate, rarely 1-septate, mostly smooth-walled and thick-walled.

Culture characteristics

Colonies on SNA 30–40 mm diam, after 2 weeks at 25 °C, pale olive to pale whitish, flat, velvety, with a regular edge, reverse light olive to light white. Colonies on PDA 30–40 mm diam, after 2 weeks at 25 °C, olive-gray to olive-green or olive-brown, powdery or flocculent, fluffy, regular, radially furrowed, lacerated or feathery, and often forming a gray-white or olive bulge in the colony kernel; reverse dark olive or dull green to black. Colonies on MEA 35–45 mm diam, after 2 weeks at 25 °C, gray-green to white or gray-white, fluffy, radially furrowed, with a whitish final edge; reverse olive-yellow to olive-gray to olive-green, with a whitish final edge. Colonies on OA 35–45 mm diam, after 2 weeks at 25°C, white to olive-green, with a pale gray final edge, velvety or fluffy, margins colorless or pale gray, glabrous, regular; reverse olive-green to dark green.

GenBank numbers

ITS: OL579742, tef1: OL581726, act: OL519779.

 Cladosporium tenuissimum Cooke, Grevillea 6: 140 (1878)

MycoBank No: 145672

Fig. 6a–i

Materials examined

China, Guizhou Province, Guiyang, Huaxi district, plantation forest of E. ulmoides, Guizhou University (26°24'16"N, 106°40'29"E), on fallen leaves of E. ulmoides, S.Y. Wang, Y. Wang & Y. Li, 13 January 2021, (HGUP 401.1; HGUP 401.2; HGUP 401.3 and HGUP 401.4, living cultures GUCC 401.2; GUCC 401.3; GUCC 401.4 and GUCC 401.5) (new substrate record).

Figure 6. 

Cladosporium tenuissimum (GUCC 401.2, new substrate record from Guizhou Province). a–d colonies on SNA, PDA, MEA and OA (left: above, right: reverse) e–h secondary ramoconidia and conidia on SNA i conidia on SNA. Scale bars: 10 µm (e–i).

Description

Saprobic on fallen leaves of Eucommia ulmoides. Sexual morph: Not developed. Asexual morph: Hyphomycetous. Mycelium abundant, superficial and submerged, overgrowing whole culture dishes, hyphae straight to slightly sinuous, septate, branching, light olive-green to olive-brown, smooth-walled, 1.5–6 µm wide. Conidiophores 13–100 × 2.5–4.5 µm (x̄= 60.8 × 3.6 µm; n = 10), erect, branching, light olive-green, smooth and thin walled. Conidia 2.5–7.5 × 2–4 µm (x̄= 4.9 × 3.2 µm; n = 30), in simple and branched acropetal chains, mostly light olive-green, aseptate, mostly smooth- and thin-walled, variable in size and shape, subglobose, ellipsoid-ovoid, obovoid, fusiform. Secondary ramoconidia 5.5–23 × 2.5–5.5 µm (x̄= 0.9 × 3.8 µm; n = 30), pale olive-green, narrowly ellipsoid to cylindrical-oblong or subcylindrical, sometimes septate and sometimes aspetate (1-septate appear at maturity), smooth- and thin-walled.

Culture characteristics

Colonies on SNA 50–55 mm diam, after 2 weeks at 25 °C, pale olive to pale white, flat, velvety, with a regular edge, reverse light olive to light white. Colonies on PDA 40–55 mm diam, after 2 weeks at 25 °C, smoke-gray to light olive-gray or olive to light olive-gray, reverse leaden-gray, gray-olive at edge both surface and reverse, woolly or felty, broad edge, regular, growth low convex, without protuberant exudates, occasionally reverse formed a sunflower like shape in the middle. Colonies on MEA 40–50 mm diam, after 2 weeks at 25 °C, olive-gray or gray, fluffy; reverse olive-green to dark olive, with an olive-yellow to gray-white edge, radially furrowed. Colonies on OA 40–60 mm diam, after 2 weeks at 25 °C, gray-white or iron-gray to gray-olive, fluffy to felty; reverse olive-brown to olive.

GenBank numbers

ITS: OL579746, tef1: OL504967, act: OL519776 (GUCC 401.2); ITS: OL579745, tef1: OL505077 (GUCC 401.3); ITS: OL579744, tef1: OL581724, act: OL519777 (GUCC 401.4); ITS: OL579743, tef1: OL581725, act: OL519778 (GUCC 401.5).