Phylogeny and taxonomy of two new Plectosphaerella (Plectosphaerellaceae, Glomerellales) species from China

Abstract The genus Plectosphaerella is the largest genus in the family Plectosphaerellaceae. Some species are plant pathogens, whereas others are soil-borne. Seven Plectosphaerella isolates were collected from various locations in the southwest of China. Using multi-locus phylogenetic (LSU, ITS, EF1α, RPB2) analyses combined with morphological characteristics, two new species, Plectosphaerella guizhouensissp. nov. and Plectosphaerella nauculasporasp. nov. are described, illustrated and compared with related species.

During the investigation of keratinolytic fungi from different soils in China, seven isolates in the genus Plectosphaerella were obtained in Guizhou Province, China. The aim of our project was to identify these isolates, based on combined molecular phylogeny and morphological characteristics.

Isolates and Morphology
Soil samples were collected from Qianlingshan Park (26°60'N, 106°69'E), Guiyang city and the affiliated hospital of Zunyi Medical University (27°70'N, 106°94'E), Zunyi city, Guizhou Province, China by Zhi-Yuan Zhang on 10 Sept. 2016. Samples were collected 3-10 cm below the soil surface and placed in Ziploc plastic bags. Isola-tion and purification of strains were undertaken according to methods described by Zhang et al. (2019). Sterile chicken feathers and human hairs were combined with the soil samples. Samples were placed in sterile Petri dishes, which were moistened with ddH 2 O. The baited soil sample Petri dishes were incubated at 25 °C for 1 month and remoistened as necessary. Two grams of sample were added to test tubes containing 9 ml of ddH 2 O. The mixture was then diluted to 1:10 4 and 1 ml of suspension was evenly spread on plates containing Sabouraud's dextrose agar (SDA, 10 g of peptone, 40 g of dextrose, 20 g of Agar, 1 litre of ddH 2 O) with anti-bacterial chloramphenicol and cycloheximide medium. Plates were incubated at 25 °C for 5 d. The axenic strains were then transferred to potato dextrose agar (PDA, Bio-way, China) plates for purification and to test-tube slants for storage at 4 °C.
Type collections of the novel species are deposited in the Mycological Herbarium of the Institute of Microbiology, Chinese Academy of Sciences, Beijing, China (HMAS). The ex-type living cultures and other strains of our study are deposited in the China General Microbiological Culture Collection Center (CGMCC) and the Institute of Fungus Resources, Guizhou University (GZAC). The axenic strains were incubated on PDA and Czapek agar (CA, Bio-way, China) at 25 °C in darkness. Macroscopic characterisation was undertaken after 14 d of incubation and the colony colours (surface and reverse) were observed. Preparations were mounted in ddH 2 O to study the mycelial morphology, conidiogenous cells, conidial structures and other microstructures from PDA cultures. Photomicrographs of diagnostic structures were made using an OLYMPUS BX53 microscope equipped with differential interference contrast (DIC) optics, an OLYMPUS DP73 high-definition colour camera and cellSens software v.1.18.

Phylogenetic Analyses
The DNA sequences, generated in this study, were assembled using Lasergene software (version 6.0, DNASTAR). Sequence data, mostly from Giraldo and Crous (2019), were downloaded from NCBI GenBank for molecular phylogenetic analyses (Table 1). Two sequences of Brunneochlamydosporium nepalense (isolates CBS 277.89 and CBS 971.72) were chosen as outgroup taxa. Sequences of each locus were aligned through MAFFT v.7.407 (Katoh and Standley 2013), using the default parameters and manually corrected in MEGA 6.06 (Tamura et al. 2013). The aligned sequences of multiple loci were concatenated by SequenceMatrix v.1.7.8 (Vaidya et al. 2011).
Maximum likelihood (ML) analyses were constructed with IQ-TREE v. 1.6.11 (Nguyen et al. 2015). The best-fit model of substitution for each locus was estimated using IQ-TREE's ModelFinder function (Kalyaanamoorthy et al. 2017) under the Bayesian Information Criterion (BIC). The selected models were TIMe+R2 for LSU, TNe+R2 for ITS, TIM2+F+R3 for EF1α and TN+I+G4 for RPB2. Bootstrap analyses was performed using the ultrafast bootstrap approximation (Minh et al. 2013) with 1,000 replicates and a bootstrap support (BS) ≥ 95% was considered as statistically significant.
For Bayesian Inference (BI), a Markov Chain Monte Carlo (MCMC) algorithm was used to generate phylogenetic trees with Bayesian probabilities using MrBayes v.3.2 (Ronquist et al. 2012) for the combined sequence datasets. The selection of the best-fit nucleotide substitution model for each locus was calculated by the Akaike Information Criterion (AIC) with Modeltest v.3.7 (Posada and Crandall 1988). The GTR+I+G model was selected for all datasets (LSU, ITS, EF1α, RPB2). Two runs were executed simultaneously for 5,000,000 generations and sampled every 500 generations. After the BI analyses, both runs were examined with Tracer v.1.5 (Drummond and Rambaut 2007) to determine burn-in and check for convergence. The final tree was submitted to TreeBASE, submission ID: 24412 (http://www.treebase.org).

Phylogenetic analyses
Fifty-five strains (including the seven with new sequence data) were included in our multi-locus dataset (Table 1), which comprised 2536 positions, of which 322 were phylogenetically informative (35 of LSU, 54 ITS, 76 EF1α, and 157 RPB2). Tree topology of the Bayesian analyses was similar to that of the Maximum likelihood analyses.
Notes. Based on multi-locus phylogenetic analyses (Figure 1, see Results) and similar morphological characteristics, the three strains are regarded as the same species, which cluster together very well and form a single clade separated from other species of Plectosphaerella ( Figure 1). Morphologically, Plectosphaerella guizhouensis differs from others species by the fusiform or cylindrical conidia, non-septate conidia (average 4 × 1.5 μm) and separate conidia (5.5 × 2 μm) (see Key). Therefore, based on combined phylogenetic and morphological evidence, P. guizhouensis is identified as a new species of Plectosphaerella. Etymology. From "naucula", referring to the navicular conidia.
Notes. Phylogenetically, our two isolates CGMCC 3.19656 and CGMCC 3.19657 cluster together very well and form a single clade separated from the other species of Plectosphaerella (Figure 1). Morphologically, Plectosphaerella nauculaspora is the only species that produces navicular conidia in this genus. Therefore, based on both morphological and phylogenetic evidence, P. nauculaspora is proposed as a novel species.

Discussion
In the present study, seven strains of Plectosphaerella fungi were isolated from soil in the Guizhou Province, China. Multi-locus phylogenetic analyses in combination with morphological data were used for identification. Our study resulted in the description of two new species, P. guizhouensis (3 isolates) and P. nauculaspora (2 isolates). In addition, our two isolates CGMCC 3.19654 and CGMCC 3.19655 closely clustered with P. plurivora and their morphological characters are similar to the original description P. plurivora (Carlucci et al. 2012).
Plectosphaerella spp. have diverse life styles and habitat sources -including pathogens of several plants, endophytes of plants, pathogens of animals (mainly involving Austropotamobius pallipes and Oratosquilla oratoria) and saprophytes on soil (Alderman and Polglase 1985, Palm et al. 1995, Domsch et al. 2007, Duc et al. 2009, Carlucci et al. 2012, Liu et al. 2013, Su et al. 2017, Liang et al. 2017, Raimondo and Carlucci 2018, Giraldo and Crous 2019. Although Plectosphaerella spp. were initially isolated from plants (from healthy or symptomatic tissue), subsequent studies found that they also widely distributed on soils and do not necessarily exhibit host specificity (Carlucci et al. 2012, Raimondo and Carlucci 2018, Giraldo and Crous 2019. However, P. oratosquillae can only be isolated from animals and it exhibits host specificity (Duc et al. 2009). Likewise, some species (mainly P. oligotrophica and P. humicola) have so far only been isolated from soils. In comparison with these previous studies, our two new species and one known species of Plectosphaerella were obtained from the soil beside a park road by the baiting technique (a method specifically designed for isolating keratinophilic fungi, Zhang et al. 2019). More studies are needed to assess whether our new species could be isolated from other habitats.
At present, more and more studies use combined data from morphological characteristics and molecular phylogeny for identifying new species (e.g. Carlucci et al. 2012, Liu et al. 2013, Su et al. 2017, Giraldo and Crous 2019, Phookamsak et al. 2019. Throughout the years, several loci have been used in the phylogenetic analyses of Plectosphaerella and its allies, containing ITS, LSU, EF1α, β-tubulin, CaM and RPB2 (Zare et al. 2007, Duc et al. 2009, Carlucci et al. 2012, Liu et al. 2013, Su et al. 2017). Giraldo and Crous (2019) revised the Plectosphaerellaceae and their results suggested that the phylogeny based on LSU+ITS+EF1α+RPB2 can be used for resolving intergeneric and interspecific relationships within the family Plectosphaerellaceae. As a result, we also used the LSU+ITS+EF1α+RPB2 dataset for phylogenetic analyses of Plectosphaerella.
Key to the species of Plectosphaerella