Additions to the Knowledge of the Genus Pezicula (Dermateaceae, Helotiales, Ascomycota) in China

Simple Summary Species of the genus Pezicula are endophytes, phytopathogens, or saprobes, and some of them have biocontrol potential, promoting plant growth and resistance to environmental stress. The species diversity of Pezicula in China was reviewed. Two new species (P. ellipsoids and P. fusispora) and two new Chinese records (P. acericola and P. carpinea) were discovered using morphological and molecular approaches. Descriptions and illustrations of macroscopic and microscopic features were provided for the new and newly found taxa. Pezicula aurantiaca was excluded from the fungal catalogue of China. Together with the previously reported taxa (P. cinnamomea, P. ericae, P. heterochroma, P. magnispora, P. melanigena, P. neosporulosa, P. ocellata, P. rhizophila, P. cf. rubi, and P. subcarnea), 14 Pezicula species are currently known from China. The results provide updated information and improve our understanding of the genus. Abstract We describe two new species of Pezicula (Dermateaceae, Ascomycota), P. ellipsoides and P. fusispora, which are discovered in China. Pezicula ellipsoides sp. nov. is distinct in producing 2–3 stipitate apothecia on a basal stroma with a light yellow hymenium, broadly ellipsoid 0–1(–2)-septate ascospores, and divergent DNA sequence data. Pezicula fusispora sp. nov. is characterized by sessile apothecia, 0.3–0.8 mm in diam, a yellowish hymenium, J+ asci 135–170 × 15–21 μm, multiseptate ascospores, 33–48 × 7–10.3 μm, and growing on rotten bamboo. In addition, Pezicula acericola and P. carpinea are reported for the first time from China, and 11 Pezicula species previously reported from China are reviewed and briefly noted, of which one was a misidentified species. Phylogenetic analyses inferred from ITS nrDNA sequences confirm the placement of P. ellipsoides and P. cinnamomea in the genus Pezicula.


Introduction
Pezicula Tul. & C. Tul. belongs to the family Dermateaceae Fr. (Helotiales, Leotiomycetes), which was established in 1869 and typified by P. carpinea (Pers.) Tul. ex Fuckel [1]. The sexual stages of the genus are often found as bright-colored, fleshy, pruinose or pulverulent, sessile to short-stipitate apothecia on woody substrates [2]. The asexual stages of Pezicula were originally placed in Cryptosporiopsis Bubák & Kabát, which is treated as a later synonym according to the 'one fungus-one name' rule of the International Code of Nomenclature for algae, fungi, and plants [3]. Pezicula species are endophytes, phytopathogens, or saprobes, and some of them have the abilities of antagonizing plant pathogens, producing compounds with antimicrobial activities, promoting plant growth, and increasing plant resistance to environmental stress [2,[4][5][6][7]. Several regional investigations of the genus have been carried out [8][9][10][11]. A monographic treatment of Pezicula was published by Verkley in 1999, in which 26 species were accepted [2]. Eleven taxa were further introduced, ten species were transferred to Pezicula from other genera, and one name was treated as synonym thereafter [3,5,[12][13][14][15][16][17][18]. One hundred and sixty-two epithets are listed under Pezicula in the Index Fungorum online database [1]. In recent years, morphological characters combined with PCR-based restriction fragment length polymorphism (RFLP) patterns of ribosomal DNA as well as DNA sequence analyses were used for reliable species identification. Forty-six species are currently recognized in the genus.
In China, the first report of Pezicula species was attributed to Teng (1963) based on specimens of P. ocellata (Pers.) Seaver collected from Ningxia and Qinghai provinces [23]. Additional species have been recorded from different regions [7,9,12,13,[24][25][26][27][28][29][30]. During our studies on the species diversity of Dermateaceae in China, herbarium specimens were reexamined, and recent collections of the genus were identified on the basis of morphological features and molecular data. The occurrence of 11 Pezicula species in China are summarized and reviewed based on the literature. In addition, two new species are described, and two new records are added to the fungal catalogue of China.

Morphological Observations
The study was based on newly collected materials and specimens preserved in the Herbarium Mycologicum Academiae Sinicae (HMAS). Macroscopic characters were recorded according to the field notes or observed under a SZX7 stereomicroscope (Olympus Corporation, Tokyo, Japan). Dried apothecia were rehydrated with distilled water and sectioned at a thickness of 15−20 µm with a YD-1508A freezing microtome (Yidi Medical Equipment Co., Ltd., Jinhua, Jiangsu, China). Iodine reactions of ascus apical apparatus were tested in Melzer's reagent and Lugol's solution with or without 3% KOH pretreatment [31]. Microscopic examinations and measurements were taken from dried apothecia under a BH-2 microscope (Olympus Corporation, Tokyo, Japan). Microscopic photographs were taken using an Axiocam 305 color microscope camera (Zeiss Corporation, Göttingen, Germany) attached to an Axioskop 2 Plus microscope (Zeiss Corporation, Göttingen, Germany).

DNA Extraction, PCR, and Sequencing
Genomic DNA was extracted from dried apothecia using a Plant Genomic DNA Kit (TIANGEN Biotech. Co., Beijing, China). PCR reaction mixtures had a final volume of 30 µL, containing 15 µL 2 × Taq MasterMix (CWBiotech. Co., Beijing, China), 1.5 µL of each primer (10 mM), 2 µL DNA, and 10 µL deionized water. The nuclear ribosomal internal transcribed spacer (ITS) region was amplified using the primers ITS1 and ITS4 [32]. PCR reactions were carried out in an Applied Biosystems 2720 thermocycler (Foster City, CA, USA) under the following conditions: 94 • C for 5 min, followed by 35 cycles of 94 • C for 30 s, 53 • C for 30 s and 30 s at 72 • C, and a final extension of 72 • C for 10 min. PCR products were purified and sequenced at Tianyi Huiyuan Bioscience and Technology Co. Ltd. (Beijing, China).

Sequence Assembly, Alignment, and Phylogenetic Analyses
Newly generated sequences were assembled using BioEdit 7.0.9.0 [33] and submitted to GenBank. ITS sequences of representative Pezicula species and two outgroup taxa were retrieved from GenBank (Table 1). The sequence dataset was aligned with MUSCLE [34] and manually adjusted where necessary in BioEdit 7.0.9.0 [33]. Newly generated sequence data are marked with **. Types are indicated with the superscript "T".
The ITS matrix was analyzed using Maximum Likelihood (ML) and Bayesian inference (BI) methods. ML analysis was conducted in RAxML 8.0 [35] using a GTRGAMMA model and tested with 1000 bootstrap replicates. BI analysis was performed using MrBayes 3.1.2 [36]. The best-fit nucleotide substitution model was estimated by MrModeltest 2.3 [37] with the Akaike information criterion. Two parallel runs of four simultaneous chains of Markov Chain Monte Carlo were performed for 2,000,000 generations with trees sampled every 100 generations. The first quarter of the sampled trees were discarded as the burn-in phase, and the remaining trees were used for calculating posterior probabilities (PP) in the majority rule consensus tree. The phylogenetic trees were visualized in FigTree v1.4.4 [38].
Notes: In the BLASTN search of the ITS region of P. ellipsoides in NCBI, P. cinnamomea (CBS 242.60, MH857970) shares the highest sequence similarity of 96.55% (503/521), followed by P. brunnea (CBS 120291, NR_155652) and P. acericola (CBS 248.97, KR859101), both with 96.35% (502/521) similarity. The phylogenetic analyses confirmed the placement of P. ellipsoides in Pezicula and its distinction from any other species of the genus.

New Records for China
Notes: Pezicula acericola is widely distributed in Europe and North America [2]. The macroscopic and microscopic features of the Chinese materials coincide with those from other countries. The ITS sequence of the Chinese collection (HMAS 33726) has a 98.89% similarity with those of the Canadian materials (KR859094-KR859101). This is a new record for China. The two voucher specimens (HMAS 33726 and HMAS 33749) were originally misidentified as P. aurantiaca, which is clearly different in possessing sessile apothecia that are mostly solitary or 2-3(-4) on a basal stroma, asci J− in Melzer's reagent, and ascospores smaller in size (14.5-23 × 6-10 µm) [2].
Notes: Pezicula carpinea is the type species of the genus, which was previously reported from Europe and North America [2,5]. The specific epithet of the species refers to its common occurrence on Carpinus sp. The sexual and asexual characteristics of the species have been well-documented and illustrated in previous studies [2,5]. The features of the Chinese collection are concordant with those from other regions of the world. The overmatured ascospores were recorded as yellow-brown and muriform [2], but such ascospores were not found in the Chinese material due to the different stage of development. Apothecia erumpent, mostly 2-15 on a basal stroma, discoid, margin slightly raised, drying concave, substipitate to short-stipitate, 0.5-2.0 mm in diam; hymenium pruinose, ochraceous when dry; receptacle concolorous with hymenium. Ectal excipulum of textura angularis to textura globulosa, 25-40 μm thick, cells hyaline to brown, isodiametric or nearly so, axis of cells arranged at a high to right angle to the outer surface, 5-20 μm in diam. Medullary excipulum of textura intricata, 110-220 μm thick, hyphae hyaline to light brown, 4-5.5 μm wide. Subhymenium composed of interwoven hyphae, 40-55 μm thick, Notes: The species was reported from Guangxi Province in China [26]. It occurs on broad-leaved trees as well as conifers [2].
Pezicula ericae ( Known distribution: Asia (China), Europe (Norway), North America (USA # ). Notes: Pezicula ericae was originally described in the USA as an endophytic fungus from roots of ericaceous plants [10]. Subsequently, strains of the species were isolated from orchids [39,40], aspen [11], pine [41], spruce [42], fir [43], and other hosts [6,44], in addition to ericaceous plants [45][46][47]. The species is able to promote plant growth [48], improve the drought resistance ability of plants [49], and synthesize compounds with antimicrobial activities against phytopathogenic bacteria and fungi [6,44] Known distribution: Asia (China # ). Notes: The species was originally described from Sichuan Province and is distinctive in producing large, muriform ascospores (46-58 × 14.5-20 µm) and growing on herbaceous stems [12]. Further distribution was not reported. The type specimen of P. magnispora is too scanty to extract DNA, and sequence data are not available. Known distribution: Asia (China), Europe (Austria # ). Notes: Pezicula melanigena was originally isolated from roots of healthy and declining 80-120-yr-old oak trees (Quercus robur and Q. petraea) in Austria and is only known from its asexual stage. It was also stated that the intensity of culture pigmentation, growth rate, size of macroconidia, color of chlamydospores, and shape of setae are variable among isolates of the species [50]. The fungus was detected in forest soil samples from northeastern China via a high-throughput sequencing method [28]. Pezicula  Notes: The species was originally introduced to science based on the materials from the Netherlands and China [13], and the Chinese samples were all endophytes of Abies beshanzuensis from Zhejiang Province. Subsequently, a strain was isolated from soil in Korea [51]. It is also known as an endophytic fungus of Pseudowintera colorata, an endemic medicinal plant of New Zealand [52]. The strain VDBF-2 isolated from Vaccinium dunalianum in Yunnan Province showed strong antagonistic effects against plant pathogens and wood-decaying fungi [30]. Based on comparative and pan-genomic analyses of the endophytic P. neosporulosa, an example of convergent genomic adaptation to endophytism was revealed, mostly determined by enzymes involved in carbohydrate metabolism and secondary metabolite biosynthesis [53].

Discussion
In this study, two novel species and two new Chinese records of Pezicula were discovered based on morphological features, and the positions of two of them, P. ellipsoides and P. acericola, were further confirmed by phylogenetic inference. Eleven species of the genus previously recorded from China were reviewed, of which one misidentified name should be excluded. Up to now, 14 Pezicula species are known from the country, among which nine were known as saprobes [12,[23][24][25][26], four were plant endophytes [7,13,30,45], and one was even reported in soil [28] but might also be plant-associated.
As far as the lifestyles of Pezicula species are concerned, they are supposed to be initially endophytic in living plant tissues but switch to saprophytic when their hosts die, and those which are weakly phytopathogenic may cause diseases when their hosts are under stress [2,5]. Some of the endophytic species show antifungal effects against plant pathogens, some may produce antibiotics and fungicidal or herbicidal compounds [2,4,6,7,30,[56][57][58][59], and others possess the ability to promote plant growth [7,48]. In view of the antimicrobial activity and plant growth promotion of Pezicula, they might have application potential in plant disease control and increasing crop yield.
In most cases, morphological characteristics are sufficient to distinguish Pezicula species, especially those that have both sexual and asexual stages. DNA sequence data are very helpful in discovering new species and understanding relationships among taxa within the group. ITS, LSU, rpb2, tub2, and translation elongation factor-1 alpha (tef-1α) gene sequences have been used in the taxonomic and phylogenetic studies of the genus [5,13,14,18]. Based on the results of the previous studies and our analyses, the variations of the ITS region appear to be able to distinguish Pezicula species correctly. Additional research should be carried out regarding species diversity, the connection of sexual and asexual stages, the selection of supplementary DNA barcodes, and the exploration of potential uses of the members of the genus. Knowing the species diversity is the first step in gaining this knowledge. We expect to find additional Pezicula species in our future surveys in different regions of the country.

Conclusions
The species diversity of the genus Pezicula in China was explored. Two new species and two new Chinese records of the genus were discovered, described, and illustrated. The previously reported taxa were reviewed with one excluded from the fungal catalogue of China, resulting in 14 species that are currently known from the country.