Morphological and phylogenetic analyses reveal two new species of Sporocadaceae from Hainan, China

Species of Sporocadaceae have often been reported as plant pathogens, endophytes or saprophytes and are commonly isolated from a wide range of plant hosts. The isolated fungi were studied through a complete examination, based on multilocus phylogenies from combined datasets of ITS/ tub2 / tef1 , in conjunction with morphological characteristics. Nine strains were isolated from Ficus microcarpa , Ilex chinensis and Schima superba in China which represented four species, viz ., Monochaetia schimae sp. nov. , Neopestalotiopsis haikouensis sp. nov. , Neopestalotiopsis piceana and Pestalotiopsis licualicola . Neopestalotiopsis piceana was a new country record for China and first host record from Ficus macrocarpa. Pestalotiopsis licualicola was first report from Ilex chinensis in China.


Introduction
Xylariales, a group of fungi distributed worldwide, whose members show considerable variability in a number of characteristics, including position of ascomata, presence and type of sterile tissues, and habit (Barr 1990). The Sporocadaceae (type genus: Sporocadus Corda) is a well-de ned family in the Xylariales ). Based on phylogenetic analyses and morphological comparison, Sporocadaceae has delimited thirty genera including Monochaetia (Sacc.) Allesch., Neopestalotiopsis Maharachch. et al., Pestalotiopsis Steyaert, Pseudopestalotiopsis Maharachch. et al., etc., which is generally congruent with the classi cation system proposed by Nag Raj (1993) prior to the DNA phylogeny era. Species of Sporocadaceae are endophytic, plant pathogenic or saprobic, and associated with a wide range of host plants.
Initially, pestalotia-like asexual morphs were classi ed in Amphisphaeriaceae by Samuels et al. (1987), accommodating 36 genera (Hawksworth et al. 1995). Its ordinal level of classi cation, the Amphisphaeriales, was introduced by Eriksson & Hawksworth (1986), but treated as a synonym of Xylariales one year later by Eriksson & Hawksworth (1987). Hawksworth et al. (1995) followed and supported this classi cation by molecular data. The order was recently resurrected to include Amphisphaeriaceae, Clypeosphaeriaceae and another four novel families derived from Amphisphaeriaceae, however, the sequence dataset was largely incomplete and some of the introduced families were not well supported statistically (Senanayake et al. 2015). Subsequently, Bartaliniaceae, Discosiaceae, Pestalotiopsidaceae and Robillardaceae (Crous et al. 2015) were synonymized and revived the older family name Sporocadaceae to accommodate them, together with the Amphisphaeriaceae and Phlogicylindriaceae, Sporocadaceae was accommodated in the Xylariales, however, Amphisphaeriales was not accepted due to a lack of phylogenetic support in their analysis (Jaklitsch et al. 2016).
Presently, agreement on the classi cation and delimitation of the family itself seems to have been reached after intense debate. Fungi in the Sporocadaceae (e.g. Bartalinia, Pestalotia, Pestalotiopsis, Robillarda, Seimatosporium, Seiridium and Truncatella) possess common asexual morphological characters related to their acervular conidiomata, conidiogenesis and conidia ). To date, most phylogenetic studies addressing genera of Sporocadaceae have been based solely on ITS and LSU sequences (Barber et al. 2011;Tanaka et al. 2011;Jaklitsch et al. 2016), or on concatenated datasets of more genes but with incomplete datasets (Senanayake et al. 2015;Wijayawardene et al. 2016). Consequently, the taxonomic concept of, and generic delimitation within Sporocadaceae remain unclear.
Most of the genera of Sporocadaceae contain over-lapping morphological characters of conidia such as the number of median cells, colour of median cells, presence of apical and basal appendages (Jeewon et al. 2002). In this study, we made a collection of the established genera Pestalotiopsis, Neopestalotiopsis and Monochaetia species on leaves of Schima superba, Ficus microcarpa and Ilex chinensis in Hainan Province, China and the new species is established with descriptions, illustrations and molecular data based on ITS, TUB2 and TEF1-α loci.

Specimen collection and morphological descriptions
The samples were collected from Hainan Province, China. The strains were isolated from diseased leaves of Schima superba, Ficus microcarpa and Ilex chinensis using tissue isolation from surface sterilized leaf tissues were conducted following the protocol of Gao et al. (2014). Tissue fragments (0.5 × 0.5 cm) were taken from the margin of leaf lesions and surface-sterilized by consecutively immersing in 75% ethanol solution for 30 s, 5% sodium hypochlorite solution for 1 min, and then rinsing in sterile distilled water for 30 s. The pieces were dried with sterilized paper towels and placed on potato dextrose agar (PDA). All the plates were incubated at biochemical incubator at 25°C for 3-4 days, then hyphae were picked out of the periphery of the colonies and inoculated onto new PDA plates. Photographs of the colonies were taken at 7 days and 15 days using a Powershot G7X mark digital camera. Micromorphological characters were observed using Olympus SZX10 stereomicroscope and Olympus BX53 microscope, all tted with Olympus DP80 high de nition colour digital cameras to photo-document fungal structures. All fungal strains were stored in 10% sterilized glycerin at 4 ℃ for further studies. The holotype specimens are deposited in the Herbarium of Plant Pathology, Shandong Agricultural University (HSAUP). Ex-type cultures are deposited in the Shandong Agricultural University Culture Collection (SAUCC). Taxonomic information of the new taxa was submitted to MycoBank (http://www.mycobank.org).
DNA extraction, PCR ampli cation, and DNA sequencing Genomic DNA was extracted from fungal mycelium on PDA, using a modi ed cetyltrimethylammonium bromide (CTAB) protocol as described in Guo et al. (2000). The internal transcribed spacer regions with intervening 5.8S nrRNA gene (ITS), part of the betatubulin gene region (TUB2), and partial translation elongation factor 1-alpha (TEF1-α) genes were ampli ed and sequenced by using primers pairs ITS4/ITS5 (White et al. 1990 PCR was performed using an Eppendorf Master Thermocycler (Hamburg, Germany). Ampli cation reactions were performed in a 50 µL reaction volume, which contained 25 µL Green Taq Mix (Vazyme, Nanjing, China), 2 µL of each forward and reverse primer (10 µM) (Tsingke, Beijing, China), and 2 µL template genomic DNA in ampli er, and were adjusted with distilled deionized water to a total volume of 50 µL. PCR parameters were as follows: 94°C for 5 min, followed by 35 cycles of denaturation at 94°C for 30 s, annealing at a suitable temperature for 30 s, extension at 72°C for 1 min and a nal elongation step at 72°C for 7 min. Annealing temperature for each gene were 55°C for ITS, 54°C for TUB2, 52°C for TEF1-α. The PCR products were visualised on 1% agarose electrophoresis gel. Sequencing was done bi-directionally, conducted by the Tsingke Biotechnology Company Limited (Shanghai, China). Consensus sequences were obtained using MEGA 7.0 or MEGA-X (Kumar et al. 2016). All sequences generated in this study were deposited in GenBank (Table 1). Isolates marked with "T" are ex-type or ex-epitype strains.

Phylogenetic analyses
Newly generated sequences in this study were aligned with additional related sequences downloaded from GenBank ( ITS, HKY+G for TUB2 and GTR+I+G for TEF1-α were selected and incorporated into the analyses. The MCMC analysis of the three concatenated genes run for 2,650,000 generations, resulting in 2651 trees. The initial 662 trees representative of the analysis burn-in phase was discarded, while the remaining trees were used to calculate posterior probabilities in the majority rule consensus trees ( Fig. 1; rst value: PP ≥ 0.50 shown). The alignment contained a total of 1453 unique site patterns (ITS: 269, TUB2: 670, TEF1-α: 514). The ML tree topology con rmed the tree topologies obtained from the BI analyses, and therefore, only the ML tree is presented (Fig. 1).
ML bootstrap support values (≥ 50%) and Bayesian posterior probability (≥ 0.50) are shown as rst and second position above nodes, respectively. The 81 strains were assigned to 59 species clades based on the three gene loci phylogeny (Fig. 1)

MycoBank 841382
Etymology: Named after the host collection, Haikou City.    (Fig. 1). Morphologically, our strains were similar to N. piceana, which was originally described with an asexual morph on wood of Picea sp., Cocos nucifera and fruit of Mangifera indica in China, but the sexual morph of N. piceana was undetermined. We therefore identify our strains as N. piceana. Phylogram of Sporocadaceae based on combined ITS, TUB2 and TEF1-α genes. The BI and ML bootstrap support values above 0.50 BYPP and 50% are shown at the rst and second position, respectively. The tree is rooted to Bartalinia robillardoides (CBS 122705), ex-type or ex-epitype cultures are indicated in bold face. Strains from the current study are in red. Some branches were shortened to t them to the page -these are indicated by two diagonal lines with the number of times a branch was shortened indicated next to the lines.    Pestalotiopsis licualacola (SAUCC210087). a diseased leaf of Ilex chinensis b surface of colony after 7 days on PDA c reverse of colony after 7 days on PDA d conidiomata e-g conidiogenous cells with conidia h-j conidia. Scale bars: 10 μm (e-j).