﻿Multi-gene phylogeny and morphology of two new Phyllosticta (Phyllostictaceae, Botryosphaeriales) species from China

﻿Abstract Phyllosticta (Phyllostictaceae, Botryosphaeriales) includes plant pathogens, endophytes and saprobes, occurring on various hosts worldwide. During the present study, isolates associated with leaf spots were obtained from the hosts Quercusaliena and Viburnumodoratissimum, and identified based on morphological features and phylogenetic inference from the analyses of five loci (ITS, LSU, tef1, act and gapdh). Results supported the introduction of two novel species, namely Phyllostictaanhuiensis and P.guangdongensis. Phylogenetically, P.anhuiensis and P.guangdongensis formed two well-separated lineages in the P.concentrica and P.capitalensis species complexes, distinguishing from all presently accepted species in this genus by DNA sequence data. Morphologically, P.anhuiensis and P.guangdongensis have the typical structure of the genus Phyllosticta, and differed from their closely related species by the length of the conidial appendage.


Introduction
The genus Phyllosticta was established by Persoon (1818) and classified in Phyllostictaceae (Botryosphaeriales) Wijayawardene et al. 2020). Initially, Phyllosticta was placed in the Phyllostictaceae (Fries 1849). In a multi-locus phylogeny in the Dothideomycetes, Schoch et al. (2006) placed Phyllosticta into Botryosphaeriaceae (Botryosphaeriales), which was agreed upon by  and Liu et al. (2012). Subsequently, Slippers et al. (2013) reinstated the Phyllostictaceae to accommodate Phyllosticta in terms of phylogenetic relationships. Recently, Pseudofusicoccum was added in this family based on the morphological characters of the conidia covered by a mucous sheath and molecular evidence . The asexual morph of Phyllosticta is characterized by pycnidial conidiomata containing aseptate conidia surrounding with a mucoid layer and bearing a single apical appendage (van der Aa 1973; van der Aa and Vanev 2002; Wikee et al. 2011). The sexual morph of Phyllosticta is characterized by erumpent ascomata, 8-spored, clavate to broadly ellipsoid asci, ellipsoid to limoniform ascospores (van der Aa 1973; Wikee et al. 2011). Following the implementation of "one fungus one name" nomenclature rules, the name Phyllosticta (asexual state) was used over Guignardia (sexual state) and Leptodothiorella (spermatial state) (Glienke et al. 2011;Wikee et al. 2011).
The Phyllosticta species identification solely delimited by morphology and host association may be difficult to assess (Wikee et al. 2011;Su and Cai 2012). Many species are difficult to distinguish due to slight morphological variation, and the mucoid layer or appendage will be absent or invisible in some species (van der Aa and Vanev 2002;Jin 2011;Wikee et al. 2011). Besides, the host range of Phyllosticta is unclear; some species exhibit the broadest host range while others do not (Wikee et al. 2011;Rashmi et al. 2019;Norphanphoun et al. 2020). To overcome the lack of morphological features and host range, phylogenetic approaches based on molecular loci were used to resolve the classification and identification of Phyllosticta species (Baayen et al. 2002;Wulandari et al. 2009;Wong et al. 2012;Wikee et al. 2013a). Based on the phylogenetic analyses of a combined ITS, LSU, tef1, act and gapdh sequence data, the current taxonomic classification of Phyllosticta comprises six species complexes i.e., P. capitalensis, P. concentrica, P. cruenta, P. owaniana, P. rhodorae and P. vaccinii species complexes (Norphanphoun et al. 2020). Currently, the polyphasic approach involving phylogenetic, morphological, and other analyses is used to clarify species boundaries (Norphanphoun et al. 2020;Zhang et al. 2022).
Members of Phyllosticta species are known as pathogenic, endophytic, or rarely saprobic fungi associated with a variety of plants and have a worldwide distribution (van der Aa and Vanev 2002; Glienke et al. 2011;Wikee et al. 2011;Jiang et al. 2021;Wang et al. 2023). As pathogens, Phyllosticta species cause spots on the leaves or fruits of many economical plants (e.g., Musa spp., Citrus spp. and Vitis spp.), leading to substantial economic losses Wong et al. 2012;Wikee et al. 2013b;Tran et al. 2017). As endophytes, some species were found associated with leaf spots but did not cause any symptom in pathogenicity tests, e.g., P. oblongifoliae was isolated from leaf spots of Garcinia oblongifolia, P. pterospermi was isolated from leaf spots of Pterospermum heterophyllum, and P. capitalensis was isolated from leaf spots of Citrus spp. (Wikee et al. 2013b;Tran et al. 2019;Zhang et al. 2022). In this study, two novel fungal species named P. anhuiensis and P. guangdongensis, were isolated from diseased leaves of Quercus aliena in Anhui Province and Viburnum odoratissimum in Guangdong Province, respectively. This paper describes these species based on molecular evidence and morphological characteristics.

Isolation and morphological observations
Samples of Quercus aliena and Viburnum odoratissimum showing necrotic spots were obtained and collected from Anhui and Guangdong Provinces. Samples were surfacesterilized in 75% ethanol for 30 s, then sterilized in 1.5% sodium hypochlorite for 1 min, followed by three rinses with sterilized water and dried on sterilized filter paper, and cut into small sections (3 × 3 mm) from the margins of infected tissues. The sections were plated onto potato dextrose agar (PDA) plates and incubated at 25 °C. Hyphal tips from the edge of emerging colonies were transferred on fresh PDA plates and purified by single-spore culturing (Choi et al. 1999). The cultures and dried specimens of the new isolates have been deposited with the China Forestry Culture Collection Center (CFCC; http://cfcc.caf.ac.cn/) and the herbarium of the Chinese Academy of Forestry (CAF; http://museum.caf.ac.cn/).
Colony features of cultures on PDA medium, synthetic low-nutrient agar (SNA), and malt extract agar (MEA) were recorded after 14 d incubation at 25 °C. After conidiomata appeared, fungal structures (including conidia, conidiogenous cells, and appendage) were measured and captured at least 50 measurements using a Nikon Eclipse 80i compound microscope with differential interference contrast optics.

Phylogenetic analyses
Newly generated in this study were combined using SeqMan v. 7.1.0, and reference sequences (Table 1) were downloaded from GenBank, according to the recent publication (Hattori et al. 2020;Norphanphoun et al. 2020;Crous et al. 2021;Bhunjun et al. 2022;Nguyen et al. 2022;Tan and Shivas 2022;Zhang et al. 2022). Alignments were done by MAFFT v. 7.036 (https://maft.cbrc.jp/alignment/server/) using default settings and manually improved using MEGA v.7.0 (Kumar et al. 2016). The phylogenetic analyses of the combined five loci (ITS, LSU, tef1, act and gapdh) were performed by maximum likelihood (ML) and Bayesian inference (BI). The ML research was conducted with the CIPRES web portal (Miller et al. 2017) using RAxML v. 8.2.12 (Stamatakis 2014) under the GTR+GAMMA model with 1000 bootstrap iterations. The BI analyses was performed by MrBayes 3.1.2 (Ronquist and Huelsenbeck 2003). MrModelTest v. 2.3 (Nylander 2004) was used to determine the best-fit evolution model for each locus. Bayesian posterior probabilities (BYPP) were evaluated by Markov Chain Monte Carlo sampling (MCMC). Four Markov chains were performed for 2 million generations in two independent runs until the split deviation frequencies decreased below 0.01, and sampling every 100 generations. The first 25% of sampled trees were discarded as burn-in, and the remaining ones were used to calculate BYPP. Trees were visualized in FigTree 1.4 (http://tree.bio.ed.ac.uk/software/ figtree), and the ML bootstraps (ML-BS) ≥ 50% and BYPP ≥ 0.9 were presented on nodes of the ML tree.
Culture characters. Colonies on PDA flat, slow growing, grayish-green in the center, and dark green at margin reaching 85 mm diameter after two weeks. Colonies on MEA slow growing, yellow in the center, white at undulate the margin, reaching a 20-25 mm diameter after two weeks. Colonies on SNA flat, slow growing, grayishgreen, reaching a 25-30 mm diameter after two weeks.

Discussion
Phyllosticta is a species-rich genus with more than 3211 records listed in the Index Fungorum (http://www.indexfungorum.org). For the Phyllosticta species identification, molecular data have proven useful in resolving species relationships (Okane et al. 2003;Su and Cai 2012;Guarnaccia et al. 2017;Norphanphoun et al. 2020;  et al. 2022). ITS is a genetic marker for genus level, and combining it with additional loci (LSU, tef1, act and gapdh) is enough for species-level resolution (Jayawardena et al. 2019;Norphanphoun et al. 2020). In this study, based on the phylogenetic analyses of presently accepted species using five loci (ITS, LSU, tef1, act and gapdh), there are six species complexes and 93 species accepted in Phyllosticta (Table 1), viz., P. capitalensis species complex (including 33 species), P. concentrica species complex (including 28 species), P. cruenta species complex (including 22 species), P. owaniana species complex (including six species), P. rhodorae species complex (including two species), and P. vaccinii species complex species complex (including two species). P. anhuiensis and P. guangdongensis formed two well separated clades in the P. concentrica and P. capitalensis species complexes, distinguishing from all accepted species in this genus by DNA sequences data.
Morphologically, our isolates have the typical structure of Phyllosticta (van der Aa and Vanev 2002). The asexual morph of species in the P. concentrica species complex is characterized by globose or ellipsoid to obvoid conidia enclosed in a thin persistent sheath with an apical mucoid appendage (Norphanphoun et al. 2020). The asexual morph of species in the P. capitalensis species complex are characterized by ellipsoid or ellipsoid to obovoid, ovoid, obpyriform conidia with a mucoid sheath with an apical mucoid appendage (Norphanphoun et al. 2020). Our isolates include the essential characteristics of their species complexes, and differ from their closest relatives by the size ranges of conidia and appendage (Motohashi et al. 2008;Glienke et al. 2011).
In this study, we introduced two novel species from forestry trees. Previously, many Phyllosticta species were found in economic hosts, and with the investigation and study of Phyllosticta, many Phyllosticta will be found on forestry trees and this will improve our understanding of the species diversity.