Phylogeny of Graphostromatacea with Three Species Isolated in China

Qirui Li (  lqrnd2008@163.com ) Guizhou medical university https://orcid.org/0000-0001-8735-2890 Xiaofeng Gong Guizhou science and technology information center Xu Zhang Guizhou Medical University Yinhui Pi Guizhou Medical University Sihan Long Guizhou Medical University Youpeng Wu Guizhou Medical University Xiangchun Shen Guizhou Medical University Yingqian Kang Guizhou Medical University Jichuan Kang Guizhou University

Nummularia Tul. & C. Tul. was introduced in 1863 (Tulasne and Tulasne 1863). The name, Biscogniauxia Kuntze was used for the sexual morph of genus (Pouzar, 1979). Biscogniauxia was reviewed by Ju et al. (1998), and forty-nine taxa were accepted. In their study, morphological differences between similar genera were discussed, and a key to the species of Biscogniauxia was provided. The morphological characteristics of Biscogniauxia are as follows: stromata widely effuse with separate ostioles at the surface; perithecia mostly in a layer, but sometimes polystichous; asci 8-spored, cylindrical, with or without an amyloid apical ring; ascospores uniseriate, rarely biseriate, ellipsoid, and brown, with or without germ slits (Ju et al. 1998; Vasilyeva et al. 2007).
Camillea was erected to include taxa with applanate, carbonaceous, cylindrical, or broadly conic-truncate, bipartite stromata with light coloured ascospores (Fries 1849 accepted it as a genus of Graphostromataceae, which was followed by Daranagama (2018).
During investigation of Xylariales from Chian, three taxa possessing features of Graphostromataceae were collected. Based on morphological and molecular data, we identi ed them as Biscogniauxia glaucae, Graphostroma guizhouensis, Camillea broomeana. Detail descriptions were given in this paper. Meanwhile, we discuss the systematic classi cation of Grapholomataceae and suggest that Vivantia and Cryptostroma corticale (Ellis & Everh.) P.H. Greg. & S. Waller should be included in this family.

Collection, isolation and morphology
Specimens with black spots on their surfer were collected from Guizhou province, China. Materials were placed into paper bags with desiccant silica gel and taken to laboratory for examinations. Specimen collection information including location and collection time was recorded. Pure cultures were isolated using single-spore isolation (Chomnunti et al. 2014). Cultures were stored in screw cap centrifuge tubes containing potato-dextrose agar (PDA) medium at 4°C and were preserved in 2 mL screw cap centrifuge tubes with 10% glycerol at − 20°C. Macroscopic characteristics were observed using Olympus SZ61 stereomicroscope and photographed with a tted Canon 700D digital camera ). The contents of the stomata were picked out and mounted in water and Melzer's reagent for anatomical examination. Characteristics of asci, ascospores, and ascus apical apparatus were photographed using a Nikon digital camera (700D) tted to a microscope (Nikon Ni). The dimensions of 30 ascospores, 20 asci, and 20 ascus apical apparatus were measured with Tarosoft ® image framework (v. 0.9.0.7).
Microphotographs were modi ed appropriately without changing features, and were arranged as a plate using Adobe Photoshop CS6. The specimens were deposited in herbarium of Guizhou medical university (GMB), herbarium of Guizhou agricultural college (GACP) and herbarium of Kunming Institute of Botany (KUN), and living cultures were deposited in Guizhou medical university culture collection (GMBC).

DNA extraction, PCR ampli cation and sequencing
Pure cultures were grown on PDA at 25°C for 2 weeks. The fungal mycelia were scratched off with sterilized scalpel. Genomic DNA was extracted from fresh fungal mycelia using the BIOMIGA fungus genomic DNA extraction kit (GD2416, Biomiga, USA), following the manufacturer's instructions. DNA extractions were stored at − 20°C. Region of internal transcribed spacers (ITS) was ampli ed with primers ITS4 and ITS1 (White et al. 1990; Gardes and Bruns 1993). Partial β-tubulin (TUB2) was ampli ed with primers Bt2a and Bt2b (Glass and Donaldson 1995). The second largest subunit of the RNA polymerase II (RPB2) was ampli ed using primers (RPB2-5F, -7cF, and − 7cR) introduced by Liu et al. (1999). The PCR primer pair ACT-512F/ACT-783R (Carbone and Kohn 1999) was used to amplify portions of the α-actin gene (ACT). The components of a 25 µL volume PCR mixture were as follows: double distilled water 9.5 µL, PCR Master Mix 12.5 µL, 1 µL of each primer, 1 µL template DNA. Quali ed PCR products were checked with 1.5% agarose gel electrophoresis stained with GoldenView and sent to Sangon Co., China, for sequencing. The PCR conditions were: initial denaturation step at 95°C for 2 min, 35 cycles of 95°C for

Phylogenetic analyses
Graphostromataceae taxa and closely related families were included in the phylogenetic analyses with two strains of Hypoxylon rickii (MUCL 53309) and H. pulicicidum (MUCL49879) as outgroup (Fig. 1). Datasets of ITS, RPB2, ACT and TUB2 were aligned respectively, and were combined to form a dataset of ITS-RPB2-TUB2-ACT. The dataset of ITS-RPB2-TUB2-ACT consisted 67 taxa, including the two sequences from two outgroup taxa, and contained 2785 alignable characters, 1347 constant characters, 1248 phylogenetically informative characters, and 190 parsimony-uninformative variable characters. Final ML optimization likelihood is -43391.566516. Topology of the complete RAxML and MrBayes trees were consistent. The tree showed that Camillea, Obolarina, Graphostroma and Cryptostroma were nested within Biscogniauxia with a well-supported (100/1). Sequences of Biscogniauxia glaucae showed a close relationship with B. formosana with bootstrap (100/1), and showed as a distinct branch. GMBC0218 was nested in group of Camillea, which showed a very close kinship with Camillea obularia (Fr.) Laessøe

Note
Here we would like to propose a new species of Graphostroma, although Graphostroma guizhouensis shows similar features with G. platystomum. A comparison of the ITS region DNA sequence data between G. guizhouensis and G. platystomum (CBS 270.87, type material of G. platystomum) revealed base pair differences of 7% which supports establishment of G. guizhouensis as a new taxon. We speculate that Graphostroma platystomum should be a group containing some cryptic species. Their morphological characteristics are very similar, but their DNA sequences are quite different. And DNA sequences should be a main diagnostic feature for the genus of Graphostroma.

Disscussion
Nummularia was introduced in 1863 to accommodate the fungi which have attened dis-shaped stromata reminiscent of coins. Obolarina, Camillea and Biscogniauxia all possess Nummularialike anamorphs. Biscogniauxia was adopted for the genus (Pouzar 1979). Obolarina was separated from Biscogniauxia with Obolarina dryophila (Tul. & C. Tul.) Pouzar (= Nummularia dryophila Tul. & C. Tul.) as the type species (Pouzar 1986). Obolarina dryophila possesses spiral germination slits of rather large spores which differ from those of Biscogniauxia. Obolarina and Biscogniauxia have almost the same morphological characteristics, such as the morphology of the stromata, asci, ascspores. Ju et al. (1998) reviewed the Biscogniauxia and presented a key that contained genera with close a nities. They gured out that the main difference between Obolarina and Biscogniauxia is that Obolarina lacks an ascal apical ring. Camillea is characterized by dark brown or black and carbonaceous bipartite stromata, asci with blue apical rings in Melzer's iodine reagent and hyaline or light brown ornamented ascospores lacking a germ slit (Laeaessøe et al. 1989). These characteristics of Camillea can clearly distinguish it from Obolarina and Biscogniauxia.
The anamorphic fungi Cryptostroma corticale introduced as type species of Cryptostroma can cause sooty bark disease of Acer pseudoplatanus L. (Gregory et al. 1951). Stroma was descripted as two layers of " oor stroma" and "roof stroma" by Gregory et al. (1951). Pirozynski (1974) established a new genus, Graphostroma to accommodate G. platystomum, and placed it among Xylariaceae. Molecular systematic studies indicated the close a nity of Graphostroma and Biscogniauxia (Smith et al. 2003). Based on ITS rDNA sequences, Koukol et al. (2015) treated C. corticale as a Hypoxyloideae clade of Xylariaceae which closed to G. platystomum and Biscogniauxia. In this study, we get the similar result. Cryptostroma was listed in Xylariales genera incertae sedis (Wijayawardene et al. 2020). Here, we propose to place Cryptostroma in Graphostromataceae.
Vivantia was accepted in the Amphisphaeriaceae to include species with features of bipartite, carbonaceous, applano-pulvinate stromata, cylindrical asci with apical ring bluing in Melzer's reagent and two-celled, subhyaline ascospores without germ slit (Rogers et al. 1996). Two-layers, carbonaceous stromata and Nodulisporiurn anamorphs indicate its a nity with Biscogniauxia. Vivantia was listed in the family of Graphostromataceae without any evidences (Wijayawardene et al. 2020). In this paper we provides evidences and would like accept Cryptostroma and Vivantia in Graphostromataceae, based on their common features, bipartite stromata, asci with apical ring bluing in Melzer's reagent, light-colored ascospores, Nodulisporium or Xylocladium like anamorphs.
Molecular phylogenetic analysis indicated that species of Graphostromataceae form monotypic family with high value (100/1), and Biscogniauxia is not a monotypic genus. Biscogniauxia was divided into 8 clades. Obolarina, Camillea and Graphostroma clusted in the branch of Biscogniauxia, have close relationship with Biscogniauxia. Obolarina, Camillea and Graphostroma form a monophyletic group respectively. Biscogniauxia mediterranea and B. rosacearum (clade 1) indicate a close relationship with Obolarina. Nevertheless, Biscogniauxia mediterranea have an apical ring of ascus bluing in Melzer's iodine reagent and dark brown ascospores (Ju et al. 1998). Biscogniauxia_rosacearum was published as an endophytic fungus without description of sexual morph. Biscogniauxia granmoi and B. marginata (clade 6) have close genetic relationship with Cryptostroma and Graphostroma. However, the ascospores of B. granmoi and B. marginata are brown which distinct from those of Cryptostroma and Graphostroma (Ju et al. 1998, Laessøe et al. 1999. In terms of the morphological characteristics of ascospores, the differences between Camillea, Graphostroma, Cryptostroma and Biscogniauxia are very obvious. However, phylogeny showed that Camillea, Graphostroma, and Cryptostroma were in the branch of Biscogniauxia. We speculate that the morphological diagnostic features currently used are insu cient for the systematic classi cation of Biscogniauxia and its related genera. Biscogniauxia may be divided into some genera to maintain the uniformity of genus characteristics. Compared with the published morphological species, there are relatively few species with available DNA sequences of Biscogniauxia and Camillea, which leads to the confusion of kinship of Camillea, Obolarina and Cryptostroma. We will not make arbitrary divisions of this genus for the time being. We look forward to more available DNA sequences or new diagnostic features to solve the systematic classi cation problem of Biscogniauxia. Figure 1 Phylogeny of Graphostromataceae and its allies obtained from a Maximum Likelihood analysis of the combines of ITS, RPB2, TUB2 and ACT using RAxML-HPC BlackBox software online. Hypoxylon rickii (MUCL 53309) and H. pulicicidum (MUCL49879) were taken as outgroup. Strains numbers were followed by their names. Bayesian posterior probabilities >0.95 and bootstrap support values for maximum likelihood (ML) higher than >75% are marked above the nodes; an en-dash ("-") indicates a value <0.95 (PP) or <75% (BS). The bold branches indicate the support values are 100/1. Biscogniauxia glaucae (GMB0007, holotype) a Material. b Ascomata on the surface of host. c Section of ascomata. d-j Asci with ascospores (stained in Melzer's reagent). h-j Ascospores. l Ascus apex with a J+ apical apparatus. Scale bars: b, c=200 μm, d-g=10 μm, h-l=5 μm