MycoKeys 97: I 9 (2023) er-reviewed open-access journal oes Be doi: 10.3897/mycokeys.97.102653 RESEARCH ARTICLE , 03 MycoKkeys https://mycokeys.pensoft. net Launched to accelerate biodiversity research Morphology and molecular analyses reveal three new species of Botryosphaeriales isolated from diseased plant branches in China Lu Lin', Yukun Bai', Meng Pan', Chengming Tian', Xinlei Fan! I The Key Laboratory for Silviculture and Conservation of Ministry of Education, Beijing Forestry University, Beijing 100083, China Corresponding author: Xinlei Fan (xinleifan@bjfu.edu.cn) Academic editor: C. Sharma Bhunjun | Received 25 February 2023 | Accepted 10 April 2023 | Published 26 April 2023 Citation: Lin L, Bai Y, Pan M, Tian C, Fan X (2023) Morphology and molecular analyses reveal three new species of Botryosphaeriales isolated from diseased plant branches in China. MycoKeys 97: 1-19. https://doi.org/10.3897/ mycokeys.97.102653 Abstract The Botryosphaeriales represents an ecologically diverse group of fungi, comprising endophytes, saprobes, and plant pathogens. In this study, taxonomic analyses were conducted based on morphological character- istics and phylogenetic analyses of multi-gene sequence data from four loci (ITS, LSU, tefl-«, and tub2). Thirteen isolates obtained from Beijing and Yunnan Province were identified as seven species of Botry- osphaeriales, including Aplosporella javeedii, Dothiorella alpina, Phaeobotryon aplosporum and Ph. rhois, and three previously undescribed species, namely Aplosporella yanqingensis, Dothiorella baihuashanensis, and Phaeobotryon platycladi. Additionally, the new records of Dothiorella alpina from the host species Populus szechuanica, Phaeobotryon aplosporum from Juglans mandshurica, and Phaeobotryon rhois from Populus alba var. pyramidalis are included. Keywords Aplosporella, dieback, Dothiorella, Phaeobotryon, phylogeny, taxonomy Introduction The Botryosphaeriales C.L. Schoch, Crous & Shoemaker is an ecologically diverse fun- gal order comprising endophytes, saprobes, and plant pathogens (Schoch et al. 2006; Ekanayaka et al. 2016; Phillips et al. 2019). Slippers et al. (2013) provided molecular Copyright Lu Lin et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 2 Lu Lin et al. / MycoKeys 97: 1-19 (2023) and morphological evidence to show that the Botryosphaeriales included six families (Aplosporellaceae Slippers, Boissin &¢ Crous, Botryosphaeriaceae Theiss. & Syd., Mel- anopsaceae A.J.L. Phillips, Slippers, Boissin & Crous, Phyllostictaceae Fr., Planistro- mellaceae M.E. Barr, and Saccharataceae Slippers, Boissin & Crous). Then, Wyka and Broders (2016) introduced Septorioideaceae Wyka & Broders., Yang et al. (2017) in- troduced two new families (Endomelanconiopsisaceae Tao Yang & Crous and Pseudo- fusicoccumaceae Tao Yang & Crous). However, Phillips et al. (2019) argued that only six families (Aplosporellaceae, Botryosphaeriaceae, Melanopsaceae, Phyllostictaceae, Planistromellaceae and Saccharataceae) could be accepted in Botryosphaeriales, with reducing Endomelanconiopsisaceae, Pseudofusicoccumaceae, and Septorioideaceae to the synonymy under Botryosphaeriaceae, Phyllostictaceae, and Saccharataceae, respec- tively. In the present study, thirteen isolates were classified as three genera (Aplosporella Speg., Botryosphaeria Ces. & De Not., and Phaeobotryon Theiss. & Syd.) in two fami- lies (Aplosporellaceae and Botryosphaeriaceae). Aplosporellaceae was introduced by Slippers et al. (2013) to accommodate two genera viz. Aplosporella and Bagnisiella Speg. However, Slippers et al. (2013) suggested that Aplosporella and Bagnisiella should be synonymized based on their close phyloge- netic relationships and their remarkably similar multiloculate sporocarps. Ekanayaka et al. (2016) agreed with this and provided evidence that the sexual morph of Aplosporella thailandica Ekanayaka, Dissanayaka, Q. Zhao & K.D. Hyde resembles Bagnisiella. Phillips et al. (2019) formally placed Bagnisiella as a synonym of Aplosporella. Sharma et al. (2017) introduced Alanomyces Roh. Sharma in Aplosporellaceae based on four loci phylogeny. Therefore, two genera (Alanomyces and Aplosporella) can be accepted in Aplosporellaceae. The morphological characters of Aplosporellaceae are multiloculate ascostromata, septate pseudoparaphyses, aseptate and ellipsoid to ovoid ascospores, and ellipsoid to subcylindrical and hyaline to pigmented conidia (Slippers et al. 2013; Phillips et al. 2019). Botryosphaeriaceae was introduced by Theissen and Sydow (1918) for three genera (Botryosphaeria, Phaeobotryon, and Dibotryon Theiss. & Syd.). Over the years the fam- ily and genera have undergone several taxonomic revisions and updates. Currently, the Botryosphaeriaceae has approximately 100 verified species in 24 genera, according to DNA sequence data (Phillips et al. 2013; Slippers et al. 2013; Yang et al. 2017; Xiao et al. 2021; Zhang et al. 2021). Botryosphaeria has uniloculate and clustered ascostro- mata and septate pseudoparaphyses (Phillips et al. 2019). In the phylogenetic tree of Botryosphaeriaceae, hyaline or colored conidia or ascospores are distributed randomly (Slippers et al. 2013). A large number of new species have been described in recent years, which indicated that the diversity of Botryosphaeriaceae was worthy of further exploration (Bezerra et al. 2021; Zhang et al. 2021; Sun et al. 2022). With the modern taxonomic approaches applying, more than 30 novel species have been identified in the last five years (Zhang et al. 2021; Rathnayaka et al. 2022; Sun et al. 2022; Wang et al. 2023). Considering the important economic status of Botryosphaeriales, a survey to explore more hidden species of Botryosphaeriales was considered imperative. Thus, a survey on the diversity of Botryosphaeriales on diseased branches was conducted in Beijing and Yunnan Province from 2021 to 2022. In this Three new species of Botryosphaeriales in China 2 study, we introduce three new species, in which Aplosporella yangingensis and Phaeobot- ryon platycladi were collected from Platycladus orientalis and Dothiorella baihuashanen- sis were collected from Juniperus chinensis in China. Moreover, the newly discovered Dothiorella alpina from Populus szechuanica, Phaeobotryon aplosporum from Juglans mandshurica, and Ph. rhois from Populus alba var. pyramidalis are featured. Materials and methods Fungal isolation Fresh specimens (woody branches and twigs with canker or dieback symptoms) were randomly collected in Beijing and Yunnan Province from the summer of 2021 to the autumn of 2022. The specimens were packed in kraft paper bags and transferred to the laboratory for fungal isolation following Jiang et al. (2022). Isolates were obtained by removing the spore mass from conidiomata to sterilised distilled water using sterilised needle, and generating single spore colonies on potato dextrose agar (PDA: 200 g potatoes, 20 g dextrose, 20 g agar per L) at 25 °C in the dark. After three to five days, hyphal tips were transferred to new PDA plates twice to obtain a pure culture. The cultures are deposited in the China Forestry Culture Collection Center (CFCC; http:// www.cfcc-caf.org.cn/), and the specimens in this study are deposited in the Museum of the Beijing Forestry University (BJFC). Morphology Morphological observations were conducted based on conidiomata produced on in- fected plant tissues. The conidiomata were manually sectioned using a double-edged blade and examined under a dissecting microscope for macroscopic and microscopic characterization, while conidiomata structure and size were imaged with a Leica ster- eomicroscope (M205) (Leica Microsystems, Wetzlar, Germany). Conidia and other microstructures were selected randomly for observation using a Nikon Eclipse 80i microscope (Nikon Corporation, Tokyo, Japan) equipped with a Nikon digital sight DSRi2 high-definition colour camera with differential interference contrast (DIC). Fifty conidia were measured per species, and 30 measurements were taken of other morphological structures. Colony characters i.e. colours and texture on PDA and MEA (malt extract agar; 30 g malt extract, 5 g mycological peptone, 15 g agar per L) at 25 °C were observed and noted over 14 days. The colony colours were determined based on the colour charts of Rayner (1970). DNA extraction, amplification and sequencing The fresh mycelium from PDA was scraped and put it in a 1.5 mL centrifuge tube for genomic DNA extraction which used the modified CTAB (cetyltrimethylammo- nium bromide) method (Doyle and Doyle 1990). For initial species confirmation, the 4 Lu Lin et al. / MycoKeys 97: 1-19 (2023) internal transcribed spacer (ITS) region was sequenced using the primer pairs ITS1/ ITS4 (White et al. 1990) for all isolates. The BLAST tool (https://blast.ncbi.nlm.nih. gov/Blast.cgi) was used to compare the resulting sequences with those in GenBank. After confirmation to the genus level, additional partial loci were amplified, including the nuclear ribosomal large subunit (LSU), the partial translation elongation factor l-alpha (tef/-«), and partial beta-tubulin (w62) using the primer pairs LROR/LR5 (Vilgalys and Hester 1990), EF1-728F/EF1-986R (Carbone and Kohn 1999), and Bt2a/Bt2b (Glass and Donaldson 1995), respectively. The additional combination of T1 and Bt2b (Glass and Donaldson 1995; O’Donnell and Cigelnik 1997) was used in case of amplification failure of the primer Bt2a and Bt2b. ‘The genes used in different genera and the amplification conditions are listed in Table 1. The PCR mixture for all regions consisted of 1 uL DNA template, 1 uL each 10 uM primer, 10 pL T5 Super PCR Mix (containing Taq polymerase, dNTP and Mg”, Beijing TisingKe Biotech Co., Ltd., Beijing, China), and 7 wL sterile water. PCR products were electrophoresed in 1% agarose gel and the DNA was sequenced by the SinoGenoMax Company Lim- ited (Beijing, China). The forward and reverse reads were edited and assembled with Seqman v. 7.1.0 in the DNASTAR Lasergene core suite software (DNASTAR Inc., Madison, Wisconsin USA). All sequences generated in this study were submitted to GenBank (Suppl. material 1). Phylogenetic analyses The sequences obtained in this study were supplemented with additional sequences ob- tained from GenBank (Suppl. material 1) based on BLAST searches and from relevant published literature on the related genera (Bezerra et al. 2021; Wijayawardene et al. 2021; Xiao et al. 2021; Zhang et al. 2021; Peng et al. 2023). The individual data-sets of each gene region were aligned separately using MAFFT v. 6.0 (Katoh and Standley 2013) and trimmed at both terminal ends in MEGA v. 6.0 (Tamura et al. 2013). Maxi- mum Likelihood (ML) analyses were conducted for the single gene sequence data sets (ITS and zef1-« regions for Aplosporella; YTS, tefl-a, and tub2 regions for Dothiorella; ITS, LSU, and tef1-« regions for Phaeobotryon). Then the combined data set of each genus of all gene regions were used for multi-gene phylogenetic analyses including Maximum Likelihood (ML) and Bayesian Inference (BI) analyses. Alanomyces indica (CBS 134264), Lasiodiplodia americana (CFCC 50065), and Alanphillipsia aloeicola (CBS 138896) were selected as the outgroup taxa for Aplosporella, Dothiorella, and Phaeobotryon analyses respectively. Table |. Genes used in this study with PCR primers and optimal annealing temperature. Locus PCR primers PCR: thermal cycles: (Annealing temp. in bold) Genus ITS ITS1/ITS4 (95 °C: 30 s, 51 °C: 30 s, 72 °C: 1 min) x 35 cycles — Aplosporella, Dothiorella, Phaeobotryon LSU LROR/LRS5 (95 °C: 45 s, 55 °C: 45 s, 72 °C: 1 min) x 35 cycles Phaeobotryon tefl-a EF1-728F/EF1-986R (95 °C: 15 s, 55 °C: 20 s, 72 °C: 1 min) x 35 cycles Aplosporella, Dothiorella, Phaeobotryon tub2 Bt2a/Bt2b (95 °C: 30 s, 55 °C: 30 s, 72 °C: 1 min) x 35 cycles Dothiorella T1/Bt2b Three new species of Botryosphaeriales in China 5 Maximum Likelihood (ML) analyses were conducted using PhyML v. 3.0 (Guin- don et al. 2010), employing a GTR model of site substitution with 1000 bootstrap replicates (Stamatakis 2014). Bayesian Inference (BI) analyses were conducted based on the DNA dataset from the results of the MrModeltest v. 2.4 (Nouri et al. 2004) using a Markov Chain Monte Carlo (MCMC) algorithm in MrBayes v. 3.1.2 (Ron- quist and Huelsenbeck 2003). Two MCMC chains were run from random trees for 1,000,000 generations, resulting in a total of 10,000 trees. ‘The first 25% of trees sam- pled were discarded as the burn-in phase of each analysis. The posterior probabilities (BPP) were calculated from the remaining trees (Rannala and Yang 1996). Phyloge- netic trees were shown using Fig Tree v .1.4.4 (Rambaut 2018) and processed by Adobe Illustrator 2019. Results Phylogenetic analyses The BLAST results indicated that the 13 isolates in this study resided in Aplosporella, Dothiorella, and Phaeobotryon. Datasets for the three genera, the number of char- acters of each gene with gaps and the substitution models used for BI analyses are provided in Table 2. The topologies of BI analyses did not significantly differ from the ML analyses. Species of Aplosporella Five isolates clustered into two phylogenetic groups for the individual genes (ITS and tef1-x), as well as the combined gene dataset (Fig. 1). In ML analysis based on the com- bined gene dataset, the matrix had 221 distinct alignment patterns. Estimated base frequencies are as follows: A = 0.211117, C = 0.277509, G = 0.253698, T = 0.257676; substitution rates: AC = 3.242352, AG = 4.568839, AT = 2.135067, CG = 2.137396, CT = 5.690231, GT = 1.000000; gamma distribution shape parameter: « = 0.217402. The isolates CFCC 58330, 58329, and 58412 resided in a clade with Ap/losporella javeedii (ML/BI = 98/1.00), while the isolates CFCC 58791 and 58792 formed an individual clade distinct from the other species in Ap/osporella (ML/BI = 100/1.00). Table 2. Substitution models used for Bayesian analyses in this study. Analyses Number of outgroup Substitution models used for Bayesian analyses/ ingroup sequences Number of characters with gaps ITS LSU tefl tub2 Aplosporella 2-genes 24 Alanomyces indica SYM+G /553 = GTR+G /417 = CBS 134264 Dothiorella 3-genes 66 Lasiodiplodia americana GTR+1+G /494 - GTR+G /322. GTR+I+G CFCC 50065 /448 Phaeobotryon 3-genes 36 Alanphillipsia aloeicola =GTR+I /488 HKY+I/562 HKY+G/299 Fa CBS 138896 6 Lu Lin et al. / MycoKeys 97: 1-19 (2023) 93/0.99-— Aplosporella macropycnidia CGMCC 37726 Aplosporella macropycnidia CGMCC 37725 -|' Aplosporella sophorae CPC 29688 Aplosporella africana CBS 121777 Aplosporella africana CBS 121778 100/1.00; Aplosporella yalgorensis MUCC512 Aplosporella yalgorensis MUCC511 98/1.00 Anlosporella prunicola STE-U 6326 9" Aplosporella prunicola CBS 121167 100/1.00) Aplosporella papillata CBS 121780 Aplosporella papillata CBS 121781 100/1.00 ; Aplosporella yangingensis CFCC 58791 Aplosporella yangingensis CFCC 58792 Aplosporella javeedii CFCC 58412 -| Aplosporella javeedii CFCC 58330 Aplosporella javeedii CFCC 58329 Aplosporella javeedii CFCC 50054 Aplosporella javeedii CFCC 50052 961.00, APlosporella hesperidica CBS 732.79 Aplosporella hesperidica CBS 208.37 Aplosporella thailandica MFLU 16-0615 Aplosporella artocarpi CPC 227991 Aplosporella ginkgonis CFCC 89661 Aplosporella ginkgonis CFCC 52442 Alanomyces indica CBS 134264 85/1.00 80/1.00 |98/7.00 0.03 Figure |. Phylogram of Aplosporella resulting from a maximum likelihood analysis based on combined ITS and tef7 loci. Numbers above the branches indicateML bootstrap values (ML-BS = 70%) and Bayes- ian Posterior Probabilities (BPP > 0.9). The tree is rooted with Alanomyces indica CBS 134264. Ex-type isolates are in bold. Isolates from the present study are marked in blue. Species of Dothiorella Three isolates clustered in two clades for the individual genes (ITS, tef1-x, and tub2), as well as the combined gene dataset (Fig. 2). In ML analysis based on the combined gene dataset, the matrix had 478 distinct alignment patterns. Estimated base frequencies are as follows: A = 0.203201, C = 0.315247, G = 0.248158, T = 0.233395; substitution rates: AC = 0.994643, AG = 2.280369, AT = 1.123589, CG = 0.895887, CT = 4.309165, GT = 1.000000; gamma distribution shape parameter: « = 0.210467. The isolate CFCC 58299 grouped with Do. alpina (ML/BI = 84/0.95), while the isolates CFCC 58549 and 58788 formed a distinct clade from the other species (ML/BI = 100/1.00). Species of Phaeobotryon Five isolates clustered into three clades for the individual genes (ITS, LSU, and tef1-«), as well as the combined gene dataset (Fig. 3). In ML analysis based on the combined gene dataset, the matrix had 223 distinct alignment patterns. Estimated base frequencies are as Three new species of Botryosphaeriales in China Dothiorella omnivora CBS 124716 Dothiorella omnivora CBS 124717 Dothiorella omnivora CBS 392.80 100/1.90| Dothiorella omnivora CBS 188.87 86/-| | Dothiorella omnivora CBS 242.51 98/1.00. || _ Dothiorella vidmadera CBS 621.74 Pall Dothiorella vidmadera CBS 725.79 100/1.004 |, Dothiorella sempervirentis CBS 124718 Dothiorella sempervirentis CBS 124719 Dothiorella iberica CBS 115041 100/1.00.. | Dothiorella iberica CBS 113188 saris Dothiorella iberica CBS 113189 4 Dothiorella parva CBS 125580 Dothiorella parva CBS 124721 70/1.00 Dothiorella parva CBS 124720 86/1.00 Dothiorella sarmentorum |MI 63581b 76)- Dothiorella americana CBS 128309 Dothiorella prunicola CBS 124723 0.28 Dothiorella iranica CBS 124722 1001.00) Dothiorella baihuashanensis CFCC 58549 Dothiorella baihuashanensis CFCC 58788 Dothiorella sp. CBS 121785 100/1.00 | Dothiorella sp. CBS 121783 eROO Dothiorella sp. CBS 121784 Pe, Dothiorella santali MUCC 509 so0rt00 Dothiorella moneti MUCC 505 ; Dothiorella pretoriensis CBS 130404 Dothiorella thripsita CBS 125445 Dothiorella casuarinae CBS 120688 97/1.00 Dothiorella casuarinae CBS 120689 Dothiorella casuarinae CBS 120690 100/1.00 ; Dothiorella capri-amissi CBS 121878 Dothiorella capri-amissi CBS 121763 Dothiorella rosulata CBS 121761 Dothiorella rosulata CBS 121762 87/0.91| Dothiorella rosulata CBS 121760 79/0.99|! Nothiorella rosulata CBS 500.72 Dothiorella mangifericola CBS 124727 Dothiorella citricola CBS 124729 Dothiorella citricola CBS 124728 81/1.00 xe 84/0.9 400/1.00 Dothiorella alpina CFCC 58299 Dothiorella alpina CGMCC 3-18001 98/0.96 | | Dothiorella yunnana CGMCC 3-17999 Dothiorella yunnana CGMCC 3-18000 Dothiorella plurivora CBS 124724 Dothiorella viticola CBS 117009 96/1.00 ' Dothiorella viticola DAR 80529 92/0.96 Dothiorella longicollis CBS 122068 100/1.00!' Dothiorella longicollis CBS 122067 100/1.00 Dothiorella longicollis CBS 122066 Dothiorella brevicollis CBS 130411 94/0.90| Dothiorella oblonga CBS 121765 400/1.00 | Dothiorella oblonga CBS 121766 aoe 40 Dothiorella dulcispinae CBS 130413 Dothiorella dulcispinae CBS 121764 Dothiorella thailandica CBS 133991 100/0.99| Dothiorella striata CBS 124731 99/1.00 |' Dothiorella striata CBS 124730 Dothiorella neclivorem DAR 80992 - Dothiorella uruguayensis CBS 124908 Dothiorella vinea-gemmae DAR 81012 Lasiodiplodia americana CFCC 50065 95/1.00 0.05 Figure 2. Phylogram of Dothiorella resulting from a maximum likelihood analysis based on combined ITS, tef2 and tub2 loci. Numbers above the branches indicateML bootstrap values (ML-BS = 70%) and Bayesian Posterior Probabilities (BPP > 0.9). The tree is rooted with Lasiodiplodia americana CFCC 50065. Ex-type isolates are in bold. Isolates from the present study are marked in blue. 8 Lu Lin et al. / MycoKeys 97: 1-19 (2023) Phaeobotryon negundinis CAA 799 Phaeobotryon negundinis CAA 797 Phaeobotryon negundinis CAA 798 99/1.00| Phaeobotryon negundinis MFLUCC 15-0436 Phaeobotryon negundinis CPC 34752 Phaeobotryon negundinis CPC 33384 Phaeobotryon negundinis CPC 33388 Phaeobotryon platycladi CFCC 58800 Phaeobotryon platycladi CFCC 58799 Phaeobotryon cupressi CBS 124701 Phaeobotryon cupressi CBS 124700 Phaeobotryon aplosporum CFCC 53774 Phaeobotryon aplosporum CFCC 53773 Phaeobotryon aplosporum CFCC 53776 99/1.00| Phaeobotryon aplosporum CFCC 53775 Phaeobotryon aplosporum CFCC 58596 100/1.00' Phaeobotryon aplosporum CFCC 58784 \,, Phaeobotryon rhois CFCC 89662 Phaeobotryon rhois CFCC 89663 Phaeobotryon rhois CFCC 58679 Phaeobotryon rhoinum CFCC 52449 Phaeobotryon rhoinum CFCC 52450 100/1.09 Phaeobotryon mamane CBS 122980 Phaeobotryon mamane CPC 12442 Phaeobotryon ulmi PB 11f Phaeobotryon ulmi CBS 174.63 Phaeobotryon ulmi CBS 114123 Phaeobotryon ulmi CMH299 Phaeobotryon ulmi CBS 123.30 Phaeobotryon ulmi CBS 138854 Phaeobotryon ulmi 94-13 Alanphillipsia aloeicola CBS 138896 x2 0.02 Figure 3. Phylogram of Phaeobotryon resulting from a maximum likelihood analysis based on combined ITS, LSU, and tef1 loci. Numbers above the branches indicateML bootstrap values (ML-BS = 70%) and Bayesian Posterior Probabilities (BPP > 0.9). The tree is rooted with Alanphillipsia aloeicola CBS 138896. Ex-type isolates are in bold. Isolates from the present study are marked in blue. follows: A = 0.223233, C = 0.267753, G = 0.277657, T = 0.231357; substitution rates: AC = 0.862696, AG = 2.117465, AT = 0.455729, CG = 1.132740, CT = 4.957268, GT = 1.000000; gamma distribution shape parameter: « = 0.272408. The isolate CFCC 58679 grouped with Ph. rhois (ML/BI = 100/1.00). The isolates CFCC 58596 and 58784 formed a unique lineage distinct from, but related to Ph. aplosporum as their clos- est relatives (ML/BI = 99/1.00). The isolates CFCC 58799 and 58800 formed a clade of their own separating them from other Phaeobotryon lineages (ML/BI = 100/1.00). Three new species of Botryosphaeriales in China ) Taxonomy Based on DNA sequences and morphology, seven species belonging to three genera were identified. Of these, Aplosporella javeedii, Dothiorella alpina, Phaeobotryon aplosporum, and Ph. rhois are known species. The remaining three species are identified as new spe- cies (Aplosporella yangingensis, Dothiorella baihuashanensis, and Phaeobotryon platycladi) and described below. Collect information and notes of all seven species were provided. Aplosporella javeedii Jami, Gryzenh., Slippers & M.J. Wingf., Fungal Biol. 118(2): 174 (2013) Description. See Fan et al. 2015. Materials examined. Cuina, Yunnan Province, Kunming City, Panlong District, Jinma County, Bailongsi Town, 25°3'44"N, 102°45'22"E, on dead branches of Popu- lus canadensis, 11 August 2022, Lu Lin & Ziqiang Wu (BJFC CF20230101, living culture CFCC 58330). Beijing City, Mentougou District, G109 National Highway, 40°3'2"N, 115°52'58"E, on dead branches of Populus beijingensis, 25 August 2022, Lu Lin & Xinlei Fan (BJFC CF20230102, living culture CFCC 58329). Changping Dis- trict, Liucun Town, Wangjiayuan Village, 40°10'23"N, 116°4'9"E, on dead branches of Populus alba var. pyramidalis, 22 September 2022, Lu Lin & Xinlei Fan (BJFC CF20230103, living culture CFCC 58412). Notes. Aplosporella javeedii was first discovered on Celtis africana and Searsia lancea in South Africa (Jami et al. 2014). Fan et al. (2015), Zhu et al. (2018), and Pan et al. (2019) expanded the host range of Aplosporella javeedii to more than ten host families in China. This species has not been reported outside South Africa and China. Aplosporella yangingensis L. Lin & X.L. Fan, sp. nov. MycoBank No: 847680 Fig. 4 Etymology. Named after the collection site of the type specimen, Yanging District in Beijing City. Description. Conidiomata pycnidial, immersed to semi immersed, erumpent from bark surface, multilocular, 650-1500 um in diam. Disc straw to green- ish olivaceous, circular to ovoid, 350-650 um in diam, with one central ostiole per disc. Ostioles inconspicuous, sometimes covered below disc by lighter en- tostroma, 100-300 um in diam. Locules multiple, irregularly arranged, sub- divided frequently by invaginations with common walls. Conidiophores re- duced to conidiogenous cells. Conidiogenous cells hyaline, phialidic, 6.0-— 1325.« 2:0-3.0° um ‘av. = S.D. = 10:7" = 2:0) 2:5°2-0.2? um). Paraphyses?pre- sent, hyaline, smooth-walled, septate, unbranched, 26.5—37.5 x 2.0—-3.0 um (av. £8.D.=32.0£3.5x2.4+0.3um).Conidiaaseptate,smooth, ellipsoid tosubcylindrical, 10 Lu Lin et al. / MycoKeys 97: 1-19 (2023) Figure 4. Aplosporella yangingensis (BJFC CF20230104) A, B habit of conidiomata on twig C transverse section of conidiomata D longitudinal section through a conidioma E, F conidiogenous cells and para- physes G conidia H colony on PDA at 14 days I colony on MEA at 14 days. Scale bars: 2 mm (A); 1 mm (C); 500 um (B, D); 10 um (E-G). brown when mature, 16.0—21.5 x 6.0-9.5 um (av. £S.D.=18.5£1.3 x 7.7 £0.7 um). Sexual morph not observed. Culture characters. Colonies on PDA spreading, white to pale grey, covering a 90 mm plate after 14 days at 25 °C. Colonies on MEA spreading, uniform with ap- pressed aerial mycelium and crenate edge, upper white, reverse pale luteous covering a 90 mm plate after 14 days at 25 °C. Materials examined. Cuina, Beijing City, Yanging District, Yeyahu National Wetland Park, 40°24'55.43"N, 115°50'26.42"E, on branches of Platycladus orientalis, 25 July 2022, Yukun Bai & Xinlei Fan (holotype BJFC CF20230104, ex-holotype culture CFCC 58791); 40°24'55.46"N, 115°50'26.42"E, on branches of Platycladus orientalis, 25 July 2022, Yukun Bai & Xinlei Fan (paratype BJFC CF20230105, ex- paratype culture CFCC 58792). Notes. In the multi-gene analyses, A. yanqgingensis is distinct and forms a moder- ately supported lineage clade (Fig. 1). In the ITS tree, A. yangingensis shows a close relationship with a clade containing A. africana FJ.J. Van der Walt, Slippers & G.J. Marais, A. macropycnidia Dou & Y. Zhang ter, A. papillata FJ.J. Van der Walt, Slip- pers & GJ. Marais, A. prunicola Damm & Crous, A. sophorae Crous & Thangavel, and A. yalgorensis K.M. Taylor, P.A. Barber & T.I. Burgess. However, it differs from A. africana by longer conidia (18.5 x 7.7 vs. 14 x 8.5 um) (Slippers et al. 2014), dif- fers from A. macropycnidia by shorter paraphyses (32.0 x 2.4 vs. 38.4 x 2.9 um) (Dou et al. 2017), differs from A. papillata by larger conidiogenous cells (10.7 x 2.5 vs. 7.4 x 2 um) (Slippers et al. 2014), and differs from A. prunicola and A. yalgorensis by smaller conidia (18.5 x 7.7 vs. 20.2 x 11 for A. prunicola and 19.9 x 10.7 for A. yal- gorensis) (Damm et al. 2007; Taylor et al. 2009). Besides, A. yangingensis differs from Three new species of Botryosphaeriales in China hi A. sophorae by 25/528 in ITS region. Therefore, A. yangingensis is introduced herein as a novel species. This is a new record of species in Ap/osporella occurring on the host genus Platycladus. Dothiorella alpina (Y. Zhang ter. & Min Zhang) Phookamsak & Hyde, Asian Journal of Mycology 3(1): 168 (2020) = Spencermartinsia alpina Y. Zhang ter. & Ming Zhang, Mycosphere 7(7): 1058 (2016). Description. See Hyde et al. 2020. Materials examined. Curna, Yunnan Province, Diging Tibetan Autonomous Pre- fecture, Shangri-La City, Sanba County, East Ring Road, 27°36'18"N, 100°1'19"E, on dead branches of Populus szechuanica, 9 August 2022, Lu Lin & Min Lin (BJFC CF20230106, living culture CFCC 58299). Notes. Dothiorella alpina was first introduced by Zhang et al. (2016a) as Spen- cermartinsia alpina, which has dark brown and 1-septate conidia. Hyde et al. (2020) transfer S. alpina to Dothiorella based on phylogenetic analyses of a concatenated data- set (ITS+tef1/-«) and morphological similarity. Dothiorella alpina was recorded on Cirus unshiu in Hunan Province, China, and Platycladus orientalis and Ipomoea sp. in Yun- nan Province, China. In this study, a new record of Do. alpina from the host Populus szechuanica is included. Dothiorella baihuashanensis L. Lin & X.L. Fan, sp. nov. MycoBank No: 847681 Fig. 5 Etymology. Named after the collection site of the type specimen, Baihuashan Natural Scenic Area in Beijing City. Description. Conidiomata pycnidial, superficial or immersed, separate, ovoid, 350-500 ym in diam, occasionally aggregated into botryose clusters. Disc black, 200-300 um in diam. Ostioles single, central, papillate. Conidi- ophores reduced to conidiogenous cells. Conidiogenous cells hyaline, holoblas- tic, cylindrical to subcylindrical or broadly lageniform, 7.5-16.0 x 3.5-6.5 um (av. = S.D. = 11.7 + 2.2 x 4.6 = 0.7 um). Conidial-septate, hazel to blackish brown, mostly truncate at the base and constricted at the septum or with a thickening at the base of the septum, moderately thick-walled, ovoid or oblong to ellipsoidal, 22.5-35.0 x 11.0-19.0 pm (av. = S.D. = 27.9 = 2.9 x 14.3 = 2.2 pm). Culture characters. Colonies on PDA spreading, covering a 90 mm plate after 14 days at 25 °C, upper white to pale grey, reverse buff to dark grey. Colonies on MEA spreading, covering a 90 mm plate after 14 days at 25 °C, uniform with appressed aerial mycelium and crenate edge, upper white to pale grey, reverse honey to dark grey. 12 Lu Lin et al. / MycoKeys 97: 1-19 (2023) verse section of a conidioma D longitudinal section through a conidioma E, F conidiogenous cells G conidia H colony on PDA at 14 days I colony on MEA at 14 days. Scale bars: 1 mm (A); 200 pm (B-D); 10 um (E-G). Materials examined. Cuina, Beijing City, Mentougou District, Qingshui County, Baihuashan Natural Scenic Area, 39°50'18.21"N, 115°34'21.13"E, on dead branches of Juniperus chinensis, 23 August 2022, Lu Lin & Xinlei Fan (holotype BJFC CF20230107, ex-holotype culture CFCC 58549); 39°50'18.16"N, 115°34'21.24"E, on dead branches of Juniperus chinensis, 23 August 2022, Lu Lin & Xinlei Fan (para- type BJFC CF20230108, ex-paratype culture CFCC 58788). Notes. The isolates CFCC 58549 and 58788 in this study formed a distinct linage in the phylogenetic trees of each individual gene (ITS, tef7/-x, and tub2) and the com- bined gene dataset (Fig. 2). They were isolated from the branches Juniperus chinensis. Dothiorella iberica was also recorded to host genus Juniperus (Alves et al. 2013). How- ever, these two species are not closely related in our phylogenetic analysis. Phaeobotryon aplosporum M. Pan & X.L. Fan, Mycol. Prog. 18(11): 1356 (2019) Description. See Pan et al. 2019. Materials examined. Cutna, Beijing City, Mentougou District, Qingshui Coun- ty, Baihuashan Natural Scenic Area, 39°51'11"N, 115°32'37"E, on dead branches of Juglans mandshurica, 23 August 2022, Lu Lin & Xinlei Fan (BJFC CF20230112, liv- ing culture CFCC 58596; BJFC CF20230113, living culture CFCC 58784). Notes. Phaeobotryon aplosporum was first discovered from Rhus typhina and Syzygium aromaticum (Pan et al. 2019). It can be distinguished from other species in Phaeobotryon by its aseptate conidia (Pan et al. 2019). In this study, the conidia formed on the specimen BJFC CF20230112 are dark brick when mature, aseptate, (16.5-20.0 x 6.0-9.0 um (av. + S.D. = 18.3 = 1.1 x 7.5 = 0.8 um), which overlap with the morphological characteristics described by Pan et al. (2019). Phylogenetically, Three new species of Botryosphaeriales in China 6) the isolates CFCC 58596 and 58784 were clustered in a clade with Ph. aplosporum with high statistical support (ML/BI = 99/1). Therefore, the isolates CFCC 58596 and 58784 are identified as Ph. aplosporum. The current study extends its host range to Juglans mandshurica. Phaeobotryon platycladi L. Lin & X.L. Fan, sp. nov. MycoBank No: 847682 Fig. 6 Etymology. Named after the host genus, Platycladus. Description. Conidiomata pycnidial, scattered, subglobose to globose, erumpent, exuding faint yellow translucent conidial droplets from central ostioles, unilocu- lar, 150-250 um diam. Disc black, 80-200 um in diam. Ostioles single, central, papillate, 21-35 pm. Conidiophores reduced to conidiogenous cells. Conidiog- enous cells hyaline, smooth, thin-walled, cylindrical, holoblastic, phialidic, pro- liferating internally with visible periclinal thickening, 5.5-14.0 x 2.5-4.0 um (av. + S.D. = 10.2 + 2.5 x 3.2 = 0.4 um). Conidia initially hyaline, oval, both ends broadly rounded, aseptate, rarely becoming 1-septate, 23.0-31.0 x 9.5-12.5 um (av. + $.D. = 26.2 + 2.5 x 10.8 + 0.8 pm). Culture characters. Colonies on PDA spreading, upper white to buff, reverse buff to isabelline covering a 90 mm plate after 14 days at 25 °C. Colonies on MEA spreading, stratiform, with appressed aerial mycelium and crenate edge, upper white to isabelline, reverse buff to hazel, covering a 90 mm plate after 14 days at 25 °C. Materials examined. Cuina, Beijing City, Haidian District, National Botanic Gardens, 39°59'42.41"N, 116°12'47.24"E, on dead branches of Platycladus orientalis, E Ke’ —= = — Figure 6. Phaeobotryon platycladi (BJFC CF20230110) A, B habit of conidiomata on twig C transverse sec- tion of a conidioma D longitudinal section through a conidioma E=G conidiogenous cells and conidia H col- ony on PDA at 14 days I colony on MEA at 14 days. Scale bars: 2 mm (A); 200 um (B—D); 10 um (E-G). 14 Lu Lin et al. / MycoKeys 97: 1-19 (2023) 4 August 2022, Yukun Bai & Xinlei Fan (holotype BJFC CF20230110, ex-holotype culture CFCC 58799); 39°59'42.43"N, 116°12'47.46"E, on dead branches of Platycla- dus orientalis, 4 August 2022, Yukun Bai & Xinlei Fan (paratype BJFC CF20230111, ex-paratype culture CFCC 58800). Notes. Phaeobotryon platycladi is monophyletic with Ph. cupressi in the phyloge- netic tree without a significant statistical support. Conidial sizes of the two species overlap, but there are differences in 6/488 in ITS region, 3/556 in LSU region, and 18/293 in tefl-« gene with gaps. Phaeobotryon rhois C.M. Tian, X.L. Fan & K.D. Hyde, Phytotaxa 205 (2): 95 (2015) Description. See Fan et al. 2015. Materials examined. Cuina, Beijing City, Yanging District, Zhangshanying County, 40°28'33"N, 115°49'58"E, on dead branches of Populus alba var. pyramidalis, 16 September 2022, Lu Lin & Chengming Tian (BJFC CF20230109, living culture CFCC 58679). Notes. Phaeobotryon rhois was first discovered on Rhus typhina distributed in Ningxia Province, China (Fan et al. 2015). Pan et al. (2019) reported this species from Dioscorea nipponica, Platycladus orientalis and Rhamnus davurica in Beijing, China. The current study extends its host range to Populus alba var. pyramidalis. Discussion In this study, a total of 13 isolates are identified as seven species of Botryosphaeri- ales, including three new species (Aplosporella yangingensis, Dothiorella baihuashan- ensis, and Phaeobotryon platycladi) and four known species (A. javeedii, Do. alpina, Ph. aplosporum, and Ph. rhois). All three new species were isolated from coniferous trees: A. yangingensis and Ph. platycladi from Platycladus orientalis and Do. baihuashan- ensis from Juniperus chinensis. Furthermore, the new records of Do. alpina from the host species Populus szechuanica, Ph. aplosporum from Juglans mandshurica, and Ph. rhois from Populus alba var. pyramidalis are included. The fungi of Botryosphaeriales play various ecological roles, such as sapro- trophs, endophytes, or plant pathogens (Phillips et al. 2005, 2008, 2013; Luque et al. 2016). Some fungi exhibit strong pathogenicity, leading to severe diseases in different parts of various plants, such as Botryosphaeria dothidea, which can cause apple ring rot of stems and fruits (Zhang et al. 2016b), as well as poplar cankers (Li et al. 2019), and the dieback and leaf spot diseases of Euonymus japonicus (Lin et al. 2023). Sometimes their ecological roles change, such as Diplodia sapinea, which is both an endophytic and a plant pathogenic fungus (Slippers et al. 2013). In this article, all species were isolated from diseased plant tissues, and their pathogenicity remains to be verified. Three new species of Botryosphaeriales in China 15 In this study, both Dothiorella and Phaeobotryon belong to Botryosphaeriaceae. Slippers et al. (2013) mentioned that some morphological features within Botryospha- eriaceae are not always stable, such as pigment production of conidia. These features might have already existed before the diversification of the group and have under- gone further changes later (Slippers et al. 2013). In this study, only aseptate conidia were observed in Phaerobotryon platycladi, and they may become pigmented with age. Moreover, whether septate or not seem to be an unstable characteristic throughout the genus Phaerobotryon. Phillips et al. (2013) mentioned that in most cases, the co- nidia of Phaeobotryon have two septa when mature. However, both the Phaeobotryon aplosporum observed in this study and the one described by Pan et al. (2019) have pig- mented but without septa. Phaeobotryon rhoinum also shows pigmented and aseptate conidia (Daranagama et al. 2016). Other species of Phaeobotryon with pigmented and septate conidia are either saprobic or pathogenic, but Ph. aplosporum and Ph. rhoinum are both pathogenic (Rathnayaka et al. 2023). The phylogenetic state analysis of the trophic pattern, conidial colour, and separation of Botryosphaeriales conducted by Rathnayaka et al. (2023) indicate that this may correspond to nutritional mode. Acknowledgements This research was funded by the National Natural Science Foundation of China (32101533), National Science and Technology Fundamental Resources Investigation Program of China (2021FY100900). We are grateful to Xiaohong Liang, Jing Han (the Experimental Teaching Centre, College of Forestry, Beijing Forestry University) for providing installed scientific equipment through the whole process. Lu Lin is grate- ful for the assistance of Ziqiang Wu (Southwest Forestry University) and Min Lin dur- ing the specimen collection, also Yixuan Li and Aoli Jia (Beijing Forestry University) during this study. Xinlei Fan would like to acknowledge the support of strain preserva- tion of Chungen Piao and Minwei Guo (China Forestry Culture Collection Centre, Chinese Academy of Forestry, Beijing). References Alves A, Barradas C, Phillips AJL, Correia A (2013) Diversity of Botryosphaeriaceae species as- sociated with conifers in Portugal. European Journal of Plant Pathology 135(4): 791-804. https://doi.org/10.1007/s10658-012-0122-2 Bezerra JDP, Crous PW, Aiello D, Gullino ML, Polizzi G, Guarnaccia V (2021) Genetic diver- sity and pathogenicity of Botryosphaeriaceae species associated with symptomatic citrus plants in Europe. Plants 10(3): e492. https://doi.org/10.3390/plants 10030492 Carbone I, Kohn LM (1999) A Method for designing primer sets for speciation studies in filamentous Ascomycetes. Mycologia 91(3): 553-556. https://doi.org/10.1080/0027551 4.1999.12061051 16 Lu Lin et al. / MycoKeys 97: 1-19 (2023) Damm U, Fourie PH, Crous PW (2007) Aplosporella prunicola, a novel species of anamorphic Botryosphaeriaceae. Fungal Diversity 27: 35-43. Daranagama DA, Thambugala KM, Campino B, Alves A, Bulgakov TS, Phillips AJL, Liu XZ, Hyde KD (2016) Phaeobotryon negundinis sp. nov. (Botryosphaeriales) from Russia. Myco- sphere 7(7): 933-941. https://doi.org/10.5943/mycosphere/si/1b/2 Dou ZP, Lu M, Wu JR, He W, Zhang Y (2017) A new species and interesting records of Aplosporella from China. Sydowia 69: 1-7. Doyle JJ, Doyle JL (1990) Isolation of plant DNA from fresh tissue. Focus 12: 13-15. https:// doi.org/10.2307/2419362 Ekanayaka AH, Dissanayake AJ, Jayasiri SC, To-anun C, Jones EBG, Zhao Q, Hyde KD (2016) Aplosporella thailandica; a nove species revealing the sexual-asexual connection in Aplo- sporellaceae (Botryosphaeriales). Mycosphere 7(4): 440-447. https://doi.org/10.5943/ mycosphere/7/4/4 Fan XL, Hyde KD, Liu JK, Liang YM, Tian CM (2015) Multigene phylogeny and morphol- ogy reveal Phaeobotryon rhois sp. nov. (Botryosphaeriales, Ascomycota). Phytotaxa 205(2): 90-98. https://doi.org/10.11646/phytotaxa.205.2.2 Glass NL, Donaldson GC (1995) Development of primer sets designed for use with the PCR to amplify conserved genes from filamentous ascomycetes. Applied and Environmental Microbiology 61(4): 1323-1330. https://doi.org/10.1128/aem.61.4.1323-1330.1995 Guindon S, Dufayard JF, Lefort V, Anisimova M, Hordijk W, Gascuel O (2010) New al- gorithms and methods to estimate maximum-likelihood phylogenies: Assessing the per- formance of PhyML 3.0. Systematic Biology 59(3): 307-321. https://doi.org/10.1093/ sysbio/syq010 Hyde KD, de Silva NI, Jeewon R, Bhat DJ, Phookamsak R, Doilom M, Boonmee S, Jaya- wardena RS, Maharachchikumbura SSN, Senanayake IC, Manawasinghe IS, Liu NG, Abeywickrama PD, Chaiwan N, Karunarathna A, Pem D, Lin CG, Sysouphanthong P, Luo ZL, Wei DP, Wanasinghe DN, Norphanphoun C, Tennakoon DS, Samarakoon MC, Jayasiri SC, Jiang HB, Zeng XY, Li JF, Wijesinghe SN, Devadatha B, Goonasekara ID, Brahmanage RS, Yang EF, Aluthmuhandiram JVS, Dayarathne MC, Marasinghe DS, Li WJ, Dissanayake LS, Dong W, Huanraluek N, Lumyong S, Liu JK, Karunarathna SC, Jones EBG, Al-Sadi AM, Xu JC, Harishchandra D, Sarma VV (2020) AJOM new records and collections of fungi: 1-100. Asian Journal of Mycology 3(1): 22-294. https://doi. org/10.5943/ajom/3/1/3 Jami F, Slippers B, Wingfield MJ, Gryzenhout M (2014) Botryosphaeriaceae species overlap on four unrelated, native South African hosts. Fungal Biology 118(2): 168-179. https://doi. org/10.1016/j.funbio.2013.11.007 Jiang N, Voglmayr H, Xue H, Piao CG, Li Y (2022) Morphology and phylogeny of Pestalo- tiopsis (Sporocadaceae, Amphisphaeriales) from Fagaceae leaves in China. Microbiology Spectrum 10(6): 03272-22. https://doi.org/10.1128/spectrum.03272-22 Katoh K, Standley DM (2013) MAFFT multiple sequence alignment software version 7: Improvements in performance and usability. Molecular Biology and Evolution 304(4): 772-780. https://doi.org/10.1093/molbev/mst010 Three new species of Botryosphaeriales in China 7 Li GQ, Li BJ, Li BQ, et al. (2019) First report of Botryosphaeria dothidea causing canker on Populus tomentosa in China. Plant Disease 103(4): e693. https://doi.org/10.1094/PDIS- 08-18-1451-PDN Lin L, Pan M, Gao H, Tian CM, Fan XL (2023) The Potential Fungal Pathogens of Euony- mus japonicus in Beijing, China. Journal of Fungi 9(2): e271. https://doi.org/10.3390/ j0f9020271 Luque J, Gomez LD, Melgarejo P (2016) Botryosphaeria species associated with apple stem rot in southern Spain. European Journal of Plant Pathology 145(1): 205-217. https://doi. org/10.1007/s10658-015-0836-9 Nouri MT, Lawrence DP, Holland LA, Doll DA, Kallsen CE, Culumber CM, Nylander JAA (2004) MrModeltest v2. Distributed by the Author; Evolutionary Biology Center, Uppsala University, Uppsala. O’Donnell K, Cigelnik E (1997) Two divergent intragenomic rDNA ITS2 types within a monophyletic lineage of the fungus Fusarium are nonorthologous. Molecular Phylogenet- ics and Evolution 7(1): 103-116. https://doi.org/10.1006/mpev.1996.0376 Pan M, Zhu HY, Bezerra JD, Bonthond G, Tian CM, Fan XL (2019) Botryosphaerialean fungi causing canker and dieback of tree hosts from Mount Yudu in China. Mycological Progress 18: 1341-1361. https://doi-org/10.1007/s11557-019-01532-z Peng C, Shi TY, Xia GC, Tian CM (2023) A New Species of Phaeobotryon from Galls of Juni- perus formosana Branches in China. Journal of Fungal Research 21(1—3): 31-41. https:// doi.org/10.13341/j.jfr.2023.0004 [in Chinese] Phillips A, Alves A, Correia A, Luque J (2005) Two new species of Botryosphaeria with brown, 1-septate ascospores and Dothiorella anamorphs. Mycologia 97(2): 513-529. https://doi. org/10.1080/15572536.2006.11832826 Phillips AJL, Alves A, Abdollahzadeh J, Slippers B, Wingfield MJ, Groenewald JZ, Crous PW (2013) The Botryosphaeriaceae: Genera and species known from culture. Studies in My- cology 76: 51-167. https://doi.org/10.3114/sim0021 Phillips AJL, Hyde KD, Alves A, Liu J-KJ (2019) Families in Botryosphaeriales: A phyloge- netic, morphological and evolutionary perspective. Fungal Diversity 94(1): 1-22. https:// doi.org/10.1007/s13225-018-0416-6 Rambaut A (2018) FigTree v.1.4.4. http://tree.bio.ed.ac.uk/software/figtree/ Rannala B, Yang Z (1996) Probability distribution of molecular evolutionary trees: A new method of phylogenetic inference. Journal of Molecular Evolution 43(3): 304-311. https://doi.org/10.1007/BF02338839 Rathnayaka AR, Chethana KT, Phillips AJ, Jones EG (2022) Two new species of Botryospha- eriaceae (Botryosphaeriales) and new host/geographical records. Phytotaxa 564(1): 8-38. https://doi.org/10.11646/phytotaxa.564.1.2 Rathnayaka AR, Chethana KWT, Phillips AJL, Liu JK, Samarakoon MC, Jone EBG, Karunar- athna SC, Zhao CL (2023) Re-evaluating Botryosphaeriales: Ancestral state reconstruc- tions of selected characters and evolution of nutritional modes. Journal of Fungi 9(2): e184. https://doi.org/10.3390/jof9020184 Rayner RW (1970) A Mycological Colour Chart. Commonwealth Mycological Institute, London. 18 Lu Lin et al. / MycoKeys 97: 1-19 (2023) Ronquist E Huelsenbeck JP (2003) MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19(12): 1572-1574. https://doi.org/10.1093/bioinformatics/btg180 Schoch CL, Shoemaker R, Seifert K, Hambleton S, Spatafora JW, Crous PW (2006) A multi- gene phylogeny of the Dothideomycetes using four nuclear loci. Mycologia 98(6): 1041-— 1052. https://doi.org/10.1080/15572536.2006.11832632 Sharma R, Kulkarni G, Sonawane MS (2017) Alanomyces, a new genus of Aplosporellaceae based on four loci phylogeny. Phytotaxa 297(2): 168-175. https://doi.org/10.11646/ phytotaxa.297.2.4 Slippers B, Boissin E, Phillips AJL, Groenewald JZ, Lombard L, Wingfield MJ, Postma A, Burgess T, Crous PW (2013) Phylogenetic lineages in the Botryosphaeriales: A systematic and evolutionary framework. Studies in Mycology 76(1): 31-49. https://doi.org/10.3114/ sim0020 Slippers B, Roux J, Wingfield MJ, Van der Walt FJJ, Jami K Mehl JWM, Marais GJ (2014) Confronting the constraints of morphological taxonomy in the Botryosphaeriales. Persoo- nia 33(1): 155-168. https://doi.org/10.3767/003158514X684780 Stamatakis A (2014) RAxML version 8: A tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics 30(9): 1312-1313. https://doi.org/10.1093/bioinfor- matics/btu033 Sun JE, Meng CR, Phillips AJL, Wang Y (2022) Two new Botryosphaeria (Botryosphaeriales, Botryosphaeriaceae) species in China. MycoKeys 94: 1-16. https://doi.org/10.3897/my- cokeys.94.91340 Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGAG: Molecular evolution- ary genetics analysis version 6.0. Molecular Biology and Evolution 30(12): 2725-2729. https://doi.org/10.1093/molbev/mst197 Taylor K, Barber PA, Hardy GESJ, Burgess TI (2009) Botryosphaeriaceae from tuart (Euca- lyptus gomphocephala) woodland, including descriptions of four new species. Mycological Research 113(3): 337-353. https://doi.org/10.1016/j.mycres.2008.11.010 Theissen E, Sydow H (1918) Vorentwiirfe zu den Pseudosphaeriales. Annales Mycologici 16: 1-34. Vilgalys R, Hester M (1990) Rapid genetic identification and mapping of enzymatically am- plified ribosomal DNA from several Cryptococcus species. Journal of Bacteriology 172(8): 4238-4246. https://doi.org/10.1128/jb.172.8.4238-4246.1990 Wang CB, Yang J, Li Y, Xue H, Piao CG, Jiang N (2023) Multi-gene phylogeny and mor- phology of two new Phyllosticta (Phyllostictaceae, Botryosphaeriales) species from China. MycoKeys 95: 189-207. https://doi.org/10.3897/mycokeys.95.100414 White TJ, Bruns T, Lee S, Taylor JW (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ (Eds) PCR protocols: a guide to methods and applications. New York. https://doi. org/10.1016/B978-0-12-372180-8.50042-1 Wijayawardene NN, Phillips AJL, Tibpromma S, Dai DQ, Selbmann L, Monteiro JS, Apt- root A, Flakus A, Rajeshkumar KC, Coleine C, Pereira DS, Fan X, Zhang L, Maharach- chikumbura SSN, Souza ME, Kukwa M, Suwannarach N, Rodriguez-Flakus P, Ashtekar Three new species of Botryosphaeriales in China 19 N, Dauner L, Tang LZ, Jin XC, Karunarathna SC (2021) Looking for the undiscovered asexual taxa: Case studies from lesser studied life modes and habitats. Mycosphere 12(1): 471-490. https://doi.org/10.5943/mycosphere/12/1/17 Wyka SA, Broders KD (2016) The new family Septorioideaceae, within the Botryosphaeriales and Septorioides strobi as a new species associated with needle defoliation of Pinus strobus in the United States. Fungal Biology 120(8): 1057-1066. https://doi.org/10.1016/j.fun- bio.2016.04.005 Xiao XE, Wang W, Crous PW, Wang HK, Jiao C, Huang K Pu ZX, Zhu ZR, Li HY (2021) Species of Botryosphaeriaceae associated with citrus branch diseases in China. Persoonia 47(1): 106-135. https://doi.org/10.3767/persoonia.2021.47.03 Yang T, Groenewald JZ, Cheewangkoon R, Jami F, Abdollahzadeh J, Lombard L, Crous PW (2017) Families, genera, and species of Botryosphaeriales. Fungal Biology 121(4): 322- 346. https://doi.org/10.1016/j.funbio.2016.11.001 Zhang M, He W, Wu JR, Zhang Y (2016a) Two new species of Spencermartinsia (Botry- osphaeriaceae, Botryosphaeriales) from China. Mycosphere 7(7): 942-949. https://doi. org/10.5943/mycosphere/si/1b/4 Zhang Y, Guan X, Wang G, Yang X, Li B, Xu Y (2016b) First report of Botryosphaeria dothidea causing apple ring rot of stems and fruits in China. Plant Disease 100(4): e865. https://doi. org/10.1094/PDIS-09-15-1094-PDN Zhang W, Groenewald JZ, Lombard L, Schumacher RK, Phillips AJL, Crous PW (2021) Eval- uating species in Botryosphaeriales. Persoonia 46(1): 63-115. https://doi.org/10.3767/ persoonia.2021.46.03 Zhu HY, Tian CM, Fan XL (2018) Studies of botryosphaerialean fungi associated with canker and dieback of tree hosts in Dongling Mountain of China. Phytotaxa 348(2): 63-76. https://doi.org/10.11646/phytotaxa.348.2.1 Supplementary material | Strains used in the molecular analyses in this study Author: Lu Lin Data type: table (Excel spreadsheet) Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODDbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/mycokeys.97.102653.suppl1