683 MycoKeys MycoKeys 106: 225-250 (2024) DOI: 10.3897/mycokeys.106.122890 Research Article New species and records of Botryosphaeriales (Dothideomycetes) associated with tree dieback in Beijing, China Yingying Wu™®, Cheng Peng’, Rong Yuan'®, Mingwei Zhang', Yang Hu2, Chengming Tian'® 1 The Key Laboratory for Silviculture and Conservation of the Ministry of Education, Beijing Forestry University, Beijing 100083, China 2 The Forestry Protection Station of Tonghzou Strict in Beijing, Beijing 101100, China Corresponding author: Chengming Tian (chengmt@bjfu.edu.cn) OPEN Qaceess Academic editor: Ning Jiang Received: 13 March 2024 Accepted: 2 June 2024 Published: 27 June 2024 Citation: Wu Y, Peng C, Yuan R, Zhang M, Hu Y, Tian C (2024) New species and records of Botryosphaeriales (Dothideomycetes) associated with tree dieback in Beijing, China. Mycokeys 106: 225-250. https://doi. org/10.3897/mycokeys. 106.122890 Copyright: © Yingying Wu et al. This is an open access article distributed under terms of the Creative Commons Attribution License (Attribution 4.0 International - CC BY 4.0). Abstract Botryosphaeriales species are important pathogens that have worldwide distribution. In this study, 23 Botryosphaeriales strains were isolated from 13 host species during a dieback disease survey in Beijing, China. Based on morphological and phylogenet- ic analyses, six Botryosphaeriales species were identified, including two new species named Dothiorella hortiarborum sp. nov. and Phaeobotryon fraxini sp. nov., and four new host records: Aplosporella ginkgonis from Cotinus coggygria var. cinereus, A. javeedii from Acer miyabei, Acer truncatum, Forsythia suspensa, Lagerstroemia indica, Sambucus williamsii, Syringa vulgaris, Ulmus pumila, Xanthoceras sorbifolium, A. yangingensis from Acer truncatum, and Do. acericola from Forsythia suspensa, Ginkgo biloba, and Syringa oblata. This study enriches the species diversity associated with tree dieback in Beijing, China, and contributes to the further study of the taxonomy of this order. Key words: Dothiorella, morphology, Phaeobotryon, phylogeny, taxonomy Introduction Botryosphaeriales species are important plant pathogens commonly found on the trunks and branches of woody plants (Phillips et al. 2013; Lawrence et al. 2017; Zhu et al. 2018; Zhang et al. 2021). They are associated with branch canker, dieback, decline, and death, with consequences for the ecological and economic value of the forest (Slippers and Wingfield 2007; Phillips et al. 2013; Mohali-Castillo 2023). Botryosphaeriales species occur on a wide range of hosts, in the form of endophytes on woody plants and herbs, lichens, and even seaweed leaves in marine environments, suggesting that they have great po- tential for research value (Yang et al. 2017; Akinsanmi et al. 2019; Zhang et al. 2021; Mohali-Castillo 2023; Rathnayaka et al. 2023). Phylogenetic analyses of DNA sequence data have an enormous influence on the systematics and taxonomy of the order Botryosphaeriales, including re- defining families and genera and identifying new species (Phillips et al. 2019; Mohali-Castillo 2023). Schoch et al. (2006) combined SSU, LSU, tef7-a, and rpb2 to first propose the order Botryosphaeriales, which contains only a sin- gle family of Botryosphaeriaceae. Minnis et al. (2012) supplemented the DNA sequence data of Planistromellaceae with phylogenetic analyses combining 225 Yingying Wu et al.: New species and records of Botryosphaeriales SSU, ITS, LSU, and rpb7, which introduced the family into the Botryosphaeri- ales. Wikee et al. (2013) reintroduced the Phyllostictaceae, grouped under Botryosphaeriales, to accommodate Phyllosticta using intronic genes (ITS, act, and tefl-a) and highly conserved coding regions of genes (LSU and GPDH). Slippers et al. (2013) added three new families, Aplosporellaceae (Aplosporella and Bagnisiella), Melanopsaceae (Melanops), and Saccharataceae (Sacchara- ta), to Botryosphaeriales based on DNA sequence data of six loci (SSU, LSU, ITS, tef1-a, tub2, and mtSSU). Wyka and Broders (2016) introduced Septori- oideaceae based on morphological and molecular evidence. Yang et al. (2017) mentioned that the LSU-rpb2 combination could effectively classify taxa at the family and genus levels, and rpb2 in combination with ITS, tef7-a, and tub2 added additional resolution for species delimitation. For this reason, they com- bined the five fragments ITS, tef1-a, tub2, LSU, and rpb2 to propose two new families, Endomelanconiopsisaceae and Pseudofusicoccumaceae. Therefore, Botryosphaeriales contained a total of nine families. However, Phillips et al. (2019) reassessed the families of Botryosphaeriales in terms of morphology of the sexual morphs and phylogenetic relationships of ITS and LSU sequence data, ultimately concluding that the order contained only six families (Aplospo- rellaceae, Botryosphaeriaceae, Melanopsaceae, Phyllostictaceae, Planistro- mellaceae, and Saccharataceae), with Endomelanconiopsisaceae, Pseudofu- sicoccumaceae, and Septorioideaceae as synonyms of existing families. Up to date, six families and 32 genera are accepted in Botryosphaeriales (https:// www.outlineoffungi.org/). Of these, Botryosphaeriaceae is rich in species diver- sity, high in pathogenicity, and widely distributed. Botryosphaeriaceae was first established by Theissen and Sydow (1918), containing three genera: Botryosphaeria, Dibotryon, and Phaeobotryon. Mor- phologically, Botryosphaericeae species are distinctive from other families in Botryosphaeriales by their large, ovoid to oblong, usually hyaline, aseptate ascospores (Phillips et al. 2013). Liu et al. (2012) assumed that ascospores could become pigmented and septate with age. Conidia in the asexual state of Botryosphaericeae are diverse in morphological characteristics (Phillips et al. 2005). Phylogenetically, however, there is a random distribution of hyaline or colored conidia or ascospores in the phylogenetic tree of Botryosphaericeae (Slippers et al. 2013). Therefore, accurate identification of species in the family by a single circumscription is not suitable. Currently, 22 genera and more than 200 species are contained within the family (https://www.outlineoffungi.org/). Recently, many new species have been introduced in the Botryosphaeriaceae, especially in the genera Dothiorella and Phaeobotryon (Jia et al. 2023; Li et al. 2023; Lin et al. 2023a; Wu et al. 2023). Saccardo (1880) first established Dothiorella and designated Do. pyrenophora as the type species. Up to now, some scholars have made systematic revisions of Dothiorella to establish a more stable phylogenetic relationship (Dissanayake et al. 2016; Dissanayake et al. 2020; Zhang et al. 2021). The distinctive features of the genera are that the conidia are colored in the early stages of development, and with 1-septate, the sexual form of ascospores is brown and septate (Sena- nayake et al. 2023). The type species of the genus Phaeobotryon is P. cercidis, which is characterized by 2-septate brown ascospores with conical apiculate-el- liptic to oblong or obovoid shapes at both ends and hyaline or brown conidia (Phillips et al. 2013; Fan et al. 2015b; Zhu et al. 2018; Pan et al. 2019). MycoKeys 106: 225-250 (2024), DOI: 10.3897/mycokeys.106.122890 296 Yingying Wu et al.: New species and records of Botryosphaeriales In recent years, multiple studies have revealed that new species of Botryos- phaeriales infest branches and trunks. Pan et al. (2019) found that Phaeobo- tryon rhois and Diplodia quercicola were detrimental to Rhus typhina and Quer- cus variabilis separately in Yudu Mountain, Beijing. Aplosporella yangingensis and Dothiorella baihuashan are mainly recorded on Pinaceae or Cupressaceae (Lin et al. 2023a). Lasiodiplodia regiae caused the canker and dieback of ap- ple trees (Wang et al. 2023). These studies suggest that Botryosphaeriales is rich in species diversity and has the potential to continue to be explored for new species. During the investigation of plant pathogens in Beijing, a higher number of diseased plant branches caused by Botryosphaeriales fungi were found. This study used phylogenetic analysis and morphological comparisons to describe new species and new host records, enriching the fungal taxa with- in Botryosphaeriales. Materials and method Sample collection and fungal isolation A survey on dieback diseases was conducted from March to November 2023 in the Tongzhou District of Beijing, China. A total of thirteen tree species were examined, namely Acer miyabei, A. truncatum, Cotinus coggygria var. cinere- us, Forsythia suspensa, Fraxinus chinensis, Ginkgo biloba, Lagerstroemia indica, Sambucus williamsii, Styphnolobium japonicum, Syringa oblata, Syringa vulgaris, Ulmus pumila, and Xanthoceras sorbifolium. Twenty specimens showing typical dieback symptoms (Fig. 1) with typical conidiomata and/or ascomata were col- lected. All samples were placed in paper bags and transported to the laboratory. Specimens with typical conidiomata pycnidial were selected for isolation. Re- moving the spore mass from conidiomata and generating single spore colonies or plating superficially sterilized diseased tissue on potato dextrose agar plates (PDA; containing 200 g potatoes, 20 g dextrose, and 20 g agar per liter) and in- cubating Petri dishes at 25 °C in the dark for 2-3 d. When colonies just formed, they transferred to fresh PDA Petri dishes (Crous et al. 2019). All specimens were deposited at the Museum of Beijing Forestry University (BUFC), and all cultures were preserved at the China Forestry Culture Collection Center (CFCC). Morphological observation Cultures were incubated on PDA at 25 °C in a 12-h day/night regime (Crous et al. 2019). After 14 days, the colonies were measured, and characteristics based on the color, shape, and sparseness of the aerial mycelium of the pathogen colonies were observed and recorded. Conidiomata were manually sectioned with a double-edged razor blade. Observations were conducted using a Leica DM 2,500 dissecting microscope (Wetzlar, Germany) and a Nikon Eclipse 80i compound microscope, equipped with differential interference contrast (DIC) illumination. Images were captured using a Nis DS-Ri2 camera with the Nikon Nis-Elements F4.30.01 software. Conidial length was measured from the base of the basal cell to the base of the apical appendage, while conidial width was measured at its widest point. A randomized selection of conidia was used for measurement (n = 50). MycoKeys 106: 225-250 (2024), DOI: 10.3897/mycokeys.106.122890 907 Yingying Wu et al.: New species and records of Botryosphaeriales Figure 1. Disease symptoms associated with Botryosphaeriales species collected from Tongzhou District, Beijing, China A Xanthoceras sorbifolium B Fraxinus chinensis C Lagerstroemia indica D Sambucus williamsii E Styphnolobium japoni- cum F Forsythia suspensa. DNA extraction, PCR amplification, and sequencing Genetic DNA was extracted using the cetyltrime-thylammonium bromide (CTAB) method when the mycelium was well spread on the PDA. DNA sam- ples were stored at -20 °C. The PCR reaction primers (forward and reverse) and amplification conditions are detailed in Table 1. Polymerase chain reaction (PCR) amplification was run on a PTC-200 Thermal Cycler amplifier from Bio- Rad, USA. The PCR amplification systems were all 20 uL, including 10 uL of Mix (Promega), 7 UL of double deionized water, 1 pL each of pre- and post-primers, and 1 uL of DNA template. PCR products were assayed by electrophoresis on 2% agarose gels. Amplified PCR products were sent to a commercial sequenc- ing provider (Tsingke Biotechnology Co. Ltd., Beijing, China). MycoKeys 106: 225-250 (2024), DOI: 10.3897/mycokeys.106.122890 228 Yingying Wu et al.: New species and records of Botryosphaeriales Table 1. Genes used in this study with PCR primers. Locus PCR primers PCR: thermal cycles: (Annealing temp. in bold) References ITS ITS1/ITS4 (95 °C: 30s, 51 °C: 30 s, 72 °C: 1 min) x 35 cycles White et al. 1990 LSU LROR/LR5 (95 °C: 45s, 55 °C: 30s, 72°C: 1 min) x 35 cycles | Vilgalys and Hester 1990 tefl-a | EF1-728F/EF1-986R | (95 °C: 15s, 55 °C: 30s, 72 °C: 1 min) x 35 cycles | Carbone and Kohn 1999 tub2 Bt2a/Bt2b (95 °C: 30 s, 55 °C: 30 s, 72 °C: 1 min) x 35 cycles | Glass and Donaldson 1995 Phylogenetic analyses The sequences obtained were assembled using SeqMan v. 7.1.0 software, and reference sequences from related publications (Phillips et al. 2019; Li et al. 2023; Lin et al. 2023a; Wu et al. 2023) were retrieved from the National Center for Biotechnology Information (NCBI; https://www.ncbi.nIm.nih.gov). All sequences generated in this study were submitted to GenBank (Table 2). Sequences were aligned in MAFFT v. 7 at the web server (https://mafft.cbre. jp/alignment/server/) (Katoh and Standley 2013; Katoh et al. 2019) and further adjustments and editing of the sequences were made with MEGA v. 6 (Tamura et al. 2013). Maximum parsimony (MP), maximum likelihood (ML), and Bayes- ian inference (BI) were selected to construct phylogenetic trees using PAUP v. 4.0610, PhyML 3.0, and MrBayes V3.1.2 (Huelsenbeck and Ronquist 2001; Swofford 2003; Silvestro and Michalak 2012). Phylograms were visualized with FigTree v. 1.4.0 (http://tree.bio.ed.ac.uk/software/figtree/) and additional ed- ited with Adobe Illustrator CS v. 5 (Adobe Systems Inc., USA). Maximum-par- simony bootstrap values (MPBP) and maximum-likelihood bootstrap values (MLBP) = 50% and Bayesian posterior probabilities (BYPP) = 0.90 are shown for each tree. Maximum parsimony analysis was performed using the tree bisection and reconnection (TBR) branch swapping algorithm with a heuristic search op- tion of 1000 random-addition sequences (Swofford 2003). Max trees were set to 5000 branches of zero length, and all parsimonious trees were saved. Other measures calculated were tree length (TL), consistency index (Cl), retention index (RI), and rescaled consistency (RC) (Swofford 2003). Maxi- mum likelihood analysis was performed with the GTR GAMMA model of site substitution, including estimation of gamma-distributed rate heterogeneity and a proportion of invariant sites (Guindon et al. 2010). The branch support from MP and ML analysis was evaluated with a bootstrapping (BS) method of 1 000 replicates (Hillis and Bull 1993). The Bayesian inference analysis employing a Markov chain Monte Carlo (MCMC) algorithm was performed with Bayesian posterior probabilities (Rannala and Yang 1996). The mod- el of nucleotide substitution was estimated by MrModeltest v.2.3 (Posada and Crandall 1998), and a weighted Bayesian analysis was considered. Two MCMC chains were run starting from random trees for 1,000,000 genera- tions and stopped when the average standard deviation of split frequencies fell below 0.01; the trees were sampled every 100" generation. The first 25% of trees were discarded as the burn-in phase of each analysis, and the Bayesian posterior probabilities (BPP) were calculated using the remaining 7,500 trees. MycoKeys 106: 225-250 (2024), DOI: 10.3897/mycokeys.106.122890 999 Yingying Wu et al.: New species and records of Botryosphaeriales Table 2. Isolates of Aplosporella, Dothiorella, and Phaeobotryon used in the molecular analyses in this study. Notes: NA: not applicable, Strains in this study are marked in bold, T: ex-type strains. Species Aplosporella africana A. africana A. artocarpi A. ginkgonis A. ginkgonis A. ginkgonis A. hesperidica A. hesperidica A. javeedii A. javeedii A. javeedii A. javeedii A. javeedii A. javeedii A. javeedii A. javeedii A. javeedii A. javeedii A. javeedii A. javeedii A. javeedii A. javeedii A. javeedii A. javeedii A. macropycnidia A. macropycnidia A. papillata A. papillata A. prunicola A. prunicola A. sophorae A. thailandica A. yalgorensis A. yalgorensis A. yangingensis A. yangingensis A. yangingensis A. yanqgingensis Alanomyces indica Dothiorella alpina Do. acacicola Do. acericola Do. acericola Do. acericola Do. acericola Strain CBS 121777° CBS 1217778" CPC 22791" CFCC 52442" CFCC 89661" CFCC 70746 CBS 732.79" CBS 208.37 CFCC 50054" CFCC 50052 CFCC 58330 CFCC 58329 CFCC 58412 CFCC 70733 CFCC 70734 CFCC 70735 CFCC 70736 CFCC 70737 CFCC 70739 CFCC 70740 CFCC 70741 CFCC 70742 CFCC 70744 CFCC 70745 CGMCC 3.17725" CGMCC 3.17726 CBS 121780° CBS 121781 CBS 121167° STE-U 6326 CPC 29688" MFLU 16-0615" MUCC5117 MUCC512 CFCC 587917 CFCC 58792" CFCC 70743 CFCC 70738 CBS 134264" CGMCC 3-180017 CBS 141295' KUMCC 18-0137" CFCC 70755 CFCC 70760 CFCC 70761 Host Acacia mellifera Acacia mellifera Artocarpus heterophyllus Rhus typhina Rhus typhina Cotinus coggygria var. cinereus Citrus aurantium Citrus sinensis Juniperus chinensis Gleditsia sinensis Populus canadensis Populus beijingensis Populus alba var. pyramidalis Styphnolobium japonicum Forsythia suspensa Forsythia suspensa Ulmus pumila Acer truncatum Sambucus williamsii Acer miyabei Lagerstroemia indica Xanthoceras sorbifolium Syringa vulgaris Ulmus pumila Cerasus yedoensis Cerasus yedoensis Acacia tortillas Acacia tortillas Prunus persica var. nucipersica Prunus persica var. nucipersica Sophora microphylla Dead stems Acacia cochlearis Eucalyptus gomphocephala Platycladus orientalis Platycladus orientalis Acer truncatum Acer truncatum Soil Platycladus orientalis Acacia mearnsii Acer palmatum Forsythia suspensa Ginkgo biloba Syringa oblata MycoKeys 106: 225-250 (2024), DOI: 10.3897/mycokeys.106.122890 Origin Namibia Namibia Thailand China China China Buenos Aires Zimbabwe China China China China China China China China China China China China China China China China China China South Africa South Africa South Africa South Africa New Zealand North Thailand Australia Australia China China China China India China Réunion China China China China ITS KF766196 EU101316 KM006450 MH133916 KM030583 PP188498 KX464083 JX681069 KP208840 KP208838 0Q651161 0Q651162 0Q651163 PP188499 PP188500 PP188501 PP188502 PP188503 PP188504 PP188505 PP188506 PP188507 PP188508 PP188509 KT343648 KT343649 EU101328 EU101329 KF766147 EF564375 KY173388 KX423536 EF591926 EF591927 0Q651164 0Q651165 PP188510 PP188511 HF563622 KX499645 KX228269 MK359449 PP188520 PP188521 PP188522 GenBank accession numbers tef1-a EU101360 EU101361 KM006481 MH133950 KM030597 PP541796 NA NA KP208846 KP208844 0Q692921 0Q692922 0Q692923 PP541797 PP541798 PP541799 PP541800 PP541801 PP541802 PP541803 PP541804 PP541805 PP541806 PP541807 KX011176 KX011177 EU101373 EU101374 NA NA NA KX423537 EF591977 EF591978 0Q692924 0Q692925 PP541808 PP541809 AB872219 KX499651 KX228376 MK361182 PP766251 PP766252 PP766253 tub2 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA PP566659 PP566660 PP566661 LSU NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA 230 Yingying Wu et al.: New species and records of Botryosphaeriales Species Strain Host Do. albiziae MFLUCC 22-0057' Albizia lebbeck Do. alpina CFCC 58299' Populus szechuanica Do. americana CBS 128309' Vitis species and Vitis vinifera Do. baihuashanensis CFCC 58549" Juniperus chinensis Do. baihuashanensis CFCC 58788" Juniperus chinensis Do. brevicollis CBS 130411 = CMW 36463' Acacia karroo Do. californica CBS 119635 Laurus nobilis Do. californica CBS 141587 Umbellularia californica Do. camelliae CMGCC 3.24158" Camellia oleifera Do. capri-amissi CBS 121763 = CMW 25403 Acacia erioloba = CAMS 11587 Do. capri-amissi CBS 121878 = CMW 25404 Acacia erioloba = CAMS 11597 Do. casuarinae CBS 120688 = CMW 4855" Casuarina sp. Do. casuarinae CBS 120689 = CMW 4856 Casuarina sp. Do. casuarinae CBS 120690 = CMW 4857 Casuarina sp. Do. citricola CBS 124728 = ICMP 16827 Citrus sinensis Do. citricola CBS 124729 = ICMP 16828" Citrus sinensis Do. citrimurotticola BES = CGMCC3.20392' Citrus unshiu Do. citrimurotticola BE8 = CGMCC3.20394 Citrus reticulatachen x C. sinensis Do. diospyricola CBS 145972 Diospyros mespiliformis Do. dulcispinae CBS 121764 = CMW 25406 Acacia mellifera = CAMS 1159 Do. dulcispinae CBS 130413 = CMW 36460° Acacia karroo Do. eriobotryae CBS 140852° Eriobotrya japonica Do. franceschinii CBS 147722 Rhamnus alaternus Do. guttulata MFLUCC 17-0242 Alnus glutinosa Do. heterophyllae CMW 46458° Acacia heterophylla Do. hortiarborum CFCC 70756" Fraxinus chinensis Do. hortiarborum CFCC 70757 Fraxinus chinensis Do. hortiarborum CFCC 70758 Lagerstroemia indica Do. hortiarborum CFCC 70759 Lagerstroemia indica Do. iberica CBS 113188 = DA-1 Quercus suber Do. iberica CBS 113189 = DE-14 Quercus ilex Do. iberica CBS 115041 = CAP 145° Quercus ilex Do. irannica CBS 124722 = CJA 153 = Olea europaea IRAN 1587C™ Do. koae CMW 48017' Acacia koa Do. lampangensis MFLUCC 18-0232' Rutaceae Do. longicollis CBS 122066 = CMW 26164 Terminalia sp. Do. longicollis CBS 122067 = CMW 26165 Lysiphyllum cunninghamii Do. longicollis CBS 122068 = CMW 26166' Lysiphyllum cunninghamii Do. magnoliae CFCC51563" Magnolia grandiflora Do. mangifericola CBS 124727' Mangifera indica Do. mangifericola IRAN 1584C Mangifera indica Do. moneti WAC 13154 = MUCC 505° Acacia rostellifera Do. neclivorem DAR 80992" Vitis vinifera Do. oblonga CBS 121765 = CMW 25407 Acacia mellifera = CAMS 11627 Do. oblonga CBS 121766 = CMW 25408 Acacia mellifera = CAMS 1163 Do. obovata MFLUCC22-0058" Pavonia odorata MycoKeys 106: 225-250 (2024), DOI: 10.3897/mycokeys.106.122890 Origin Thailand China USA: Missouri China China South Africa Turkey USA China South Africa South Africa Australia Australia Australia New Zealand New Zealand China China South Africa Namibia South Africa Spain Italy Italy Réunion China China China China Spain Spain Spain Iran, Golestan Hawaiian Is. Thailand Australia Australia Australia China Iran Iran Australia Australia South Africa South Africa Thailand ITS ON751762 0Q651166 HQ288218 0Q651167 0Q651168 JQ239403 MT587396 KX357188 0Q190531 EU101323 EU101324 DQ846773 DQ846772 DQ846774 EU673322 EU673323 MW880663 MW880661 MT587398 EU101299 JQ239400 KT240287 OP999677 KY797637 MN103794 PP188523 PP188524 PP188525 PP188526 AY573198 AY573199 AY573202 KC898231 MH447652 MK347758 EU144052 EU144053 EU144054 KY111247 KC898221 MT587407 EF591920 KJ573643 EU101300 EU101301 ON751763 tef1-a ON799588 0Q692932 HQ288262 0Q692933 0Q692934 JQ239390 MT592108 KX357211 0Q241464 EU101368 EU101369 DQ875331 DQ875332 DQ875333 EU673289 EU673290 MW884166 MW884164 MT592110 EU101344 JQ239387 KT240262 0Q067247 NA MH548348 PP723042 PP723043 PP723044 PP723045 EU673278 AY573230 AY573222 KC898214 MH548338 MK340869 EU144067 EU144068 EU144069 KY213686 KX464614 MT592119 EF591971 KJ573640 EU101345 EU101346 ON799589 GenBank accession numbers tub2 ON799590 0Q692926 HQ288297 0Q692927 0Q692928 JQ239371 MT592579 KX357165 0Q275064 KX464850 KX464851 DQ875340 DQ875339 DQ875341 KX464852 KX464853 MW884195 MW884193 MT592581 KX464854 JQ239373 MT592582 NA NA MH548324 PP566662 PP566663 PP566664 PP566665 EU673097 KX464855 EU673096 KX464856 MH548327 MK412874 KX464857 KX464858 KF 766130 NA NA NA EF591954 KJ577551 KX464862 KX464863 ON799591 LSU NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA 231 Yingying Wu et al.: New species and records of Botryosphaeriales Do. Do. Do. Do. Do. Do. Species omnivora omnivora omnivora omnivora omnivora parva Do. parva Do. parva Do. plurivora Do. pretoriensis Do. Do. Do. Do. Do. Do. Do. Do. Do. Do. Do. Do. Do. Do. Do. Do. Do. Do. Do. Do. Do. Do. Do. Do. Do. Do. Do. Do. Do prunicola rhamni rosulata rosulata rosulata rosulata santali saprophytica sarmentorum sempervirentis sempervirentis sp. sp. sp. striata striata styphnolobii symphoricarpicola tectonae thailandica thripsita ulmacea uruguayensis vidmadera vidmadera vinea-gemmae viticola westralis . yunnana Do. yunnana Do. zanthoxyli CBS 124721 = CBS 124730 = CBS 124731 = CBS 392.80 CBS 124716 = CJA 241 = IRAN 1573C CBS 242.51 CBS 188.87 CBS 124720 = CJA 27 = IRAN 1579C™ CBS 125580 CBS 124724 = CJA 254 = IRAN 1557C™ CBS 130404 = CMW 36480° CBS 124723 = CAP 187 = IRAN 1541C' MFLUCC 14-0902" CBS 121760 = CMW 25389 = CAMS 14447 CBS 121761 = CMW 25392 = CAMS 1147 CBS 121762 = CMW 25395 = CAMS 1150 CBS 500.72 WAC 13155 = MUCC 5097 MFLUCC 23-0210 IMI 63581b IRAN 1581C = CBS 124719 IRAN 1583C = CBS 124718 = CJA 264° CBS 121783 = CMW 25432 = CAMS 1187 CBS 121784 = CMW 25430 = CAMS 1185 CBS 121785 = CMW 25433 = CAMS 1188 Cr01" CPC 33923" MFLUCC18-0232' CBS 133991 = CPC 21557 = MFLUCC 11-0438 CBS 125445 = BRIP 51876a" CBS 141414" CBS 124908 = CMW 26763' CBS 621.74 CBS 725.79" DAR 81012" CBS 117009° WA10NO017 CGMCC 3-17999" CGMCC 3-18000 CMGCC 3.241597 CJA 35 ICMP 16819 ICMP 16824" Juglans regia Juglans regia Corylus sp. Corylus sp. Corylus avellana Citrus sp. Acacia karroo Prunus dulcis Rhamnus cathartica Acacia karroo Acacia mellifera Acacia mellifera Medicago sativa Santalum acuminatum Ulmus sp. Cupressus sempervirens Cupressus sempervirens Acacia mearnsii Acacia mearnsii Acacia mearnsii Citrus sinensis Citrus sinensis Styphnolobium japonicum Symphoricarpos Tectona grandis Dead bamboo culm Acacia harpophylla Ulmus laevis Hexachlamis edulis Pyrus communis Pyrus malus Vitis vinifera Vitis vinifera Vitis vinifera Camellia sp. Camellia sp. Zanthoxylum bungeanum MycoKeys 106: 225-250 (2024), DOI: 10.3897/mycokeys.106.122890 S Origin Iran Italy France Iran Iran Austria Iran South Africa Portugal outh European Russia Namibia South Africa South Africa South Africa Australia Thailand UK: England Iran Iran South Africa South Africa South Africa New Zealand New Zealand Crym Italy Thailand Thailand Australia Germany Uruguay Switzerland Switzerland Australia Spain Australia China China Sichuan GenBank accession numbers tef1-a CBS 124717 = CJA 214 = Juglans regia Iran KC898233 | KC898216 | KX464865 IRAN 1570C tub2 KC898232 | KC898215 | KX464864 EU673317 EU673316 KC898234 KX464123 KC898217 EU673119 KX464866 KX464867 KC898225 | KC898208 | KX464874 JQ239405 | JQ239392 | JQ239376 EU673313 MF398893 KF766227 EU101293 EU101319 EU673318 EF591924 KX464124 | KX464616 | KX464868 EU673280 MF398945 EU101335 EU101338 EU101364 EU673100 NA KX464877 KX464878 KX464879 EU673118 EFS91.958 ORS27239 | OR532455 | OR532454 AY573212 EU673102 KC898237 | KC898220 | KX464885 KC898236 EU101333 EU101331 EU101334 EU673320 KC898219 EU101378 EU101376 EU101379 KX464884 KX464859 KX464860 KX464861 NA MH880849 MK069594 JX646796 | JX646861 | JX646844 MT587415 EU080923 KX464129 KX464130 KJ573644 AY905554 HM009376 KX499643 KX499644 0Q190536 KJ573641 10573681 ‘aeons HM8s00511 0Q241468 KX464888 KJ577552 EU673104 NA NA NA 0Q275069 NA 232 Yingying Wu et al.: New species and records of Botryosphaeriales Species Neofusicoccum luteum Neofusicoccum parvum Phaeobotryon aplosporum P. aplosporum P. aplosporum P. aplosporum P. aplosporum P cupressi P cupressi P. fraxini P. fraxini P juniperi P juniperi P mali P mali P mali P mali P mali P mamane P. mamane P. negundinis P. negundinis P. negundinis P. negundinis P. negundinis P. negundinis P. negundinis P platycladi P platycladi P rhoinum P rhoinum P rhois | P rhois P rhois P spiraeae P spiraeae P spiraeae P ulmi P ulmi P ulmi P ulmi P ulmi P ulmi P ulmi Alanphillipsia aloeicola CBS 562.927 CMW 9081' CFCC 53774 CFCC 53775" CFCC 53776 CFCC 58596 CFCC 58784 CBS 124700 = CBS 124701 = IRAN 1455C™ IRAN 1458C CFCC 70762" CFCC 70763 JU001 7 JU005 XJAU 2930" XJAU 2772 XJAU 2782 XJAU 3094 XJAU 3100 CBS 122980 = CPC 12440" CPC 12442 CAA 797 CAA 798 CAA 799 CPC 33384 CPC 33388 CPC 34752 MFLUCC 15-0436" CFCC 58799" CFCC 58800 CFCC 52449 CFCC 52450° CFCC 89662 = CCTCC AF2014017° 94- 1 CBS 114123 = UPSC 2552 CBS 138854 = CPC 24264" CBS 123.30 = ATCC 24443 CBS 174.63 CMH 299 PB_11f CBS 138896 = CPC 23674" Actinidia deliciosa Populus nigra Syzygium aromaticum Rhus typhina Rhus typhina Juglans mandshurica Juglans mandshurica Cupressus sempervirens Cupressus sempervirens Fraxinus chinensis Fraxinus chinensis Juniperus formosana Juniperus formosana Malus pumila Juglans regia Malus ‘Royalty’ Elaeagnus angustifolia Rhus typhina Sophora chrysophylla Sophora chrysophylla Acer negundo Ligustrum vulgare Forsythia intermedia Acer nugundo Dead stem Acer negundo Acer negundo Platycladus orientalis Platycladus orientalis Rhus typhina Rhus typhina Rhus typhina Ulmus pumila Ulmus laevis Ulmus sp. Ulmus glabra House dust Ulmus glabra Aloe sp. MycoKeys 106: 225-250 (2024), DOI: 10.3897/mycokeys.106.122890 Origin New Zealand New Zealand China China China China China Iran Iran China China China China China China China China China USA USA Russia Ukraine Ukraine Ukraine Russia China China China China ITS MH862376 AY236943 MN215837 MN215838 0Q651169 0Q651170 FJ919672 FJ919671 PP188527 PP188528 EU673332 EU673333 KX061513 KX464690 KX464968 GenBank accession numbers LSU NA AY236888 | AY236917 NA MN215836 | MN205996 FJ919661 FJ919660 OP941637 | OP948218 OP941638 | OP948219 0948218 | MW326854 MW509520 | MW509518 | MW326853 | MW509520 0P 948219 | MW326852 “ws09517 MW326858 | MW509517 MW326878 | MW509518 KX061514 | KX061508 KX061515 | KX061509 MT587542 MT587543 | MT592277 MT587544 KU820970 0Q651172 N N N N N N N N N N N N N N N N A A A A A A A A A A A A A A A A A N NA KM030584 | KM030598 CFCC 89663 = CCTCC Rhus typhina China KM030585 | KM030599 NA AF2014016 CFCC 586797 Populus alba var. China 0Q651171 | 0Q692929 NA 00652542 pyramidalis NA Ulmus glabra MT587539 | MT592273 KP004444 | MT592027 Poland South Africa MN215871 MN215872 MN215873 0Q652540 0Q652541 KX464538 KX464539 PP177348 PP177349 0P941644 0P941645 MW367101 MW367094 MW367092 MW367100 MW367093 EU673248 DQ377899 NA NA NA MT587323 MT587324 MT587325 NA 0Q652543 0Q652544 MH133940 MH133941 KM030591 KM030592 NA MT587320 MT587321 DQ377861 MT587322 NA NA KP004472 233 Yingying Wu et al.: New species and records of Botryosphaeriales Result Phylogenetic analysis The BLAST results indicated that the 23 isolates resided in Ap/osporella, Do- thiorella, and Phaeobotryon (14 for Aplosporella, 7 for Dothiorella, and 2 for Phaeobotryon). Separate phylogenetic trees for each of the three genera were constructed in this study. In Aplosporella, the combined ITS and tef1-a dataset consists of 944 charac- ters, including alignment gaps (508 for ITS and 436 for tef1-a), of which 794 are constant and 60 are variable parsimony uninformative characters. MP analysis with the remaining 90 parsimony-informative characters resulted in one equal- ly parsimonious tree: tree length (TL) = 230; consistency index (Cl) = 0.817; retention index (RI) = 0.896; and rescaled consistency index (RC) = 0.732. In ML analysis based on the combined gene dataset, the matrix had 193 distinct alignment patterns. Estimated base frequencies are as follows: A = 0.217607, C = 0.264598, G = 0.259539, T = 0.258256, AC = 2.784746, AG = 2.845183, AT = 1.353935, CG = 1.848853, CT = 4.935430, GT = 1.000000, gamma distribu- tion shape parameter: a = 0.157110, and likelihood value of In: -2 499.855852. The maximum likelihood (ML) and Bayesian methods (BI) for phylogenetic analyses have the same topology and terminal clades. Fourteen isolates were distributed in Aplosporella, aggregated with three known species, A. javeedii, A. yangingensis, and A. ginkgonis, separately (Fig. 2). The single gene tree for ITS and tef1-a of Aplosporella is shown in Suppl. material 1. In Dothiorella, sequences of the combined ITS, tef1-a, and tub2 were aligned; the dataset consists of 1,319 characters, including alignment gaps (534 for ITS, 369 for tef1-a, and 416 for tub2), of which 905 are constant and 107 are variable par- simony uninformative characters. MP analysis with the remaining 307 parsimo- ny-informative characters resulted in one equally parsimonious tree: tree length (TL) = 1,282; consistency index (Cl) = 0.477; retention index (RI) = 0.824; and rescaled consistency index (RC) = 0.394. In ML analysis based on the combined gene dataset, the matrix had 601 distinct alignment patterns. Estimated base fre- quencies are as follows: A = 0.206208, C = 0.312741, G = 0.250328, T = 0.230723, AC = 0.833804, AG = 2.174710, AT = 1.041501, CG = 0.791470, CT = 3.735830, GT = 1.000000, gamma distribution shape parameter: a = 0.215045, and likeli- hood value of In: -8 567.497788. Three of the seven isolates were of the known species Dothiorella acericola, and the other four isolates formed a separate clade for designation as new species based on phylogenetic analysis (Fig. 3). The sin- gle gene tree for ITS, tef7-a, and tub2 of Dothiorella is shown in Suppl. material 2. In Phaeobotryon, the combined ITS, LSU, and tef7-a dataset consists of 1,394 characters, including alignment gaps (494 for ITS, 333 for LSU, and 567 for tef1-a), of which 1,218 are constant and 56 are variable parsimony uninforma- tive characters. MP analysis with the remaining 120 parsimony-informative characters resulted in one equally parsimonious tree: tree length (TL) = 259; consistency index (Cl) = 0.799; retention index (RI) = 0.913; and rescaled con- sistency index (RC) = 0.730. In ML analysis based on the combined gene data- set, the matrix had 239 distinct alignment patterns. Estimated base frequen- cies are as follows: A = 0.224820, C = 0.266099, G = 0.277247, T = 0.231833, AC = 0.602998, AG = 2.181745, AT = 0.500445, CG = 0.607508, CT = 4.549533, GT = 1.000000, gamma distribution shape parameter: a = 0.020014, and likelihood MycoKeys 106: 225-250 (2024), DOI: 10.3897/mycokeys.106.122890 034 Yingying Wu et al.: New species and records of Botryosphaeriales value of In: -3 357.887099. Eight isolates were assigned to Phaeobotryon, one isolate aggregated with P mali, and two isolates stood alone, not branching off from known species, representing a new species (Fig. 4). The single gene tree for ITS, LSU, and tef7-a of Phaeobotryon is shown in Suppl. material 3. Aplosporella javeedii CFCC 50052 Aplosporella javeedii CFCC 70734 Aplosporella javeediti CFCC 50054 Aplosporella javeedii CFCC 58330 Aplosporella javeedii CFCC 58412 Aplosporella javeedii CFCC 58329 Aplosporella javeedii CFCC 70742 Aplosporella javeedii CFCC 70739 Aplosporella javeedii CFCC 70736 o7/o7/ip APlosporella javeedit CFCC 70744 Aplosporella javeedii CFCC 70741 Aplosporella javeedii CFCC 70737 Aplosporella javeedii CFCC 70735 ! Aplosporella javeedii CFCC 70745 Aplosporella javeedii CFCC 70740 Aplosporella javeedii CFCC 70733 Aplosporella sophorae CPC 29688 aia Aplosporella macropycnidia CGMCC 3.17725 | 82/90/0.98] & Aplosporella macropycnidia CGMCC 3.17726 8B/90/I—_ |. Aplosporella africana CBS 121777 74/73/0.98 Aplosporella africana CBS 121778 98/100/1 Aplosporella yalgorensis MUCC 511 Aplosporella yalgorensis MUCC 512 Aplosporella prunicola CBS 121167 ?1196/0.99! Anlosporella prunicola STE-U 6326 71/76/0.91 100/100/1; Aplosporella papillata CBS 121780 Aplosporella papillata CBS 121781 Aplosporella yangingensis CFCC 58792 Aplosporella yangingensis CFCC 58791 90/95/- 00/100) 4 nlosporella yangingensis CFCC 70738 Aplosporella yangingensis CFCC 70743 Aplosporella hesperidica CBS 208.37 Aplosporella hesperidica CBS 732.79 60/76/1 Aplosporella thailandica MFLU 16-0615 f Aplosporella artocarpi CPC 22791 aia Aplosporella ginkgonis CFCC 70746 Aplosporella ginkgonis CFCC 52442 Aplosporella ginkgonis CFCC 89661 Alanomyces indica CBS 134264 59/72/- 63/70/0.95 Outgroup 0.03 Figure 2. Phylogram generated from RAXML analysis based on ITS with tef1-a sequence data of Aplosporella isolates. The tree was rooted in Alanomyces indica (CBS 134264). The MP, ML (2 50%), and BI (2 0.9) bootstrap supports are given near the nodes, respectively. Isolates from this study are marked in blue, and ex-type strains are marked in bold. MycoKeys 106: 225-250 (2024), DOI: 10.3897/mycokeys.106.122890 235 Yingying Wu et al.: New species and records of Botryosphaeriales Dothiorella acericola CFCC 70755 99/1001 | Dothiorella acericola CFCC 70761 cove Dothiorella acericola KUMCC 18-0137 “ese Dothiorella acericola CFCC 70760 Dothiorella plurivora CBS 124724 80/97/Q.99f Dothiorella hortiarborum CFCC 70758 85/99/, a Dothiorella hortiarborum CFCC 70759 Dothiorella hortiarborum CFCC 70756 56/33) ' Dothiorella hortiarborum CFCC 70757 55/5¢ Pp Dothiorella alpina CFCC 38299 LTL Dothiorella alpina CGMCC 3-18001 Dothiorella yannana CGMCC 3-17999 | Dothiorella magnoliae CFCC51563 Dothiorella yunnana CGMCC 3-18000 87/95/I Dothiorella rosulata CBS 121762 68/85/Q.92} Dothiorella rosulata CBS 121761 ESE [I Dothiorella rosulata CBS 121760 Dothiorella rosulata CBS 500.72 La Tr Dothiorella mangifericola IRAN 1584C 752/0.97— Dothiorella mangifericola CBS 124727 79/861 Dothiorella citricola CBS 124729 anete Dothiorella citricola CBS 124728 be 96/100/1 fg9/95/9— Dothiorella westralis WA10NO01 5070/0 Dothiorella viticola CBS 117009 66/76/1 Dothiorella brevicollis CBS 130411 54/37/- sv9s/I" Dothiorella striata CBS 124730 Dothiorella striata CBS 124731 Dothiorella heterophyllae CMW46458 =, Dothiorella saprophytica MFLUCC 23-0210 . Dothiorella longicollis CBS 122068 Dothiorella longicollis CBS 122067 Dothiorella longicollis CBS 122066 Dothiorella diospyricola CBS 145972 Dothiorella lampangensis MFLUCC 18-0232 Dothiorella obovata MFLUCC22-0058 Dothiorella tectonae MFLUCC18-0232 Dothiorella neclivorem DAR 80992 Pace 6p Dothiorella citrimurotticola CGMCC3.20392 abt aoe Dothiorella citrimurotticola CGMCC3.20394 Score Dothiorella uruguayensis CBS 124908 : Dothiorella oblonga CBS 121766 Dothiorella oblonga CBS 121765 15/05) LOMO. Dothiorella dulcispinae CBS 121764 100/100/1 Dothiorella dulcispinae CBS 130413 Dothiorella thailandica CBS 133991 Dothiorella albiziae MFLUCC 22-0057 ' Dothiorella vinea-gemmae DAR 81012 Dothiorella omnivora CBS 392.80 Dothiorella omnivora CBS 124716 89/98/9,98) Dothiorella omnivora CBS 124717 , Dothiorella omnivora CBS 188.87 775/024" Dothiorella omnivora CBS 242.51 58/86/09] ful Dothiorella vidmadera CBS 725.79 Dothiorella vidmadera CBS 621.74 79/95/6911] ) Dothiorella parva CBS 124721 Dothiorella parva CBS 125580 Dothiorella guttulata MFLUCC 17-0242 67/67/2981 | Dothiorella parva CBS 124720 Dothiorella sarmentorum IMI 63581b Dothiorella styphnolobii Cr01 Dothiorella americana CBS 128309 Dothiorella iberica CBS 115041 Dothiorella iberica CBS 113188 147, Dothiorella iberica CBS 113189 uae) L,I Dothiorella symphoricarpicola CPC 33923 79/77/09 ha Dothiorella californica CBS 119635 Dothiorella californica CBS 141587 ido) Dothiorella sempervirentis IRAN 1581C Dothiorella sempervirentis IRAN 1583C ae 3997/4 Dothiorella eriobotryae CBS140852 : 96/100/1 Dothiorella rhamni MFLUCC 14-0902 Dothiorella franceschinii CBS 147722 Dothiorella prunicola CBS 124723 Dothiorella santali WAC 13155 56/84. Dothiorella koae CMW 48017 Dothiorella moneti WAC 13154 TOI SS Dothiorella thripsita CBS 125445 100/100/1 Dothiorella pretoriensis CBS 130404 Dothiorella sp. CBS 121784 oo/1004| Dothiorella sp. CBS 121785 56/71/0.53 Dothiorella sp. CBS 121783 Dothiorella casuarinae CBS 120689 00/100 Dothiorella casuarinae CBS 120688 90/98/1 Dothiorella casuarinae CBS 120690 Dothiorella acacicola CBS 141295 187/008 100/100/1 Dothiorella capri-amissi CBS 121878 100/100/1 Dothiorella capri-amissi CBS 121763 100/100/1_ 4 Dothiorella baihuashanensis CFCC 58549 x2 Dothiorella baihuashanensis CFCC 58788 Dothiorella iranica CBS 124722 100/100/1 Dothiorella camelliae CMGCC 3.24158 Dothiorella zanthoxyli CMGCC 3.24159 Dothiorella ulmacea CBS 141414 100/100/1 Neofusicoccum parvum CMW9081 Neofusicoccum luteum CBS 562.92 Outgroup 0.03 Figure 3. Phylogram generated from RAXxML analysis based on ITS, tefl-a, and tub2 sequence data of Dothiorella iso- lates. The tree was rooted in Neofusicoccum luteum (CBS 562.92) and Neofusicoccum parvum (CMW9081). The MP ML (= 50%), and BI (= 0.9) bootstrap supports are given near the nodes, respectively. Isolates from this study are marked in blue, and ex-type strains are marked in bold. MycoKeys 106: 225-250 (2024), DOI: 10.3897/mycokeys.106.122890 Yingying Wu et al.: New species and records of Botryosphaeriales Phaeobotryon negundinis CAA797 Phaeobotryon negundinis CAA798 Phaeobotryon negundinis CAA799 Phaeobotryon negundinis CPC 33388 96/95/1| Phaeobotryon negundinis CPC 33384 Phaeobotryon negundinis CPC 34752 Phaeobotryon negundinis MFLUCC 15-0436 95/99/1) Phaeobotryon juniperi JU 001 Phaeobotryon juniperi JU 005 i 0/100/11 Phaeobotryon cupressi CBS 124701 Phaeobotryon cupressi CBS 124700 94/100/1) Phaeobotryon platycladi CFCC 58800 Phaeobotryon platycladi CFCC58799 Phaeobotryon spiraeae CFCC 53926 87/98/0.97| Phaeobotryon spiraeae CFCC 53927 Phaeobotryon spiraeae CFCC 53925 99/100/1 Phaeobotryon mamane CPC 12442 Phaeobotryon mamane CPC 12440 Phaeobotryon rhoinum CFCC 52450 Phaeobotryon rhoinum CFCC 52449 98/100/1 Phaeobotryon aplosporum CFCC 53774 Phaeobotryon aplosporum CFCC 53776 86/99/1___| Phaeobotryon aplosporum CFCC 53775 Phaeobotryon aplosporum CFCC 58596 S3/520 BarranOe Phaeobotryon aplosporum CFCC58784 61/75/- | Phaeobotryon mali XJAU 2782 62/71/0.9N Phaeobotryon mali XJAU 2930 Phaeobotryon mali XJAU 2772 Phaeobotryon mali XJAU 3100 ena Phaeobotryon mali XJAU 3094 ., Phaeobotryon rhois CFCC 89662 ae Phaeobotryon rhois CFCC 89663 100/100/1) Phaeobotryon fraxini CFCC 70762 Phaeobotryon fraxini CFCC 70763 Phaeobotryon ulmi CBS 123.30 Phaeobotryon ulmi CMH299 Phaeobotryon ulmi PB_11f 66/65/|' Phaeobotryon ulmi CBS 138854 99/99/1 Phaeobotryon ulmi CBS 174.63 Phaeobotryon ulmi CBS 114123 Phaeobotryon ulmi 94-13 Alanphillipsia aloeicola CBS 138896 Outgroup 66/62/0.96 0.02 Figure 4. Phylogram generated from RAxML analysis based on ITS, LSU, and tef1-a sequence data of Phaeobotryon iso- lates. The tree was rooted in Alanphillipsia aloeicola (CBS 138896). The MP, ML (= 50%), and BI (= 0.9) bootstrap supports are given near the nodes, respectively. Isolates from this study are marked in blue, and ex-type strains are marked in bold. MycoKeys 106: 225-250 (2024), DOI: 10.3897/mycokeys.106.122890 997 Yingying Wu et al.: New species and records of Botryosphaeriales Taxonomy Aplosporella ginkgonis C.M. Tian, Z. Du & K.D. Hyde, Mycosphere 8(2): 1249 (2017) Description. See Du et al. 2017. Material examined. CHINA, Beijing City, Tongzhou District, Majugiao Wet- land Park, 39°46'12"N, 116°37'12"E, on the disease branches of Cotinus coggygria var. cinereus, 2 May 2023, Y.Y. Wu, BJUFC-S1931, living culture CFCC 70746. Notes. Aplosporella ginkgonis was first reported in Gansu Province, China, causing canker and dieback disease in Ginkgo biloba and Morus alba (Du et al. 2017). Zhu et al. (2018) and Li et al. (2023) discovered the species on Rhus typhina and Zanthoxylum bungeanum, respectively, extending its host range. In the present study, one isolate (CFCC 70746) was identified as A. ginkgonis based on the phylogenetically highly supported clade with 99% MP, 95% ML, and 0.94 BYPP values (Fig. 2) and morphological characteristics. This is the first report of A. ginkgonis on Cotinus coggygria var. cinereus. Aplosporella javeedii Jami, Gryzenh., Slippers & M.J. Wingf., Fungal Biology 118(2): 174 (2013) Description. See Fan et al. 2015. Material examined. CHINA, Beijing City, Tongzhou District, Hougezhuang Plain Forest, 29°50'24"N, 116°54'00"E, on the dead branches of Styphnolo- bium japonicum, 8 April 2023, C.M. Tian, S.J. Li & Y.Y. Wu, BUFC-S1932, liv- ing culture CFCC 70733; ibid. on the dead branches of Forsythia suspensa, BJFC-S1933, living culture CFCC 70734; ibid. on the dead branches of For- sythia suspensa, BJFC-S1934, living culture CFCC 70735; ibid. on the dead branches of Ulmus pumila, BJFC-S1935, living culture CFCC 70736; CHINA, Beijing City, Tongzhou District, Central Green Forest Park, 39°52'16'N, 116°42'04"E, from branches of Acer truncatum, 12 April 2023, C.M. Tian, Y.M. Liang, C. Peng, Y. Hu & Y.Y. Wu, BUFC-S1936, living culture CFCC 70737; CHINA, Beijing City, Tongzhou District, Central Green Forest Park, 39°52'16'N, 116°42'04"E, on the dead branches of Sambucus williamsii, 19 April 2023, C.M. Tian, C. Peng, R. Yuan, M.W. Zhang & Y.Y. Wu, BUFC-S1937, living culture CFCC 70739; ibid. on the dead branches of Acer miyabei, BJFC-S1938, liv- ing culture CFCC 70740; ibid. on the dead branches of Lagerstroemia indica, BJFC-S19339, living culture CFCC 70741; ibid. on the dead branches of Xantho- ceras sorbifolium, BJFC-S1940, living culture CFCC 70742; China, Beijing City, Tongzhou District, Majugiao Wetland Park, 39°46'12"N, 116°37'12"E, from branches of Syringa vulgaris, 2 May 2023, Y.Y. Wu, BUFC-S1941, living culture CFCC 70744, ibid. on the dead branches of Ulmus pumila, BJFC-S1942, living culture CFCC 70745. Notes. Ap/osporella javeedii was initially reported on Celtis africana and Sear- sia lancea in South Africa (Jami et al. 2014). Fan et al. (2015a) recorded this spe- cies in China for the first time, associating it with the canker or dieback disease MycoKeys 106: 225-250 (2024), DOI: 10.3897/mycokeys.106.122890 038 Yingying Wu et al.: New species and records of Botryosphaeriales of five hosts: Albizia julibrissin, Broussonetia papyrifera, Gleditsia sinensis, Ju- niperus chinensis, and Styphnolobium japonicum. Aplosporella javeedii is wide- spread on host plants of more than 10 families (Fan et al. 2015a; Zhu et al. 2018; Pan et al. 2019; Lin et al. 2023a). In this study, we report new host records for this species, including Acer miyabei, Acer truncatum, Forsythia suspensa, Lagerstroemia indica, Sambucus williamsii, Syringa vulgaris, Ulmus pumila, and Xanthoceras sorbifolium. Aplosporella yangingensis L. Lin & X.L. Fan, MycoKeys 97: 9 (2023) Description. See Lin et al. 2023a. Material examined. CHINA, Beijing City, Tongzhou District, Central Green Forest Park, 39°52'16"N, 116°42'04"E, on the dead branches of Acer trun- catum, 12 April 2023, C.M. Tian, Y.M. Liang, C. Peng, Y. Hu & Y.Y. Wu, BJFC-S1943, living culture CFCC 70743; ibid. BUFC-S1944, living culture CFCC 70738. Notes. Aplosporella yangingensis was first discovered on the branches of Platycladus orientalis in Beijing (Lin et al. 2023a). In this study, the two iso- lates (CFCC 70738 and CFCC 70743) from Acer truncatum formed a clade with 100% MP, 100% ML, and 1.00 BYPP values in the multi-locus phyloge- netic tree with A. yangingensis (Fig. 2). Compared with the description of Lin et al. (2023a), this study has shorter conidia and thinner conidiogenous cells (11.0-16.5 x 6.0-9.0 um vs. 16.0-21.5 x 6.0-9.5 um and 5.0-20.5 x 1.0- 2.0 um vs. 6.0-13.5 x 2.0-3.0 um). Thus, these isolates were identified as A. yangingensis, and herewith we are providing a new host record for A. yan- gingensis, Acer truncatum. Dothiorella acericola Phookamsak, Tennakoon & K.D. Hyde, Fungal Diversity 95: 78 (2019) Description. See Pan et al. 2021. Material examined. CHINA, Beijing City, Tongzhou District, Hougezhuang Plain Forest, 29°50'24"N, 116°54'00"E, on the dead branches of Forsythia sus- pensa, 8 April 2023, C.M. Tian, S.J. Li & Y.Y. Wu, BUFC-S1948, living culture CFCC 70755; CHINA, Beijing City, Tongzhou District, Majugiao Wetland Park, 39°46'12"N, 116°37'12"E, on the dead branches of Ginkgo biloba, 2 May 2023, Y.Y. Wu, BUFC-S1949, living culture CFCC 70760; ibid. on the dead branches of Syringa oblata, BJFC-S1950, living culture CFCC 70761. Notes. Based on phylogenetic analyses (Fig. 3), three isolates in this study clustered with Dothiorella acericola and formed a clade with 99% MP, 100% ML, and 1.00 BYPP values. Dothiorella acericola is reported to be associated with the canker disease of Acer palmatum in China (Phookamsak et al. 2019). Pan et al. (2021, 2023) found that Do. acericola infests Ziziphus jujuba and Koelreu- teria paniculata branches. The fungus was also recorded on dead branches of Euonymus japonicus (Lin et al. 2023b). This is the first discovery of this fungus in the host families Oleaceae and Ginkgoaceae. MycoKeys 106: 225-250 (2024), DOI: 10.3897/mycokeys.106.122890 939 Yingying Wu et al.: New species and records of Botryosphaeriales Dothiorella hortiarborum Y.Y. Wu & C.M. Tian, sp. nov. MycoBank No: 851826 FIG=) Etymology. “Hort” means “garden,” and “arbor” means “tree” in Latin. Collected from Fraxinus chinensis and Lagerstroemia indica, both of which are landscap- ing and greening trees. Holotype. CHINA, Beijing City, Tongzhou District, Central Green Forest Park, 39°52'16"N, 116°42'04"E, on the dead branches of Fraxinus chinensis, 19 April 2023, C.M. Tian, C. Peng, R. Yuan, M.W. Zhang & Y.Y. Wu (holotype BUFC-S1951, ex-type cultures CFCC 70756). Description. Sexual morph: Not observed. Asexual morph: Conidiomata pycnidial, scattered to aggregated, immersed to semi-immersed in bark, glo- bose to subglobose, dark gray to black, unilocular, 260-450 um diam. Disc black, ovoid, 310-330 um diam. Ostioles single, light gray, circular, central, papillate, 30-45 um diam. Locules single, black, oval, 100-380 um, Conidio- phores reduced to conidiogenous cells. Conidiogenous cells: hyaline, smooth, thin-walled, holoblastic, cylindrical to subcylindrical, 4.5-11.0 x 2.0-4.0 um (av. + S.D.= 6.8 + 1.3 x 2.9 + 0.5 um). Conidia initially hyaline, then producing light yellow pigmentation, uneven surface, thick-walled, dark brown when matrues, 1-septate, constricted at the septum, smooth, ovoid with a broadly rounded apex, truncate base. 10.0-19.0 x 6.0-11.0 um (av. + S.D.= 14.9 + 2.6 x 8.1 + 1.0 um). Figure 5. Dothiorella hortiarborum (BJFC-S1951) A, B habit of conidiomata on branch C transverse section of conidioma D longitudinal section through conidioma E, F conidiogenous cells and conidia G top (left) and bottom (right) sides of colonies on potato dextrose agar (PDA) H, I conidia. Scale bars: 1000 ym (A); 200 um (B-D); 10 um (E-F, H-1). MycoKeys 106: 225-250 (2024), DOI: 10.3897/mycokeys.106.122890 240 Yingying Wu et al.: New species and records of Botryosphaeriales Culture characters. Colonies on PDA with aerial mycelium gray-green, thick and dense, fluffly, margin with undulate and irregular, reverse with inky blue pig- ment accumulation, reaching 60 mm diam in 7 days at 25 °C. Other material examined. CHINA, Beijing City, Tongzhou District, Central Green Forest Park, 39°52'16'N, 116°42'04"E, on the dead branches of Fraxinus chinensis, 19 April 2023, C.M. Tian, C. Peng, R. Yuan, M.W. Zhang & Y.Y. Wu, BUFC-S2366, liv- ing culture CFCC 70757; CHINA, Beijing City, Tongzhou District, Central Green For- est Park, 39°52'16'"N, 116°42'04'E, on the dead branches of Lagerstroemia indica, 19 April 2023, C.M. Tian, C. Peng, R. Yuan, M.W. Zhang & Y.Y. Wu, BUFC-S1952, living culture CFCC 70758; ibid. BJFC-S236/7, living culture CFCC 70759. Notes. Dothiorella hortiarborum formed an independent clade with 87% MP 97% ML, and 0.99 BYPP values and is distinct from Do. acericola and Do. pluriv- ora in the multi-locus analyses (Fig. 3). Morphologically, Do. hortiarborum can be distinguished from Do. acericola by shorter conidia (Phookamsak et al. 2019) and Do. plurivora by smaller conidia (10.0-19.0 x 6.0-11.0 um vs. 22.3-22.7 x 10.8- 11.2 um) (Abdollahzadeh et al. 2014). Additionally, Do. hortiarborum differs from Do. acericola in tef1-a (five bp difference from 170 characters, with 97.1% simi- larity, including no gaps) sequences, and Do. plurivora in tef1-a (one bp difference from 254 characters, with 99.6% similarity, including one gap), tub2 (three bp dif- ference from 370 characters, with 99.2% similarity, including one gap) sequences. Phaeobotryon fraxini Y.Y. Wu & C.M. Tian, sp. nov. MycoBank No: 851827 Fig. 6 Etymology. Named after the host, Fraxinus chinensis. Holotype. CHINA, Beijing City, Tongzhou District, Central Green Forest Park, 39°52'16"N, 116°42'04"E, on the dead branches of Fraxinus chinensis, 19 April 2023, C.M. Tian, C. Peng, R. Yuan, M.W. Zhang & Y.Y. Wu (holotype BUFC-S1953, ex-type cultures CFCC 70762). Description. Sexual morph: Not observed. Asexual morph: Conidiomata pycnid- ial, scattered, occasionally aggregated, superficial or immersed, globose, dark brown to black, unilocular, 200-360 um diam. Disc inconspicuous. Ostioles single, brown or black, circular, central, papillate, 40-85 um diam. Locules single, globose, 100-170 um, Conidiophores reduced to conidiogenous cells. Conidiogenous cells hyaline, smooth, thin-walled, holoblastic, cylindrical, formed from the cells lining the inner walls of the locules, 7.0-14.0 x 1.0-5.0 um (av. + S.D.= 10.6 + 2.0 x 3.1 + 0.8 um). Conidia initially hyaline, smooth, thin-walled, then gradually producing light yellow pigment, becoming yellow or light brown, occasionally with bubbles, mature with 1-septate, brownish yellow to dark brown, oblong, obtuse, rounded at both ends, 13.0-20.0 x 7.0-10.0 um (av. + S.D.= 17.6 + 1.3 x 8.7 + 0.7 um). Culture characters. Colonies on PDA with aerial gray-white mycelium, thick and dark black at the edge, thin and paler in color in the center, fluffly, entire margin, re- verse with black pigment accumulation, reaching 60 mm diam in 7 days at 25 °C. Other material examined. CHINA, Beijing City, Tongzhou District, Central Green Forest Park, 39°52'16"N, 116°42'04"E, on the dead branches of Fraxinus chinensis, 19 April 2023, C.M. Tian, C. Peng, R. Yuan, M.W. Zhang & Y.Y. Wu, BJFC-S2368, living culture CFCC 70763. MycoKeys 106: 225-250 (2024), DOI: 10.3897/mycokeys.106.122890 oA Yingying Wu et al.: New species and records of Botryosphaeriales C longitudinal section through conidioma D, E conidiogenous cells and conidia F top (left) and bottom (right) sides of colonies on potato dextrose agar (PDA) G-L conidia. Scale bars: 500 um (A); 200 um (B, C); 10 um (D, E, G-L). Notes. Based on multi-locus phylogenetic analysis, the two isolates cluster separately in a high-supported clade with 100% MP, 100% ML, and 1.00 BYPP value (Fig. 4). In the phylogenetic analysis, Phaeobotryon fraxini showed a close relationship to P mali and P. rhois. These three species could be distinguished based on ITS, tef1-a, and LSU loci from P. mali by nineteen bp (6/465 in ITS; 10/184 in tef1-a; 3/559 in LSU) and P. rhois by twenty-two bp (7/465 in ITS; 12/184 in tef1-a; 3/559 in LSU). Moreover, P. fraxini differs from P mali and P. rhois in having smaller conidia (13.0-20.0 x 7.0-10.0 um vs. 22.0-31.5 x 12-16.5 um for P mali and 20-25 x 10-12 um for P rhois) (Fan et al. 2015b; Jia et al. 2023) (Table 3). Therefore, P. fraxini is introduced as a novel species. Table 3. Comparison of species in Phaeobotryon. Species Phaeobotryon aplosporum P mali P cupressi P. fraxini P juniperi P mamane P. negundinis P platycladi P rhoinum P rhois P spiraeae P ulmi MycoKeys 106: 225-250 (2024), DOI: 10.3897/mycokeys.106.122890 Host Rhus typhina Malus pumila Cupressus sempervirens Fraxinus chinensis Juniperus formosana Sophora chrysophylla Acer negundo Platycladus orientalis Rhus typhina Rhus typhina Spiraea salicifolia Ulmus laevis Location China China Iran China China USA Russia China China China China Germany Conidial size 17-19 x 5.5-7 22.0-31U5 <4 2516.5 24.1525 *12.2-12:5 13-20 x 7-10 245 = 27.5% 412.0—13:5 35-38 x 14-15 16-24.5 x 7.9-11.5 23.0-31.0 x 9.5-12.5 19-21 x 7.5-9 20-25 x 10-12 2315-28 585-135 28.5-32.5 x 16.5-18.5 Septation aseptate 1-septate 1(-2)-septate 1-septate 1-septate 1(-2)-septate aseptate aseptate or 1-septate 1-septate 1-septate aseptate aseptate or 1-septate Reference Pan et al. 2019 Jia et al. 2023 Abdollahzadeh et al. 2009 This study Peng et al. 2023 Phillips et al. 2008 Daranagama et al. 2016 Lin et al. 2023a Zhu et al. 2018 Fan et al. 2015b Jin and Karunarathna 2021 Zhang et al. 2021 242 Yingying Wu et al.: New species and records of Botryosphaeriales Discussion In this paper, 23 Botryosphaeriales isolates were identified as six species based on multi-locus phylogenetic analyses. These species included two new species, namely Dothiorella hortiarborum and Phaeobotryon fraxini, and four new hosts: Aplosporella ginkgonis on Cotinus coggygria var. cinereus; A. javee- dii on Acer miyabei; Acer truncatum; Forsythia suspensa; Lagerstroemia indica; Sambucus williamsii, Syringa vulgaris; Ulmus pumila; Xanthoceras sorbifolium; A. yangingensis on Acer truncatum; and Do. acericola on Forsythia suspensa; Ginkgo biloba; and Syringa oblata. The six fungal species identified in this study involve a total of 13 different hosts, which elucidates the wide range of hosts of Botryospaeriales. Aplosporella is the type genus of Aplosporellaceae (Slippers et al. 2013). The distinctive morphological feature of Aplosporella species is that both as- cospores and conidia are aseptately hyaline to pigmented (Slippers et al. 2013; Phillips et al. 2019). In this study, a total of three new host record species of the genus were identified, including A. ginkgonis, A. javeedii, and A. yangingensis. Aplosporella javeedii has the highest isolation rate and the widest host range, involving five orders of host plants, including Dipsacales, Fabales, Lamiales, Myrtales, and Rosales. Currently, this species is mainly found in warm temper- ate and tropical regions (Fan et al. 2015a; Zhu et al. 2018), and further explo- ration is needed to determine whether the geographic range of A. javeedii is related to climate. Dothiorella was considered a synonym of Diplodia based on a broad mor- phological concept (Crous and Palm 1999). Phillips et al. (2005) compared the morphological characteristics again and found that the conidia of Dothiorella were brown, with 1-septate in early development, and the conidia still adhered to the conidiogenous cells. In contrast, the conidia of Diplodia become black and septate after being excreted from the conidiomata. Crous et al. (2006) con- firmed these morphological differences. Therefore, Dothiorella is regarded as an independent genus in the Botryosphaeriaceae. In this study, the conidia of Do. hortiarborum are transparent and aseptate when attached to conidioge- nous cells. After being released by the conidiomata, the conidia bear yellowish pigment or become brown with a 1-septate. In recent years, many new species of Dothiorella have been published with conidial morphology similar to Do. hor- tiarborum (Li et al. 2023; Lin et al. 2023a; Wu et al. 2023). These suggest that the morphological characteristics of Dothiorella are not always stable. Thus, it is not accurate to rely solely on the morphology of conidia for Dothiorella; combining phylogenetic analysis and the size of conidia of neighboring spe- cies is necessary. Dothiorella species have been reported on more than 20 host plants in China (https://fungi.ars.usda.gov/). This study has expanded its host range in Oleaceae plants (Do. acericola in Forsythia suspensa, Ginkgo biloba and S. oblata, and Do. hortiarborum in Fraxinus chinensis). Currently, many Dothiorella species have been recorded from Fraxinus, distrib- uted mainly in regions such as Europe and North America (Table 4). In this study, a new species, Do. hortiarborum, from F. chinensis, was introduced in China. Howev- er, based on morphological and DNA sequence data, Do. hortiarborum shows sig- nificant differences from other species in Fraxinus. Phylogenetic analysis showed that Do. hortiarborum belongs to a different lineage from Do. omnivora, Do. sp., MycoKeys 106: 225-250 (2024), DOI: 10.3897/mycokeys.106.122890 943 Yingying Wu et al.: New species and records of Botryosphaeriales Table 4. Comparison of species from Fraxinus in Dothiorella. Specise Host Location Conidial size Septation Reference Dothiorella concaviuscula Fraxinus viridis USA 4-6 x 2.5-3 no description Jepson 1896 Do. fraxini Fraxinus sp. Belgium 26-30 x 12 1-septate Saccardo 1892 Do. fraxinicola Fraxinus sp. USA 18-30 x 6-7 no description Ellis and Everhart 1895 Do. hortiarborum Fraxinus chinensis China 10.0-19.0 x 6.0-11.0 1-septate This study Do. omnivora Fraxinus excelsior Bosnia 19.3-25.5 x 7.5-10.6 1-septate Linaldeddu et al. 2016 Do. sp. Fraxinus excelsior Bosnia, Herzegovina 11-14x 6-8 2-4-septate Zlatkovié et al. 2016 Do. vidmadera Fraxinus ornus Australia 21.2-21.9 x 9.6-9.8 1-septate Pitt et al. 2013 and Do. vidmadera (Fig. 3), while distinguishing them based on the size of conidia and the number of septates (Table 4). Do. concaviuscula, Do. fraxini, and Do. frax- inicola were not available for sequence information due to their earlier publication; however, Do. hortiarborum can also be easily distinguished from them based on their documented conidia size. In addition, Do. lagerstroemiae and Do. hortiarbo- rum were both isolated from Lagersiroemia alba, but its conidia were significantly smaller than Do. hortiarborum (8.3-10 x 3.5—4 um vs. 10.0-19.0 x 6.0-11.0 um). Phaeobotryon species have more overlapping morphological characters, with 1(—2) septate or aseptate conidia and similar pigmentation variations. For example, P cupressi and P juniperi have overlapping sizes of conidia (24.1- 25 x 12.2-12.5 um vs. 24.5-27.5 x 12.0-13.5 pm), P rhoinum and P rhois are derived from the same host and geographic origin, and the conidia have 1-sep- tate (Table 3). So, morphology combined with phylogenetics to further clarify the affinities between species is essential. Furthermore, Phaeobotryon species were reported on a variety of hosts and considered to be potential or oppor- tunistic pathogens (Weiland et al. 2016; Zhu et al. 2020; Ilyukhin and Ellouze 2023; Jia et al. 2023). In this study, P fraxini was isolated only from dead Frax- inus chinensis; more extensive specimen collection was needed to confirm its distribution characteristics and pathogenicity. Although Botryosphaeriales recorded many fungi on Index Fungorum (https://www.indexfungorum.org/), only some species are now recognized. Mainly due to the early records of many species, the lack of model specimens, or the low quality of specimens, it is difficult to obtain strains and DNA data. Therefore, more detailed sampling is needed to revise the classification system of related taxa in Botryosphaeriales. Additional information Conflict of interest The authors have declared that no competing interests exist. Ethical statement No ethical statement was reported. Funding This study is financed by National Natural Science Foundation of China (Project No.: 32371887), Survey of Insect and Pathogen Diversity in Beijing Municipal Administra- tive Center. MycoKeys 106: 225-250 (2024), DOI: 10.3897/mycokeys.106.122890 oA Yingying Wu et al.: New species and records of Botryosphaeriales Author contributions Conceptualization, Yingying Wu and Chengming Tian; data curation, Yingying Wu; funding acquisition, Chengming Tian; investigation, Yingying Wu, Cheng Peng, Rong Yuan, Mingwei Zhang, Yang Hu; project administration, Chengming Tian; resources, Yingying Wu, Cheng Peng, Rong Yuan, Mingwei Zhang, Yang Hu; supervision, Chengming Tian; writing-original draft, Yingying Wu; writing-review and editing, Yingying Wu, Cheng Peng, and Chengming Tian. All authors have read and agreed to the published version of the manuscript. 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Antonie van Leeuwenhoek 109(4): 543-564. https://doi.org/10.1007/s10482-016-0659-8 Supplementary material 1 Aplosporella Authors: Yingying Wu, Cheng Peng, Rong Yuan, Mingwei Zhang, Yang Hu, Chengming Tian Data type: pdf Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) 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.106.122890.suppl1 Supplementary material 2 Dothiorella Authors: Yingying Wu, Cheng Peng, Rong Yuan, Mingwei Zhang, Yang Hu, Chengming Tian Data type: pdf Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) 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.106.122890.suppl2 Supplementary material 3 Phaeobotryon Authors: Yingying Wu, Cheng Peng, Rong Yuan, Mingwei Zhang, Yang Hu, Chengming Tian Data type: pdf Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) 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.106.122890.suppl3 MycoKeys 106: 225-250 (2024), DOI: 10.3897/mycokeys.106.122890 250