A Morpho-Molecular Re-Appraisal of Polystigma Fulvum and P. Rubrum (Polystigma, Polystigmataceae)

Phytotaxa 422 (3): 209–224 ISSN 1179-3155 (print edition) https://www.mapress.com/j/pt/ PHYTOTAXA Copyright © 2019 Magnolia Press Article ISSN 1179-3163 (online edition) https://doi.org/10.11646/phytotaxa.422.3.1

A morpho-molecular re-appraisal of Polystigma fulvum and P. rubrum (Polystigma, Polystigmataceae)

DIGVIJAYINI BUNDHUN1,2, RAJESH JEEWON3, MONIKA C. DAYARATHNE1,4,5, TIMUR S. BULGAKOV6, ALEXANDER K. KHRAMTSOV7, JANITH V.S. ALUTHMUHANDIRAM8, DHANDEVI PEM1, CHAIWAT TO- ANUN2 & KEVIN D. HYDE1,4,5* 1 Center of Excellence in Fungal Research, Mae Fah Luang University, Chiang Rai 57100, Thailand 2 Division of Plant Pathology, Department of Entomology and Plant Pathology, Faculty of Agriculture, Chiang Mai University, Chiang Mai 50200, Thailand 3 Department of Health Sciences, Faculty of Science, University of Mauritius, Reduit, Mauritius 4 World Agroforestry Centre East and Central Asia Office, 132 Lanhei Road, Kunming 650201, China 5 Key Laboratory for Plant Biodiversity and Biogeography of East Asia (KLPB), Kunming Institute of Botany, Chinese Academy of Sci- ence, Kunming 650201, Yunnan, China 6 Russian Research Institute of Floriculture and Subtropical Crops, 2/28 Yana Fabritsiusa Street, Sochi 354002, Krasnodar region, Rus- sia 7 Department of Botany, Belarusian State University, 4 Nezavisimosti Av., Minsk 220030, Belarus 8 Beijing Key Laboratory of Environment Friendly Management on Fruit Diseases and Pests in North China, Institute of Plant and Envi- ronment Protection, Beijing Academy of Agriculture and Forestry Sciences, Beijing, China * Corresponding author: KEVIN D. HYDE, [email protected]

Abstract

Collections of eleven Prunus specimens infected with Polystigma species from Belarus and Russia yielded two existing taxa: Polystigma fulvum (sexual morph) and Polystigma rubrum (asexual morph). DNA based phylogenies of large subunit nuclear rDNA (LSU) and nuclear ribosomal internal transcribed spacer (ITS) are provided for the first time for Polystigma fulvum and its placement is confirmed within Polystigmataceae. The concatenated LSU and ITS DNA sequence data for P. rubrum, analyzed to infer its potential relationship to other Polystigma species, also show that strains of P. rubrum are segregated into two subclades with sufficient genetic differences. No significant differences in morphology or morphometry among the strains of P. rubrum between the two subclades, especially vis-à-vis the conidiogenous cell and conidium sizes are observed (P>0.05). Subspecies concept of P. rubrum complex is discussed.

Key words: biotrophs, molecular phylogeny, Rosaceae, taxonomy, Xylariomycetidae

Introduction

The family Polystigmataceae Höhn. comprises obligate fungal biotrophic species which cause leaf spots almost exclusively on living leaves of the economically important Prunus species (Rosaceae) across the Euro-Asiatic regions (Cannon 1996, Suzuki et al. 2008, Habibi et al. 2015, 2017, Roberts et al. 2018). However, Polystigmataceae has been considered a synonym of Phyllachoraceae Theiss. & P. Syd. and all members of Phyllachoraceae were accommodated in Polystigmataceae (Dennis 1968, Müller & von Arx 1973). Hawksworth et al. (1983) later raised Polystigmataceae to ordinal level, Polystigmatales O.E. Erikss., with a single family Phyllachoraceae. Consequently, all species of Polystigma DC. were transferred to Phyllachoraceae. An additional reason for placing Polystigma species within Phyllachoraceae was based on their morphological resemblances to members of the Phyllachora Nitschke ex Fuckel. These included thin-walled clavate asci and hyaline aseptate ascospores. However, Polystigma species equally have brightly colored stromata and mostly undergo sympodial proliferation compared to the black stromata and percurrent conidiogenesis of Phyllachora species (Cannon 1996). A multi-gene phylogeny (ITS, SSU and LSU) of Polystigma amygdalinum P.F. Cannon and P. rubrum (Pers.) DC., with related taxa, indicated that Polystigma could not be accommodated in Phyllachoraceae (order Phyllachorales, subclass Sordariomycetidae), since the genus is related to Xylariales (subclass Xylariomycetidae), from which it

Accepted by Sinang Hongsanan: 8 Oct. 2019; published: 30 Oct. 2019 209 evolved almost 90 million years ago in the late Cretaceous (Habibi et al. 2015, Habibi & Banihashemi 2017). This has been accepted by Dayarathne et al. (2017) who re-instated Polystigmataceae, with Polystigma as the sole genus within Xylariales. Morphological similarities linking Polystigma to Phyllachora, as mentioned above, might have been the result of convergent evolution (Habibi et al. 2015). The deciduous nature of Prunus leaves mainly determines the reproductive state of Polystigma species (Cannon 1996). Generally, conidiomata develop in stromata within the host tissue during the summer, followed by gradual ascomatal formation in the autumn (Cannon 1996). The ascomata mature during winter after leaf fall and mature ascospores are released in the spring to infect young leaves (Cannon 1996, Suzuki et al. 2008, Habibi & Banihashemi 2017). Their ascospores do not possess any germ pore or slit and they produce appressoria upon germinating (Mehrotra & Aneja 1990). Almond red leaf blotch and plum red leaf spots have been reported to be caused by P. amygdalinum and P. rubrum respectively, with their hosts being consequently prematurely defoliated (Ashkan & Asadi 1974, Banihashemi 1990, Saad & Masannat 1997, Suzuki et al. 2008). Polystigma rubrum was also reported to seriously attack the plum cultivars ‘Green gage’ and ‘Cacanska najbolja’ in the Institute of Agriculture in Kyustendil, Bulgaria during the period 1997–1999 (Borovinova 2001). Polystigma is typified by P. rubrum (Lamarck & de Candolle 1815). The latter is widely distributed around temperate areas, with more collections reported from the United Kingdom (Cannon 1996). The geographical distribution of P. rubrum in Britain, however, was recently observed to become more restricted to western, coastal regions as compared to northern Europe (Roberts et al. 2018). Interestingly, P. rubrum was more frequent in leaves already infected by the gall mite Eriophyes prunispinosae. The hypothesis was that leaf galls damaged the leaf surface or modified the plant developmental process and favored infection by P. rubrum (Roberts et al. 2018). Most taxonomic work on Polystigma have been based primarily on morphology (Cannon 1996, Suzuki et al. 2008). To date, only two species, P. rubrum and P. amygdalinum, have DNA sequence data (Habibi et al. 2015, Dayarathne et al. 2017). In this study, we provide DNA sequence data for the first time for P. fulvum Pers. ex DC., an important pathogenic species. In addition, phylogenetic relationships of Polystigma species are investigated and their taxonomic affinities, especially with respect to their subspecies concepts are discussed with additional strains from P. rubrum.

Materials & Methods

Sample collection and morphological studies Samples of Prunus leaves bearing disease symptoms were collected from various sites of Rostov region of Russia (FIGURE 1) and from Sychevichi Village, Belarus between 8th of October and 21st of November 2017 and brought to the laboratory in paper bags. Specimens were examined using a Motic dissecting microscope (SMZ 168). Free-hand sections of fruiting bodies were prepared to examine the shapes of ascomata and conidiomata along with the fruiting bodies’ wall structure. Squash mounts were also prepared in water and stained with Melzer’s reagent for micro- morphology. Fungal characters were determined with a Nikon Eclipse E600 microscope and digital images captured with a Nikon DS-U2 and Cannon 750D camera. Lengths and widths were measured using the Tarosoft (R) Image Frame Work software. Images used for photo plates were processed with Adobe Photoshop CS6 v. 12.0 software (Adobe Systems, USA). Single spore isolation was carried out as described in Chomnunti et al. (2014) initially using malt extract agar (MEA). As germination was not observed, potato dextrose agar (PDA) and water agar (WA) were used to stimulate growth both at room temperature and 18 °C (Vijaykrishna et al. 2004, Liu et al. 2010). However, all attempts were unsuccessful. Herbarium materials were deposited in the Mae Fah Luang University (MFLU) Herbarium, Chiang Rai, Thailand as well as the Herbarium of Cryptogams Kunming Institute of Botany Academia Sinica (KUN-HKAS). Faces of fungi number for P. fulvum was registered according to Jayasiri et al. (2015). Species concepts are discussed following Jeewon & Hyde (2016) and the Genealogical concordance phylogenetic species recognition (GCPSR) model by Taylor et al. (2000).

DNA extraction, PCR amplification and sequencing Genomic DNA was extracted directly from fruiting bodies using a DNA extraction kit (D3591- 01, Omega Bio-Tek) following the manufacturer’s protocol. DNA amplification was performed by polymerase chain reaction (PCR). DNA sequence data were obtained from the partial sequences of two loci: the large subunit nuclear rDNA (28S, LSU) and

210 • Phytotaxa 422 (3) © 2019 Magnolia Press BUNDHUN ET AL. the internal transcribed spacer (ITS) region (ITS1-5.8S nrDNA-ITS2). The LSU was amplified with LR0R (Rehner & Samuels 1994) and LR5 (Vilgalys & Hester 1990) while the ITS region was amplified using primer pair ITS5 and ITS4 (White et al. 1990). Amplifications were performed in 25 μl of PCR reaction mixtures composed of 16.2 μl ddH2O, 0.3 μl of TaKaRa Ex-Taq DNA polymerase, 2.5 μl of 10x Ex-Taq buffer, 3.0 μl of dNTPS, 1 μl of genomic DNA and 1 μl of each primer. DNA sequence data for β-tubulin and RNA polymerase II subunit (RPB2) were attempted but without success. The PCR thermal cycle program involved an initial denaturation step of 3 min at 95 °C, followed by 34 cycles of denaturation for 30 sec at 95 °C and 30 sec of annealing along with 1 min elongation at 72 °C, with a final extension of 10 min at 72 °C. The annealing temperatures used in the thermal cycler program were 52 °C for LSU and 58 °C for ITS. Sequencing of the PCR fragments was carried out with the primers mentioned above at the Beijing Biomed Gene Technology co. LTD, China. Acquired nucleotide sequence data from this study have been deposited in GenBank (TABLE 1).

FIGURE 1. Polystigma rubrum on different Prunus hosts. a Prunus cerasifera b Prunus domestica c Prunus stepposa. Scale bars: a–c = 1 cm.

TABLE 1. Strains and related GenBank accession numbers of taxa included in this study. Sequences generated in this study are in brown bold and the type strains are in bold. Taxon Voucher/Culture GenBank Accessions LSU ITS Arecophila bambusae HKUCC 4794 AF452038 - Acremonium sclerotigenum CBS 124.42 NG_057139 LC144892 Amphirosellinia fushanensis HAST 91111209 - GU339496 Amphirosellinia nigrospora HAST 91092308 - GU322457 Annulohypoxylon annulatum CBS 140775 KY610418 KY610418 Annulohypoxylon truncatum CBS 140778 KY610419 KY610419 Astrocystis concavispora MFLUCC 14-0174 KP340545 KP297404 Atrotorquata spartii MFLUCC 13-0444 NG_057064 - Barrmaelia rhamnicola CBS 142772 MF488990 NR_153497 Biscogniauxia arima WSP 122 - EF026150 Brunneiperidium gracilentum MFLUCC: 14-0011 KP340542 KP297400 Cainia anthoxanthis MFLUCC 15-0539 KR092777 NR_138407 Calceomyces lacunosus CBS 633.88 KY610476 KY610397 Camillea obularia ATCC 28093 KY610429 KY610384 Camillea tinctor HAST 363 - JX507806 Collodiscula bambusae GZU H0102 KP054280 KP054279 Collodiscula fangjingshanensis GZU H0109 KR002591 KR002590 Coniocessia nodulisporioides CBS 281.77 AJ875224 MH861061 Creosphaeria sassafras STMA 14087 KY610468 KY610411 Daldinia andina CBS 114736 KY610430 AM749918 Daldinia bambusicola CBS 122872 KY610431 KY610385 Diatrype disciformis CBS 197.49 DQ470964 - Discoxylaria myrmecophila JDR 169 - GU322433 Entoleuca mammata JDR 100 - GU300072 Entonaema liquescens ATCC 46302 KY610443 KY610389 Euepixylon sphaeriostomum JDR 261 - GU292821 Eutypa lata CBS 208.87 DQ836903 DQ006927 ...continued on the next page

Polystigma fulvum and P. rubrum Phytotaxa 422 (3) © 2019 Magnolia Press • 211 TABLE 1. (Continued) Taxon Voucher/Culture GenBank Accessions LSU ITS Hypomontagnella monticulosa MUCL 54604 KY610487 KY610404 Hypomontagnella submonticulosa CBS 115280 KY610457 KC968923 Hyponectria buxi UME 31430 AY083834 - Hypoxylon fragiforme MUCL 51264 KM186295 KC477229 Hypoxylon griseobrunneum CBS 331.73 KY610483 KY610402 Jackrogersella cohaerens CBS 119126 KY610497 KY610396 Jackrogersella multiformis CBS 119016 KY610473 KC477234 Kretzschmaria deusta CBS 163.93 KY610458 KC477237 Lopadostoma americanum CBS 133211 KC774568 NR_132027 Melogramma campylosporum MBU* JF440978 JF440978 Nemania abortiva BISH 467 - GU292816 Nemania maritima STMA 04019 KY610414 KY610414 Obolarina dryophila MUCL 49882 GQ428316 GQ428316 Oxydothis metroxylonis MFLUCC 15-0283 KY206764 KY206775 Oxydothis palmicola MFLUCC 15-0806 KY206765 KY206776 Podosordaria mexicana WSP 176 - GU324762 Podosordaria muli WSP 167 - GU324761 Polystigma amygdalinum EA-1* - KC756360 Polystigma amygdalinum MA2* - KC756364 Polystigma amygdalinum M4* - KC756362 Polystigma fulvum MFLU 18-0261 MK429727 MK429738 Polystigma rubrum MFLU 15-3091 MF981079 KY594023 Polystigma rubrum K(M)197843 - MG768911 Polystigma rubrum MFLU 18-0262 MK429728 MK429739 Polystigma rubrum MFLU 18-0263 MK429729 MK429740 Polystigma rubrum MFLU 18-0264 MK429730 MK429741 Polystigma rubrum MFLU 18-0265 MK429731 MK429742 Polystigma rubrum MFLU 18-0266 MK429732 MK429743 Polystigma rubrum MFLU 18-0267 MK429733 MK429744 Polystigma rubrum MFLU 18-0268 MK429734 MK429745 Polystigma rubrum MFLU 18-0269 MK429735 MK429746 Polystigma rubrum MFLU 18-0270 MK429736 MK429747 Polystigma rubrum MFLU 18-0271 MK429737 MK429748 Polystigma sp. Rub1* - KC966927 Poronia punctata CBS 656.78 KY610496 KT281904 Pseudomassaria sepincoliformis CBS 129022 JF440984 JF440984 Pyrenopolyporus hunteri MUCL 52673 KY610472 - Pyrenopolyporus laminosus MUCL 53305 KY610485 KC968934 Pyriformiascoma trilobatum MFLUCC: 14-0012 KP340543 KP297402 Rhopalostroma angolense CBS 126414 KY610459 KY610420 Rosellinia aquila MUCL 51703 KY610460 KY610392 Rosellinia corticium MUCL 51693 KY610461 KY610393 Rostrohypoxylon terebratum CBS 119137 DQ840069 DQ631943 Ruwenzoria pseudoannulata MUCL 51394 KY610494 KY610406 Sarcoxylon compunctum CBS 359.61 KY610462 KT281903 Sarocladium oryzae CBS 180.74 HG965047 HG965026 Stilbohypoxylon elaeicola JDR 173 - EF026148 Stilbohypoxylon quisquiliarum JDR 172 - EF026119 Thamnomyces dendroidea CBS 123578 KY610467 FN428831 Trichoderma amazonicum IB50 JN939814 HM142358 Uncultured fungus clone 126_NA11_P33_P16* - KC966204 Vialaea minutella BRIP 56959 KC181924 KC181926 Xylaria hypoxylon CBS 122620 KY610495 KY610407 Xylaria polymorpha MUCL 49884 KY610464 KY610408

212 • Phytotaxa 422 (3) © 2019 Magnolia Press BUNDHUN ET AL. Abbreviations and acronyms: ABS—Aberystwyth University Herbarium, Wales, UK; ATCC—American Type culture collection; BISH—Herbarium Pacificum, Bishop Museum, Honolulu, Hawaii, U.S.A; BRIP—Queensland Plant Pathology Herbarium Collection; CBS—Culture collection of the Centraalbureau voor Schimmelcultures, Fungal Biodiversity Centre, Utrecht, The Netherlands; DSM— Herbarium of University of Dar es Salaam, Tanzania; HAST—Herbarium, Biodiversity Research Center, Academia Sinica, Taipei; HKUCC—Hong Kong University Culture Collection; IB—Iberia, Peru; JDR—Personal herbarium and strain collection of J.D.R., Pullman, WA; K(M)—Mycological Herbarium, Royal Botanical Gardens Kew; MA—Real Jardín Botánico herbarium, Madrid, Spain; MFLU—Herbarium of Mae Fah Luang University, Chiang Rai, Thailand; MFLUCC—Mae Fah Luang University Culture Collection, Chiang Rai, Thailand; MUCL—Culture Collection Mycothèque de Université Catholique de Louvain; STMA—Herbarium/ culture collection of M.S., Wupperta; UME—Herbarium of Umeå University, Sweden; WSP—Herbarium of Washington State University, Pullman, WA; *Strain designation from GenBank.

Sequence alignment and phylogenetic analyses Sequences generated from the different primers of the LSU gene and ITS region used in this study were analyzed with other sequences obtained from GenBank along with recently published relevant phylogenies (Habibi et al. 2015, Daranagama et al. 2016, 2018, Dayarathne et al. 2017, Li et al. 2017, Wendt et al. 2018 ). Sequence data were aligned by MAFFT v. 7 with the web server (https://mafft.cbrc.jp/alignment/server/). They were then edited and improved manually when necessary using BioEdit v. 7.0.5.2 (Hall 1999). Phylogenetic analyses of the aligned data were based on Maximum Likelihood (ML) and Bayesian analyses (BI) with details as outlined by Tang et al. (2007, 2009). The evolutionary models for analyses were selected independently for each locus using MrModeltest v. 2.3 (Nylander 2004) under the Akaike Information Criterion (AIC). The GTR + I + G model was set as best-fit model for both LSU and ITS and included in the analyses. RAxML-HPC2 on XSEDE (v. 8.2.8) (Stamatakis et al. 2008, Stamatakis 2014) in CIPRES Science Gateway platform (Miller et al. 2010) was used to generate the ML trees using GTR + I + G model of evolution and bootstrap support by running 1000 pseudo replicates. Bayesian analysis was executed in MrBayes v. 3. 1. 2 (Huelsenbeck & Ronqvist 2001) through Markov Chain Monte Carlo (MCMC) sampling to calculate the Posterior probabilities (PP) (Rannala & Yang 1996, Zhaxybayeva & Gogarten 2002). Six Markov chains were run in parallel for 17M generations with trees being sampled every 100th generation. The distribution of log-likelihood scores was examined to determine the stationary phase for each search and to decide whether additional runs were required to reach convergence, using the program Tracer 1.5 (Rambaut & Drummond 2007). First 20% of generated trees were discarded and the remaining 80% were used to calculate PP of the majority rule consensus tree. The resulting trees were viewed in FigTree v. 1.4.0 (Rambaut 2012) and annotated in Microsoft PowerPoint (2013).

Genealogical concordance phylogenetic species recognition (GCPSR) analysis Strains of P. rubrum were analyzed using the GCPSR model. A pairwise homoplasy index (PHI) (Philippe & Bryant 2006) test was conducted in SplitsTree4 (Huson 1998, Huson & Bryant 2006) as described by Quaedvlieg et al. (2014) with aim of determining the recombination level within the phylogenetically closely related taxa using a two-locus combined dataset. A significant recombination is present in the dataset if the pairwise homoplasy index is below a 0.05 threshold (Фw < 0.05). The relationships among the taxa of P. rubrum were visualized by constructing a split graph, using both the LogDet transformation and splits decomposition options (FIGURE 5).

Results

Phylogenetic analyses Phylogenetic analyses of combined LSU and ITS sequence data were conducted to examine the phylogenetic placement of the Polystigma strains within Xylariomycetidae (TABLE 1). The combined gene dataset comprised seventy-nine taxa from Xylariales, including eleven Polystigma strains collected and three from Hypocreales, namely, Acremonium sclerotigenum (CBS 124.42), Sarocladium oryzae (CBS 180.74) and Trichoderma amazonicum (IB50), as outgroup taxa. Two different alignments conforming to each individual gene (trees not shown) and a combined alignment of the two genes were analyzed in this study. After exclusion of ambiguous regions and introns, the combined dataset included 1575 positions (LSU: 1–817; ITS: 818–1575), including gaps. The phylogenetic trees from different datasets were similar in topology. The best scoring RAxML tree, based on the combined aligned data, is used as the representative tree and illustrated in FIGURE

Polystigma fulvum and P. rubrum Phytotaxa 422 (3) © 2019 Magnolia Press • 213 2. The bootstrap support values for ML, equal to or greater than 70% (black) and Bayesian PP equal to or greater than 0.95 (blue) are given above the nodes (FIGURE 2). RAxML analysis yielded the best scoring tree (FIGURE 2) with a final ML optimization likelihood value of -22565.475101. The matrix comprised 968 distinct alignment patterns, with 32.17% of undetermined characters or gaps. Estimated base frequencies were: A = 0.245456, C = 0.238904, G= 0.276039, T = 0.239602 and substitution rates: AC = 1.539585, AG = 2.577562, AT = 1.431987, CG = 1.060273, CT = 5.024115, GT = 1.000000; proportion of invariable sites I = 0.368030; gamma distribution shape parameter α = 0.589748. All Polystigma species grouped together (Clade A) within Xylariales with strong bootstrap support (100% ML/ 1.00 PP) close to Hyponectria buxi. Polystigma fulvum (MFLU 18-0261) was basal to all other Polystigma species with good statistical support (100% ML/1.00 PP). Strains of P. rubrum clustered with strains of P. amygdalinum and an uncultured fungus (clone 126_NA11_P33_P16) with 82% ML support. Strains of P. rubrum constituted one strongly supported clade (96% ML/ 1.00 PP) which was divided into two subclades, A1 and A2. Subclade A1 had bootstrap support of 82% ML and consisted of eight strains of P. rubrum collected in this study along with Polystigma sp. (Rub 1) and P. rubrum K(M)197843. The second well-supported subclade, A2 (97% ML/ 1.00 PP), included two P. rubrum strains (MFLU 18-0262 and MFLU 18-0263) from this study, grouping with P. rubrum MFLU 15-3091. A PHI test revealed no significant recombination event (Φw > 0.05) among the strains of P. rubrum (FIGURE 5). A three-gene phylogeny (SSU, LSU and ITS) was initially analyzed in the study and tree topology was similar to the two-gene phylogenetic tree with similar support at the different clades. However, a two-gene phylogeny has been used and illustrated (FIGURE 2), since only 15 out of 82 taxa (<20%) had SSU sequence data. The SSU sequence data for P. fulvum (MFLU 18-0261, MK429749), P. rubrum (MFLU 18-0263, MK429750; MFLU 18-0265, MK429751; MFLU 18-0269, MK429752 and MFLU 18-0271, MK429753) have been deposited in the GenBank. Some contentious taxa such as ‘Polystigma’ pusillum were excluded from our phylogenetic analyses since they were distantly related to the other Polystigma species. This is in agreement with Mardones et al. (2017) and Dayarathne et al. (2017) where these strains clustered in the Phyllachorales, but with uncertainty. The strain Polystigma rubrum ABS15-001 was also excluded from the phylogenetic analysis since it lacked ITS1 and its placement into one or the other clade would have been problematic.

Taxonomy

Polystigma fulvum Pers. ex DC., in de Candolle & Lamarck, Fl. franç., Edn 3 (Paris) 5/6: 164 (1815) FIGURE 3 Index Fungorum number: IF189406, Facesoffungi number: FoF 05686

Parasitic on Prunus padus L. Leaf spots developing on living leaves, scattered, irregular, swollen, composed of pigmented outer layers and a hyaline inner layer, bordered by apparently healthy leaf tissue. Sexual morph: Ascostromata irregularly-shaped, but roughly circular with uneven margin, ca. 10 mm in diameter, dark orange-brown on adaxial side and yellowish brown on abaxial surface, forming an inner hyaline layer within and throughout the leaf tissue with a high number of ascomata which appear like reddish orange spots 67–126 µm diam. on the strongly raised adaxial surface, ostioles nearly inconspicuous and somewhat sunken, though with slight protuberance on the flat or slightly concave abaxial surface. External layer of stroma 40–60 µm thick at the adaxial surface and thinner at the abaxial one. Ascomata 194–218 µm high, 230–246 µm diam (x̅ = 204.9 × 237.7 µm, n = 10), almost spherical with the walls differentiated from stromatal tissue. Ostiole 37–54 × 26–87 µm (x̅ = 43.5 × 54.5 µm, n = 8), almost inconspicuous, periphysate. Periphyses up to 18 µm long and 2–5 µm wide at the base, gradually becoming narrow at the apex. Peridium 16–31 µm thick, hyaline, adequately developed, composed of compressed cells, fusing with the stromatal tissue, both of which are filled with shiny yellow material. Paraphyses up to 78 µm long, rather sparse, gradually tapering towards the apex, very thin-walled, strongly inflated between the septa. Asci 50–110 (133) × 10–15 (18) µm (x̅ = 94.8 × 13 µm, n = 58), 8-spored, unitunicate, narrowly clavate with long stalk (to ca. 52 µm), thin-walled at every stage, the apex obtuse, 1–4 × 2–5 µm diam. (x̅ = 2.2 × 3.1 µm, n= 14) with a J-, refractive, apical annulus. Ascospores 8–15 × 3–8 µm (x̅ = 11.9 × 5 µm, n = 97), arranged biseriately or irregularly seriate, cylindrical, ellipsoidal to obovoid, hyaline, aseptate, regularly guttulate, comprising a faint gelatinous sheath, no appendage. Asexual morph: See illustrations in Cannon 1996 and Suzuki et al. 2008. Material examined:—BELARUS, Minsk region, Maladzyechna District, Sychevichi Village, 54°12′18.3″ N, 27°14′32.7″ E, near children’s health camp “Brigantina”, deciduous forest, on living and fallen leaves of Prunus padus (Rosaceae), 15 October 2017, Alexander K. Khramtsov, T-2083 (MFLU 18-0261, HKAS 104987).

214 • Phytotaxa 422 (3) © 2019 Magnolia Press BUNDHUN ET AL. FIGURE 2. Best scoring RaxML tree based on analyses of a combined LSU and ITS sequence dataset. Bootstrap support values for ML equal to or greater than 70% (black), Bayesian posterior probabilities (PP) equal to or greater than 0.95 (blue) are defined as ML/PP above and below the nodes. Species used for morphological observation in the present study are indicated in bold brown. The tree is rooted to Acremonium sclerotigenum (CBS 124.42), Sarocladium oryzae (CBS 180.74) and Trichoderma amazonicum (IB50). The scale bar represents the expected number of nucleotide substitutions per site.

Polystigma fulvum and P. rubrum Phytotaxa 422 (3) © 2019 Magnolia Press • 215 FIGURE 3. Polystigma fulvum (MFLU 18-0261). a Swollen, brown leaf spots on Prunus padus (Rosaceae). b Close up of leaf spots on host. c Appearance of immersed ascomata. d Section through ascoma. e Close-up of ostiole on the abaxial surface. f Peridium. g–i Asci. j Non-amyloid apical annulus. k–m Ascospores. n Sheath surrounding an ascospore. Scale bars: a = 1 cm, b = 200 µm, c, d= 100 µm, e, g–i = 50 µm, f = 20 µm, j–n = 10 µm.

Notes:—Polystigma fulvum is characterized by cylindric-ellipsoid to obovoid, guttulate ascospores surrounded by mucilaginous sheaths (Cannon 1996, Suzuki et al. 2008) as compared to the almost cylindric ascospores of P. amygdalinum which lack a sheath and are marginally constricted at the center. The ascospores of P. fulvum are also different from those of P. rubrum, which are seldom characterized as obovoid and lack sheaths (Cannon 1996). The ascospores of P. fulvum are distinct from those of P. deformans which are narrower at the base and are occasionally slightly constricted (Cannon 1996). Ascospores had sheaths in our study, but the width of the asci was 13 μm on average [versus 11.3 μm in Suzuki et al. (2008)]. The variations in asci size [50–110 (133) × 10–15 (18) µm in this study, versus 95–148 × 10–12(14) μm in Suzuki et al. (2008) and 98–128 × 14.5–16(18.5) µm in Cannon (1996)] may be related to the maturity of the ascomata as well as to the time of the year the diseased leaves were collected (Cannon 1996).

216 • Phytotaxa 422 (3) © 2019 Magnolia Press BUNDHUN ET AL. Polystigma rubrum (Pers.) DC., in de Candolle & Lamarck, Fl. franç., Edn 3 (Paris) 5/6: 164 (1815) FIGURE 4 ≡ Xyloma rubrum Pers., Observ. mycol. (Lipsiae) 2: 101 (1800) Index Fungorum number: IF182352; Facesoffungi number: FoF 02910

FIGURE 4. Polystigma rubrum (MFLU 18-0263). a Swollen, reddish brown leaf spots on Prunus cerasifera Ehrh.. b, c Close ups of leaf spots on host. d Vertical section through pycnidial conidioma. e Conidioma wall. f, g Conidiogenous cells and developing conidia. h–j Conidia. Scale bars: a = 1 cm, b = 5 mm, c = 100 µm, d, f = 50 µm, e, h–j = 10 µm, g = 30 µm.

Parasitic on Prunus stepposa Kotov, Pr. domestica L., Pr. cerasifera Ehrh. Leaf spots developing on living leaves, dispersed, uneven, swollen, pigmented, bordered by healthy leaf tissue. Asexual morph: Conidial stromata 5–13 mm diam., irregularly-shaped, roughly orbicular, orange to reddish-brown, becoming darker in the central region, developing within and throughout the leaf tissue, contain numerous conidiomata. Stromatal tissue comprising upper and lower plant tissue layers 35–40 μm thick whose cells are filled with shiny orange material, and an intermediate layer 180–226 μm thick composed almost completely of occluded angular to vertically elongated fungal cells. Conidiomata 84–236 µm high, 84–209 µm diam (x̅ = 103 × 203.9 µm, n = 7), roughly globose. Ostiole epigenous, barely conspicuous. Conidiomatal wall inadequately developed, not properly discernable from the stromatal tissue. Conidiogenous cells 6–29 × 0.5–3 µm (x̅ = 13.1 × 2.2 µm, n = 42) spanning the inner surface of the wall, produced

Polystigma fulvum and P. rubrum Phytotaxa 422 (3) © 2019 Magnolia Press • 217 horizontally from successive cells of short conidiophores derived from a thin layer of hyaline thin-walled cells of textura angularis, initially cylindrical but progressively tapering towards apex, which appears faintly irregular owing to successive conidial scars resulting from sympodial proliferation. Conidia 11–45 × 0.2–1.5 µm (x̅ = 20.1 × 1.0 μm, n = 34), the lower part lanceolate to fusiform, the upper part filiform, sigmoidally curved to hooked, the base ± truncate, hyaline, aseptate, apparently smooth-walled. Sexual morph: See illustrations in Cannon 1996. Material examined:—RUSSIA, Rostov region, Shakhty City, Cotton fabric microdistrict, 47°72′18.0″ N, 40°25′27.5″ E, wild trees near Grushevka River, on living leaves of Prunus cerasifera, 9 October 2017, Timur S. Bulgakov, T-2085 (MFLU 18-0263, HKAS 104978; FIGURE 4). Other materials examined:—RUSSIA, Rostov region, Rostov-on-Don City, 47°13′28.6″ N, 39°40′07″ E, street trees, on living leaves of Prunus domestica, 24 October 2017, Timur S. Bulgakov, T-2084 (MFLU 18-0262, HKAS 104980).—RUSSIA, Rostov region, Shakhty City, Cotton fabric microdistrict, 47°43′18.5″ N, 40°15′09.9″ E, torn shrubs near Grushevka River, on living leaves of Prunus stepposa, 9 October 2017, Timur S. Bulgakov, T-2086 (MFLU 18-0264, HKAS 104981).—RUSSIA, Rostov region, Shakhty City, Cotton fabric microdistrict, 47°43′18.5″ N, 40°15′09.9″ E, torn shrubs near Grushevka River, on living leaves of Prunus stepposa, 9 October 2017, Timur S. Bulgakov, T-2087 (MFLU 18-0265, HKAS 104983).—RUSSIA, Rostov region, Shakhty City, 47°43′18.5″ N, 40°15′09.9″ E, street trees, on living leaves of Prunus domestica, 9 October 2017, Timur S. Bulgakov, T-2088 (MFLU 18-0266, HKAS 104977).—RUSSIA, Rostov region, Shakhty City, 47°42′49.5″ N, 40°11′54.2″ E, street trees, on living and fallen leaves of Prunus domestica, 20 November 2017, Timur S. Bulgakov, T-2089 (MFLU 18-0267, HKAS 104984).—RUSSIA, Rostov region, Rostov-on-Don City, 47°13′22″ N, 39°40′09.9″ E, street trees, on living leaves of Prunus domestica, 24 October 2017, Timur S. Bulgakov, T-2090 (MFLU 18-0268, HKAS 104982).— RUSSIA, Rostov region, Shakhty City, 47°71′56.3″ N, 40°20′01.8″ E, street trees, on living and fallen leaves of Prunus domestica, 1 November 2017, Timur S. Bulgakov, T-2091 (MFLU 18-0269, HKAS 104986).—RUSSIA, Rostov region, Shakhty City, Alexandrovsky (Central) park, 47°42′20.1″ N, 40°12′11″ E, on living and fallen leaves of Prunus domestica, 5 November 2017, Timur S. Bulgakov, T-2092 (MFLU 18-0270, HKAS 104979).—RUSSIA, Rostov region, Shakhty City, Cotton fabric microdistrict, 47°43′18.5″ N, 40°15′09.9″ E, torn shrubs near Grushevka River, on living leaves of Prunus stepposa, 9 October 2017, Timur S. Bulgakov, T-2093 (MFLU 18-0271, HKAS 104985) (Morphometry of all strains are given in TABLE 2).

TABLE 2. Morphometry of Polystigma rubrum strains from this study. Strain height × diameter of conidiomata Length × width of Length × width of conidia (µm) (µm) conidiogenous cells (µm) (n = 34) (na = 7) (n = 42) MFLU 18-0262 153–207 × 118–143 10–25 × 0.5–2.2 18–45 × 0.3–1.2 (Subclade A2) (x̅b =178.8 × 132.9) (x̅ = 16.7 × 1.3) (x̅ = 32.9 × 0.67)

MFLU 18-0263 119–185 × 84–130 9–28 × 1–3 17–38 × 0.4–1.1 (Subclade A2) (x̅ = 147.7 × 104.3) (x̅ = 16.3 × 1.8) (x̅= 25.9 × 0.7)

MFLU 18-0264 164–236 × 178–203 8–26 × 1–2.7 16–40 × 0.2–1. (Subclade A1) (x̅ = 195.2 × 192.3) (x̅ = 15.6 × 1.7) (x̅ = 26.9 × 0.6)

MFLU 18-0265 156–202 × 102–171 6–21 × 0.9–2.6 15–36 × 0.3–1.1 (Subclade A1) (x̅ = 179.4 × 129.6) (x̅ =13.1 × 1.6) (x̅ = 27.8 × 0.5)

MFLU 18-0266 179–193 × 162–168 10–28 × 1.1–2.9 14–35 × 0.2–1.3 (Subclade A1) (x̅ = 187.3 × 164) (x̅ = 15.2 × 1.6) (x̅ = 24.8 × 0.8)

MFLU 18-0267 169–176 × 200–209 9–23 × 0.8–2.2 16–37 × 0.2–1.2 (Subclade A1) (x̅ = 172.8 × 203.9) (x̅ = 15.2 × 1.6) (x̅ = 24.8 × 0.8)

MFLU 18-0268 181–185 × 148–160 8–28 × 1–2.8 21–39 × 0.2–1.3 (Subclade A1) (x̅ = 183.7 × 152.1) (x̅ = 16.9 × 2) (x̅ = 32.2 × 0.8)

MFLU 18-0269 84–121 × 112–122 12–20 × 1.2–3 11–28 × 0.7–1.4 (Subclade A1) (x̅ = 103 × 117.9) (x̅ = 15.3 × 2) (x̅ = 20.1 × 1)

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218 • Phytotaxa 422 (3) © 2019 Magnolia Press BUNDHUN ET AL. TABLE 2. (Continued) Strain height × diameter of conidiomata Length × width of Length × width of conidia (µm) (µm) conidiogenous cells (µm) (n = 34) (na = 7) (n = 42) MFLU 18-0270 170–178 × 161–169 15–29 × 1.3–3 14–37 × 0.2–1.5 (Subclade A1) (x̅ = 174.3 × 164.2) (x̅ = 22.5 × 2.2) (x̅ = 24.3 × 0.7)

MFLU 18-0271 145–147 × 149–166 12–26 × 1.3–2.8 15–34 × 0.2–1.2 (Subclade A1) (x̅ = 145.8 × 154.5) (x̅ =17.3 × 2.1) (x̅ = 22 × 0.6)

na = number x̅b = mean

Notes:—Typification of Polystigma rubrum was once considered problematic since the only two available collections of the species, initially identified as Xyloma rubrum by Persoon, lacked sufficient collection data for its typification (Cannon 1996). The first collection was in poor condition and was speculated to contain ascomata initials of P. rubrum. The second consisted of mature stromata on diseased leaves of Prunus padus, which two successive annotations re-identified as P. fulvum. Ultimately, none of them could be referred to as either lectotype or neotype of P. rubrum (Cannon 1996). This problem was, however, recently solved when an asexual morph of P. rubrum collected from Russia was designated as a reference specimen based on strong morpho-phylogenetic support (Dayarathne et al. 2017). Stromata of P. rubrum usually produce conidia in summer and autumn on living leaves followed by initiation of sexual structure during winter and release of ascospores the subsequent spring from fallen overwintered leaves (Cannon 1996). In this study, specimen voucher MFLU 18-0267 was collected around mid of November. However, the initiation of sexual structures in the fallen leaves of the specimen was not observed. Polystigma rubrum is generally also known to have two subspecies, namely, P. rubrum subsp. rubrum and P. rubrum subsp. ussuriense (Cannon 1996). The former has a wider distribution over the Euro-Asiatic countries whereas the latter has been mentioned to originate mainly from Eastern Russia (Cannon 1996). Polystigma rubrum subsp. rubrum is distinguished from P. rubrum subsp. ussuriense by its almost black ascostromata with inconspicuous ostioles and 22–42 µm long conidia occurring mainly on Pr. domestica and Pr. spinosa (Cannon 1996).

Discussion

In this study, among the eleven diseased Prunus specimens collected, ten were infected with Polystigma rubrum and one with P. fulvum. Details, as outlined in previous publications (Cannon 1996, Suzuki et al. 2008, Habibi et al. 2015, Dayarathne et al. 2017), were compared and DNA sequence data were also analyzed to re-evaluate the taxonomy of Polystigma species. Addition of more Polystigma strains has resulted in a further resolved phylogeny from those illustrated by Dayarathne et al. (2017) and Roberts et al. (2018). Interestingly, P. rubrum strains clustered in two subclades (A1 and A2) as illustrated in FIGURE 2. Morphological observations, however, led to no significant differences between the two subclades be it the shape of the conidiomata, ostiole, conidiomatal wall, conidiogenous cells, conidia or type of conidiogenesis [P. rubrum K(M)197843 and ABS15-001 and Polystigma sp. Rub1 were not compared here due to lack of morphological details (Habibi et al. 2015, Roberts et al. 2018)]. No significant differences (P> 0.05) were equally observed in the morphometry of the P. rubrum strains between subclades A1 and A2 vis-à-vis the conidiomata, conidiogenous cell and conidium sizes (TABLES 2 & 3). Variation in conidium length between the subspecies P. rubrum subsp. rubrum and P. rubrum subsp. ussuriense in northern Europe and eastern Asia could occur due to environmental influence but this hypothesis was never tested owing to a lack of collections from central Russian areas (Cannon 1996). Our collections come from Southern Russian areas and their grouping into two separate subclades indicates that conidium size may not be adequately suitable for delineating between subspecies (TABLES 2 & 3). One could argue that the color of the stromata could be important in taxonomy. All P. rubrum strains in subclade A1 [except Polystigma sp. Rub1 for which no phenotypic detail has been provided and K(M)197843 which had orange-red stromata (Roberts et al. 2018)] had dark reddish brown to almost black stromata while the strains [including P. rubrum MFLU 15-3091 (Dayarathne et al. 2017)] in subclade A2 had reddish brown stromata with darker central regions. Polystigma rubrum ABS15-001 possessed orange-red stromata (Roberts et al. 2018). The difference in stromata color,

Polystigma fulvum and P. rubrum Phytotaxa 422 (3) © 2019 Magnolia Press • 219 however, as per our observations in Rostov region (Russia) is mostly accounted for by the age and the maturity of the stromata and host plants. The stromata are usually bright orange during the middle of summer but they become darker starting late summer to late autumn. The color further changes from orange to bright red, then to dark red, reddish brown and even dark brown to almost black, especially, after leaf falling. The visible stromata color also depends on the host plant, such that stromata on the leaves of Pr. domestica are often darker than the ones on the leaves of Pr. cerasifera and Pr. stepposa (FIGURE 1). Base pair comparison of the ITS regions revealed >2.5% nucleotide difference between groups A1 and A2. This is above the threshold of 1.5% recommended by Jeewon & Hyde (2016). Even the insignificant recombination event (Фw > 0.05) indicates a difference between the strains in subclade A1 and subclade A2 (FIGURE 5), which could infer that these can be considered as different species. However, to avoid any taxonomic confusion, we maintain them as the same species. We reckon that future studies may even establish new species within P. rubrum complex based on multi-gene phylogeny derived from protein coding genes, as has been reported in the case of Colletotrichum (Weir et al. 2012, Hyde et al. 2014, Jayawardena et al. 2016a, b, Ma et al. 2018, Damm et al. 2019).

TABLE 3. Relationship between subclades A1 and A2 vis-à-vis conidiomata, conidiogenous cell and conidium sizes. Features (µm) Subclades = Mean ± SD P-value* (ANOVA) Conidiomata height Subclade A1 = 167.7 ± 29.9 0.852 Subclade A2 = 163.3 ± 22.0 Conidiomata diameter Subclade A1 = 159.8 ± 28.8 0.098 Subclade A2 = 118.6 ± 20.2 Conidiogenous cell length Subclade A1 = 16.4 ± 2.8 0.958 Subclade A2 = 16.5 ± 2.4 Conidiogenous cell width Subclade A1 = 1.9 ± 0.3 0.191 Subclade A2 = 1.6 ± 0.4 Conidium length Subclade A1 = 25.4 ± 3.7 0.224 Subclade A2 = 29.4 ± 4.9 Conidium width Subclade A1 = 0.7 ± 0.2 0.741 Subclade A2 = 0.7 ± 0.02 *P-value is considered significant if P<0.05

FIGURE 5. Results of the pairwise homoplasy index (PHI) test of Polystigma rubrum strains using both LogDet transformation and splits decomposition. PHI test results (Φw) < 0.05 indicate significant recombination within the dataset.

220 • Phytotaxa 422 (3) © 2019 Magnolia Press BUNDHUN ET AL. Polystigma fulvum was basal to all other Polystigma species and the phylogeny supports it as an independent lineage (FIGURE 2). Therefore, it warrants species status. The latter herein is characterized by dark orange-brown stromata with sunken ostioles protuberating faintly on the abaxial surface and guttulate ascospores with a gelatinous sheath. This tallies with the original morphological description provided by Cannon (1996) and Suzuki et al. (2008). In the present study, the uncultured fungus (clone 126_NA11_P33_P16) was sister to P. amygdalinum with 94% bootstrap support (FIGURE 2). This uncultured fungus was recovered from soil in Alaska (Timling et al. 2014) and presumed to represent P. fulvum since Alaskan riverbanks are significantly surrounded by Pr. padus (Roberts et al. 2018). Comparison of the ITS region of the uncultured fungus with the ITS region of the P. fulvum examined in this study revealed more than 25 base pair differences out of 478 (5.2%) base pairs. Careful considerations should, therefore, always be taken before names are given to such kind of unidentified species collected from environmental samples and being deposited in GenBank (Jeewon et al. 2017, 2018, Hongsanan et al. 2018). In addition, the authenticity of the DNA sequences deposited in GenBank also needs to be properly checked to avoid erroneous taxonomic assumptions (Nilsson et al. 2006). The grouping of the uncultured fungus (clone 126_NA11_P33_P16) with the Polystigma species also indicates that it belongs to the genus Polystigma and that the diversity of Polystigma species from Prunus hosts could be higher and other Polystigma species can potentially infect leaves of Pr. padus in addition to P. fulvum.

Acknowledgements

The authors are thankful for the financial support from the Thailand Research grants entitled the future of specialist fungi in a changing climate: baseline data for generalist and specialist fungi associated with ants, Rhododendron species and Dracaena species (grant no: DBG6080013) and Impact of climate change on fungal diversity and biogeography in the Greater Mekong Subregion (grant no: RDG6130001) for supporting this study. Dr R. Jeewon is grateful to the University of Mauritius and Mae Fah Luang University for enabling research collaboration. Dr Indunil C. Senanayake, Dr Ruvishika Jayawardena, Dr Dhanushka N. Wanasinghe and Binu Samarakoon are acknowledged for their valuable suggestions and support.

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