fungal biology 121 (2017) 322e346

journal homepage: www.elsevier.com/locate/funbio

Families, genera, and species of

Tao YANGa, Johannes Z. GROENEWALDa, Ratchadawan CHEEWANGKOONb,**, Fahimeh JAMIc, Jafar ABDOLLAHZADEHd, Lorenzo LOMBARDa, Pedro W. CROUSa,b,c,e,* aCBS-KNAW Fungal Biodiversity Centre, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands bDepartment of Entomology and , Faculty of Agriculture, Chiang Mai University, Chiang Mai 50200, Thailand cDepartment of Microbiology and Plant Pathology, Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria 0002, South Africa dDepartment of Plant Protection, Agriculture Faculty, University of Kurdistan, P.O. Box 416, Sanandaj, Iran eMicrobiology, Department of Biology, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands article info abstract

Article history: Members of Botryosphaeriales are ecologically diverse, but most commonly associated with Received 26 July 2016 leaf spots, fruit and root rots, die-back or cankers of diverse woody hosts. Based on mor- Received in revised form phology and DNA sequence data, the Botryosphaeriales have to date been shown to contain 8 November 2016 eight families, with an additional two, Endomelanconiopsisaceae (Endomelanconiopsis) and Accepted 14 November 2016 Pseudofusicoccumaceae (Pseudofusicoccum) being newly described in this study. Furthermore, Available online 21 November 2016 Oblongocollomyces is introduced as new , while Spencermartinsia is reduced to synon- Corresponding Editor: ymy under . Novel species include pyri (Pyrus sp., the Netherlands), Dip- Kerry O’Donnell lodia citricarpa (Citrus sp., Iran), (Vitis vinifera, Italy), L. sterculiae (Sterculia oblonga, Germany), pistaciarum (Pistacia vera, USA), N. buxi (Buxus sempervirens, Keywords: France), N. stellenboschiana (Vitis vinifera, South Africa), and hawaiiensis (Protea Canker pathogens laurifolia, Hawaii). New combinations are also proposed for pistaciae (associ- Multi-locus sequence typing (MLST) ated with fruit rot of Pistacia vera)inNeofusicoccum, and Sphaeria gallae (associated with galls Systematics of Quercus)inDiplodia. The combination of large subunit of the nuclear ribosomal RNA gene (LSU)-rpb2 proved effective at delineating taxa at family and generic level. Furthermore, rpb2 also added additional resolution for species delimitation, in combination with ITS, tef1 and tub2. In this study we analysed 499 isolates, and produce an expanded phyloge- netic backbone for Botryosphaeriales, which will help to delimit novelties at species, genus and family level in future. ª 2016 British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Introduction a diverse ecology, and can be saprobic, endophytic, or plant pathogenic (Slippers & Wingfield 2007; Jami et al. 2014; Species of Botryosphaeriales are distributed worldwide, and oc- Slippers et al. 2014; Trakunyingcharoen et al. 2015; Crous cur on a wide range of different host plants. They have et al. 2016), causing a range of disease symptoms including

* Corresponding author. CBS-KNAW Fungal Biodiversity Centre, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands. ** Corresponding author. E-mail addresses: [email protected] (R. Cheewangkoon), [email protected] (P.W. Crous). http://dx.doi.org/10.1016/j.funbio.2016.11.001 1878-6146/ª 2016 British Mycological Society. Published by Elsevier Ltd. All rights reserved. Families, genera, and species of Botryosphaeriales 323

leaf spots, fruit and root rots, die-back or cankers (Phillips et al. to resolve their in light of current molecular- 2013; Sarr et al. 2014; Trakunyingcharoen et al. 2014). In addi- based knowledge in Botryosphaeriales. Furthermore, the cur- tion, some species are even known to cause opportunistic in- rent taxonomy of Botryosphaeriales is revisited by employing fections in humans (de Hoog et al. 2000). new genes with appropriate evolutionary rates, and finding The genus is based on B. dothidea, which was morphological characters to support or refute the currently epitypified by Slippers et al. (2004), thereby fixing the generic accepted classification. application of the name. Schoch et al. (2006) subsequently in- troduced the order Botryosphaeriales (), which Materials and methods at the time contained a single family, .In a further study Minnis et al. (2012) resolved the status of Planis- Isolates tromellaceae (Kellermania), showing it to also belong to this or- der, which was also confirmed by Liu et al. (2012), who Isolates used in this study were obtained from CBS, Utrecht, delineated several additional unresolved lineages within The Netherlands, which included available ex-type strains of Botryosphaeriales. To this extent Wikee et al. (2013) resurrected described species (Supplementary Table S1). To induce sporu- the Phyllostictaceae (), while Slippers et al. (2013) in- lation, isolates were inoculated onto sterile pine needles and troduced the new families Aplosporellaceae ( and placed on 2 % water agar (PNA; Smith et al. 1996), potato dex- Bagnisiella), Melanopsaceae (Melanops), and Saccharataceae (Sac- trose agar (PDA), malt extract agar, oatmeal agar (OA; Crous charata), and Wyka & Broders (2016) introduced Septorioidea- et al. 2009b), and incubated at 25 C under near-ultraviolet light ceae (Septorioides). for 2e4 wks. Other than Botryosphaeria, several additional genera have over the years been recognised as belonging to Botryophaeria- ceae. Liu et al. (2012) examined numerous generic type speci- DNA extraction, amplification, and sequencing mens, and based on morphology accepted 29 genera in the family. However, based on the species known from culture Total genomic DNA was extracted from 7-d-old axenic cul- and supported by DNA data, Phillips et al. (2013) only recog- tures, grown on MEA at room temperature, using the Ultra- nised 17 genera and 110 species. The amendment of article Clean Microbial DNA isolation kit (Mo Bio Laboratories, 59 of the International Code of Nomenclature for algae, fungi California, USA) following the protocols provided by the man- and plants, and the One ¼ One Name initiative ufacturer. Partial gene sequences were determined for the (Wingfield et al. 2012; Crous et al. 2015) again saw several gen- large subunit of the nuclear ribosomal RNA gene (LSU), DNA- era being merged, most notably Botryosphaeria (¼), directed RNA polymerase II second largest subunit (rpb2), the Kellermania (Planistroma, Panistromella), Phyllosticta translation elongation factor 1-alpha (tef1), the internal tran- (¼), and Sphaeropsis ()(Crous scribed spacer 1 and 2 including the intervening 5.8S nrDNA b et al. 2014, 2015a; Wijayawardene et al. 2014; Rossman et al. gene (ITS), and the -tubulin gene (tub2) using the primers 2015). Furthermore, a revision of the Tiarosporella complex listed in Table 1. Amplification protocols followed Pavlic et al. saw the number of genera rise to 22 within Botryosphaeriaceae (2009), Phillips et al. (2013) and Slippers et al. (2013). Amplicons (Crous et al. 2015c). were sequenced in both directions using a BigDye Terminator The first single-gene phylogeny with extended sampling v. 3.1 Cycle Sequencing Kit (ThermoFisher Scientific) according led to the recognition of 10 botryosphaeria-like clades (Crous to the manufacturer’s instructions. A consensus sequence was et al. 2006), which were later recognised as a family based on manually generated for each set of forward and reverse se- multi-gene data, separating the Botryosphaeriaceae (as Botryos- quences in MEGA v. 6 (Tamura et al. 2013). Additional se- phaeriales) from the and (Schoch et al. quences were obtained from GenBank using megaBLAST e 2006). The discovery of new families in this order has since re- searches (accessions KX463943 KX465087; Supplementary lied on DNA phylogenetic data (Minnis et al. 2012; Slippers et al. Table S1). 2013; Wikee et al. 2013; Wyka & Broders 2016), supported by morphological characteristics. However, the taxonomic status Phylogenetic analyses of several lineages remained unresolved at family, generic or species level. Based on the blast searches, strains were binned into generic The CBS-KNAW Fungal Biodiversity Centre (CBS) culture groups and a subset was used to create an overview phylog- collection in Utrecht, The Netherlands, contains numerous eny. Sequences were aligned using MAFFT v. 7 (Katoh & Botryosphaeriaceae cultures, many of which were subject of Standley 2013) and refined manually after which the multiple past studies (e.g. Crous et al. 2006, 2015c; Phillips et al. 2013; alignments were concatenated following the combinations Slippers et al. 2013, Sarr et al. 2014) or were deposited by other listed for each set in Table 2. The datasets were subjected to authors in the culture collection as part of their own manu- Maximum Likelihood (ML), Maximum Parsimony (MP) and scripts (e.g. Begoude et al. 2010; Jami et al. 2013; Linaldeddu Bayesian Inference (BI) analyses. et al. 2015, to list but a few). However, more than 100 cultures The ML analysis was performed using RAxML v. 8 (rando- in the culture collection remain listed without a species epi- mised accelerated (sic) ML for high performance computing; thet; the largest number of these only being listed as Botryos- Stamatakis 2014) through the RAxML blackbox website phaeria sp. (http://embnet.vital-it.ch/raxml-bb/) to obtain an additional The main aim of the present study was to re-examine measure of branch support. The option for the gamma model strains of the unidentified Botryosphaeriales in the CBS, and of rate heterogeneity was selected and the proportion of 324 T. Yang et al.

Table 1 e Details of primer combinations used during this study for generic amplification and sequencing. Locus Primer name Sequence 50/30 Tm (C) Reference

ITS ITS5 GGA AGT AAA AGT CGT AAC AAG G 52 White et al. (1990) ITS4 TCC TCC GCT TAT TGA TAT GC 52 White et al. (1990) LSU LR0R ACC CGC TGA ACT TAA GC 52 Rehner & Samuels (1994) LR5 TCC TGA GGG AAA CTT CG 52 Vilgalys & Hester (1990) tef1 EF1-728F CAT CGA GAA GTT CGA GAA GG 54 Carbone & Kohn (1999) EF1-986R TAC TTG AAG GAA CCC TTA CC 54 Carbone & Kohn (1999) tub2 Bt-2a GGT AAC CAA ATC GGT GCT GCT TTC 52 Glass & Donaldson (1995) Bt-2b ACC CTC AGT GTA GTG ACC CTT GGC 52 Glass & Donaldson (1995) rpb2 RPB2-6F TGG GGK WTG GTY TGY CCT GC 60 / 58 / 54 Liu et al. (1999) fRPB2-7cR CCC ATR GCT TGY TTR CCC AT 60 / 58 / 54 Liu et al. (1999) RPB2bot6F GGT AGC GAC GTC ACT CCC 60 / 58 / 54 Sakalidis et al. (2011) RPB2bot7R GGA TGG ATC TCG CAA TGC G 60 / 58 / 54 Sakalidis et al. (2011)

Table 2 e Statistical information of the different analyses performed in this study. Analysis Substitution models used for Bayesian analysis/unique site patterns

LSU rpb2 ITS tef1 tub2

Overview GTRþIþG/161 GTRþIþG/404 ee e Diplodia eeHKYþG/89 HKYþG/118 HKYþG/130 Dothiorella eeGTRþIþG/102 GTRþG/207 GTRþIþG/125 Lasiodiplodia 3-gene eeGTRþI/45 HKYþG/88 GTRþI/62 Lasiodiplodia 2-gene eeHKYþG/50 HKYþIþG/107 e Neofusicoccum 4-gene e GTRþI/140, GTRþIþG/122 HKYþG/145 GTRþI/131 Neofusicoccum 2-gene eeGTRþIþG/113 HKYþG/164 e Phaeobotryon/ HKYþI/32 e HKYþI/32 HKYþG/124 e Saccharata eeSYMþG/64 HKYþIþG/65 GTRþG/65 Tiarosporella-like eeK80þI/55 HKYþI/199 GTRþIþG/155 Analysis Statistics for the parsimony analyses

Number of strains Number of Number of Number of Number of (incl. outgroup(s) included parsimony-informative parsimony-uninformative constant sites characters characters

Overview 100 1450 460 122 868 Diplodia 90 1275 264 101 910 Dothiorella 71 1285 397 50 838 Lasiodiplodia 3-gene 28 1170 104 129 937 Lasiodiplodia 2-gene 72 733 114 54 565 Neofusicoccum 4-gene 60 1885 322 240 1323 Neofusicoccum 2-gene 199 847 268 51 528 Phaeobotryon/Barriopsis 10 1417 140 162 1115 Saccharata 15 1614 97 453 1064 Tiarosporella-like 19 1032 366 93 573 Analysis Tree length Consistency index (CI) Retention index (RI) Rescaled CI (RC) Number of saved trees

Overview 2724 0.358 0.812 0.291 72 Diplodia 655 0.704 0.930 0.654 1000 Dothiorella 1202 0.606 0.923 0.560 1000 Lasiodiplodia 3-gene 385 0.706 0.735 0.520 2 Lasiodiplodia 2-gene 347 0.648 0.873 0.566 1000 Neofusicoccum 4-gene 1068 0.668 0.845 0.564 384 Neofusicoccum 2-gene 675 0.665 0.957 0.636 100 Phaeobotryon/Barriopsis 442 0.869 0.760 0.661 1 Saccharata 664 0.920 0.735 0.676 10 Tiarosporella-like 971 0.759 0.829 0.629 2 Families, genera, and species of Botryosphaeriales 325

Fig 1 e The first of 72 equally most parsimonious trees obtained from a heuristic search with 100 random taxon additions of the combined LSU and rpb2 sequence alignment of selected families in the Botryosphaeriales. The scale bar represents the number of changes and bootstrap support values of MP (MP-BS) and RAxML analysis (ML-BS) and Bayesian posterior prob- ability values (PP) are shown at the deeper nodes or nodes representing distinct genera (MP-BS/ML-BS/PP). Ex-type strains and are indicated in bold font and genera and families are indicated to the right of the tree in blocks of different colours. The three possible family boundaries for the Botryosphaeriaceae are indicated by numbered white circles. Thickened lines rep- resent those branches present in the parsimony strict consensus tree. Some branches were shortened to facilitate layout of the tree. The tree was rooted to Phaeosphaeria ammophilae (CBS 114595; Pleosporales, Phaeosphaeriaceae).

invariable sites was estimated by the software. The robust- The MP analyses were performed in PAUP (Phylogenetic ness of the analysis was evaluated by bootstrap support Analysis Using Parsimony, v. 4.0b10; Swofford 2003)with (MLBS) with the number of bootstrap replicates automatically phylogenetic relationships estimated by heuristic searches determined by the software. with 100 random taxon additions. The tree-bisection- 326 T. Yang et al.

Fig 1 e (continued).

reconnection option was used, with the branch swapping Prior to the Bayesian analyses, MrModeltest (Nylander 2004) option set to ‘best trees’ only. All characters were weighted was used to determine evolutionary model for each partition equally and alignment gaps were treated as fifth state. (Table 2). The Bayesian analyses were performed using MrBayes Measures calculated for parsimony included tree length v. 3.2.8 (Ronquist & Huelsenbeck 2003) with the Markov Chain (TL), consistency index (CI), retention index (RI) and Monte Carlo (MCMC) algorithm and two sets of four chains rescaled consistence index (RC) (Table 2). Bootstrap analy- started in parallel from a random tree topology were employed ses (MPBS, Hillis & Bull 1993) were based on 1000 with the heating parameter set at 0.3. The MCMC analysis ran replications. until the average standard deviation of split frequencies came Families, genera, and species of Botryosphaeriales 327

below 0.01, with trees saved every 100 or 1000 generations, Oblongocollomyces, and two novel families, Endomelanconiopsi- depending on the matrix size. The first 25 % of saved trees saceae (Endomelanconiopsis) and Pseudofusicoccumaceae (Pseudo- were discarded as the ‘burn-in’ phase and posterior probabilities fusicoccum), which are described below. The remaining (PP) determined from the remaining trees. analyses treated Diplodia (Fig 2), Dothiorella (including Spencer- All resulting trees were printed using Geneious v. 5.5.4 martinsia; Fig 3), Lasiodiplodia (Fig 4), Neofusicoccum (Fig 5), (Kearse et al. 2012); layout of trees was done using Adobe Illus- Phaeobotryon and Barriopsis (Fig 6), Saccharata (Fig 7) and trator CS5. Novel sequences derived in this study were depos- tiarosporella-like genera (Fig 8). The Diplodia phylogeny ited in GenBank and the alignments and trees in TreeBASE (Fig 2) revealed three taxonomic novelties which are treated (Study 20109; www.treebase.org/treebase/index.html). below, and a further four isolates most likely representing novel species but which are not described in the present pa- Evaluation of loci for species resolution per. Species of Spencermartinsia clustered inside Dothiorella (Fig 3) and therefore new combinations in Dothiorella are pro- The ability of loci to distinguish the species included in each vided for those species in Spencermartinsia. The Lasiodiplodia alignment was evaluated as described by Gomes et al. (2013) phylogeny (Fig 4) revealed two taxonomic novelties which and Alvarez et al. (2016). In short, neighbour joining (NJ) trees are treated below, and one additional isolate that most likely with the HKY85 substitution model were generated for each represents novel species but which will not be described in locus of the alignments used to generate Figs 1e8 and the present paper. The Neofusicoccum phylogeny (Fig 5) S1eS2. The trees were then manually evaluated for its ability revealed three taxonomic novelties which are treated below, to resolve species as distinct from one another. A species was and a further 10 isolates most likely representing novel spe- only counted if it was distinct from its closest relatives and if cies but which are not described in the present paper. The Sac- the species clade contained all strains of that species included charata phylogeny (Fig 7) revealed one taxonomic novelty in the analysis. Species names were applied to the trees based which is treated below, and a further two isolates most likely on the phylogenetic analyses performed in the present study, representing novel species but which are not described in the but also based on prior studies where in some cases more, or present paper. No novelties were found in the phylogenies of other, loci were employed for species recognition than the Phaeobotryon and Barriopsis (Fig 6) and tiarosporella-like genera ones used here. These are indicated by additional taxon (Fig 8). The potentially novel taxa that were not described in names after the accession number in Figs 1e8 and S1eS2. this study were sterile and we refrained from describing these as new based only on fixed nucleotide positions. We believe that species of Botryosphaeriaceae are common enough that Morphological characteristics fresh, sporulating cultures of these sterile cultures are bound to be recollected in the near future, allowing for them to be Fungal structures were mounted in clear lactic acid (85 % v/v) properly described and illustrated. In addition, the majority and studied under a Nikon SMZ25 stereo-microscope, and of these potentially novel taxa are represented by single line- with a Zeiss Axio Imager 2 light microscope using differential ages, and a larger sampling of these is required to confirm the interference contrast (DIC) illumination and a Nikon DS-Ri2 fixed nucleotide positions used for a description based on DNA camera and software. Thirty observations were made of data alone. each taxonomically informative structure where possible, and for spores the 95 % percentiles are presented, with ex- Evaluation of loci for species resolution tremes given between brackets. Colony characters and pig- ment production were noted after 1 wk of growth on PDA, The evaluation of the success of the individual loci to resolve MEA and OA incubated at 25 C in the dark. Colony colours species revealed that the most optimal locus is related to the (surface and reverse) were rated according to the colour chart genus in question, and also to some degree, the sampling of Rayner (1970). Reference specimens were deposited in the within the genus (Table 3). In the NJ overview phylogeny CBS fungarium, and nomenclature and descriptions of taxo- (data not shown), rpb2 was able to discriminate all genera in- nomic novelties in MycoBank (Crous et al. 2004). cluded in the phylogeny, whereas LSU only managed to achieve 80 % success. This was mainly due to species of the Results genera Diplodia, Lasiodiplodia, and Sphaeropsis not clustering into monophyletic lineages but sometimes intermixing or Phylogenetic analyses forming separate lineages. For several genera, such as Diplo- dia, Lasiodiplodia, Neofusicoccum (2-gene alignment), and Sac- In total, eight multigene phylogenies (Figs 1e8) were gener- charata, ITS and tef1 did not perform exceptionally well on ated, with an additional two unpublished phylogenies each their own to resolve the species phylogenies and species rec- containing a two-gene alignment of a larger set of isolates ognition requires the use of an additional locus such as tub2. for Lasiodiplodia and Neofusicoccum (data not shown, see Tree- Interestingly, in the 4-gene Neofusicoccum alignment, ITS and BASE). Statistics and models used for the different datasets tef1 did not perform so poorly (85 % versus 69 % for ITS and are shown in Table 2. The most parsimonious trees from 73 % versus 55 % for tef1, 4-gene versus 2-gene respectively). each alignment are presented here, with the support values In this case, the sample size appears to influence resolution; from the RAxML and Bayesian analyses plotted in addition in the 2-gene alignment a larger sampling per species was in- at the nodes. A phylogeny of all the families and genera are cluded, resulting in more interspecific diversity which de- provided in Fig 1 and revealed one novel genus, creases the intraspecific variation. However, in Lasiodiplodia 328 T. Yang et al. Families, genera, and species of Botryosphaeriales 329

this effect was the strongest in the ITS analysis where the Culture characteristics: Colonies covering dish in 7 d, with cot- smaller sampling of the 3-gene alignment allowed for 50 % tony aerial mycelium. Colonies surface on MEA glaucous species recognition versus 25 % in the 2-gene alignment, grey, reverse dark olivaceous; on PDA surface and reverse whereas the success rate was identical for tef1 between the grey olivaceous; on OA surface grey olivaceous. two alignments. This implies that the intraspecific variation Material examined: The Netherlands: Wijdenes, on Pyrus sp., for this locus is lower in Lasiodiplodia compared to the intra- collector and date unknown, (holotype CBS H-22718, culture specific variation for ITS. In Dothiorella, both ITS and tef1 per- ex-type CBS 121862 ¼ PD 03708098). formed quite well, and tub2 less so. Saccharata is one of the Notes: Diplodia pyri has larger conidia than that of D. pyricola few genera where none of the loci performed well on their (on Pyrus communis, Chile; conidia ellipsoid to subovate, 1- own; species in this genus are therefore best resolved based septate, 22e24 10e12 mm; Spegazzini 1921). It is also easily on a combined phylogeny rather than on a phylogeny derived distinguished from the hyaline conidia of Dothiorella pyricola from any single locus. Only S. capensis and S. kirstenboschensis (on Pyrus sp., Pakistan, conidia cylindrical-fusoid, were resolved by all individual loci, while isolates of Saccharata 15.5e20 4.5 mm) and Dothiorella pyri (on Pyrus communis, sp. 1, S. hawaiiensis and S. proteae were intermingled in both Poland, conidia ellipsoid to ovate, 11e15 7e8.5 mm) the ITS and tef1 NJ phylogenies (data not shown). For the (Aderhold 1905; Ahmad 1971). remaining alignments, almost all loci were highly successful Diplodia citricarpa Abdollahz. & Crous, sp. nov. e MycoBank in resolving species and genera. The only exceptions were MB817634; Fig 10. that ITS failed to resolve Eutiarosporella africana and E. tritici Etymology: Name refers to the host genus from which it was in the tiarosporella-like alignment and that tub2 could not isolated, Citrus. properly resolve the generic relationships. For example Mara- sasiomyces clustered inside Eutiarosporella, and the two species Conidiomata on PNA pycnidial, globose, erumpent, sepa- of Tiarosporella, although separate from the other included rate, dark brown to black, unilocular, up to 250 mm diam, species in the alignment, did not form a monophyletic lineage. with central ostiole; wall of 3e6 layers of dark brown, thick- Taxonomy walled textura angularis. Paraphyses not observed. Conidiophores reduced to conidiogenous cells or with a supporting cell. Con- Based on the phylogenetic analyses conducted in this study, idiogenous cells subcylindrical, proliferating several times per- several new families, genera and species were delineated. currently at apex, discrete, indeterminate, hyaline, smooth, These morphological descriptions are provided below. arising from the inner wall of the locule, 6e15 4e5 mm. Con- idia solitary, subcylindrical, at first aseptate, becoming 1- Botryosphaeriaceae Theiss. & Syd., Ann. Mycol. 16: 16. 1918. septate with age, granular, medium brown, finely verruculose, Type genus: Botryosphaeria Ces. & De Not. apex obtuse, base truncate, scar 3e4 mm diam, (19e)22e25 (e27) 9(e10) mm. Diplodia pyri Tao Yang & Crous, sp. nov. e MycoBank MB817633; Fig 9. Culture characteristics: Colonies covering dish in 2wk, with Etymology: Name refers to the host genus from which it was fluffy aerial mycelium. Colonies on MEA, PDA, and OA surface isolated, Pyrus. dark mouse grey, reverse fuscous black. Material examined. Iran: Hormozgan, Minab, Hajikhademi, on Conidiomata pycnidial, stromatic, globose, immersed, be- twigs of Citrus sp., 3 Mar. 2007, J. Abdollahzadeh & A. Javadi coming erumpent, separate, dark brown to black, unilocular, (holotype IRAN 14274F, isotype CBS H-22719, culture ex-type up to 300 mm diam, with central papillate neck up to 400 mm CBS 124715 ¼ CJA 131 ¼ IRAN 1578C); CBS 124714 ¼ IRAN long, and 150 mm diam; wall of 6e8 layers of dark brown, 1510C. thick-walled textura angularis. Ostiole central, circular, Notes: Diplodia citricarpa represents a novel species on Citrus, 50e70 mm diam. Conidiophores reduced to conidiogenous cells. with larger conidia than that of D. citri (Citrus, Italy, conidia el- Conidiogenous cells lageniform to subcylindrical, proliferating lipsoid, 1-septate, 18e21 8e10 mm; Saccardo 1884), and D. cit- several times percurrently at apex, discrete, indeterminate, ricola (Citrus, Australia, conidia elongated ellipsoid, 1-septate, hyaline, smooth, arising from the inner wall of the locule, 6e8 2.5e3.5 mm; McAlpine 1899). Finally, conidia are also 7e15 3e6 mm. Conidia ellipsoid to subclavate, straight to smaller than those of D. citrina (Citrus, India, conidia ellipsoid, slightly curved, at first aseptate, hyaline, granular, smooth, 1-septate, 20e25 10e13 mm; Sydow et al. 1916). becoming 1 (e2)-euseptate, walls 1e1.5 mm diam, outer sur- face of wall appearing pitted, apex obtuse, base truncate, Diplodia gallae (Schwein.) Crous, comb. nov. e MycoBank scar 3e4 mm diam, (21e)24e28 (e35) (11e)13e15 (e18) mm. MB817673.

Fig 2 e The first of 1000 equally most parsimonious trees obtained from a heuristic search with 100 random taxon additions of the combined tef1, ITS and tub2 sequence alignments of Diplodia. The scale bar represents the number of changes and bootstrap support values of MP (MP-BS) and RaxML analysis (ML-BS) and Bayesian posterior probability values (PP) are shown at the nodes (MP-BS/ML-BS/PP). Ex-type strains and taxonomic novelties are indicated in bold font and the species are delimited with coloured blocks. Thickened lines represent those branches present in the parsimony strict consensus tree. One branch was shortened to facilitate layout of the tree. The tree was rooted to Lasiodiplodia gonubiensis and L. theobromae (CBS 115812, CBS 164.96). 330 T. Yang et al. Families, genera, and species of Botryosphaeriales 331

50/65/0.55 CBS 131.33

68/-/- CBS 287.47 CBS 142.52 CBS 110495 CBS 141.49 CBS 175.26 CBS 164.96

-/-/0.99 CBS 124.13 CBS 176.26 -/-/0.54 99/100/1 Lasiodiplodia jatrophicola CBS 111.13 -/51/0.99 73/66/1 CBS 113520 Lasiodiplodia mahajangana CBS 124060 Lasiodiplodia vitis CBS 456.78 Lasiodiplodia parva CBS 177.89 Lasiodiplodia hormozganensis -/69/0.97 CBS 356.59 Lasiodiplodia euphorbicola

95/89/1 CBS 342.78 Lasiodiplodia sterculiae CBS 447.62 96/95/1 CBS 116459 Lasiodiplodia pseudotheobromae

78/66/- CBS 122519 Lasiodiplodia margaritacea

100/100/1 CBS 116355 Lasiodiplodia gonubiensis CBS 115812 -/-/0.97 CBS 121103 Lasiodiplodia plurivora CBS 122065 Lasiodiplodia sp. 1 CBS 118740 Lasiodiplodia rubropurpurea CBS 118739 Lasiodiplodia venezuelensis

100/100/1 CBS 110492 Lasiodiplodia crassispora CBS 118741 CBS 122528 phoenicum 10

Fig 4 e The first of two equally most parsimonious trees obtained from a heuristic search with 100 random taxon additions of the combined tub2, tef1 and ITS sequence alignment of Lasiodiplodia. The scale bar represents the number of changes and bootstrap support values of MP (MP-BS) and RaxML analysis (ML-BS) and Bayesian posterior probability values (PP) are shown at the nodes (MP-BS/ML-BS/PP). Ex-type strains and taxonomic novelties are indicated in bold font and the species are delimited with coloured blocks. Thickened lines represent those branches present in the parsimony strict consensus tree. The tree was rooted to Neodeightonia phoenicum (CBS 122528).

Basionym: Sphaeria gallae Schwein., Trans. Am. phil. Soc., New Aplosporella gallae (Schwein.) Petr. [as ’Aplosporella’], Hedwigia Series 4(2): 207 (1832) [1834]. 65: 273 (1925). Synonyms: Macroplodia gallae (Schwein.) Kuntze, Revis. gen. pl. Botryodiplodia gallae (Schwein.) Petr. & Syd., Die Gattung der (Leipzig) 3(2): 492 (1898). Pyrenomyzeten, Sphaeropsideen und Melanconieen: 152 (1926).

Fig 3 e The first of 1000 equally most parsimonious trees obtained from a heuristic search with 100 random taxon additions of the combined tef1, ITS and tub2 sequence alignment of Dothiorella. The scale bar represents the number of changes and bootstrap support values of MP (MP-BS) and RaxML analysis (ML-BS) and Bayesian posterior probability values (PP) are shown at the deeper nodes or nodes representing distinct genera (MP-BS/ML-BS/PP). Ex-type strains and taxonomic novel- ties are indicated in bold font and the species are delimited with coloured blocks. Thickened lines represent those branches present in the parsimony strict consensus tree. The tree was rooted to Neofusicoccum luteum (CBS 110299, CBS 110497). 332 T. Yang et al.

99/100/1 CBS 123645 Neofusicoccum umdonicola 98/95/1 CBS 123644 CBS 121.26 80/85/1 90/97/1 Neofusicoccum ribis CBS 115475 CBS 124923 100/100/1 85/68/0.99 CBS 124924 Neofusicoccum batangarum CBS 128008 100/100/1 -/49/0.93 Neofusicoccum occulatum CBS 256.80 CBS 123635 Neofusicoccum cordaticola 98/100/1 71/76/0.99 CBS 123634 77/88/1 CBS 123639 Neofusicoccum kwambonambiense CBS 123640

98/-/1 CMM1285 Neofusicoccum brasiliense CBS 138823 -/-/0.98 CBS 124493 Neofusicoccum parvum -/-/0.51 CBS 124491 CBS 318.76 Neofusicoccum sp. 3

68/-/1 CBS 719.85 Neofusicoccum algeriense -/-/0.99 CBS 110855 Neofusicoccum sp. 4 99/100/1 CBS 117452 100/100/1 Neofusicoccum andinum 94/99/1 CBS 117453 100/100/1 CBS 116131 Neofusicoccum arbuti CBS 116573 84/-/1 CBS 126655 Neofusicoccum nonquaestitum 100/100/1 CBS 114497 96/94/1 Neofusicoccum macroclavatum CBS 118223 CBS 157.26 Neofusicoccum sp. 5 100/100/1 CBS 115679 Neofusicoccum eucalypticola CBS 115791 Neofusicoccum eucalyptorum 93/90/1 CBS 118532 Neofusicoccum mangiferae 83/99/1 CBS 113083 Neofusicoccum pistaciarum -/39/0.66 CBS 113084 -/40/0.60 CBS 115403 Neofusicoccum sp. 2 100/99/1 CBS 121718 Neofusicoccum mediterraneum CBS 113089 Neofusicoccum sp. 1 96/100/1 95/98/1 -/60/0.61 CBS 125263 Neofusicoccum terminaliae CBS 125264 88/-/- CBS 122811 Neofusicoccum ursorum CBS 112878 Neofusicoccum viticlavatum CBS 595.76 Neofusicoccum pistaciae 53/96/0.94 89/91/1 CBS 110887 Neofusicoccum vitifusiforme 100/100/1 CBS 118099 Neofusicoccum corticosae CBS 120081 CBS 121112 Neofusicoccum sp. 10 87/93/1 CBS 110865 100/99/1 CBS 121115 89/-/- 89/82/0.67 CBS 139662 85/99/0.99 CBS 110864 100/99/1 Neofusicoccum stellenboschiana 100/100/1 CBS 121116 CBS 562.92 Neofusicoccum luteum 95/97/1 97/100/1 CBS 110487 83/-/0.98 CBS 122813 Neofusicoccum cryptoaustrale 62/96/0.96 CBS 121424 Neofusicoccum sp. 7 99/96/0.97 CBS 121419 Neofusicoccum sp. 8 100/100/1 100/-/1 CBS 114176 Neofusicoccum protearum CBS 111999 Neofusicoccum sp. 6 CBS 118101 Neofusicoccum sp. 9 MUCC510 Neofusicoccum pennatisporum CBS 113714 Neofusicoccum buxi CBS 100564 20

Fig 5 e The first of 384 equally most parsimonious trees obtained from a heuristic search with 25 random taxon additions of the combined tef1, ITS, rpb2, and tub2 sequence alignment of Neofusicoccum. The scale bar represents the number of changes Families, genera, and species of Botryosphaeriales 333

100/100/1 CBS 122980 P. mamane 52/75/0.78 CPC 12442 100/100/1 CBS 124700 54/55/- P. cupressi CBS 124701 Phaeobotryon CFCC 89663 83/75/0.51 P. rhois 100/100/1 CFCC 89662 CBS 123.30 Phaeobotryon sp.

100/97/1 CBS 174.26 B. fusca Barriopsis CBS 124698 B. iraniana CBS 392.80 Dothiorella omnivora 30

Fig 6 e The single most parsimonious tree obtained from a heuristic search with 100 random taxon additions of the com- bined LSU, tef1 and ITS sequence alignment of Phaeobotryon and Barriopsis. The scale bar represents the number of changes and bootstrap support values of MP (MP-BS) and RaxML analysis (ML-BS) and Bayesian posterior probability values (PP) are shown at the nodes (MP-BS/ML-BS/PP). Ex-type strains are indicated in bold font and the species are delimited with coloured blocks. Genera are shown to the right of the tree in blocks of different colours. The tree was rooted to Involcriomyces ignotus (CBS392.80).

CBS 115498 -/-/0.98 CBS 115384

CBS 119218 64/83/0.99 CBS 115206 Saccharata proteae

90/78/0.98 CBS 121410 CBS 121406

91/53/0.95 CBS 115490

100/100/0.99 CBS 111787 99/-/- Saccharata hawaiiensis CBS 114570

CBS 114569 Saccharata sp. 1

60/-/0.50 CBS 122694 Saccharata sp. 2 x 3 94/-/0.95 CBS 125546 Saccharata intermedia

CBS 122693 Saccharata capensis

CBS 123537 Saccharata kirstenboschensis x 3 CBS 732.79 Aplosporella hesperidica 60

Fig 7 e One of 10 equally most parsimonious trees obtained from a heuristic search with 100 random taxon additions of the combined tef1, ITS, and rpb2 sequence alignment of Saccharata. The scale bar represents the number of changes and boot- strap support values of MP (MP-BS) and RaxML analysis (ML-BS) and Bayesian posterior probability values (PP) are shown at the nodes (MP-BS/ML-BS/PP). Ex-type strains and taxonomic novelties are indicated in bold font and the species are de- limited with coloured blocks. Thickened lines represent those branches present in the parsimony strict consensus tree. The tree was rooted to Aplosporella sp. (CBS 732.79).

and bootstrap support values of MP (MP-BS) and RaxML analysis (ML-BS) and Bayesian posterior probability values (PP) are shown at the nodes (MP-BS/ML-BS/PP). Ex-type strains and taxonomic novelties are indicated in bold font and the species are delimited with coloured blocks. Thickened lines represent those branches present in the parsimony strict consensus tree. The tree was rooted to Botryosphaeria dothidea (CBS 100564). 334 T. Yang et al.

99/100/0.98 DAR 82492 86/100/0.98 DAR 82491 E. darliae 100/100/0.98 CBS 118530 100/100/0.98 DAR 82490 100/55/0.89 E. pseudodarliae DAR 82489 91/70/0.98 CBS 118719 E. tritici 92/62/0.97 CBS 110392 Eutiarosporella sp. Eutiarosporella

98/100/0.97 DAR 82485 E. tritici-australis 100/100/0.99 DAR 82486

100/100/0.98 CBS 133854 100/100/0.97 E. africana 100/100/0.83 CBS 135850 100/100/0.96 CBS 130405 E. urbis-rosarum CBS 130406 98/100/0.98 CBS 118718 M. karoo Marasasiomyces 100/100/0.98 CBS 532.76 S. madreeya Sakireeta CBS 109699 Tiarosporella sp. Tiarosporella 100/100/0.98 CBS 114650 T. paludosa CBS 110496 Sphaeriopsis porosa CBS 100163 Sphaeriopsis visci 60

Fig 8 e One of two equally most parsimonious trees obtained from a heuristic search with 100 random taxon additions of the combined tef1, ITS, and tub2 sequence alignment of the tiarosporella-like group of genera. The scale bar represents the number of changes and bootstrap support values of MP (MP-BS) and RaxML analysis (ML-BS) and Bayesian posterior prob- ability values (PP) are shown at the nodes (MP-BS/ML-BS/PP). Ex-type strains are indicated in bold font and the species are delimited with coloured blocks. Thickened lines represent those branches present in the parsimony strict consensus tree. Genera are shown to the right of the tree in blocks of different colours. The tree was rooted to Sphaeropsis visci and Sphaeropsis porosa (CBS 100163 and CBS 110496, respectively).

Sphaeropsis gallae (Schwein.) A.W. Archer, Annls mycol. 25:37 correlate with the strains clustering in Diplodia. The three cul- (1927). tures used by A.W. Archer (presumably from the USA) to intro- Additional synonyms listed in Petrak & Sydow (1927). duce the combination in Sphaeropsis, were treated here, but Material examined: Unknown: on galls of Quercus sp., A.W. Ar- unfortunately, all three (deposited in CBS in 1925) remained cher, CBS 212.25, CBS 211.25, CBS 213.25. sterile. Nevertheless, a new combination is required for this Notes: Sphaeria gallae was described by D. von Schweinitz to fungus in Diplodia. This pathogen has in the past been referred name a fungus associated with galls on Quercus in the USA. to in literature under this name as D. gallae (Schwein.) Cooke The fungus was subsequently placed in several different gen- (Wollenweber & Hochapfel 1941), although as far as we could era, namely Aplosporella, Botryodiplodia, Macroplodia, and establish, the combination has never been officially Sphaeropsis, all of which suggest it has dark conidia, which published.

Table 3 e Summary of the number of species/genera distinguished per locus for each alignment used in this study. Analysis LSU rpb2a ITS tef1 tub2

Overview e genera 80 % (20/25) 100 % (25/25) ee e Overview e species 59 % (46/78) 91 % (71/78) ee e Diplodia ee 55 % (11/20) 65 % (13/20) 70 % (14/20) Dothiorella ee 83 % (25/30) 93 % (28/30) 67 % (20/30) Lasiodiplodia 3-gene ee 50 % (8/16) 56 % (9/16) 88 % (14/16) Lasiodiplodia 2-gene ee 25 % (8/32) 56 % (18/32) e Neofusicoccum 4-gene e 58 % (21/36) 85 % (35/41) 73 % (30/41) 63 % (26/41) Neofusicoccum 2-gene ee 69 % (29/42) 55 % (23/42) e Phaeobotryon/Barriopsis e genera 50 % (1/2) e 100 % (2/2) 100 % (2/2) e Phaeobotryon/Barriopsis e species 100 % (6/6) e 100 % (6/6) 100 % (6/6) e Saccharata e 43 % (3/7) 57 % (4/7) 57 % (4/7) e Tiarosporella-like e genera ee 82 % (9/11) 100 % (11/11) 100 % (11/11) Tiarosporella-like e species ee 100 % (4/4) 100 % (4/4) 25 % (1/4)

a Not all isolates of Neofusicoccum amplified for this locus. Families, genera, and species of Botryosphaeriales 335

Fig 9 e Diplodia pyri (CBS 121862). (A). Colony sporulating on PNA. (B). Conidiogenous cells. (C). Young, hyaline conidia. (D). Mature, brown, 1-septate conidia. Scale bars: A [ 300 mm, all others [ 10 mm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Dothiorella a reliable feature at generic level, and that these clades are best treated as a single genus, Dothiorella. The genus Dothiorella was resurrected by Phillips et al. (2005) to accommodate species with conidia that turn brown while still Dothiorella citricola (A.J.L. Phillips & Abdollahz.) Tao Yang & attached to their conidiogenous cells. Furthermore, Phillips Crous, comb. nov. e MycoBank MB817674. et al. (2008) introduced Spencermartinsia to accommodate Basionym: Spencermartinsia citricola A.J.L. Phillips & Abdollahz., dothiorella-like species with apiculate ascospores. This deci- Persoonia 32: 7 (2014). sion was supported by a phylogeny based on ITS and tef1, Description and illustration: Abdollahzadeh et al. (2014). and a rather limited taxon sampling (Phillips et al. 2013). Dothiorella mangifericola Tao Yang & Crous, nom. nov. e Slippers et al. (2013) employed a six-gene phylogeny and MycoBank MB817675. more isolates, but found that additional loci and isolates could Synonym: Spencermartinsia mangiferae Abdollahz. et al., Persoo- no longer separate Dothiorella from Spencermartinsia. Likewise nia 32: 9 (2014). in the present study, we found that a three-gene phylogeny Description and illustration: Abdollahzadeh et al. (2014). again included Spencermartinsia in Dothiorella. From these re- Notes: Because the name Dothiorella mangiferae is already occu- sults we conclude that apiculate ascospores, which evolved pied, a new name is proposed for this species. in several genera in the family (see Phillips et al. 2013) is not

Fig 10 e Diplodia citricarpa (CBS 124715). (A). Colony sporulating on PNA. (BeE). Conidiogenous cells. (F). Mature, brown, 1- septate conidia. Scale bars: A [ 250 mm, all others [ 10 mm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) 336 T. Yang et al.

Dothiorella plurivora (Abdollahz. et al.) Tao Yang & Crous, Paraphyses intermingled among conidiogenous cells, hyaline, comb. nov. e MycoBank MB817676. cylindrical, aseptate, unbranched, ends obtuse, up to 60 mm Basionym: Spencermartinsia plurivora Abdollahz. et al., Persoo- long, 2e3 mm diam. Conidiophores reduced to conidiogenous nia 32: 9 (2014). cells. Conidiogenous cells hyaline, smooth, ampulliform to sub- Description and illustration: Abdollahzadeh et al. (2014). cylindrical, proliferating percurrently to form 1e3closely spaced apical annellations, 5e15 5e8 mm. Conidia ellipsoi- Dothiorella rosulata (F.J.J. Van der Walt et al.) Tao Yang & dal, apex obtuse, base truncate, scar 4e5 mm diam, widest Crous, comb. nov. e MycoBank MB817677. at the middle or upper third, thick-walled (2 mm diam), ini- Basionym: Spencermartinsia rosulata F.J.J. Van der Walt et al., tially hyaline, granular and aseptate and remaining so for Persoonia 33: 164 (2014). a long time, becoming 1-septate and dark brown after release Description and illustration: Slippers et al. (2014). from the conidiomata, with melanin deposits on the inner Dothiorella viticola A.J.L. Phillips & J. Luque, Mycologia 97: 1118 surface of the wall arranged longitudinally giving a striate ap- (2006) [2005]. pearance to the conidia, (25e)26e28 (e32) (12e)15e16 Synonyms: A.J.L. Phillips & J. Luque, (e17) mm. Mycologia 97: 1118 (2006) [2005]. Spencermartinsia viticola (A.J.L. Phillips & J. Luque) A.J.L. Phillips Culture characteristics: Colonies covering dish in 7 d, with cot- et al., Persoonia 21: 51 (2008). tony aerial mycelium. Colonies on MEA, surface glaucous Description and illustration: Luque et al. (2005). grey, reverse dark olivaceous; on PDA surface and reverse grey olivaceous; on OA surface olivaceous buff. Dothiorella westralis (W.M. Pitt et al.) Tao Yang & Crous, comb. Material examined: Italy: Sicily, from canker symptoms of Vitis nov. e MycoBank MB817678. vinifera, date unknown, S. Burruano (holotype CBS H-22720, Basionym: Spencermartinsia westralis (as westrale) W.M. Pitt culture ex-type CBS 124060). et al., Australas. Pl. Path. 44: 48 (2015). Notes: Rodrıguez-Galvez et al. (2017) present data to distinguish Description and illustration: Pitt et al. (2015). 15 different species of Lasiodiplodia. Lasiodiplodia vitis, which Lasiodiplodia vitis Tao Yang & Crous, sp. nov. e MycoBank represents a novel species on Vitis vinifera in Italy, is clearly MB817635; Fig 11. distinct from the taxa presently recognised on this host (Van Etymology. Name refers to the host genus from which it was Niekerk et al. 2006; Phillips et al. 2013), as most reports have isolated, Vitis. been attributed to L. theobromae in the past (Burruano et al. 2008). Conidiomata stromatic, formed on PNA in culture, unilocu- Lasiodiplodia sterculiae Tao Yang & Crous, sp. nov. e Myco- late, up to 400 mm diam, dark brown to black; wall of 6e10 Bank MB817636; Fig 12. layers of brown textura angularis, immersed in the host tissue, Etymology: Name refers to the host genus from which it was becoming erumpent when mature, developing a central pa- isolated, Sterculia. pillate neck up to 200 mm long, with ostiole 50e70 mm diam.

Fig 11 e Lasiodiplodia vitis (CBS 124060). (A). Colony sporulating on PNA. (B). Paraphyses. (C). Conidiogenous cells. (D). Young, hyaline conidia. (E). Mature, brown, 1-septate conidia, intermixed with immature conidia. Scale bars: A [ 400 mm, all others [ 10 mm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) Families, genera, and species of Botryosphaeriales 337

Fig 12 e Lasiodiplodia sterculiae (CBS 342.78). (A). Colony sporulating on PNA. (BeF). Conidiogenous cells giving rise to conidia. (G). Mature, thick-walled conidia, some with hyaline appendage. Scale bars: A [ 300 mm, all others [ 10 mm.

Conidiomata pycnidial, immersed to semi-superficial, sepa- establish, this is the first species of Lasiodiplodia other than L. rate, globose, dark brown, unilocular, to 300 mm diam; wall of theobromae described from this host. dark brown, thick-walled textura angularis, paler and thinner- Neofusicoccum buxi Crous, sp. nov. e MycoBank MB817679; walled towards the conidiogenous region, often with dark Fig 13. brown superficial hyphae over the surface. Ostiole central, sin- Etymology: Name refers to the host genus from which it was gle, papillate. Conidiophores hyaline, smooth, subcylindrical, isolated, Buxus. 0e2-septate, branched below or not, 8e20 2.5e3.5 mm, aris- ing from the inner layers of cells lining the locule. Conidioge- Conidiomata solitary, immersed, becoming erumpent, nous cells hyaline, smooth, subcylindrical to doliiform, brown, up to 250 mm diam, with central ostiole, splitting epi- 7e12 2.5e3.5 mm, discrete, indeterminate, proliferating per- dermis with irregular rupture, exuding conidia in a white mu- currently with one or two distinct annellations, or proliferat- coid mass; wall consisting of 3e5 layers of brown textura ing at the same level giving rise to periclinal thickenings. angularis. Conidiophores lining the inner layer of the conidioma, Conidia hyaline when young, and not turning brown, even af- unbranched, subcylindrical, hyaline, smooth, 0e1-septate, ter 2 mo on SNA, thick-walled, obovoid to ellipsoid, straight, 15e30 3e8 mm. Conidiogenous cells hyaline, integrated, termi- broadly rounded at the apex, base truncate, (12e)14e16 nal, subcylindrical, proliferating several times percurrently (e17) (8e)10e11 (e12) mm; outer layer frequently forming near apex, rarely with minute periclinal thickening, a swollen, thin-walled appendage-like bulge on 25 % of the 15e25 5e7 mm. Conidia hyaline, granular, smooth, thin- conidia observed. Paraphyses not observed. walled, subcylindrical, straight to slightly curved, widest in the middle or in the upper third, apex subobtuse, base sub- Culture characteristics: Colonies covering dish in 7 d, with truncate, scar 3e5 mm diam, with minute marginal frill, sparse aerial mycelium. Colonies on MEA, surface greyish se- (25e)30e38 (e45) (6.5e)7e8(e9) mm. synasexual pia, reverse purplish grey; on PDA surface greyish sepia, re- morph not observed. verse pale grey olivaceous; on OA surface grey olivaceous. Material examined: Germany: Braunschweig, on Sterculia Material examined: France: Provence, near Pont du Gard, on leaf oblonga, Mar. 1978, S. Bruhn (holotype CBS H-13230, culture of Buxus sempervirens, 24 Oct. 1974, H.A. van der Aa (holotype ex-type CBS 342.78). CBS H-12162; CBS H-12166, culture ex-type CBS 116.75). Sweden: Notes: Lasiodiplodia sterculiae was originally deposited in CBS as Uppsala, Uppsala Botanical Garden, on Buxus sempervirens,12 representative of Aplosporella sterculiae (Sterculia tomentosa, Nov. 1985, O. Constantinescu, CBS 113714. Erythrea, conidia ellipsoid, chestnut brown, 24 9.6 mm; Notes: Dothiorella candollei causes leaf spots on Buxus sempervirens Saccardo & Trotter 1931), but actually represents a species of (Batdorf 1995). The two cultures cited above have been deposited Lasiodiplodia with much smaller conidia. As far as we could in the CBS under the name Dothiorella candollei. However, the 338 T. Yang et al.

Fig 13 e Neofusicoccum buxi (CBS 116.75). (A). Conidiomata in vivo. (B, C). Conidiogenous cells. (D). Mature, hyaline, aseptate conidia. Scale bars: A [ 250 mm, all others [ 10 mm.

basionym, Sphaeropsis candollei was never validly published, as it Conidiomata solitary, woolly due to abundant external hy- contained reference to the type of Sphaeria buxi (Alb. & Schwein.) phae, erumpent, stromatic, brown, up to 500 mm diam on DC., the epithet that should have been used for this fungus. PNA, with central ostiole, exuding conidia in a white mucoid Since then, the name has become confused with a different fun- mass; wall consisting of 3e5 layers of brown textura angularis. gus occurring on Buxus, namely Hyponectria buxi (Alb. & Conidiophores lining the inner layer of the conidioma, subcylin- Schwein.) Sacc. A new name is thus required for the Neofusicoc- drical, hyaline, smooth, 0e1-septate, 15e30 3e5 mm, un- cum species associated with leaf spots of Buxus, which is intro- branched or branched below the apical septum. duced here as N. buxi. Conidiogenous cells hyaline, integrated, terminal, subcylindri- cal, rarely ampulliform, proliferating several times percur- Neofusicoccum pistaciae (Zachos et al.) Crous, comb. nov. e rently near apex, rarely with minute periclinal thickening, MycoBank MB817680. 12e20 3e4 mm. Conidia hyaline, granular, smooth, thin- Basionym: Camarosporium pistaciae Zachos et al., Annls Inst. walled, fusoid-ellipsoidal, straight to slightly curved, widest phytopath. Benaki, N.S. 11: 57 (1974). in the middle or in the upper third, apex subobtuse, base sub- Material examined: Greece: near Thessaloniki, on fruit of Pista- truncate, scar 2.5e3 mm diam, with minute marginal frill, (19e) cia vera, 1972, D.G. Zachos (isotype CBS H-499, culture ex- 23e26 (e27) 5e6(e6.5) mm. Dichomera synasexual morph not isotype CBS 595.76). observed. Note: The ex-isotype strain of Camarosporium pistaciae clusters in Neofusicoccum, and thus a new combination is required for Culture characteristics: Colonies covering dish in 7 d, with cot- this species. tony aerial mycelium. Colonies on MEA, surface and reverse Neofusicoccum pistaciarum Tao Yang & Crous, sp. nov. e dirty white; on PDA surface and reverse olivaceous grey; on MycoBank MB817637; Fig 14. OA surface grey olivaceous. Etymology: Name refers to the host genus from which it was Material examined: USA: California, Kern County, on Pistacia isolated, Pistacia. vera, 12 Apr. 2012, T.J. Michailides (holotype CBS H-22722, cul- ture ex-type CBS 113083 ¼ CPC 5263).

Fig 14 e Neofusicoccum pistaciarum (CBS 113083). (A). Colony sporulating on PNA. (B, C). Conidiogenous cells. (D). Mature, Hyaline, aseptate conidia. Scale bars: A [ 500 mm, all others [ 10 mm. Families, genera, and species of Botryosphaeriales 339

Notes: Inderbitzin et al. (2010) generated a six-gene phylogeny Etymology: Name refers to the elongated conidiomatal necks. to highlight the extraordinary high species diversity of Botryos- phaeriaceae on almond in California. In the same paper he also Conidiomata pycnidial, globose to subglobose, covered in included several isolates from Pistacia vera, from which he re- short hyphae, superficial, immersed or semi-immersed, de- ported Botryosphaeria dothidea, , Lasiodiplodia the- veloping very long necks, aggregated in clusters; wall of 3e6 obromae, Neofusicoccum mediterraneum, and N. parvum. layers of brown textura angularis. Paraphyses intermingled Neofusicoccum pistaciarum represents yet an additional species among conidiogenous cells, hyaline, smooth, flexuous, asep- from this host, which appears distinct from those species tate, rarely branched, with obtuse apices. Conidiophores re- presently recognised (Phillips et al. 2013). duced to conidiogenous cells. Conidiogenous cells hyaline, holoblastic, cylindrical to ampuliform, proliferating at the Neofusicoccum stellenboschiana Tao Yang & Crous, sp. nov. e same level to form periclinical thickenings or rarely proliferat- MycoBank MB817638; Fig 15. ing percurrently. Conidia honey coloured to black-brown, 0e1 Etymology: Name refers to Stellenbosch, the town in South (e3)-septate, smooth, hilum truncate, moderately thick- Africa where this fungus was collected. walled, straight to slightly irregularly curved, variable in shape. Conidiomata pycnidial, brown, superficial on PNA, globose, solitary, to 150 mm diam; wall of 3e6 layers of brown textura Type species: Oblongocollomyces variabilis (F.J.J. van der Walt, angularis. Conidiophores reduced to conidiogenous cells. Coni- et al.) Tao Yang & Crous. diogenous cells hyaline, subcylindrical to doliiform, with 1e4 Notes: Oblongocollomyces differs from Sphaeropsis s.str. by form- percurrent proliferations near apex, 10e13 2e3 mm. Conidia ing extremely long conidiomatal necks on PNA, or being ag- hyaline, fusoid, base subtruncate, aseptate, forming two septa gregated in dense clusters on OA, and having conidia that before germination, smooth with granular contents, (17) can be up to 3-septate. The long-necked, aggregated conidio- 19e21 (22) (4.5e)5.5e6 mm. Spermatogonia and Dichomera mata is a feature that is more common in the family, but atyp- synasexual morph not observed. ical for Sphaeropsis or Diplodia.

Culture characteristics: Colonies covering dish in 7 d, with cot- Oblongocollomyces variabilis (F.J.J. van der Walt, et al.) Tao tony aerial mycelium. Colonies on MEA, PDA, and OA surface Yang & Crous, comb. nov. e MycoBank MB817640; Fig 16. and reverse dirty white. Basionym: Sphaeropsis variabilis F.J.J. van der Walt, et al., Per- soonia 33: 164. 2014. Material examined: South Africa: Western Cape Province, Stel- lenbosch, on Vitis vinifera, date unknown, F. Halleen (holotype Conidiomata pycnidial, globose to subglobose, covered in CBS H-22723, culture ex-type CBS 110864 ¼ CPC 4598). short hyphae, superficial, immersed or semi-immersed, up Notes: In the study of Botryosphaeriaceae grapevine disease to 390 mm diam; wall of 3e6 layers of brown textura angularis; pathogens occurring in South Africa, Van Niekerk et al. conidiomata on PNA developing necks up to 1.5 mm long, on (2004) treated this isolate as part of the Neofusicoccum australe OA however, necks absent and conidiomata aggregated in complex, commenting on differences in virulence observed a stroma in clusters of up to 15. Paraphyses intermingled among isolates of this species. With the benefit of additional among conidiogenous cells, hyaline, smooth, flexuous, up to loci employed in this study, we were now able to show that 60 mm long, 3e4 mm diam, aseptate, rarely branched, with ob- this isolate was actually closely related to N. australe, but rep- tuse apices. Conidiophores reduced to conidiogenous cells. Con- resents a distinct species, named here as N. stellenboschiana. idiogenous cells hyaline, holoblastic, cylindrical to ampuliform, Oblongocollomyces Tao Yang & Crous, gen. nov. e MycoBank (5.5e)6e11 (e16.5) 2.5e5.5 (e7.5) mm, proliferating at the MB817639. same level to form periclinical thickenings or rarely

Fig 15 e Neofusicoccum stellenboschiana (CBS 110864). (A). Colony sporulating on PNA. (B, C). Conidiogenous cells. (D). Mature, hyaline, aseptate conidia. Scale bars: A [ 150 mm, all others [ 10 mm. 340 T. Yang et al.

Fig 16 e Oblongocollomyces variabilis (CBS 121774). (A). Colony sporulating on PNA, showing long pycnidial neck. (B). Colony sporulating on OA. (C). Paraphyses. (DeF). Conidiogenous cells. (G). Mature, brown, 1-septate conidia. Scale bars: A, B [ 400 mm, all others [ 10 mm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

proliferating percurrently to form one or two annellations. Type genus: Endomelanconiopsis E.I. Rojas & Samuels. Conidia honey coloured to black-brown, 0e1(e3)-septate, Notes: Endomelanconiopsisaceae resides between Pseudofusicoc- granular with central guttule, smooth, hilum truncate, cumaceae and Melanopsaceae (Fig 1). The family presently con- 3e4 mm diam, moderately thick-walled, straight to slightly ir- tains a single genus with two species (Phillips et al. 2013). regularly curved, cylindrical to ovoid or occasionally reniform The latter two families have no members bearing conidia or allantoid or subclavate, (24e)26.5e33.5 (e37) (8e)10.5e14 with germ slits, a feature which appears to be restricted to (e17) mm. the Endomelanconiopsisaceae.

Pseudofusicoccumaceae Tao Yang & Crous, fam. nov. e Myco- Culture characteristics: See Slippers et al. (2014). Bank MB817642. Material examined: Namibia: Windhoek, on Acacia karroo, Feb. 2006, F.J.J. van der Walt & J. Roux (holotype PREM 59637, cul- Conidiomata large, immersed to superficial, uni-to multiloc- ture ex-type CBS 121774 ¼ CMW 25419). ular, covered with hyphae; wall of dark brown textura angula- Notes: With the description of S. variabilis, Slippers et al. (2014) ris. Conidiophores reduced to conidiogenous cells. Conidiogenous commented on the fact that this species was allied to Phaeo- cells hyaline, smooth, cylindrical, proliferating percurrently to botryon, Barriopsis, and Sphaeropsis, but eventually chose to al- form distinct annellations, or proliferating at the same level, locate it to Sphaeropsis based on its typical conidia with obtuse giving rise to periclinal thickenings. Conidia hyaline, thin-to apices and truncate bases. As shown here, however, the rea- slightly thick-walled, aseptate, granular, cylindrical, rarely el- son for this deviating morphology, lies in the fact that it repre- lipsoid, straight to slightly curved, surrounded by a persistent sents a distinct genus. mucous sheath.

Type genus: Pseudofusicoccum Mohali, Slippers & M.J. Wingf. Endomelanconiopsisaceae Tao Yang & Crous, fam. nov. e Notes: Morphologically the family, which is typified by Pseudo- MycoBank MB817641. fusicoccum, is similar to Fusicoccum asexual morphs of Botryos- phaeria (Botryosphaeriaceae), but distinct in that conidia tend to Conidiomata stromatic, immersed, peridermal to subepider- be more cylindrical in shape, and are encased in a persistent mal, separate, irregularly multilocular; walls composed of mucoid sheath. pale brown, thin-walled textura angularis. Dehiscence irregu- lar. Conidiophores reduced to conidiogenous cells. Conidiogenous Saccharataceae Slippers, Boissin & Crous, Stud. Mycol. 76: 41. cells holoblastic, determinate, discrete, cylindrical, tapered to- 2013. wards the apices, hyaline, smooth, thin-walled. Conidia asep- Type genus: Saccharata Denman & Crous. tate, pyriform to limoniform, dark brown, thick-walled, Saccharata hawaiiensis Tao Yang & Crous, sp. nov. e Myco- smooth, base often protruding and papillate, often with a cen- Bank MB817643; Fig 17. tral guttule and a single germ slit. Families, genera, and species of Botryosphaeriales 341

Etymology: Name refers to the Island of Hawaii, where this fun- gus was collected. Discussion

Conidiomata pycnidial, eustromatic, to 450 mm diam, im- In this study, a large number of cultures were analysed to pro- mersed, subepidermal, separate, dark brown, uni-to multi- duce an expanded phylogenetic backbone for Botryosphaer- locular, walls consisting of dark brown textura angularis, ostio- iales, to help delimit novelties at species, genus and family late. Fusicoccum-like synasexual morph. Conidiophores hya- level. The majority of the cultures included here were initially line, smooth, branched, subcylindrical, 1e3-septate, formed characterised based on their macro-morphology and/or using from the inner layers of the locule, 20e60 3e4.5 mm, inter- limited DNA sequence data available at that time. This study mingled with hyaline, septate paraphyses that are branched, stresses the impact DNA sequence data have in providing 3e8-septate, up to 90 mm long, 3e4 mm diam. Conidiogenous cells more accurate identifications of Botryosphaeriales, which has discrete or integrated, hyaline, smooth, cylindrical, entero- consequences for cultivation and trade of plant material blastic, proliferating percurrently with numerous apical per- (Crous et al. 2015b, 2016). current annellations, 10e20 2.5e3.5 mm. Conidia hyaline, thin-walled, aseptate, smooth, fusiform, widest in the middle Identification of unknown cultures or upper third of the conidium, with subobtuse apex and e e e e e e m a truncate base, (17 )24 30 ( 38) (4 )5 7(8) m. One-hundred-and-twenty-six cultures in the CBS culture col- Diplodia-like synasexual morph occurring in same conidio- lection, which were not identified to species level, were re- mata as the fusicoccum-like morph. Conidiophores hyaline, examined during the course of this study. The largest part e smooth, branched, cylindrical, 1 3-septate, formed from the (97 cultures), originally listed as ‘Botryosphaeria sp.’, were e e m inner layers of the conidioma, 15 25 2 3 m. Conidiogenous found to represent mainly species of Diplodia [24 cultures: Di. cells phialidic, discrete or integrated, hyaline, smooth, cylin- corticola (1 culture), Di. pyri (1 culture), Di. seriata (21 cultures) drical, determinate, with prominent periclinal thickening, and a Diplodia sp. (1 culture)] and Neofusicoccum [69 cultures: e e m 6 10 2 3 m. Conidia medium brown, thick-walled, finely N. algeriense (1 culture), N. australe (18 cultures), N. macroclava- verruculose, guttulate, aseptate, subcylindrical to narrowly el- tum (3 cultures), N. parvum (13 cultures), N. pistaciarum (5 cul- e lipsoid with rounded, but somewhat thickened ends, (8 ) tures), N. protearum (3 cultures), Neofusicoccum sp. 1 (3 e e e m 10 13 ( 15) 2.5 3.5 m. cultures), Neofusicoccum sp. 2 (11 cultures), Neofusicoccum sp. 4 (3 cultures), Neofusicoccum sp. 6 (1 culture), Neofusicoccum Culture characteristics: Colonies covering dish in 7 d, with sp. 8 (7 cultures), and N. stellenboschiana (1 culture)], as well sparse to cottony aerial mycelium. Colonies on MEA, PDA, as one culture each of Lasiodiplodia crassispora, Lasiodiplodia and OA dirty white. mahajangana, phaseolina, and Saccharata hawai- Material examined: USA: Hawaii, Manii Floral, on Protea laurifolia iensis. Twelve ‘Dothiorella sp.’ cultures were identified as Do. cv. ‘Rose mink’, 12 Feb. 1999, P.W. Crous (holotype CBS H- iranica (1 culture), Do. omnivora (2 cultures), Do. parva (2 cul- ¼ 22724, culture ex-type CBS 111787 CPC 2268). tures), Do. prunicola (1 culture), Do. sempervirentis (2 cultures), Notes: The genus Saccharata presently contains four species as well as N. luteum (1 culture), N. occulatum (1 culture) and that occur on host plants indigenous to South Africa, such N. parvum (2 cultures). Seven Spencermartinsia cultures were as Encephalartos and genera of (Crous et al. 2008, identified as Do. citricola (2 cultures), Do. mangifericola (1 cul- 2009a), but seem to have also been exported along with these ture), Do. plurivora (2 cultures), and Do. striata (2 cultures). plants to other countries (Crous et al. 2013). One further exam- The remaining 10 cultures represent Aplosporella hesperidica ple is Saccharata hawaiiensis, which was collected from South (was Aplosporella sp.), Bagnisiella sp. (was Aplosporella sp.), African Protea cultivated in Hawaii. Cophinforma eucalypti (was Pseudofusicoccum sp.), Diplodia

Fig 17 e Saccharata hawaiiensis (CBS 111787). (A). Colony sporulating on OA. (B, C). Conidiogenous cells. (D). Mature, hyaline, aseptate conidia, and smaller, pigmented diplodia-like conidia of the synasexual morph. Scale bars: A [ 450 mm, all others [ 10 mm. 342 T. Yang et al.

citricarpa (was Diplodia sp.), Diplodia sp. 1 (was Diplodia sp.), Septorioides was treated as a new monotypic genus typified Dothiorella omnivora (was Botryodiplodia sp.), Dothiorella parva by Septorioides pini-thunbergii in Botryosphaeriaceae. However, (was sp.), Eutiarosporella sp. (was Diplodia sp.), N. vitifusi- this genus has since been shown to also cluster in a separate forme or N. corticosae (was Neofusicoccum sp.), and Tiarosporella family, namely Septorioideaceae (Wyka & Broders 2016). sp. (was Tiarosporella sp.). Several of these previously uniden- tified species were found to represent taxonomic novelties, Genera of Botryosphaeriaceae namely Di. citricarpa, Di. pyri, N. stellenboschiana, S. hawaiiensis, and an additional number of sterile cultures still awaiting fur- Within the Botryosphaeriaceae, a combination of SSU-ITS-LSU- ther description at species level. tef1-tub2 provided high support for the delimitation of genera (Phillips et al. 2013). In the present study, genera could be Families of Botryosphaeriales clearly delimited based on the LSU-rpb2 dataset. However, Dothiorella and Spencermartinsia clustered in the same clade The Botryosphaeriales phylogeny presented here agrees well intermixing with one another. The two genera were separated with general phylogenies in previous studies (Liu et al. 2012; based on the presence of apiculi on ascospores of Spencermar- Slippers et al. 2013; Phillips et al. 2013; Wyka & Broders 2016). tinsia (Phillips et al. 2008, 2013), although this separation was The topologies of the phylogenetic trees generated from ML, seen as tentative (Slippers et al. 2014). Because Spencermartinsia MP, and Bayesian analyses were highly similar and especially rendered Dothiorella paraphyletic in our study, we decided to the generic and family clades received high support values in reduce them to a single genus. Furthermore, Sphaeropsis varia- all analyses. Our two-gene (LSU and rpb2) phylogeny supports bilis (Slippers et al. 2014) was found to cluster separate from the same generic clades as the four-gene and six-gene phylog- Sphaeropsis. Morphologically it was found to be distinct, hav- enies employed by Liu et al. (2012) and Slippers et al. (2013), re- ing conidiomata that were either aggregated in dense clusters spectively. However, some rearrangements in the backbone on OA, or with extremely long necks on PNA, leading us to in- were observed, such as the position of Pseudofusicoccum versus troduce it as a new genus, Oblongocollomyces. Endomelanconiopsis at a more basal position and the position of Neodeightonia at the more terminal position. Previous studies Species complexes have included Pseudofusicoccum and Endomelanconiopsis as part of the family Botryosphaeriaceae [the four-gene, five-gene Phillips et al. (2013) recommended that at least two loci (ITS- and six-gene phylogenies of Liu et al. (2012), Phillips et al. tef1) be used for species separation within Botryosphaeriaceae. (2013) and Slippers et al. (2013), respectively]. This scenario is However, the range of variation within a species becomes indicated by ‘node 1’ (MP-BS/ML-BS/PP: 98/97/1) in our phylog- more apparent as additional isolates are added to the dataset eny (Fig 1); the same node received 55/-/0.95 (MP-BS/ML-BS/PP) (also see above). In this regard, in addition to ITS-tef1 sequence support, 100/100 (MP-BS/ML-BS) support, and 89/0.99 (ML-BS/ data, data from tub2, rpb2 and other loci have at times been PP) support, in the phylogenies of Liu et al. (2012), Phillips found necessary to provide convincing support to separate et al. (2013), and Slippers et al. (2013), respectively. A more re- cryptic species. strictive scenario would be to exclude the two basal genera, In this study, more than 200 Neofusicoccum strains were Pseudofusicoccum and Endomelanconiopsis, from Botryosphaeria- studied. The phylogeny derived from the ITS-tef1 dataset pre- ceae. This scenario indicated by ‘node 2’ (MP-BS/ML-BS/PP: sented a polytomy in the N. parvum/N. ribis species complex. 76/98/1 support) in Fig 1; the same node received 73/84/1 Therefore, an ITS-tef1-tub2-rpb2 dataset was generated after (MP-BS/ML-BS/PP) support (Endomelanconiopsis was not in- selecting representatives from each species clade. This phy- cluded in that study), 100/100 (MP-BS/ML-BS) support, and logeny provided higher support values for each species clade 99/1 (ML-BS/PP) support, in the phylogenies of Liu et al. in general, and resolved the polytomy. Sakalidis et al. (2011) di- (2012), Phillips et al. (2013), and Slippers et al. (2013), respec- vided N. parvum/N. ribis complex into eight species based on tively. An even more conservative scenario would also ex- the same gene combination. clude Neofusicoccum and Dothiorella from Botryosphaeriaceae In another species complex, namely Diplodia seriata, several (Fig 1; scenario indicated by ‘node 3’, MP-BS/ML-BS/PP: 92/98/ minor sub-clades were detected within the major ITS phylog- 1 support); however, this node was only present in the study eny. Furthermore, the phylogenetic relationships among D. of Phillips et al. (2013) and was poorly supported (MP-BS/ML- seriata, D. pinea, and D. scrobiculata were not well resolved by BS: -/71). The third scenario would result in a situation where ITS sequence data (Phillips et al. 2007). In subsequent studies many genera would each receive an own family, which would tef1 was combined with ITS to resolve species (Phillips et al. not be an ideal situation. The second scenario prompted us to 2012, 2013; Alves et al. 2014). In the present study, a combina- describe two novel families to accommodate Pseudofusicoccum tion of ITS-tef1-tub2 was used. Compared to the trees gener- and Endomelanconiopsis. Although these two genera are indi- ated based on ITS-tef1, those from ITS-tef1-tub2 were better vidually strongly supported, they are consistently placed basal supported, and provided a more stable topology. to ‘Botryosphaeriaceae’, and their relation to each other and to For Lasiodiplodia, the ITS-tef1, the loci that have been the the other Botryosphaeriaceae genera change depending on the most commonly used in literature, revealed (Fig. S1, see Tree- type of analysis and the sampling of included loci [also see BASE) that some recently described species could not be sep- phylogenies in Liu et al. (2012), Phillips et al. (2013) and arated as distinct, for example L. jatrophicola/L. iraniensis Slippers et al. (2013)]. Pseudofusicoccum and Endomelanconiopsis (similar to the result of Linaldeddu et al. 2015), L. euphorbi- are also morphologically distinct from other genera in Botryos- cola/L. marypalme (Machado et al. 2014; Netto et al. 2014), and phaeriaceae. In the analysis of Quaedvlieg et al. (2013), L. crassispora/L. pyriformis (Burgess et al. 2006; Slippers et al. Families, genera, and species of Botryosphaeriales 343

2014). Unfortunately cultures from those species were not also been used successfully in different groups of fungi at fam- available to us for study and therefore future studies employ- ily level, such as Teratosphaeriaceae (Quaedvlieg et al. 2013), ing additional gene regions would be required to resolve these Didymellaceae (Chen et al. 2015), Nectriaceae (Lombard et al. uncertainties. Inclusion of tub2 in the concatenated alignment 2015), and at species level, e.g. Ramularia (Videira et al. 2015), greatly improves the resolution of the resulting tree. Based on Alternaria (Woudenberg et al. 2015), Microdochium only ITS and tef1, Sakalidis (2011) identified two putative hy- (Hernandez-Restrepo et al. 2016), Talaromyces (Yilmaz et al. brids in Lasiodiplodia. More recently, Cruywagen et al. (2017) 2016), and Chaetomium (Wang et al. 2016). When rpb2 was showed that phylogenies based on five individual genes employed across the kingdom Fungi, however, results were were incongruent and the authors concluded that several pre- less encouraging (see Stielow et al. 2015), most likely due to viously described Lasiodiplodia species are in fact hybrids. The a saturation of informative sites which decreases phyloge- authors advocate the use of at least four gene loci to judge netic information in that apparent distances largely underes- whether a species could be recognised as distinct. Likewise, timate the real genetic distances (Philippe et al. 2011). Rodrıguez-Galvez et al. (2017) identified a putative hybrid be- tween L. citricola and L. parva during their survey of Lasiodiplo- dia species associated with dieback of mango in Peru. Conclusions

Gene regions at family and genus level There is an important distinction between ‘describing’ and ‘delimiting’ species. DNA phylogeny seeks more to aid in delim- e In the present study a combination of the LSU and rpb2 gene iting species to highlight genetically distinct groups exhibit- regions proved to be useful loci to be employed at family level ing levels of sequence divergence suggestive of a new taxon. in Botryosphaeriales; the combination of these two loci pro- The combination of molecular support with morphology has vided phylogenies similar to those of the four-gene, five- generally proven helpful to describe these new taxa (Crous gene and six-gene phylogenies of Liu et al. (2012), Phillips et al. 2015a,b,c,d). In Botryosphaeriales, taxon circumscription et al. (2013) and Slippers et al. (2013), respectively. Further- has suffered from insufficient taxon sampling, and too few in- more, these gene regions also proved to be highly suited for formative loci. In the present study, the combination of LSU- the delineation of genera within the respective families, lead- rpb2 has proven efficient at family and generic level. In addi- ing to the introduction of one new genus. At species level, tion, rpb2 has also proven to work well for species definition, a combination of at least the ITS and tef1 gene regions proved in combination with ITS, tef1 and tub2. With a robust back- adequate for species recognition in many genera, but for gen- bone phylogeny for Botryosphaeriales, it will in future be easier era such as Diplodia, Neofusicoccum, and Lasiodiplodia, addi- to delimit novelties at species, genus and family level. This ap- tional loci such as rpb2 and tub2 are required to establish proach again highlights the importance of culture collections a robust phylogeny. Based on the derived phylogenies and as treasure troves of undescribed fungal biodiversity. morphological examinations of the strains investigated, sev- eral taxonomic novelties were introduced. Conflicts of interest Different combinations of loci have in the past been used in Botryosphaeriales to resolve its taxonomy at family level. The authors report no conflicts of interest. The authors alone Schoch et al. (2006) employed SSU-LSU-tef1-rpb2, while are responsible for the content and writing of the paper. Minnis et al. (2012) used SSU-ITS-LSU-rpb1, Wikee et al. (2013) used ITS-LSU-actA-tef1-gpdh, and Slippers et al. (2013) used SSU-LSU-ITS-tef1-tub2-mtSSU. From those studies, a trend Appendix A. Supplementary data was noticeable that with an increasing number of strains and gene markers, more families, genera and species could Supplementary data related to this article can be found at be resolved in Botryosphaeriales. However, LSU proved to be http://dx.doi.org/10.1016/j.funbio.2016.11.001. the only gene region that was consistently used in all studies to introduce novel families in the order, and this also proved references to be the case in other families recently introduced in Dothideo- mycetes (e.g. Quaedvlieg et al. 2013; Crous et al. 2015a; Tanaka et al. 2015). In contrast, gene regions such as ITS, tef1 and tub2 that have several introns, are difficult to align within Abdollahzadeh J, Javadi A, Zare R, Phillips AJL, 2014. A phyloge- netic study of Dothiorella and Spencermartinsia species associ- the order (Crous et al. 2015d). These aspects have led to the un- ated with woody plants in Iran, New Zealand, Portugal and certain taxonomic placement of taxa in several studies. For in- Spain. Persoonia 32:1e12. stance Pseudofusicoccum clustered outside the Aderhold R, 1905. Einige neue Pilze. Arbeiten aus der Kaiserlichen Botryosphaeriaceae in the studies of Hyde et al. (2013) and Biologischen Anstalt fur€ Land-und Forstwirtschaft 4: 461e463. Wijayawardene et al. (2014), but basal in Botryosphaeriaceae in Ahmad S, 1971. Contributions to the fungi of West Pakistan XI. e other studies (Slippers et al. 2013; Phillips et al. 2013). In the Biologia 17:67 85. Schizo- present study, an LSU-rpb2 dataset was generated for phyloge- Alvarez LV, Groenewald JZ, Crous PW, 2016. Revising the parmaceae: Coniella and its synonyms Pilidiella and Schizoparme. netic comparisons. Since both loci represent slowly evolving Studies in Mycology 85:1e34. genes compared to the intron-rich protein-coding genes often Alves A, Linaldeddu B, Deidda A, Scanu B, Phillips A, 2014. The com- used in previous studies, it proved easier to generate a robust plex of Diplodia species associated with Fraxinus and some other alignment for families within the order. Furthermore, rpb2 has woody hosts in Italy and Portugal. Fungal Diversity 67:143e156. 344 T. Yang et al.

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