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Phylogeny and taxonomy of the genus Tubakia s. lat.

The genus Tubakia is revised on the basis of morphological and phylogenetic data. The phylogenetic affinity of Tubakia to the family Melanconiellaceae (Diaporthales) was recently postulated, but new analyses based on sequences retrieved from material of the type species of Tubakia, T. dryina, support a family of its own, viz. Tubakiaceae fam. nov. Our phylogenetic analyses revealed the heterogeneity of Tubakia s. lat. which is divided into several genera, viz., Tubakia s. str., Apiognomonioides gen. nov. (type species: Apiognomonioides supraseptata), Involutiscutellula gen. nov. (type species: Involutiscutellula rubra), Oblongisporothyrium gen. nov. (type species: Oblongisporothyrium castanopsidis), Paratubakia gen. nov. (type species: Paratubakia subglobosa), Racheliella gen. nov. (type species: Racheliella wingfieldiana sp. nov.), Saprothyrium gen. nov. (type species: Saprothyrium thailandense) and Sphaerosporithyrium gen. nov. (type species: Sphaerosporithyrium mexicanum sp. nov.). Greeneria saprophytica is phylogenetically closely allied to Racheliella wingfieldiana and is therefore reallocated to Racheliella. Particular emphasis is laid on a revision and phylogenetic analyses of Tubakia species described from Japan and North America. Almost all North American collections of this genus were previously referred to as T. dryina s. lat., which is, however, a heterogeneous complex. Several new North American species have recently been described. The new species Sphaerosporithyrium mexicanum, Tubakia melnikiana and T. sierrafriensis, causing leaf spots on several oak species found in the North-Central Mexican state Aguascalientes and the North-Eastern Mexican state Nuevo León, are described, illustrated, and compared with similar species. Several additional new species are introduced, including Tubakia californica based on Californian collections on various species of the genera Chrysolepis, Notholithocarpus and Quercus, and T. dryinoides, T. oblongispora, T. paradryinoides, and Paratubakia subglobosoides described on the basis of Japanese collections. Tubakia suttoniana nom. nov., based on Dicarpella dryina, is a species closely allied to T. californica and currently only known from Europe. Tubakia dryina, type species of Tubakia, is epitypified, and the phylogenetic position and circumscription of Tubakia are clarified. A revised, supplemented key to the species of Tubakia and allied genera on the basis of conidiomata is provided.


INTRODUCTION
Tubakia species are endophytes in leaves and twigs of many tree species, but can also cause conspicuous leaf symptoms as plant pathogens (Cohen 1999, Gonthier et al. 2006, Hashizume et al. 2008, Sieber 2007. Obvious leaf spots are dotted with minute punctiform conidiomata (pycnothyria) composed of convex scutella with radiating threads of cells fixed to the substratum by a central columella, mostly surrounded by a sheath of small fertile cells that give rise to one-celled, phialidic conidiogenous cells. The conidia are globose, subglobose, ellipsoid, broad ellipsoid-obovoid to subcylindrical or somewhat irregular in shape, aseptate, hyaline, subhyaline to pigmented. The conidiomata and the conidia are distinct and readily allow identification to generic level. Other fungal fruiting structures including crustose-pycnidial conidiomata on overwintering twigs that open by lateral to irregular dehiscence or sporodochia formed on leaf veins (Holdenrieder & Kowalski 1989, Harrington Editor-in-Chief Prof. dr P.W. Crous, Westerdijk Fungal Biodiversity Institute, P.O. Box 85167, 3508 AD Utrecht, The Netherlands. E-mail: p.crous@westerdijkinstitute.nl et al. 2012) may also be formed and have been described for T. dryina and T. iowensis. Some species produce a second type of much smaller conidia (microconidia), either in "normal" pycnothyria or in separate, mostly smaller pycnothyria. Reports of the occurrence and sporulation of Tubakia dryina as a saprobe on necrotic tissue caused by other fungi (Munkvold & Neely 1990) are unconfirmed and need further study and clarification.
Tubakia was previously assigned to the Diaporthales within Ascomycota (Yokoyama & Tubaki 1971, Yun & Rossman 2011. In a more detailed study on its phylogenetic affinity and position in the hierarchical system of the Ascomycota, Senanayake et al. (2017) placed Tubakia in the newly introduced family Melanconiellaceae. The sexual morph of "T. dryina" has been referred to as Dicarpella dryina (Belisario 1991). However, D. dryina is not the type species of Dicarpella, and at least one species, Dicarpella quercifolia was linked to Mastigosporella hyalina (≡ Harknessia hyalina; Barr 1979, Nag Raj & Di Cosmo 1981, Rossman et al. 2015. Dicarpella quercifolia, together with D. georgiana, were subsequently reallocated to the genus Wuestneiopsis (Reid & Dawsett 1990). The relation and synonymy of Dicarpella and Tubakia are, however, unclear. Clarification requires phylogenetic data for D. bina, the type species of this genus, which is only known from the type collection. Saccardo (1913) introduced the genus Actinopelte for A. japonica, a scutellate fungus found in Japan on Castanea crenata (= C. pubinervis). Saccardo (l.c.) confused the large conidia of this species with asci, which was clarified and corrected by Theissen (1913: 509) who provided a detailed discussion, description, and illustration (Theissen 1913: 508, fig. VI) of A. japonica. Höhnel (1925) revisited Actinopelte, added a new species, A. americana, and introduced the new combination A. dryina, based on Leptothyrium dryinum. Yokoyama & Tubaki (1971) discussed the history of this genus in detail, published results of comprehensive examinations of Japanese collections in vivo and in vitro, and described A. castanopsidis, A. rubra, and A. subglobosa based on Japanese collections. Since Saccardo's Actinopelte turned out to be illegitimate (Art. 53.1, later homonym of Actinopelte Stitzenb. 1861), Sutton (1973) introduced the replacement name Tubakia and reallocated all species recognised and treated in Yokoyama & Tubaki (1971) to this genus. Three additional Tubakia species have subsequently been described: T. seoraksanensis (Yun & Rossman 2011), T. iowensis (Harrington et al. 2012), and T. chinensis (Braun et al. 2014). So far, all European collections in the genus Tubakia have been assigned to a single species, T. dryina, whereas in Asia this genus has a much higher degree of species diversity. Additional undescribed species from Asia were predicted, and were detected in the course of the present studies. Braun et al. (2014) described and illustrated T. chinensis on Castanea henryi, a species with very large pycnothyria (135-200 µm diam), and globose to subglobose conidia. Based on its very small scutella, Braun et al. (2014) described and illustrated a second Tubakia species on Castanea henryi in China, tentatively referred to as Tubakia sp. The recently introduced Tubakia thailandensis (Senanayake et al. 2017) is morphologically very close to the Chinese collection on Castanea henryi and could be conspecific. According to Harrington et al. (2012), Japanese specimens referred to as T. dryina in Yokoyama & Tubaki (1971) do not belong to T. dryina s. str. The phylogeny of the Asian species of Tubakia has not yet been determined. Boroń & Grad (2017) examined numerous Polish strains referred to as T. dryina and found two different ITS groups designated as haplotypes. The diversity of Tubakia species in North America is not well known, although it is likely to contain more than just the single species listed in various publications as T. dryina. North American collections assigned to T. dryina are undoubtedly heterogeneous, i.e., T. dryina in North America is a complex of cryptic species (Harrington et al. 2012). An important contribution to resolve problems around the diversity of the T. dryina complex in North America has recently been published by Harrington & McNew (2018), including several new species based on US collections. Several Tubakia samples recently collected in Mexico on endemic oaks appear to be morphologically similar to T. dryina but are sufficiently distinct to suggest that they are undescribed species. A Tubakia causing serious oak diseases in California also appears to represent an additional undescribed species. Therefore, the present study presents a comprehensive examination and revision of Tubakia spp. in North America and worldwide, based on in vivo and in vitro morphological analyses as well as phylogenetic data. This work has been accomplished in collaboration by an international group of mycologists and phytopathologists from Asia, Europe and North America.

Isolates
Isolates included in this study were obtained from symptomatic leaves of diverse hosts, and identified as species of Tubakia based on primarily the presence of pycnothyrial conidiomata and aseptate conidia. In addition, several isolates were obtained from the culture collection of the Westerdijk Fungal Biodiversity Institute (CBS culture collection), in Utrecht, The Netherlands, the CDFA Plant Pathogen Collections (CDFA), Sacramento, USA, and from the working collection of Pedro Crous (CPC), housed at the Westerdijk Institute. Japanese isolates were obtained from NBRC (National Institute of Technology and Evaluation, Culture Collection Division, Chiba, Japan) and examined by C. Nakashima. Single conidial colonies were established from sporulating conidiomata on Petri dishes containing pine needle agar (PNA) (Smith et al. 1996), 2 % malt extract agar (MEA), potato-dextrose agar (PDA), and oatmeal agar (OA) (Crous et al. 2009b), and incubated at 22 °C under continuous nearultraviolet light to promote sporulation. Descriptions of culture characteristics on Czapek agar and potato sucrose agar refer to Yokoyama & Tubaki (1971). The cultures included in this study are listed in Table 1.
The generated sequences for each gene were subjected to megablast searches (Zhang et al. 2000) to identify closely related sequences in the NCBIs GenBank nucleotide database. For the LSU and rpb2 alignments, subsets of sequences from the alignments of Senanayake et al. (2017) and Fan et al. (2018) were used as backbones. For the tef1 alignment, tef1 sequences from Harrington & McNew (2018) were downloaded from GenBank or kindly supplied by the authors when not available in a public database. Loci were aligned with the online version of MAFFT v. 7 (Katoh & Standley 2013) after which the alignments were manually checked and improved where necessary using MEGA v. 7 (Kumar et al. 2016). Isolates missing both tef1 and tub2 sequences were excluded from the concatenated ITS/tef1/ tub2 alignments.
The phylogenetic methods used in this study included Bayesian analyses performed with MrBayes v. 3.2.6 (Ronquist et al. 2012) and Maximum Parsimony analyses performed with PAUP v. 4.0b10 (Swofford 2003) as explained in Videira et al. (2017). For the Bayesian analyses, trees were sampled every 100 generations, the temperature parameter was set to 0.25 and the stop value to 0.01. The Bayesian analyses were applied to the separate overview LSU and rpb2 alignments as well as the concatenated ITS/tef1/tub2 alignments and the expanded tef1 alignment after MrModelTest v. 2.2 (Nylander 2004) was used to determine the best nucleotide substitution model settings for each data partition.
All resulting trees were printed with Geneious v. 11.0.3 (http://www.geneious.com, Kearse et al. 2012) and the layout of the trees was done in Adobe Illustrator v. CC 2017. The alignments and respective phylogenetic trees were deposited in TreeBASE, study number 21897.

Morphology
All fungal structures were examined by means of light microscopy, using an Olympus BX50 or Zeiss Axio imager A1 microscope. Shear's liquid or distilled water and lactic acid were used as mounting media, and aniline blue (cotton blue) was used to stain colourless structures (columella, conidiogenous  (1995) structures, and conidia). If possible, measurements of 30 conidia and other structures were made at a magnification of ×1000, and the 95 % confidence intervals were determined (extreme values in parentheses). Cultures were studied on MEA, OA, PDA, and PNA. Colony colours were rated using the charts of Rayner (1970).

LSU phylogeny
The alignment contained 127 isolates representing a large majority of families known from sequence data belonging to Diaporthales. were mapped on the Bayesian tree as the second value ( Fig. 1; MP-BS >74 % shown). From the analysed characters, 481 were constant, 63 were variable and parsimony-uninformative and 160 were parsimony-informative. A strict consensus tree was calculated from the equally most parsimonious trees and the branches were mapped with a thicker line on the Bayesian tree ( Fig. 1; Length = 790, CI = 0.376, RI = 0.841, RC = 0.316). The parsimony phylogeny showed the same terminal family clades as those presented in the Bayesian phylogeny ( Fig. 1); however, the order of the clades was different, for example, in the parsimony analysis Apoharknessiaceae was the sister clade to Tubakia and not Melanconiellaceae (data not shown, available in TreeBASE). Twenty-one families are represented in the phylogenetic tree ( Fig. 1). The majority of families were supported by high posterior probability or bootstrap support values. However, Stilbosporaceae was only fully supported by the Bayesian analysis whilst Harknessiaceae did not receive significant support from either the Bayesian or the maximum parsimony analysis. Cryphonectriaceae and Melanconiellaceae each received moderate Bayesian posterior probability support but no maximum parsimony bootstrap support. The clade containing the Tubakia sequences was fully supported in both the Bayesian and maximum parsimony analyses and a novel family is proposed for this genus below. The node connecting Melanconiellaceae and Tubakiaceae was only fully supported in the Bayesian analysis but not supported in the maximum parsimony analysis. A sequence of the ex-type culture of "Greeneria" saprophytica is also included in this clade; this species was described by Tangthirasunun et al. (2014) but no Tubakia sequences were included in their phylogenetic tree. Our phylogenetic tree shows that this species has to be excluded from Greeneria and is better accommodated in Tubakiaceae. Supported by the isolated position in the LSU tree within the Tubakiaceae but outside of Tubakia s. str. and distant from all other genera of the family, this species warrants a genus of its own. Phylogenetic signals of LSU are low within Diaporthales in general and its families. Therefore, phylogenetic analyses using rpb2 sequences were also performed to distinguish whether the family Tubakiaceae contains a single genus, Tubakia, or several genera, but even the overall topology of the LSU tree suggests the heterogeneity of Tubakia s. lat.

rpb2 phylogeny
The alignment contained 130 isolates representing a large majority of families known from sequence data belonging to Diaporthales. A strain of Sordaria fimicola (CBS 723.96, GenBank DQ368647.1; Sordariales) was used as outgroup. The final alignment contained a total of 785 characters used for the phylogenetic analyses, including alignment gaps. MrModelTest recommended that the Bayesian analysis should use dirichlet base frequencies and the GTR+I+G model. The Bayesian analyses generated 6 742 trees from which 5 058 trees were sampled after 25 % of the trees were discarded as burn-in. The posterior probability values (PP) were calculated from the 5 058 trees ( Fig. 2; first value: PP >0.74 shown). The alignment contained a total of 473 unique site patterns. The Maximum Parsimony (MP) analyses generated the maximum of 20 equally most parsimonious trees and the bootstrap support values (MP-BS) were mapped on the Bayesian tree as the second value ( Fig. 2; MP-BS >74 % shown). From the analysed characters, 313 were constant, 28 were variable and parsimony-uninformative and 444 were parsimony-informative. A strict consensus tree was calculated from the equally most parsimonious trees and the branches were mapped with a thicker line on the Bayesian tree ( Fig. 2; Length = 3 934, CI = 0.237, RI = 0.792, RC = 0.188). The parsimony phylogeny showed the same overall topology as that presented in the Bayesian phylogeny (Fig. 2); however, the order of the genera in Gnomoniaceae differed slightly between the analyses (data not shown, available in TreeBASE).
Twelve families are represented in the phylogenetic tree (Fig. 2). The majority of families was supported by high posterior probability and bootstrap support values. However, Melanconiellaceae and Tubakiaceae were both only fully or highly supported by the Bayesian analysis while the node connecting Melanconiellaceae and Tubakiaceae was only fully supported in the Bayesian analysis and supported with 90 % in the maximum parsimony analysis. The rpb2 sequences demonstrate a clear substructure in Tubakia s. lat. with highly supported lineages, and therefore several novel genera are introduced in the Taxonomy section below to accommodate those lineages.

Combined ITS/tef1/tub2 phylogeny -all species
It was not possible to generate all loci for all of the included isolates, mainly due to the fact that some loci failed to amplify for some isolates, even though several attempts were made to obtain a product suitable for sequencing. To reduce the amount of missing data in the alignment, such isolates were excluded from the analyses. The alignment contained 95 isolates representing Tubakia and allied taxa, and a strain of Melanconis groenlandica (CBS 116540; GenBank KU878552.1, KU878554.1 and KU878555.1, respectively ITS/tef1/tub2) was used as outgroup. The final alignment contained a total of 1 740 characters used for the phylogenetic analyses, including alignment gaps. The Maximum Parsimony (MP) analyses generated the maximum of 1 000 equally most parsimonious     trees, the first of which is shown in Fig. 3    proposed for ITS and HKY+I+G for tef1 and tub2. The Bayesian analyses generated 12 702 trees from which 9 528 trees were sampled after 25 % of the trees were discarded as burn-in. The posterior probability values (PP) were calculated from the 9 528 trees ( Fig. 3 Eighteen species lineages/clades are represented in the phylogenetic tree (Fig. 3). The majority of species were supported by high posterior probability or bootstrap support values, especially in the basal part of the tree (Fig. 3, part 1). However, the bottom half of the second part of the phylogeny was not as well resolved as the basal part of the phylogeny, with most of the phylogenetic signal coming from the tub2 sequences.
Overall, the same species clades/lineages are observed in the individual gene trees, although the order or basal organisation sometimes changed between the different loci (data not shown). ITS was the least successful in resolving species clades (for example T. paradryinoides/dryinoides and the T. suttoniana complex). In addition, two isolates of T. japonica (MUCC2297, MUCC2298) and one of T. seoraksanensis (CPC 26553) clustered in the T. dryina clade. The tef1 phylogeny could not resolve      proposed for ITS and HKY+I+G for tef1 and tub2. The Bayesian analyses generated 2 602 trees from which 1 952 trees were sampled after 25 % of the trees were discarded as burn-in. The posterior probability values (PP) were calculated from the 1 952 trees ( Fig. 4; first value: PP >0.74 shown). The alignment contained a total of 310 unique site patterns (ITS: 63, tef1: 149, tub2: 98). The Bayesian phylogeny supported the same terminal clades as those presented in the parsimony phylogeny ( Fig. 4; data not shown, available in TreeBASE) and the same species as described above for Fig. 3.

tef1 phylogeny
A tef1 phylogeny was also generated (Fig. 5) to enable a more direct comparison of the data in the present paper with the recently published data from Harrington & McNew (2018). The alignment contained 136 isolates and a strain of Melanconis groenlandica (CBS 116540; GenBank accession numbers listed in Table 1) was used as outgroup. The final alignment contained a total of 592 characters used for the phylogenetic analyses, including alignment gaps. The Maximum Parsimony (MP) analyses generated the maximum of 1 000 equally most Saprobic, endophytes in leaves and twigs and plant pathogens causing leaf spots and twig dieback. Asexual morphs: sporodochia, crustose to pustulate pycnidioid stromatic conidiomata and superficial scutellate pycnothyria. Conidiogenous cells monophialidic, colourless, often with collarettes. Conidia formed singly, mostly globose to broad ellipsoid-obovoid, aseptate, hyaline to pigmented, often with basal frill or truncate peg-like hilum. Sexual morphs: apiognomonia-and dicarpellum-like, diaporthaloid, dark, rostrate, ostiolate perithecial ascomata, with dark stromatic layers, polyascal; asci unitunicate, 8-spored; ascospores aseptate or with a single septum near the apex, hyaline.

Notes:
The clade containing all former Tubakia species was highly supported in both Bayesian and maximum parsimony analyses ( Fig. 1; PP = 1, MP-BS = 99 %). In addition, its sister relation changed between the two analyses; in the Bayesian analysis, it was sister to Melanconiellaceae and in the maximum parsimony analysis it was sister to Apoharknessiaceae. The branch separating the Tubakiaceae clade from its closest sister clade was longer than the branches separating several other families in the phylogeny, e.g. Melanconidaceae / Gnomoniaceae and Harknessiaceae / Schizoparmaceae (Fig. 1). Analyses of LSU and above all rpb2 data and corresponding trees clearly indicate the heterogeneity of the genus Tubakia s. lat. Some lineages are included that warrant the division of Tubakia s. lat. into several genera. Etymology: Composed of the name of the genus Apiognomonia and -oides (resembling), referring to the similarity between the new genus and the latter one.

Editor-in-Chief
Prof. dr P.W. Crous, Westerdijk Fungal Biodiversity Institute, P.O. Box 85167, 3508 AD Utrecht, The Netherlands. E-mail: p.crous@westerdijkinstitute.nl Notes: Kaneko & Kobayashi (1984) introduced the name Apiognomonia supraseptata on the basis of ascomata formed on leaves of Quercus glauca incubated in a humid petri dish. Conidia were not formed. An ex-type strain (ATTC 58737) was used to retrieve a LSU rDNA sequence (GenBank AF277127, Zhang & Blackwell 2001) which was used by Harrington & McNew (2018) as basis to transfer A. supraseptata to Tubakia. They classified the mycelial colonies in culture to be tubakia-like. According to Kaneko & Kobayashi (1984), Apiognomonia has twocelled ascospores in which the top cell is larger than the bottom cell, but in A. supraseptata the top cell is smaller. Harrington & McNew (2018) postulated that A. supraseptata represents the only clearly demonstrated sexual morph of Tubakia. The allocation of Apiognomonia supraseptata to Tubakiaceae is reasonable and could be confirmed in our own analyses ( Fig. 1). Apiognomonia supraseptata is not included in the rpb2 tree due to lack of sequence data, but in the LSU (Fig. 1) and ITS trees (not shown) this species clusters distantly from all other lineages outside of the Tubakia s. str. clade, i.e., this species cannot be maintained in Tubakia s. str. Owing to its isolated position in the LSU tree within the Tubakiaceae, but distant from all other genera of this family, Apiognomonia supraseptata is best accommodated in a genus of its own. A. supraseptata and the sexual morph of Tubakia suttoniana have various morphological characters in common (rostrate perithecia, unitunicate 8-spored asci, colourless conidia), but A. supraseptata differs in forming uniseptate ascospores with a septum near the apex.
Notes: Records from Korea on Quercus serrata date from Lee et al. (1991) and Cho & Shin (2004).

Type species: Actinopelte castanopsidis
Genus of Tubakiaceae. Living as endophyte in leaves. Mycelium internal, hyaline, and external, pigmented. Asexual morphs forming crustose conidiomata on the surface of leaf litter and superficial pycnothyria on brown, necrotic areas on leaves. Pycnothyria usually circular or subcircular when viewed from above, superficial, easily removable, scutellate, fixed to the leaf by a central columella; scutellum convex to flattened, often recurved at the edge, membranous, dense, compact when young, later loose at the margin, outline regular, circular to subcircular, composed of hyphal strands, mostly branched, thickwalled, pigmented, margin often recurved at the edge, ultimate tips of the hyphal strands obtuse to rounded; conidiophores reduced to conidiogenous cells, obclavate, hyaline to pigmented, arising from small, colourless fertile cells around the central pycnothyrial columella; conidiogenous cells phialidic, sometimes forming indistinct periclinal thickenings or annellations; conidia formed singly, oblong to oblong-ellipsoid, wall thin to somewhat thickened, hyaline, smooth; microconidia not observed.
Etymology: Composed of "para-" (next to, near) and Tubakia (referring to the similarity with the latter genus).
Genus of Tubakiaceae. Living as endophyte in leaves. Mycelium internal, hyaline, and external, pigmented. Asexual morphs forming crustose conidiomata on shed leaves (litter) and superficial pycnothyria on leaf spots of living leaves. Pycnothyria usually circular or subcircular when viewed from above, superficial, easily removable, scutellate, fixed to the leaf by a central columella; scutellum convex to flattened, often recurved at the edge, membranous, dense to looser towards the periphery, outline irregular, sometimes splitting with age, subcircular, composed of hyphal strands, mostly branched, thick-walled, pigmented, ultimate tips of the hyphal strands obtuse to pointed; conidiophores reduced to conidiogenous cells, subcylindrical, oblong-obclavate, hyaline to pale olivaceous brown or pale brown, arising from small, colourless fertile cells around the central pycnothyrial columella, conidiogenous cells phialidic; conidia formed singly, globose, subglobose to broad
Notes: Actinopelte subglobosa (≡ Tubakia subglobosa) and a new closely allied Japanese species cluster together, but outside of the Tubakia s. str. clade, i.e., these species do not belong to the Tubakia core species, so that they have to be excluded from Tubakia s. str. This lineage is fully supported in the Bayesian trees based on rpb2 and LSU (Figs 1, 2) and requires a genus of its own, which is described as Paratubakia. Actinopelte castanopsidis is allied to Paratubakia, but forms a separate single species lineage, treated as separate genus, Oblongisporothyrium gen. nov. The type species of the latter genus shares scutella with margins somewhat curved inwardly and colourless conidia with Paratubakia subglobosa, type species of Paratubakia, but differs in having oblong conidia (vs. globose to subglobose in T. subglobosa). Illustrations: Yokoyama & Tubaki (1971: 65, pl. 1G, 67, pl. 2E;69, pl. 3E;73, pl. 7E-H;76, pl. 10A-H).
Notes: Paratubakia subglobosa and P. subglobosoides sp. nov. are characterised by having obtuse to acute scutellum tips, resembling scutella of Tubakia dryina and T. iowensis, but they are morphologically readily distinguishable from the latter two species by their globose, subglobose to broad ellipsoid conidia. Furthermore, they are phylogenetically clearly distinct from and not closely allied to T. dryina (Figs 1-3, 5), i.e., they cluster outside of the Tubakia s. str. clade.
Etymology: Composed of the epithet of the comparable species, Paratubakia subglobosa, and -oides (similar to).
In vitro: On MEA with optimal growth at 20 °C, attaining 38-40 mm after 14 d, margin indefinite, at first pale olivaceous, reverse pale olivaceous brown. On the dried culture prepared from the culture NBRC 9343, abundant sporulation was observed. Hosts range and distribution: On Quercus glauca, Fagaceae, Asia (Japan).

Type species: Racheliella wingfieldiana Crous & U. Braun.
Notes: A new tubakia-like species found in South Africa on Syzygium guineense clusters together with Greeneria saprophytica, described from Thailand on old shed leaves of Syzygium cumini (Fig.  1). The two taxa are undoubtedly closely allied but unfortunately no culture or DNA of Greeneria saprophytica was available to generate more sequence data for comparison. However, based on ITS, these two species are 464/553 (84 %) similar. They cluster outside of Tubakia s. str. and distant from all other recognised genera of Tubakiaceae, suggesting that they pertain to a genus of their own. The pycnothyria of the type species are reminiscent of conidiomata of other genera of Tubakiaceae. Colourless conidia are in line with other tubakioid genera excluded from Tubakia s. str. The stromatic conidiomata formed by Greeneria saprophytica clearly differ from crustose conidiomata of all other tubakioid genera in having branched, septate conidiophores with phialidic conidiogenous cells provided with flared, conspicuous collarettes with serrate margin.  fig. 2, 280, fig. 3).
In vitro: Colonies on PDA medium to dark brown from above and reverse, with medium to dense surface mycelium, flat, irregular to rhizoidal, with undulate to lobate margin, attaining 25 mm diam in 7 d at 27 °C; sporulating after 14 d. Host range and distribution: Only known from the type collection.
Notes: The position of Greeneria saprophytica in the consensus tree published in Tangthirasunun et al. (2014) clearly suggested that this species and G. uvicola, the type species of Greeneria, are not congeneric. Tubakia species were not included in the phylogenetic analyses which led to an allocation of the new species collected in Thailand on dead leaves of Syzygium cumini to Greeneria. In the current analyses, the sequences retrieved from the ex-type culture of G. saprophytica cluster within the Tubakiaceae, but outside of the Tubakia s. str. clade, i.e., this species does not belong to the latter genus (Fig. 1). A collection of "Tubakia sp." found in South Africa on Syzygium guineensis is closely allied and clusters together with G. saprophytica and the two species require a genus of their own. At first glance, Greeneria saprophytica seems to be morphologically quite distinct from species of Tubakia s. lat. However, the conidiomata of the latter species are reminiscent of those of Tubakia californica, a species that does not form any pycnothyria but only stromatic conidiomata, usually (50-)80-200(-220) µm diam, composed of basal stromatic layers and phialidic conidiogenous cells. The size of the conidiomata in Greeneria saprophytica and Tubakia californica is comparable. Aseptate pigmented conidia are common in Tubakia s. lat. A clear difference between Greeneria saprophytica and all species hitherto assigned to Tubakia lies in the characters of the conidiophores, which are longer, septate and sometimes branched in G. saprophytica [vs. shorter, unbranched, usually aseptate (conidiophores reduced to conidiogenous cells) in species of all other genera of Tubakiaceae]. In addition, the conidiogenous cells have flared collarettes with serrate margins. G. saprophytica is phylogenetically allied to Tubakia thailandensis, which differs in forming small pycnothyria, aseptate, unbranched conidiophores, and subglobose, colourless conidia. Stromatic conidiomata are not developed. G. saprophytica found in Thailand on Syzygium cumini was assigned to Greeneria (Melanconiellaceae) owing to morphological similarities of the conidiomata, but since this species pertains to the Tubakiaceae it has to be excluded from Greeneria and is reallocated together with a new species on Syzygium from South Africa to the new genus Racheliella.
In vitro: Colonies flat, spreading, with sparse aerial mycelium and feathery, lobed margins, reaching 60 mm diam after 2 wk. On MEA, PDA and OA surface isabelline with cinnamon spore masses, reverse isabelline. On PDA, surface olivaceous to isabelline, reverse honey to isabelline. Genus of Tubakiaceae. Foliicolous, saprobic (perhaps also living as endophyte). Mycelium internal. Asexual morph forming superficial pycnothyria on dead leaves; pycnothyria usually circular or subcircular when viewed from above, superficial, easily removable, scutellate, fixed to the leaf by a central columella; scutellum convex, membranous, dense, outline irregular, composed of hyphal strands, arising from a small central disc, cells thick-walled, pigmented, ultimate tips of the hyphal strands obtuse; conidiophores reduced to conidiogenous cells, small, arising below the scutellum, phialidic, with a minute collarette and wide periclinal thickening; conidia formed singly, globose, subglobose, wall thickened, hyaline, smooth; microconidia not observed.
Notes: Tubakia thailandensis was not included in the phylogenetic analyses used to generate Fig. 2 as only ITS and LSU sequence data are available for it. Based on a megablast search against the ITS sequences used in the combined alignment, the closest match was T. dryinoides with 89 % (404/452). It is also not included in the RPB2 tree, but its position in the LSU tree and a comparision with other species included in the LSU as well as the RPB2 tree clearly indicate that T. thailandensis clusters within the Tubakiaceae, but outside of the Tubakia clade, i.e., this species has to be excluded from Tubakia s. str. It forms a separate lineage in the LSU tree close to Racheliella, which justifies the classification as genus of it own. Pycnothyria of Saprothyrium are well characterised by having obtuse outer tips of the scutellum strands combined with globose-subglobose colourless conidia, and shares these traits with the genus Sphaerosporithyrium, which is, however, phylogenetically distant, clustering in basal position to Tubakia s. str. Colourless globose-subglobose conidia formed in pycnothyria distinguish Saprothyrium from all species of Tubakia s. str. in terms of morphology. Description in vivo: Saprobic on dead leaves. Conidiomata 40-50 µm high, 50-75 µm wide, pycnothyria with radiate scutella, scattered to gregarious, superficial on the substratum. Scutella convex, brown to dark brown, thick-walled cells, radiating from a central point, ultimate tips obtuse, not pointed. Conidiophores short, forming under the developing scutella. Conidiogenous cells 5-10 µm high, 2-4 µm wide, phialidic, with a minute collarette and wide periclinal thickening. Conidia globose to subglobose, 10-12.5 × 7.5-8.5 µm (on average 11.3 × 8.1 µm, n = 20), smooth, hyaline, thick-walled.
In vitro: Mycelium on PDA white when young, dark green, light grey to black from above and reverse when aged, with medium mycelium, flat, rhizoid to irregular form, lobate margin, and attaining a diam of 46 mm in 7 d at 27 °C. Notes: Saprothyrium thailandense is morphologically close to "Tubakia sp." on Castanea henryi in China described and illustrated in Braun et al. (2014). The relation between S. thailandense, collected as saprobic fungus on dead leaves in Thailand, and Tubakia sp., found in China on distinct leaf spots on Castanea henryi, is unclear. It is also unclear if the leaf spots associated with "Tubakia sp." in China are truly caused by this tubakia-like fungus. The leaves were covered with lesions of several other fungi, including Tubakia chinensis, i.e., it is possible that it represents an endophyte or saprobic species forming conidiomata on necrotic tissue caused by other fungi. Culture and sequence data are not available for the Chinese Tubakia, although urgently necessary for a comparison of the two taxa. Etymology: Composed of "sphaero-" (globose), "spori-" (spore) and "-thyrium (referring to pycnothyrium, the special conidioma type). Genus of Tubakiaceae. Living as endophyte in leaves and causing leaf spots. Mycelium internal. Asexual morph forming superficial pycnothyria on leaf spots of living leaves. Pycnothyria usually circular or subcircular when viewed from above, superficial, easily removable, scutellate, fixed to the leaf by a central columella; scutellum flattened-convex, membranous, compact, dense, uniformly pigmented, pale to medium dark brown, or with a darker central zone, outline regular, subcircular, composed of hyphal strands, mostly branched, thick-walled, pigmented, ultimate tips of the hyphal strands obtuse or even truncate; conidiophores usually reduced to conidiogenous cells, conical, ampulliform-cuspidate, subcylindrical, delicate, not very conspicuous, arising from the underside of the scutella, around the upper portion of the columella, radiating, conidiogenous cells phialidic; conidia formed singly, globose, subglobose to broad ellipsoid-ovoid, thin-walled, hyaline or with a very pale greenish to olivaceous tinge, smooth; microconidia not observed.
Notes: A new undescribed tubakia-like species was collected in Mexico on Quercus eduardi. Sequences retrieved from the new Mexican species cluster in all phylogenetic trees close to but always outside of the Tubakia s. str. clade (Figs 1-3, 5), suggesting that this species cannot be assigned to the latter genus, i.e., it warrants a genus of its own. Owing to the conidial shape and colour, the new genus Sphaerosporithyrium is morphologically comparable with Paratubakia. Pycnothyria formed by species of the latter genus differ, however, in having pointed tips of hyphal scutellum strands. Colourless globose-subglobose conidia clearly distinguish Sphaerosporithyrium from all species of Tubakia s. str. Etymology: Named after Mexico, the country where this species was collected.

Microconidia not observed in vivo.
In vitro: On MEA at 22 °C colonies attaining 67-69 mm diam after 16 d, margin undulate, with concentric rings of aerial mycelium, center white, reverse colourless, without sporulation. Notes: Sphaerosporithyrium mexicanum is well-characterised by a combination of globose, subglobose to broad ellipsoid-ovoid conidia and pycnothyria with obtuse to truncate tips of the radiating scutellum threads. The conidial shape is reminiscent of Paratubakia subglobosa conidia, but the latter species, known from Asia on Quercus glauca (≡ Cyclobalanopsis glauca), differs in having scutella with pointed tips of radiating hyphal threads. The status of S. mexicana as species in its own right is fully supported in the phylogenetic trees (Figs 1-3 Genus of Tubakiaceae. Living as endophytes in leaves and twigs, and pathogenic, forming distinct leaf lesions. Asexual morphs forming sporodochial or crustose to pustulate, pycnidioid, stromatic, unilocal conidiomata on petioles and twigs, erumpent, dehiscing by irregular fissures, and superficial pycnothyria on living, faded or shed leaves, without distinct symptoms on living leaves or forming distinct leaf spots. Pycnidia usually circular or subcircular when viewed from above, superficial, easily removable, scutellate, fixed to the leaf by a central columella; scutellum composed of loose to dense hyphal strands, mostly branched, thick-walled, pigmented, margin compact or outer portions of the radiating hyphal strands looser to free, tips rounded, truncate or pointed, margin usually not recurved; conidiophores reduced to conidiogenous cells, usually subcylindrical-conical, lageniform, hyaline to pale brown, arising from small, colourless fertile cells around the upper part of the central pycnothyrial columella, conidiogenous cells phialidic, percurrently proliferating, sometimes forming indistinct periclinal thickenings or annellations (collarettes); conidia formed singly, globose to broad ellipsoid-obovoid, sometimes subcylindrical or somewhat irregular, wall thin to somewhat thickened, smooth to faintly rough, hyaline to pigmented, apex rounded, base rounded to attenuated, sometimes with distinct frill or peg-like basal hilum. Sexual morph: Diaporthaloid, dicarpella-like, forming dark stromatic pseudoparenchymatic layers with pigmented, dark perithecia on fallen overwintered leaves, rostrate, beak short, usually lateral-eccentric, slightly protuberant, ostiolate, ostiole periphysate, peridium variable in thickness, paler than stromatic layers, polyascal; asci unitunicate, 8-spored, oblong-ellipsoid, stalk short to oblong, ascal apex with two refractive conoid structures, asci deliquescing at maturity; paraphyses lacking; ascospores more or less uniseriate, becoming irregularly biseriate, one-celled, hyaline, ellipsoid to fusiform, often inequilateral or slightly curved, wall finely ornamented, content granular-guttulate. Putative sexual morph: Dicarpella dryina (see Tubakia suttoniana).
Notes: In its traditional circumscription, Tubakia s. lat. comprises a wide range of species with characteristic superficial conidiomata composed of radiating scutella, a basal columella fixing the scutellum to the leaf surface, and colourless phialidic conidiogenous cells arising from fertile cells around the upper part of the columella bearing solitary, one-celled, colourless to pigmented conidia. However, results of phylogenetic analyses revealed the heterogeneity of Tubakia s. lat. and led to a new circumscription of Tubakia s. str. (emend.) which forms a separate well-supported clade within the family Tubakiaceae evident in the rpb2 and LSU trees (Figs 1, 2). Species clustering outside of the Tubakia clade, belonging to other lineages, are excluded and assigned to other (new) genera. Tubakia species may form different types of asexual morphs. Pycnothyria with typical scutella are characteristic, diagnostic and commonly formed. In addition, sporodochial conidiomata composed of clusters of conidiogenous cells may be developed, e.g. on leaf veins formed by T. iowensis. Crustose or pustulate pycnidioid conidiomata may be formed by several Tubakia species, including T. californica, T. dryina, and T. iowensis. In T. californica they are the only hitherto known fructification. They are mostly formed on petioles and leaf blades (often on and close to veins) of old necrotic leaves, either shed (litter) or still attached as in the case of T. californica. The classification of these conidiomata caused some uncertainty and confusion. Holdenrieder & Kowalski (1989) designed these conidiomata as "pycnidial" although they clearly described non-ostiolate conidiomata dehiscing by irregular rupture. Harrington et al. (2012) used the general term "conidioma" and added "pycnothyrium" in brackets. These conidiomata cannot be referred to as pycnidia owing to lacking ostioles, but due to stromatic wall layers and a dehiscence by irregular fissures they should rather be classified as stromatic. Description in vivo: Leaf and twig endophyte, also causing leaf spots, amphigenous, subcircular to angular-irregular, 1-18 mm diam, brown, with narrow darker margin, dark violet, brown to blackish or along necrotic leaf veins. Conidiomata (pycnothyria) amphigenous, mainly hypophyllous, superficial, scattered to gregarious, dark brown to blackish, 40-115 µm diam, about 30 µm high, circular in outline, scutellate, fixed to the leaf surface by a central columella. Scutella somewhat convex, centre with a colourless or pale disc, about 5-30 µm diam, surrounding small cells, about 2.5-3 µm diam, giving rise to radiating threads of hyphal cells, simple to often 1-4 times bifurcating, cells 5-20 µm long and 2-5 µm wide, wall about 1 µm thick, ends of the radiating threads simple or furcate, tips obtuse; central columella below the scutellum parenchymatic, colourless or pale, about 20 µm high and 25-30 µm broad; Conidiophores reduced to conidiogenous cells, delicate, formed around the upper part of the columella. Conidia solitary, broad ellipsoid(-ovoid), 9-13(-14) × 6-8.5 µm, aseptate, subhyaline or pale to medium olivaceous brown, wall thin (0.5-1 µm), smooth or almost so. Microconidia sometimes produced from same pycnothyrium as macroconidia or from smaller pycnothyria, hyaline, fusiform, curved, aseptate, (3.5-)4-7 × 2-3 mm. Crustose conidiomata forming in winter or spring, erumpent on twigs or inside of acorn caps, black, irregular in shape, 150-500 mm diam, covering layer composed of thick-walled cells, textura angularis, dehiscing marginally or by irregular fissures; conidiophores lining inside of crustose conidiomata; conidia thick-walled, hyaline to light brown, obovoid to ellipsoidal, aseptate, 9.5-14 × 5.5-7.5 mm.
In vitro: On MYEA with optimal growth at 25 °C, attaining a diam of 56 to 64 mm after 7 d, initially with a distinct ring of dense aerial mycelium, later developing concentric rings of fluffy white to grey aerial mycelium with wet conidial masses that are initially hyaline, becoming olive green then black, coalescing into large areas. Conidia in culture abundant, thick-walled, smooth to finely varicose, hyaline to dark brown, obovoid to ellipsoidal, aseptate, 9-15(-16.5) X (4-)4.5-7.5(-8) mm. Notes: Höhnel (1925) described Actinopelte americana based on a North American collection on Quercus coccinea and distinguished his new species from T. dryina by having smaller conidia. Type material of this species has been examined and shown to be morphologically rather similar to T. dryina, but distinguishable by having obtuse (non-pointed) ultimate tips of radiating hyphal scutellum threads. Attempts to locate the particular specimen of Fungi Columb. 286 that Höhnel had examined failed. Therefore, Harrington & McNew (2018) designated a duplicate from the McClatchie Herbarium in NY as lectotype, which, however, contains pycnothyria of two Tubakia spp. associated with different necrotic spots. One of these Tubakia species has relatively small conidia, as described by Höhnel (1925) for A. americana, and the tips of the radiating scutellum strands are blunt (Harrington & McNew 2018, fig 2a,  b). The second type of pycnothyria on the lectotype material has scutella with acute tips and larger conidia (Harrington & McNew 2018, fig 2c), rather matching the wide concept of T. macnabbii (Harrington & McNew 2018). According to Harrington & McNew (2018), other duplicates of Fungi Columb. 286 in NY and ISC each contained leaves with two Tubakia species. In other duplicates, e.g., BPI 390073, 390075, only the small-spored Tubakia with obtuse tips of scutellum strands has been observed. The lectotypification proposed by Harrington & McNew (2018) is reasonable, follows and maintains Höhnel's original description. Harrington & McNew (2018) managed to extract DNA form the lectotype of A. americana and a generated sequence matched that of Tubakia sp. A (Harrington & McNew 2016;Harrington et al. 2012), which they have commonly isolated as a twig and leaf endophyte in Q. macrocarpa and other Quercus spp. in Iowa, often also associated with leaf spots and necrotic veins (Harrington & McNew 2018, fig 4).
Tubakia americana is phylogenetically (Figs 3, 5) closely related to T. dryina, and both species produce crustose pycnothyria on twigs (Harrington et al. 2012), but the two species are readily distinguishable by differences in the pycnothyrial scutella (tips of the scutellum strands obtuse in T. americana and pointed in T. dryina). In culture, the mycelium of T. americana is much lighter in colour than the dark mycelium of T. dryina (Harrington et al. 2012). Species of Quercus sect. Quercus (Q. bicolor, Q. macrocarpa and Q. robur) seem to be the primary hosts of T. americana in Iowa (Harrington et al. 2012). The "T. americana" ITS cluster published in Harrington et al. (2012) is divided in two groups -the upper one composed of North American sequences represents T. americana, and the lower one is composed of allied cryptic taxa from Eurasia. The Asian taxon in this group is described herein as T. dryinoides. The European sequences based on isolates from France might represent another cryptic species, but due to insufficient sampling and lacking ecological and morphological data we prefer to maintain them in T. dryinoides, at least tentatively. ITS sequences retrieved from leaves of Quercus robur and Fagus sylvatica in Poland (haplotype 2 in Boroń & Grad 2017) seem to belong to this cryptic European taxon and suggest that it might be rather common. fully defoliate in the fall. Symptomatic trees are primarily mature trees, approximately 25 years and older; dry, brown leaves from the previous seasons' growth remain attached on branches in the spring; symptom progression in the late summer and fall is variable depending on location; in the California foothills where the disease is prominent, the new distal growth appears healthy until late August when small, brown lesions develop on the leaves, predominantly on the abaxial side; irregular, brown lesions with chlorotic borders may also occur near the leaf margins; lesions enlarge over time, and by early September, the associated lateral leaf veins become brownish black, by late September, more than half of the leaf area on affected leaves is brown and discoloration of the veins extends to the midrib; in October, many of the affected leaves become dry and uniformly brown and most leaf veins turn black; small, black, embedded crustose Tubakia conidiomata with conidia develop in late September on symptomatic petioles and leaves, often associated with the veins, but also on the leaf blade. Conidiomata crustose, pycnidioid, stromatic, scattered to loosely aggregated, subepidermal, erumpent, at first "closed" (covered by a black stromatic layer), protruding portion hemispherical to depressed-convex (hourglass-shaped), dehiscing by irregular fissures, finally exposed, widely opened, subcircular to somewhat irregular in outline, usually (50-)80-200(-220) µm diam, wall about 10-20 µm thick, black, crustose covering layer composed of large cells, mostly 4-10 µm diam, with thin to thickened walls, 0.5-1 µm wide, pale to medium dark brown, dark brown in mass, rounded, oblong to angular-irregular in outline (textura globulosa to angularis), basal layer paler; conidiogenous cell lining the base of the conidiomata, colourless, thin-walled, attenuated towards the tip, about 10 × 3-4 µm, but early collapsing and lapsing (hence detailed observations and measurements not possible); conidia solitary, broad ellipsoid, ellipsoid-ovoid to short and broad subcylindrical, straight, rarely slightly curved or somewhat irregular in shape, both ends rounded to subtruncate, basal frills or truncate peg-like bases not observed, 8-15 × 4.5-7 µm, length/width ratio 1.4-2.3 (on average 1.8), at first subhyaline to pale greenish, later greenish, pale olivaceous to brownish, wall thin, to about 0.5 µm, smooth or almost so (light microscopy), microconidia not observed (pycnothyria with free radiating scutella not developed).
In vitro: Optimal growth at 25-30°C on MEA, colonies attaining 60 mm after 7 d, dingy white to pale yellow with regular margin, becoming yellowish grey with concentric rings in reverse, conidial formation not observed. On PDA, colonies initially creamy white, becoming greyish beige and forming olivaceous brown concentric rings in reverse, margin irregular to scalloped. Conidiomata brownish black, forming in concentric rings; conidia broadly ellipsoid, ellipsoid-ovoid, straight, initially subhyaline, later becoming olivaceous brown, 10-14 × 7-9 µm (average 11.9 × 8.0), length/width ratio 1.2-1.9 (average 1.5).  Notes: Tubakia californica is an endophyte found in California on several oak species and two additional fagaceous host species. It causes symptoms and lesions on the affected tree species, crustose, stromatic, pycnidioid conidiomata may be formed, but characteristic leaf spots spread over the whole leaf blade (comparable with those formed by T. dryina and most other Tubakia spp.) and pycnothyria are not developed or have at least not yet been observed. The leaf lesions caused by T. californica are rather reminiscent of those formed by T. iowensis. In October 2014, Tubakia was cultured from Q. kelloggii trees in California, El Dorado Co., with foliar symptoms similar to those seen in Groveland. Between 2012 and 2017, this fungus was also isolated from similar leaf lesions and twig dieback of other Fagaceae species throughout California including the counties of Contra Costa, Del Norte, Humboldt, Marin, San Luis Obispo, and Shasta. Overall, symptoms seem to develop later in the season in the Sierra Nevada foothill locations (Tuolumne Co. and El Dorado Co.) than in the other California counties. The elevations of the Groveland and the El Dorado Co. sites are 956 m and about 850 m respectively, while most of the other locations were at or near sea level. In addition to colder winters, both foothill locations leaf out later in the season, which may explain the difference in the onset of disease symptoms to the valley and coastal locations. Overall, disease severity was greater on trees in low areas and on branches in the lower portion of the canopy (unpublished observations).
Owing to lacking pycnothyria, a phylogenetic approach was required to evaluate the Californian taxon. Analyses of the sequence data and their position in the phylogenetic trees clearly favour a separate species. The T. californica clade is well supported in both combined analyses ( Illustration: Braun et al. (2014: 24, fig. 1).
Description in vivo: Leaf spots amphigenous, subcircular to angular-irregular, 0.5-5 mm diam, at first dingy greenish to greyish green, later brownish to dingy greyish brown, finally grey to greyish white, with narrow darker margin, brown to reddish brown, finally darker, often slightly raised or limited by veins. Conidiomata (pycnothyria) epiphyllous, small lesions with a single pycnothyrium, larger ones with up to 18 pycnothyria, punctiform, black, circular or subcircular when viewed from above, superficial, easily removable, scutellate, fixed to the leaf by a central columella. Scutella convex, 135-200 µm diam, membranous, somewhat translucent (conidia more or less

Editor-in-Chief
Notes: Tubakia dryina, originally described as leaf-spotting fungus on Quercus robur in Italy, was previously circumscribed and applied in a very broad sense comprising collections from Asia, Europe, and North America mainly on Quercus spp., but also on some additional fagaceous genera, e.g. on Castanea and Fagus spp., and in North America on host plants belonging to numerous other plant families (Glawe & Crane 1987, Yokoyama & Tubaki 1971 ILLS 32849, Q. macrocarpa, BPI 291913-391914, Q. marilandica, ILLS 29755, 30075, 32836, 32841, Q. nigra, BPI 391918, 840864, Q. phelos, BPI 391921, 391922, Q. rubra, BPI 391917, 391928, 391929, 390077-390081, 390084, 390085, 391925-391927, 803135, 863076, Q. shumardii, ILLS 117552, Q. stellata, BPI 391930, Q. velutina, BPI 390087, 390088, 391931, 391933-391936, 863077, Q. virginiana, BPI 391932, Toxicodendron radicans [≡ Rhus radicans], ILLS 30072, Ulmus alata, ILLS 29740. First molecular examinations and analyses of T. dryina s. lat. carried out by Harrington et al. (2012) suggested that this species represents a strongly heterogeneous complex comprising several cryptic species. Some of them have recently been described by Harrington & McNew (2018), and additional ones are introduced in the present work. Host range and distribution of T. dryina s. str. is insufficiently known and probably largely unrevealed due to its basically endophytic life strategy. Documented collections usually refer to material with distinct leaf spots and developed pycnothyria. Nevertheless, T. dryina seems to be a primarily European species with Quercus robur as principal host plant on which this fungus has probably been introduced to other regions of the world, including proven cases in New Zealand and North America (USA, Harrington & McNew 2018). In the eastern USA, T. dryina s. str. occurs also on Quercus alba and Q. macrocarpa (Harrington et al. 2012, Harrington & McNew 2018. Therefore, it is currently not possible to finally decide if T. dryina being a native North American species or an introduced neomycete. However, most North American Tubakia collections previously referred to as T. dryina pertain to other species, including T. americana, T. hallii, T. iowensis, T. liquidambaris, T. macnabbii, and T. tiffanyae, but Tubakia on numerous Quercus spp. and on numerous non-fagaceous hosts in North America has not yet been properly examined, i.e., identifications based on cultures and sequence analyses are still lacking. Heredia (1993) reported "T. dryina" from Mexico on Quercus germana and Q. sartorii. These records are doubtful and belong very probably to other Tubakia species (current analyses of Tubakia in Mexico revealed several endemic species -see Sphaerosporithyrium mexicanum, Tubakia melnikiana and T. sierrafriensis).
Collections of "T. dryina" from Asia do not belong to this species. Harrington et al. (2012) emphasised that Japanese specimens referred to as T. dryina in Yokoyama & Tubaki (1971) do not agree with the concept of T. dryina s. str., which could be confirmed in the course of our own re-examinations of the collections concerned (see T. dryinoides). Zahedi et al. (2011) reported and illustrated "Tubakia dryina" on Quercus castaneifolia from North Iran (Guilan Province), which is, however, quite distinct from true T. dryina s. str. (see notes under Tubakia sp. [Excluded, doubtful and insufficiently known species]). The identity of "T. dryina s. lat." on Quercus hartwissiana in Turkey (Huseyinov & Selçuk 2001) is unclear as well.
Boroń & Grad (2017) examined T. dryina in Poland and analysed the variation of ITS data of numerous isolates. Haplotype 1 in Boroń & Grad (2017) is representative for T. dryina s. str. The published results and analyses confirm T. dryina as common and widespread species on Quercus robur and Fagus sylvatica, at least in Poland. In a single case, they found this species on Tilia cordata (Tiliaceae), which needs, however, further research and confirmation. Haplotype 2 probably belongs to European sequences of T. dryinoides (s. lat.).
The conidiogenesis of Tubakia dryina was examined in detail by Jones & Holcomb (1978) and described to be phialidic, which was later confirmed in Glawe & Crane (1987). Taylor & Clarke (1996) described the formation of internal mycelium and conidial germination, and Taylor (2001) dealt with the ultrastructure of pycnothyria. Holdenrieder & Kowalski (1989) emphasized that T. dryina may live as endophyte in healthy leaves and twigs, and Boddy & Rayner (1984) isolated this fungus from living and dead twigs in the UK.
Etymology: Named after the morphological and phylogenetic affinity to Tubakia dryina.
In vitro: on MEA with optimal growth at 20° C, attaining 30-40 mm diam after 14 d, margin scalloped, at first creamy white, forming concentric rings of olivaceous mycelium, reverse greyish white, with olivaceous edge. Conidial formation not observed. Notes: Tubakia dryinoides is morphologically similar to T. dryina, at least in terms of characters of the pycnothyria, and was referred to as T. dryina in Yokoyama & Tubaki (1971). In contrast to genuine T. dryina collections, the conidia in T. dryinoides remain hyaline or subhyaline, at least until germination (see Yokoyama & Tubaki 1971). The phylogenetic analyses corroborated a close affinity of the Japanese collections to T. dryina but clearly suggested a separate species (Figs 3-5), which confirms the doubts upon the correct assignment of Japanese collections to T. dryina by Harrington et al. (2012). Tubakia dryinoides belongs to a cluster undoubtedly composed of several species, including T. paradryinoides, which is genetically clearly distinct from T. dryinoides and morphologically easily distinguishable by its much larger conidia, 14-21 × 10-15 µm. Tubakia americana (Figs 3-5) is another closely allied species. Harrington & McNew (2018) included ITS sequences belonging to T. dryinoides in their T. americana "clade", which is, however, heterogeneous and divided into at least two clades representing T. americana and T. dryinoides. Several Chinese strains, isolated from Quercus sp. (GenBank FJ598616), Linderia glauca (GenBank JF502454) and from an unknown host as endophyte (GenBank FJ025349), all unpublished, belong to this clade and may be T. dryinoides. The identity of European strains (CBS 329.75 and CPC 33586, Quercus spp., France) belonging to this cluster is still unclear, but may indicate the presence of an additional cryptic European species. The current sampling is, however, not sufficient for a final conclusion. Above all, the morphology of pycnothyria and the colour of conidia of the European taxon are unknown (conidia colourless in T. dryinoides). Therefore, the collections concerned are tentatively maintained in T. dryinoides. ITS sequences retrieved from leaves of Quercus robur and Fagus sylvatica in Poland ("T. dryina" haplotype 2 in Boroń & Grad 2016) seem to belong to this cryptic European taxon and suggest that it might be rather common, but pycnothyria and the conidial colour of the Polish collections were not described. Description in vivo: Conidiomata (pycnothyria) superficial, hypophyllous or epiphyllous on necrotic interveinal spots and along necrotic mid and lateral leaf veins. Scutella 45-155 µm diam, radiate, composed of a series of dark brown, thick-walled cells originating from a central cell, ending in blunt to acute tips. Sporodochia superficial with only a few radiating dark hyphae, mainly hypophyllous, on necrotic interveinal tissues and along leaf veins. Conidiophores reduced to conidiogenous cells, on underside of scutella or on top of sporodochia. Conidia hyaline, turning light brown with age, smooth to slightly varicose, aseptate, obovoid to ovoid, 9.5-14.5(-16) × 7.5-10(-11) µm (mean 12.4 × 8.7 µm). Microconidia not seen on leaves.
Host range and distribution: On Quercus (alba, bicolor, macrocarpa, muehlenbergii, stellata), Fagaceae, North America (USA, Arkansas, Iowa, Kansas, Minnesota, Missouri, Wisconsin). (2018) for collections previously referred to as Tubakia sp. B (Harrington et al. 2012, Harrington & McNew 2016. Tubakia hallii is morphologically and genetically close to T. iowensis (Figs 3, 5), but the analysis of a combined dataset of ITS and tef1 sequences (Harrington & McNew 2018) showed that the two species form monophyletic sister groups, and they are morphologically differentiated. Tubakia iowensis causes characteristic oak blight characterised by forming necroses of leaf veins (Harrington et al. 2012) and has a restricted host range and distribution, whereas T. hallii is associated with leaf spots and necrotic veins, appears to have a broader host range (Quercus alba, Q. bicolor, Q. macrocarpa, Q. muehlenbergii, and Q. stellata) and geographic distribution, and crustose pycnothyria have not been observed on the petioles of overwintering leaves. Pycnothyria may be larger in T. hallii (up to 155 µm) compared to T. iowensis (up to 110 µm), but the sizes are variable and overlapping. In culture on MYEA, the production of conidia in T. hallii is not uncommon, but rare in T. iowensis (Harrington & McNew 2016). Illustrations: Harrington et al. (2012: 82, fig. 1A-N, 87, fig. 2A-L).
In vitro: On MEA with optimal growth at 25 °C, attaining 50-56 mm diam after 7 d, margin scalloped, at first white, felt-like, light grey at 10 d, forming concentric rings of aerial hyphae, reverse yellow at 10 d, with dark grey, dense "tissue" in concentric rings; conidia formed in culture ellipsoid-obovoid, 9-15.5(-18.5) × 5-8(-8.5) µm, with somewhat thickened walls, smooth to finely rough, at first colourless, later somewhat pigmented. Notes: Tubakia iowensis is a species belonging to the T. dryina complex and is morphologically similar to T. dryina s. str. Harrington et al. (2012) published results of detailed examinations of this species, described it as new species and pointed out core differences between the new species and T. dryina s. str. Although the pycnothyria of T. iowensis are difficult to differentiate from T. dryina conidiomata, the symptoms of bur oak blight are quite different. Characteristic circular to angularirregular leaf spots dispersed on the whole leaf blade are not formed with T. iowensis, as they are for T. dryina; the lesions are rather confined to and spread along the veins. Severe infections may lead to early leaf dieback and defoliation, and, finally, branch dieback. Fusiform microconidia formed in pycnothyria are narrower, only 1-2 µm wide (vs. 1.5-2.5 µm wide in T. dryina). The status of T. iowensis as a separate species has been confirmed by sequence analyses; it is fully to highly supported in both the Bayesian and maximum parsimony analyses (Fig.  3). The Mexican isolate presenting T. sierrafriensis on Quercus eduardi is related to T. iowensis/californica/suttoniana complex as well, but these species are genetically separated  and morphologically quite different: they have pycnothyrial scutella with obtuse to sometimes even truncate tips of radiating cell threads and clear differences in conidial shape. A sequence retrieved from a Tubakia culture (CPC 23753) isolated from dead leaves of Quercus sp. in Iran clusters in the T. iowensis clade. This unusual finding requires further research based on a broader sampling of Iranian collections.
In vitro: On MYEA similar to cultures of T. macnabbii, but with slower growth, a flat surface, and abundant production of conidia formed from sporodochia in concentric rings (CBS 139744 = A771 and CBS 139745 = A830, both from ISC 453303).
Notes: Pycnothyria of Leptothyriella liquidambaris are morphologically indistinguishable from those of Tubakia dryina. Therefore, it is not surprising that L. liquidambaris was previously usually considered a synonym of the latter species. However, based on culture characteristics and results of molecular sequence analyses, Harrington & McNew (2018) demonstrated that Tubakia on Liquidambar styraciflua represents a species of its own (Fig. 5).
Notes: According to Harrington & McNew (2018), Tubakia macnabbii, previously referred to as Tubakia sp. D (Harrington and McNew 2016), was the most commonly encountered Tubakia species in the eastern USA, where it appears to be indigenous and widespread on oaks belonging to sect. Lobatae, but this species was also found on Castanea spp. in Florida and Iowa. Harrington & McNew (2018) assigned Tubakia specimens from California on leaves and twigs of Quercus agrifolia, Q. wislizeni, Q. kelloggii, and Lithocarpus densiflorus, collected by S. Latham, to T. macnabbii, which belong, however, to T. californica (see discussion under the latter species). CBS 639.93, ex-type strain of Dicarpella dryina, isolated from leaves of Quercus rubra collected in a nursery in Italy, was also included in T. macnabbii, but has to be excluded (see discussion under T. suttoniana). Harrington & McNew (2018) questioned that the sexual morph described by Belisario (1991) actually pertains to Tubakia, although the ex-type strain of D. dryina clusters in the Tubakia clade and was assigned by them to T. macnabbii. Harrington & McNew (2018) distinguished Tubakia macnabbii from T. dryina by the production of crustose conidiomata on vein and leaf tissue, rather than twigs (Harrington et al. 2012), larger conidia formed in crustose conidiomata of T. macnabbii, and cultures lacking the concentric rings typical of T. dryina. The conidia of T. macnabbii from radiate scutella are slightly smaller than those of T. hallii and T. iowensis, which usually occur on oaks of Quercus sect. Quercus. Unlike T. iowensis, T. macnabbii readily produces conidia in culture. Tubakia californica, another species belonging to the T. macnabbii complex, forms quite distinct symptoms, and radiate pycnothyria are lacking. Tubakia tiffanyae is another closely allied species on red oaks, which differs from T. macnabbii by somewhat larger conidia and characteristically circular leaf spots. Tubakia suttoniana, although insufficiently known, seems to be morphologically distinguished from T. macnabbii by having obtuse tips of radiating scutellum strands and short cylindrical to subclavate microconidia with rounded apex and broadly truncate base.

Type
Notes: Tubakia melnikiana is morphologically close to and confusable with T. dryina and only distinguishable from typical collections of the latter species by having obtuse to acute ultimate tips of the radiating pycnothyrial hyphal strands and narrowly ellipsoid (-subcylindrical) to broad ellipsoid-ovoid microconidia with obtuse to rounded apex and round to short obconically truncate base. The differences in the scutella of the two species are only gradual, and microconidia are only occasionally formed. However, T. melnikiana and T. dryina are genetically clearly distinct and not closely allied . Tubakia melnikiana is closer to T. suttoniana. The T. melnikiana clade is fully supported by both the Bayesian and maximum parsimony analyses (Figs 3, 4).
Etymology: Epithet referring to the more oblong conidia in comparison with T. dryina.
In vitro: On MEA with optimal growth at 20 °C, attaining 20-25 mm diam after 14 d, margin scalloped, at first ivory white, velvety and flattened on the surface of colony, reverse pale grey. Conidial formation not observed in vitro.
Notes: Tubakia oblongispora is phylogenetically closely allied to T. dryina but morphologically quite distinct (see diagnosis; Figs 3,5). It belongs to a group of Tubakia species with obtuse, non-acute tips of the ultimate branchlets of radiating scutellum strands. Among species of this morphological group, T. oblongispora is comparable with Oblongisporothyrium castanopsidis, which differs, however, in having hyaline, broader conidia, 11-20 × 7-9.5 µm, with a length/width ratio of 1.6-2.2.
Etymology: Composed of para-(similar to) and the name of the comparable species, Tubakia dryinoides.
In vitro: On MEA with optimal growth at 20 °C, attaining 35-40 mm after 14 d, margin scalloped, straw coloured, forming concentric ring of aerial hyphae, reverse in straw coloured, forming a dark brown concentric ring. Conidial formation not observed. Notes: Tubakia paradryinoides is phylogenetically very closely allied to T. dryina and more distant from T. dryinoides and T. oblongispora (Figs 3,5), which is also reflected in the morphological characters of pycnothyria of the species involved. The pycnothyrial scutella of these species are characterised by having hyphal strands with acute ultimate tips. However, T. paradryinoides is easily distinguishable from T. dryina and T. dryinoides by its hyaline to subhyaline, much larger conidia, 14-21 × 10-15 µm (vs. at first hyaline, subhyaline, but later pale olivaceous, olivaceous brown to brownish and (7-)9-16(-18) × (5-)6-10(-10.5) µm in T. dryina, and 8.6-14.7 × 5.5-8.5 (-10) µm in T. dryinoides). The independence of T. paradryinoides as a distinct species is mainly supported by its unique tub2 sequence which influences its position in the phylogenetic tree (data not shown, see Fig. 3). Also see the notes under T. dryinoides; this assemblage of sequences could comprise several closely allied lineages that might represent additional cryptic species.
In vitro: On MEA reaching 32-44 mm diam after 10 d at 25 °C in the dark, low velutinous to fuzzy, margin uneven, whitish to pale yellow, centre darker, becoming paler towards the margin, olive brown, light olive brown to yellow, margin white, reverse wrinkled, non-sporulating; hyphae branched, septate, 3.2-4.8 µm wide, hyaline to slightly brownish in mass, some hyphae with short coils on side branches. Host range and distribution: Only known from the type collection.
In vitro: Colony covering dish in 2 wk at 25 °C with moderate aerial mycelium and feathery margins. On MEA surface smoke grey, reverse smoke grey with concentric circles of olivaceous grey. On PDA surface smoke grey, reverse smoke grey with olivaceous grey margin. On OA surface smoke grey with patches of olivaceous grey.
Hosts range and distribution: On Quercus rubra, Europe (Italy).
Notes: Dicarpella dryina was introduced as sexual morph of "Tubakia dryina" (Belisario 1991). Harrington et al. (2012) questioned that the two morphs belong together and speculated that D. dryina might instead be the sexual morph of Actinopelte americana or another related cryptic American Tubakia species. However, sequences retrieved from the ex-type strain of D. dryina cluster in the Tubakia clade and were assigned to T. macnabbii in Harrington & McNew (2018). Belisario (1990) emphasised that a comparison of cultures derived from single ascospores and the original cultures obtained from naturally infected tissue and conidia did not show any differences, i.e., the ex-type cultures represent single ascospore cultures. Taking this into account, there are currently no objective reasons to doubt that D. dryina represents a sexual morph belonging to Tubakia. Unfortunately, Belisario (1989Belisario ( , 1990 did not describe the pycnothyria associated with D. dryina in detail, but her figures (Belisario 1991: 149, figs 1-3) suggest that the terminal tips of radiating scutellum strands are obtuse-rounded and not acute as in T. dryina. The microconidia are cylindrical to short clavate with rounded apex and broadly truncate base vs. ellipsoidovoid to fusiform, attenuated towards a narrow base as those occurring in T. dryina. This confirms that the asexual morph of D. dryina does not coincide with T. dryina, and these differences do not support the conspecificity of D. dryina and T. dryina as discussed in Harrington et al. (2012). Furthermore, D. dryina (as T. suttoniana) and T. dryina are clearly confirmed as separate species in the phylogenetic trees (Figs 3, 5). Harrington & McNew (2018) de facto reduced Dicarpella dryina to synonymy with the new species Tubakia macnabbii since the ex-type culture of D. dryina (CBS 639.93) was cited and included in the protologue of T. macnabbii. The latter species, based on type material from Missouri on Quercus palustris, is undoubtedly a common North American species with wide host range (Harrington & McNew 2018). The taxonomic conclusion to include Tubakia collections from Italy on Quercus rubra in T. macnabbii was just based on phylogenetic analyses. However, the resolution of the "T. macnabbii clade" in their ITS tree is insufficient, and their tef1 tree reflects a higher degree of variation, suggesting a complex of several cryptic taxa (also see Fig. 5 in the present study). Harrington & McNew (2018) supposed that the North America T. macnabbi occurring on various hosts of Quercus sect. Lobatae had been introduced to Europe (Italy), but the cluster including the sequence retrieved from the ex-type strain of Dicarpella dryina encompasses several sequences obtained from strains isolated from European collections (Netherlands) on the European oak Quercus robur and one strain on Q. cerris in New Zealand. Furthermore, Belisario's (1990) micrographs of "T. dryina" on Q. rubra show pycnothyria with obtuse tips of radiating scutellum strands and short cylindrical to subclavate microconidia with rounded apex but broadly truncate base, which is not in agreement with morphological characters of T. macnabbii. Therefore, we prefer to keep D. dryina as separate species.
Although the ITS sequence is not distinct from sequences of the newly described Tubakia from California (T. californica sp. nov.), T. suttoniana clusters separately from T. californica in the tef1 (Fig. 5), tub2 (not shown, see TreeBASE) and combined trees (Figs 3, 4). Based on their ITS tree, Harrington & McNew (2018) assigned a strain isolated from twigs of Quercus agrifolia in California, 27 April 2012, S. Latham (A1177) to T. macnabbii. However, this strain was also included in our own analyses (CPC 31497 = CDFA#1007) and clearly belongs to T. californica, which further demonstrates the heterogeneity of T. macnabbii.
As outlined above, in the phylogenetic trees, T. suttoniana belongs to an assemblage of poorly resolved Tubakia strains from Europe (Italy, Netherlands, isolated from Quercus robur, Q, rubra and Quercus sp.) and New Zealand (isolated from Quercus cerris) that neither form a distinct cluster nor a clade, but rather reflect different lineages which might be different species. "Tubakia dryina" formed on leaf spots on Quercus cerris (Belisario 1993) and isolated as endophyte from buds and shoots of Q. cerris (Gennario et al. 2001) was also reported from Italy. Tubakia macnabbii is also part of this complex. The sample numbers are currently too small to resolve this assemblage of some taxa and more isolates are needed. Tubakia suttoniana represents a cryptic species morphologically and is phylogenetically distinct from T. dryina, but currently only known with certainty from its type material. In addition, it forms a complex of closely related species which also includes T. japonica, T. melnikiana, T. seoraksanensis and "Tubakia sp. nov. II" (Figs 3-5).
According to Art. 55.1, the name D. dryina is legitimate and applicable as basionym although published under the illegitimate genus name Dicarpella Syd. & P. Syd. (younger homonym, Art. 53.1). In the type paragraph, Belisario (1990) cited two different collections, one from Grosseto, Tuscany, the other one from Rome, and specified ROHB as herbarium in which the holotype was deposited [as "Belisario (RHOB)"], but without clear indication which of the two cited collections represents the genuine holotype. Due to this imprecise, confusing citation, the name D. dryina would be invalid according to Art. 40.1, 40.6. However, on page 148 it was mentioned that she (Belisario) first found this fungus in the Grosseto farm nursery in February 1989. Later the same fungus was collected on dead leaves in a nursery in Rome. The reference to "Belisario" may be accepted as reference to the first collection from "Grosseto" as holotype so that one can consider D. dryina to be validly published. Description in vivo: Conidiomata (pycnothyria) superficial, hypophyllous or epiphyllous, on circular leaf spots or along necrotic leaf veins. Scutella radiate, (40-)60-150 µm diam, composed of a series of dark brown, thick-walled cells originating from a central cell, ending in blunt to acute tips. Sporodochia with no scutella or poorly developed scutella epiphyllous or hypophyllous on necrotic vein tissue, light to dark brown due to masses of conidia. Conidiophores on underside of scutella or on sporodochia. Conidia hyaline, turning light brown with age, smooth to slightly varicose, aseptate, obovoid to ovoid (9-)10-15.5 × (8-)8.5-11.5(-12) µm (mean 12.9 × 9.6 µm). Microconidia sterile, hyaline, aseptate, fusiform 3-8 × 1-2.5 µm may develop from small pycnothyria, alone or along with macroconidia. Crustose conidiomata developing in late summer to fall on underside of necrotic veins and found on overwintering leaves still hanging from twigs, erumpent, brown to black, irregularly shaped, 80-580 µm diam, single to grouped, covered with dark, thick-walled cells that break open in fissures due to swelling when wet. Conidia hyaline to brown, ellipsoidal to obovate to irregularly shaped, aseptate, (11-)13-18(-20) × (6-)7-9.5 µm (mean 15.2 × 7.9 µm).
In vitro: On MYEA with optimal growth at 25 C, 40-58 mm diam after 7 d, creamy white aerial mycelium, smooth to scalloped at edge, developing concentric rings of dense mycelium, underside golden yellow to slightly darker with age, sometimes with dark cell masses on surface or subsurface. Conidiophores rare to abundant, short, hyaline, sometimes aggregated (sporodochia) on agar surface. Conidia hyaline to dark brown, thick-walled, smooth to slightly varicose, aseptate, obovoid to ellipsoidal, 10-16 × 7-11 µm.
Notes: This species was recently introduced by Harrington & McNew (2018) for Tubakia sp. C (Harrington & McNew 2016) which is closely allied to T. macnabbii but genetically separated (Fig. 5) and morphologically distinguished by having wider conidia and causing characteristic circular leaf spots with lightcoloured centres (see Harrington & McNew 2018: fig 1h) in addition to the vein necrosis.

Notes:
The generic affinity of this species is unclear. Cultures and results of molecular sequence analyses are not yet available. This species is only known from its type collection (Brazil, Para, Belem, on leaves of Byrsonima coriacea, 3 Feb. 1963, F.C. Albuquerque, BPI 391941) and has not yet been revised and reassessed. However, the described and illustrated characteristics of A. stellata do not fit into the generic concept of Tubakia. Farr (1967) described a basal membrane, unknown in true Tubakia species, a one-celled columella, and conidiogenous cells arising from the underside of the scutellum. In Tubakia spp., the columella is composed of several cells or a single cell surrounded by small, fertile, parenchymatous cells giving rise to conidiogenous cells, above all around the point of attachment of the columella at the scutellum and peripherally. Tubakia species are usually confined to fagaceous hosts in the northern hemisphere. The reallocation of A. stellata to Tubakia by Harrington & McNew (2018) was just a formal act, neither supported by culture and sequence data nor accompanied by a critical discussion of the morphology of this species. ellipsoid-ovoid, colourless or pale conidia, about 7-10 × 5-7 µm. Shape and size of the pycnothyria are reminiscent of those of Tubakia sp. on Castanea henryi in China (Braun et al. 2014: 25) and Saprothyrium thailandense, which differ, however, in having globose to subglobose conidia. There is no Tubakia species with comparable pycnothyria. Therefore, the collection on Q. castaneifolia from Iran represents probably an undescribed species, but without cultures and sequence data the generic affinity remains unclear.

DISCUSSION
The genus Tubakia was assigned to the Diaporthales (Yokoyama & Tubaki 1971, Yun & Rossman 2011, http://www.mycobank. org/ and http://www.indexfungorum.org/names/names.asp) primarily based on the description of Dicarpella dryina as the putative sexual morph of Tubakia dryina. However, the latter species is not the type species of Tubakia, and D. dryina is also not the type species of Dicarpella, which requires a more detailed discussion. Phylogenetic analyses of these species were previously not available. During the course of the present studies on Tubakia spp., ex-type cultures of Tubakia japonica, type species of Tubakia, and Dicarpella dryina could be included in phylogenetic analyses and proved to be congeneric. However, Dicarpella dryina is not conspecific with Tubakia dryina, as previously assumed and postulated in Harrington et al. (2012), and phylogenetically represents a separate species belonging to an assemblage of related Tubakia species, including T. californica, T. dryinoides, T. japonica, T. macnabbii, T. melnikiana, and T. seoraksanensis. All recognised species of Tubakia, except for T. chinensis, are known in vitro and have been included in the present molecular sequence analyses, which revealed that Tubakia s. lat., as previously circumscribed, represents a complex of cryptic genera. The question that remains is whether the genera Dicarpella and Tubakia s. str. are congeneric. A simple answer to this question is not yet possible since it requires phylogenetic data for Dicarpella bina, the type species of Dicarpella, which is hitherto only known from its type collection (Sognov et al. 2008). Based on the morphological similarity between D. bina and D. dryina, it might be possible that the two genera are, indeed, congeneric. Dicarpella bina was originally described from living leaves of Quercus agrifolia in California. Quercus agrifolia is also a common host of Tubakia californica.
On the other hand, it can also not be excluded that the type species of Dicarpella might be more distantly related to Tubakia as currently suspected or even unrelated. A final conclusion is still pending and further cultures and sequence data for D. bina are needed. In any case, the older name Dicarpella would not threaten the younger name Tubakia in case that the two genera were congeneric, since Dicarpella Syd. & P. Syd., 1913 (nom. illeg.) being a younger homonym of Dicarpella Sitzenb., 1861, i.e., Tubakia would be the correct name in any case. Barr (1978) assigned Dicarpella to the Pseudovalsaceae subfam. Pseudovalsoideae tribe Ditopelleae, and later included Dicarpella quercifolia with Mastigosporella hyalina as putative asexual morph (Barr 1979). A similar unnamed asexual morph was found in D. georgiana, which was later described as M. nyssae (Nag Raj & Di Cosmo 1981). Mastigosporella hyalina (≡ Harknessia hyalina) was the reason for previous reports that Dicarpella spp. might be associated with Harknessia spp. as asexual morphs (Cannon 2001). Monod (1983) retained Dicarpella georgiana in Gnomoniella, recognised D. bina and D. quercifolia in Dicarpella, and added D. liquidambaris-styracifluae and D. orientalis. Rossman et al. (2007) cited Harknessia spp. under diaporthalean fungi of uncertain position. However, true Harknessia spp. are not closely allied to former Dicarpella spp. with Mastigosporella asexual morphs and have been placed in a family of its own, viz. Harknessiaceae (Crous et al. 2012). Reid & Dowsett (1990) examined Dicarpella in detail, excluded D. georgiana and D. quercifolia, both associated with asexual morphs belong to Mastigosporella, and reallocated them to the new genus, Wuestneiopsis. Rossman et al. (2015) recommended to protect the name Mastigosporella and to reduce Wuestneiopsis to its synonymy. Thus, the illegitimate name Dicarpella currently comprises D. bina, D. liquidambarisstyracifluae, and D. orientalis, but to date none of them has been phylogenetically clarified. The illegitimate name Dicarpella was in need of a new genus name, but since the relation between Dicarpella and Tubakia remains unproven and unclear, any corresponding nomenclatural change would be premature.
Another question concerns the affiliation of Tubakia in the hierarchical system of the Ascomycota. The placement within the Diaporthales is clearly resolved. Tubakia is currently usually listed as diaporthalean genus of unclear family affinity (Yokoyama & Tubaki 1971, Yun & Rossman 2011, http://www.mycobank. org/ and http://www.indexfungorum.org/names/names.asp). Dicarpella was usually assigned to the Melanconidaceae s. lat., comprising up to almost 30 genera (Eriksson et al. 2001, Lumbsch & Huhndorf 2007, Maharachchikumbura et al. 2015. This affiliation is, however, disputable (see discussion above). Most genera allocated to the Melanconidaceae s. lat. have not yet been phylogenetically examined, i.e., the affinity of the genera concerned remains unclear and unproven. Melanconis, the type genus of the family Melanconidaceae, has been phylogenetically examined and the independent status of the latter family has been confirmed (Castlebury et al. 2002, Du et al. 2017. Castlebury et al. (2002) emphasised that the family Melanconidaceae should rather be confined to the genus Melanconis, and recommended to exclude the numerous other previously included genera, including Melanconiella (Voglmayr et al. 2012). Voglmayr et al. (2017) introduced the new family Juglanconidaceae for the new genus Juglanconis, which splits the genus Melanconis and Melanconidaceae s. lat. The present phylogenetic analyses indicate a relation of Tubakia to Melanconiella spp., possibly in sister positions, depending on the kind of analyses employed. Voglmayr et al. (2012) confirmed Melanconiella as a separate genus, clearly distinct from the morphologically similar genus Melanconis and not affiliated to the Melanconidaceae, and stressed that this genus does not belong to any established dothidealean family. Morphological traits of Melanconiella spp., including the characters of associated asexual morphs, do not favour Melanconiella spp. and Tubakia being placed in the same family. In contrast to Tubakia, the bark-inhabiting Melanconiella spp. are characterised by forming ectostromatic discs with a central column, perithecia with lateral non-rostrate ostioles, broad band-like paraphyses, consistently bicellular ascospores, described herein as T. californica. Tubakia gloeosporioides (≡ Actinothyrium gloeosporioides) and T. nyssae (≡ Pirostoma nyssae) are two additional combinations introduced by Harrington & McNew (2018), but cultures and sequence data are not yet available for the two taxa, and the morphological characters of the pycnothyria formed on leaves of Nyssa and Sassafras are not distinguishable from those of T. dryina s. lat. Therefore, we prefer to assign these species to the list of "Excluded, doubtful and insufficiently known species", at least tentatively until the affinity of the two taxa can be elucidated by means of cultures and sequence data.
Thus, the number of species pertaining to the family Tubakiaceae (Tubakia s. lat. in its previous circumscription) increased from eight in 2016 to the current 26 accepted here. However, examination of the individual gene trees has shown that there is some movement of isolates between species clades for ITS and tub2 (see results above), indicating some degree of gene transfer between species, which could be explained by the overlap in host species. More isolates need to be subjected to multi-gene phylogenies or other molecular analyses to understand the underlying processes and frequency at which these transfers happen.