New species, combinations and records of Thyronectria, with a key to species

The new species Thyronectria ulmi is described from Ulmus laevis and U. minor collected in Austria, the Czech Republic and Slovakia. It is morphologically and phylogenetically close to the North American T. chrysogramma, which also occurs on Ulmus and shares olive green to brown muriform ascospores, but differs from the latter by geographic distribution, narrower asci, smaller ascospores with fewer septa and DNA sequence data from seven loci (ITS and LSU regions of nu rDNA, ACT1, RPB1, RPB2, TEF1 and TUB2 genes). As in many other Thyronectria species, ascomata of T. ulmi were closely associated with Diplodia, indicating a fungicolous habit. The genus Neothyronectria is synonymised with Thyronectria based on morphological and molecular phylogenetic data, and the new combinations T. citri and T. sophorae are proposed. A key to 45 accepted species of Thyronectria is provided. The recently described T. abieticola, previously known from the Czech Republic and France, is newly reported from Austria and Slovakia; its pycnidial anamorph is recorded, described and illustrated from natural substrates for the first time. A sporodochial anamorph is reported from natural substrates for T. aurigera, a new record for Austria as well. New host and distribution records include T. rhodochlora on Acer pseudoplatanus in Austria and Fraxinus excelsior in the Czech Republic, T. sinopica on Hedera colchica in the Czech Republic and Bupleurum fruticosum in Spain, and T. zanthoxyli on Sorbus aucuparia in Belgium and Ulmus sp. in the USA. Thyronectria cucurbitula is confirmed by sequence data from Pinus strobus collected in the Czech Republic, challenging the host ranges given for T. cucurbitula (Pinus subgen. Pinus) and T. strobi (Pinus subgen. Strobus), and questioning the European and Chinese records of T. strobi.

Based on sequence data, Crous et al. (2016) established the new genus Neothyronectria with N. sophorae, a pycnidial anamorph, as generic type, which occurred on dead branches of Sophora microphylla collected in New Zealand. Based on a Bayesian analysis of LSU rDNA sequence data and the results of BLAST searches of ITS and LSU sequences, the genus Neothyronectria was considered to be closely related to but distinct from Thyronectria. Yang et al. (2019) added a second species from China, N. citri, with a teleomorph resembling Thyronectria in subglobose to globose ascomata with a distinct bright to greenish yellow scurf. Remarkably, the morphology of N. citri is notably close to the generic type of Thyronectria, T. rhodochlora, with which it shares subhyaline to hyaline muriform ascospores not budding within asci. These striking morphological similarities and the incomplete taxon and sequence sampling in the phylogenies of Crous et al. (2016) and Yang et al. (2019) call for a critical re-evaluation of the status of Neothyronectria as a distinct genus. Therefore, extended multigene phylogenies based on seven loci (ITS and LSU regions of nu rDNA and ACT1, RPB1, RPB2, TEF1 and TUB2 genes) were performed to evaluate whether Neothyronectria is congeneric with Thyronectria.
Recently, several fresh collections closely resembling the North American Thyronectria chrysogramma were made on dead branches of Ulmus spp. in Austria, the Czech Republic and Slovakia, but they differed by having smaller ascospores with fewer septa. Therefore, sequence data were produced to reveal whether these collections are conspecific with T. chrysogramma or represent a distinct undescribed species. In addition, in several fresh collections of T. abieticola, a pycnidial anamorph was found to be closely associated with ascomata, while in some recent collections of T. aurigera, a sporodochial anamorph was observed. As for these species anamorphs have not been recorded from natural substrates, pure cultures were obtained from conidia and sequenced to confirm their connection with the teleomorphs. The morphology of the ana-and teleomorphs of these collections was documented and illustrated with species descriptions provided or amended accordingly. Finally, for several Thyronectria species, new host associations and country records were confirmed by sequence data, which are reported here.

Sample sources
Isolates investigated in this study originated from ascospores or conidia of freshly collected specimens. Details of the strains including NCBI GenBank accession numbers of gene sequences used to compute the phylogenetic trees are listed in Table 1. Strain acronyms other than those of official culture collections are used here primarily as strain identifiers throughout the work. Details of the specimens used for morphological investigations are listed in the "Taxonomy" section under the respective descriptions. Herbarium acronyms are according to Thiers (2021). Most specimens have been deposited in the Fungarium of the Department of Botany and Biodiversity Research, University of Vienna (WU-Mycologicum, https:// www. jacq. org/ WU-Myc#).

Morphological observations
Microscopic preparations were mounted in water, 3% potassium hydroxide (KOH), or lactic acid (LA). Study of macromorphology was done by using a Nikon SMZ 1500 or a Nikon SMZ 18 stereomicroscope (Nelville, NY) equipped with a Nikon DS-U2 digital camera or a Keyence VHX-6000 Digital Microscope (Mechelen, Belgium). For light microscopy, a Zeiss Axio Imager.A1 compound microscope (Oberkochen, Germany), equipped with Nomarski differential interference contrast (DIC) optics and a Zeiss Axiocam 506 colour digital camera or a DIC microscope Nikon Eclipse Ni-U and a Nikon DS-Ri2 camera, was used. Photographs and measurements were taken by using the NIS-Elements D v. 3.0 or Zeiss ZEN Blue Edition software. For certain images of ascomata or condiomata, the stacking software Zerene Stacker version 1.04 (Zerene Systems LLC, Richland, WA, USA) was used. Measurements are reported as maxima and minima in parentheses and the mean plus and minus the standard deviation of a number of measurements given in parentheses.

Culture observations
Cultures were prepared and maintained as described previously (Jaklitsch 2009) except that sometimes also 3.9% potato dextrose agar (PDA: VWR, Radnor, Pennsylvania) was used for isolation. Germinating ascospores/conidia were placed on PDA or CMD (CMA: Sigma, St Louis, Missouri; supplemented with 2% (w/v) D( +)-glucose-monohydrate). Cultures used for the study of anamorph micromorphology were grown on PDA at room temperature. Microscopic observations were made in tap water except where noted. The plates were sealed with parafilm and incubated at room temperature. An ex-holotype culture of T. ulmi was deposited at the Westerdijk Fungal Biodiversity Centre (CBS-KNAW), Utrecht, The Netherlands.

Phylogenetic analyses
Published sequences of a single accession for each Thyronectria species served as basis for the sequence matrix. Representative sequences were selected from Hirooka et al.  Ma et al. (2020). The accessions were selected according to availability of markers, and if possible, ex-type sequences were used (marked with an asterisk in Fig. 1). In addition, four Nectria species were included, and Septofusidium berolinense, S. herbarum and Tilachlidium brachiatum (Tilachlidiaceae) were selected as outgroups according to Lombard et al. (2015). Available sequences were downloaded from GenBank; details on the sequences used in the phylogenetic analyses are provided in Table 1.
To reveal the phylogenetic position of the newly sequenced Thyronectria accessions, the newly generated sequences were aligned with the GenBank sequences. All alignments were produced with the server versions of MAFFT (www. ebi. ac. uk/ Tools/ mafft or http:// mafft. cbrc. jp/ align ment/ server/) and checked and refined using BioEdit v. 7.0.4.1 (Hall 1999). For phylogenetic analyses, the sequence matrices were combined; the resulting matrix contained 6235 alignment positions from seven loci (540 from ITS, 816 from LSU, 630 from ACT1, 706 from RPB1, 1096 from RPB2, 1325 from TEF1 and 1122 from TUB2).
Maximum likelihood (ML) analyses were performed with RAxML (Stamatakis 2006) as implemented in raxmlGUI 2.0 (Edler et al. 2021) using the ML + rapid bootstrap setting and the GTRGAMMA substitution model with 1000 bootstrap replicates. Substitution model parameters were calculated separately for the different gene regions included in the combined analyses. Maximum parsimony (MP) analyses were performed with PAUP v. 4.0a169 (Swofford 2002), using 1000 replicates of heuristic search with random addition of sequences and subsequent TBR branch swapping (MUL-TREES option in effect, steepest descent option not in effect). All molecular characters were unordered and given equal weight; analyses were performed with gaps treated as missing data; the COLLAPSE command was set to MAXBRLEN. Bootstrap analysis with 1000 replicates was performed in the same way, but using five rounds of random sequence addition and subsequent TBR branch swapping during each bootstrap replicate, with the COL-LAPSE command set to MINBRLEN.

Molecular phylogeny
Of the 6235 characters of the combined matrix, 1932 were parsimony informative (148 in ITS, 114 in LSU, 150 in ACT1, 300 in RPB1, 452 in RPB2, 423 in TEF1 and 345 in TUB2). The phylogram of the best ML tree (lnL = − 55,221.1187) obtained by RAxML is shown as Fig. 1. The MP analysis revealed a single tree of length 10, 896 (not shown) that had a similar topology as the ML tree. Except for minor differences, tree topologies agree well with those of Jaklitsch and Voglmayr (2014), Checa et al. (2015) and Voglmayr et al. (2016).
The genus Thyronectria is highly supported in both ML (100%) and MP (99%) analyses, but as in previous analyses (e.g. Jaklitsch and Voglmayr 2014;Checa et al. 2015;Voglmayr et al. 2016;Zeng and Zhuang 2017;Li et al. 2018;Ma et al. 2020), support of deeper backbone nodes is low or absent (Fig. 1). The ML and MP analyses reveal different phylogenetic positions of the main green-spored (T. roseovirens) clade, which in the MP analysis is sister group to the T. rhodochlora-clade (not shown). Also, the clade containing T. sinopica has a slightly different position in the MP analysis, where it is placed in the tree as the lineage branching off next after T. berolinensis. However, none of these topological differences received significant MP bootstrap support.
For a detailed description of the teleomorph, see Lechat et al. (2018).
Anamorph on the natural host: Conidiomata sporodochial, formed below epidermis of bark, scattered to gregarious, often tightly associated with pycnidia of Diplodia sp., oblong, tongue-shaped, laterally compressed, erect to prostrate, 100-450 µm high, 100-270 µm wide, dark orange to orange brown, commonly lighter coloured towards the apex, surface smooth, waxy, shiny. Stroma pseudoparenchymatous, of textura angularis of isodiametric cells 3-12 μm diam with walls 0.5 μm thick and containing a large orange guttule 2-4 µm diam, becoming gradually smaller towards outside, the outermost cell layer bearing the densely arranged phialides covering the entire stroma. Phialides terminal, solitary, cylindrical to irregularly ampulliform, (3.3-)4.9-7.3(-9.1) × (1.4-)1.9-2.6(-3) μm (n = 70). Conidia oblong to ellipsoidal, (2.5-)2.8-3.5 (-4.3) × (0.9-)1.  (Hirooka et al. 2012) may indicate a recent introduction of the species from North America. However, as the ascomata are rather inconspicuous, the species may have been overlooked. In the past 2 years, it has also been recorded several times from Central Europe (e.g. Czech Republic, Germany, Slovakia). The abundant availability of host substrate (corticated dead twigs of Fraxinus), following the rapid spread of ash dieback disease caused by Hymenoscyphus fraxineus, may have favoured the occurrence of T. aurigera. The current records are the first for Austria, and a detailed search has shown that the species is actually widespread but usually overlooked on dead corticated branches of various Fraxinus species, where it is constantly associated with effete conidiomata of Diplodia sp., indicating a fungicolous habit.
We here provide an amended description of Hirooka et al. (2012) to include additional features of the teleomorph and the characters of the sporodochial anamorph on the natural substrate. Until recently, no anamorph had been known for T. aurigera on natural substrates, which is likely due to the development of the small orange sporodochia beneath the host bark, becoming evident only after its removal. In addition, they were observed in only two collections (WU 44631 and WU 44632). The connection of the anamorph with the teleomorph has been proven by sequences obtained from conidial and ascospore isolates. Holotype: BJFC S1770 Notes: For a detailed description of the species, see Yang et al. (2019). In our phylogenetic analyses, Neothyronectria citri is embedded within a highly supported subclade of Thyronectria that also contains the generic type, T. rhodochlora (Fig. 1). In addition, the teleo-and anamorphs as described and illustrated in Yang et al. (2019) fully match Thyronectria, in particular T. rhodochlora with which it shares semi-immersed to erumpent ascomata in groups with distinct bright yellow scurf and hyaline to yellowish brown muriform ascospores not budding within asci. Therefore, N. citri is here combined in Thyronectria. The phylogenetic analyses of Yang et al. (2019) include neither members of the clade containing T. rhodochlora nor other, basal species (T. aurigera, T. chrysogramma), which evidently is the reason why Neothyronectria resolved as a separate sister clade in their analyses.
Holotype: CBS H-22880 Notes: For a detailed description of the species, see Crous et al. (2016). Neothyronectria sophorae is the generic type of Neothyronectria, which was established as a distinct genus based on a phylogenetic placement outside Thyronectria. However, the phylogenetic analyses of Crous et al. (2016) contained only four species of Thyronectria and were solely based on LSU rDNA data, resulting in insignificant topological support for its placement. In our extended multigene analyses, N. sophorae clusters with high support among other Thyronectria species. In addition, it is revealed to be closely related to the generic type, T. rhodochlora (Fig. 1). No teleomorph is known for the species, but its pycnidial anamorph fully matches the genus Thyronectria. Based on this sound evidence, N. sophorae is here transferred to Thyronectria. Polhorský,Halasů & Voglmayr,sp. nov. Figure 6,7. MycoBank: MB 841958.
Cultures and anamorph in culture: On PDA colony producing conidia immediately after germination (ca. 24 h), up to 58 mm diam after 18 days at room temperature; surface cream, without aerial hyphae, soon turning yellow-orange and slimy from the centre due to conidial masses; reverse pale orange, after several weeks sometimes producing pycnidia near the edge. Conidia forming mostly on short hyphal pegs, directly on vegetative hyphae or by microcyclic conidiation. Hyphae 1.3-3 μm broad, in old colonies forming thick-walled chlamydospores, 2.8-6.3 μm broad. Pegs on hyphal cells mostly solitary, terminal or lateral, conical or cylindrical, with balls of aggregated conidia at the apex, without visible collarettes, (0.9-)1.3-3(-4.5) × (0.9-)1-1.8(-3.1) μm (n = 30). Conidia cylindrical to ellipsoid, (3-)4.6-6.6 (-7.8) × (0.9-)1.2-2(-2.6) μm, l/w = (2.6-)3-4.2 (-5 Notes: Thyronectria ulmi is the closest relative of the North American T. chrysogramma, which also occurs on Ulmus and is associated with Diplodia sp. Both species share olive green to brown muriform ascospores, but T. ulmi differs from T. chrysogramma by geographic distribution, narrower asci, smaller ascospores with fewer septa and DNA sequence data. Remarkably, we observed that ascospores become distinctly wine red in lactic acid, which has so far not been reported for Thyronectria species. When investigating other Thyronectria species with green ascospores, this reaction was also observed in the closely related T. chrysogramma and in T. roseovirens, but not in T. asturiensis, T. giennensis and T. pistaciae, in which ascospore colour does not change at all in lactic acid. Notes: As the species is rarely illustrated, we here add illustrations of the collection from Belgium. For a detailed description of the species, see Hirooka et al. (2012;under Pleonectria zanthoxyli). Thyronectria zanthoxyli was originally described from North America, where it was primarily recorded from Zanthoxylum americanum (Hirooka et al. 2012), to which a North American record from Ulmus is here added. In Europe, T. zanthoxyli has been previously reported from Crataegus in France (Hirooka et al. 2012), and we here add a record from Sorbus aucuparia collected in Belgium. According to Hirooka et al. (2012), Thyronectria zanthoxyli is distinguished from the closely related T. rhodochlora and T. virens by mostly immersed ascomata and by distinctly curved, and from T. rhodochlora also by narrower, ascospores. Our ascospore measurements ((19.4-)21.2-23.6(-24.9) × (7.1-)7.8-  8.8(-9.7) μm, l/w = (2.3-)2.5-2.9(-3.1) (n = 50)) largely agree with those given in Hirooka et al. (2012). However, ascospores of our collections are not as distinctly curved as illustrated in Hirooka et al. (2012) and are therefore morphologically more similar to those illustrated for T. virens. Finally, the sequence data unequivocally identify our collections as T. zanthoxyli. This demonstrates that morphological distinction between these three closely related taxa can be difficult, and then, sequence data are required for reliable species identification. Notes: Hirooka et al. (2012) described Thyronectria strobi (as Pleonectria strobi) from Pinus strobus and distinguished it from the closely related T. cucurbitula primarily by sequence data and by different host ranges, with hosts from Pinus subgen. Strobus for T. strobi and from Pinus subgen. Pinus for T. cucurbitula. Both species were reported to be morphologically highly similar, with slightly shorter ascospore length ranges in T. strobi (22-64 µm), yet widely overlapping with those of T. cucurbitula (33-75 µm). Both species are therefore practically impossible to separate by teleomorph morphology alone. Differences were also reported for the anamorph in pure culture, with conidia consistently formed on ellipsoid lateral phialidic pegs in T. cucurbitula, while additionally also flask-shaped lateral pegs were observed in T. strobi (Hirooka et al. 2012). Although sequence data were generated only from North American isolates from Pinus strobus, specimens from P. flexilis and P. monticola were also listed as T. strobi by Hirooka et al. (2012).

Additional new host records
Considering that our collection from Pinus strobus actually represents T. cucurbitula, the separation of these similar species by their host range is not any more tenable. It is therefore questionable whether T. strobi occurs outside North America at all, as no such records have yet been confirmed by sequence data. Collection BPI 632658 from Dabroszyn (formerlyTamsel), Poland, identified by Hirooka et al. (2012) as T. strobi, may therefore, like our collection, actually represent T. cucurbitula, and also the Chinese record from Pinus armandii (Zeng and Zhuang 2017)  Notes: For a detailed description of the species, see Jaklitsch and Voglmayr (2014). Thyronectria rhodochlora is commonly found on Acer campestre and several other hosts, but has so far not been recorded from Acer pseudoplatanus and Fraxinus excelsior. As in all other collections of the species examined, the specimens examined here are tightly associated with an effete Diplodia sp., indicating a fungicolous habit.
Thyronectria sinopica (Fr.) Jaklitsch & Voglmayr, Persoonia 33: 206 (2014 Notes: Thyronectria sinopica is a common species with a specific occurrence on the genus Hedera (Araliaceae), in particular H. helix (Hirooka et al. 2012). We here add a confirmed record from Hedera colchica. The current Spanish collection morphologically and with its DNA sequences fully matches this Thyronectria species and extends its host range to Bupleurum from the closely related family Apiaceae.

Discussion
This publication provides additional new data on the biodiversity, host range and distribution of Thyronectria, a nectriaceous genus. Thyronectria ulmi is revealed as a new species from Central Europe, being closely related to the North American T. chrysogramma. Both species share hosts from the genus Ulmus and are fungicolous on Diplodia sp., but differ by sequence data and morphology. Remarkably, T. ulmi has been collected several times mainly on dead branches of Ulmus laevis in riverine forests of the River Danube and the Morava/March, where it appears to be widespread. Considering that mature Ulmus trees have become rare in such habitats due to Dutch elm disease (previously caused by Ophiostoma ulmi and presently by O. novo-ulmi, Kirisits and Konrad 2004), it is not surprising that the fungal biodiversity on Ulmus hosts harbours still undescribed species. That all but one collection of T. ulmi were made on mature U. laevis likely reflects the higher abundance of this host species compared to U. minor in the post-epidemic period of Dutch elm disease (Kirisits and Konrad 2004). It is nevertheless possible to find T. ulmi on U. minor and younger trees as well, as shown by one of the Czech finds from ca. 20-25 years old U. minor (PRM 955819).
Based on the investigation of recent collections, we were also able to provide new data on asexual morphs on natural substrate for several species. Detailed morphological investigations resulted in extended species descriptions and illustrations of ana-and teleomorphs of little-known species. For the first time, we here report and describe pycnidia from natural substrate for the recently described T. abieticola, for which pycnidia were so far only known from pure culture (Lechat et al. 2018). In addition, for T. abieticola, only an ITS sequence from the type culture was available, resulting in an uncertain phylogenetic placement. We here add additional markers for this species, and in our multigene phylogeny, it is revealed as the most basal taxon of the main conifericolous clade with high (92% MP) to maximum (100% ML) bootstrap support (Fig. 1).