Introduction

Thyronectria Sacc. 1875 is a phylogenetically distinct genus that is morphologically well-characterised by nectriaceous ascomata covered by a yellow scurf, long, more or less persistent apical paraphyses (Jaklitsch and Voglmayr 2014) and pycnidial anamorphs in most species. Based on multigene phylogenies and morphology, Jaklitsch and Voglmayr (2014) and Voglmayr et al. (2016) synonymised the genera Allantonectria, Mattirolia, Pleonectria and Thyronectroidea with Thyronectria, and several new species were described in recent years (Jaklitsch and Voglmayr 2014; Checa et al. 2015; Voglmayr et al. 2016; Zeng and Zhuang 2017; Lechat et al. 2018; Li et al. 2018; Ma et al. 2020). Ascospores within the genus are remarkably diverse; they can be aseptate, uniseptate, transversely multiseptate or muriform with eusepta and/or distosepta, of allantoid, ellipsoid, oblong, fusiform, globose, clavate or vermiform shapes and with hyaline, yellowish, rosy, green or brown colouration. Ascospores of several species are budding in the ascus to produce oblong to allantoid, 1-celled, hyaline ascoconidia. A common association with effete pyrenomycetes, in particular Diplodia spp., indicates a fungicolous ecology of many species (Jaklitsch and Voglmayr 2014; Voglmayr et al. 2016). Currently, the genus Thyronectria contains 43 accepted species and for 36 of these, DNA sequence data are available.

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.

Materials and methods

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#).

Table 1 Isolates and accession numbers used in the phylogenetic analyses; those in bold were isolated/sequenced in the present study
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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.

DNA extraction, PCR and sequencing

The extraction of genomic DNA, PCR and sequencing of segments of seven loci, i.e. the nu rDNA region encompassing the internal transcribed spacers 1 and 2, along with the 5.8S, and the D1-D2 domains of the LSU (ITS-LSU), α-actin (ACT1) gene, RNA polymerase II subunit 1 (RPB1) and subunit 2 (RPB2) genes, translation elongation factor 1-α (TEF1) gene, and β-tubulin (TUB2) gene, was performed as reported in Jaklitsch and Voglmayr (2014) and Voglmayr et al. (2016).

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. (2012), with additions from Jaklitsch and Voglmayr (2011, 2014), Zeng and Zhuang (2012, 2013, 2017), Checa et al. (2015), Lombard et al. (2015), Crous et al. (2016), Voglmayr et al. (2016), Lechat et al. (2018), Li et al. (2018), Yang et al. (2019) and 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.

Fig. 1
figure 1

Phylogram of the best maximum likelihood tree (lnL = -55,221.1187) revealed by RAxML from an analysis of the combined (ITS-LSU, ACT1, RPB1, RPB2, TEF1, TUB2) matrix of Thyronectria. ML and MP bootstrap support above 50% are given above or below the branches. Strain or herbarium numbers are given following the taxon names; strains in bold red were isolated and sequenced during the present study; holo-, neo- or epitype strains/specimens are marked by an asterisk. The tree was rooted with Septofusidium berolinense, S. herbarum and Tilachlidium brachiatum (Tilachlidiaceae) according to Lombard et al. (2015)

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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/alignment/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 (MULTREES 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 COLLAPSE command set to MINBRLEN.

Results

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.

Taxonomy and descriptions

Thyronectria abieticola Lechat, Gardiennet & J. Fourn., Ascomycete.org 10(1): 56 (2018). Figure 2, 3.

Fig. 2
figure 2

Thyronectria abieticola, teleomorph (BRA CR33223). a–c Stromata emerging through bark. d Transverse section through stroma and perithecium. e Ostiolar canal and periphyses in section. f, g Peridium wall in transverse section. h Apical paraphyses. i Asci. j Ascospores. (all in water except e, g in KOH). Scale bars (a–c) 300 µm; (d) 100 µm; (e, f) 20 µm; (g–j) 10 µm

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Fig. 3
figure 3

Thyronectria abieticola, anamorph (a, d–g BRA CR33223; b, c, h–p WU 44628). a Perithecia surrounded by pycnidia (arrows) on natural substrate (rehydrated). b, c Collapsed pycnidia (dry). d Pycnidium in section. e Pycnidial wall lined with conidiophores and phialides. f Conidiophores arising from pycnidial wall. g Peridium wall in face view. h Sterile hyphae. i, j Squash mount of pycnidial wall in water (i) and KOH (j). k–o Branched conidiophores with phialides. p Conidia. (all vital in water). Scale bars (a) 300 µm, (b) 400 µm, (c) 200 µm, (d) 100 µm, (e–l) 10 µm, (m–p) 5 µm

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For a detailed description of the teleomorph, see Lechat et al. (2018).

Anamorph on the natural host: Pycnidia either associated with perithecia on a common hypostroma or separate, solitary or in small groups, pyriform and dark reddish brown when turgid, collapsed and blackish when dry, 200–740 µm diam, 150–320 µm high, with a distinct apical papilla, surface smooth to slightly warted. Peridium 27–36 µm thick, of two regions: an outer region of thick-walled elongate cells orange to carmine red in water, violet in 3% KOH and an inner rigion of subhyaline to yellowish isodiametric cells 5–10 μm diam with walls up to 1 μm thick; the interior densely clothed with numerous fascicles of conidiophores. Conidiophores densely branched up to 3 times, 22–40 μm long, and 1.5–2.5 μm wide, hyaline, smooth. Phialides terminal, solitary, long cylindrical, slightly tapering towards tip, (6.5–)9.0–12.5(–14.5) × (1.1–)1.3–1.9(–2.6) μm (n = 77). Conidia oblong to ellipsoidal, (2.3–)2.7–3.3(–3.9) × (1–)1.2–1.5(–1.7) μm, l/w = (1.5–)1.9–2.5(–3.5) (n = 159), hyaline, 1-celled, straight to scarcely curved, eguttulate, smooth.

Cultures and anamorph in culture: see Lechat et al. (2018)

Distribution: Europe (Austria, Czech Republic, France, Slovakia).

Ecology: Only known from bark of Abies spp., possibly fungicolous.

Specimens examined: Austria, Oberösterreich, St. Willibald, Großer Salletwald, on dead corticated fallen branch of Abies grandis, soc. Diplodia sp., 48° 21′ 10ʺ N, 13° 42′ 50ʺ E, alt. 440 m, 16 May 2019, H. Voglmayr (WU 44628, holomorph; culture ex anamorph THYA); St. Aegidi, W of Dornedt, 48° 26′ 19ʺ N, 13° 41′ 55ʺ E, alt. 610 m, on dead corticated branches of a felled Abies alba tree, soc. Diplodia sp., 9 May 2021, H. Voglmayr (WU 44629, teleomorph). Slovakia, Poprad, Kvetnica, on fallen dead corticated twigs of Abies alba, soc. Diplodia sp., alt. 770 m, 27 Jan. 2018, A. Polhorský (BRA CR33223, holomorph); Žilina, Istebné, on upper branches and twigs of a recently fallen Abies alba tree, 49° 13′ 12ʺ N, 19° 12′ 10ʺ E, alt. 663 m, 15 Feb. 2018, A. Polhorský (BRA CR33222, holomorph).

Notes: The teleomorph of our collections fully agrees with the original description, which is therefore not repeated here, but we provide illustrations of the teleomorph to complement the illustrations of Lechat et al. (2018). Based on our recent Austrian and Slovakian collections, we here add the description of the newly recorded pycnidial anamorph on the natural substrate. Upon closer examination, the anamorph was present in three of the four collections we studied. All known collections of the species were independently made within a few years in Austria, the Czech Republic, France and Slovakia on a well-investigated host genus, Abies, indicating that T. abieticola may represent a recent introduction, the origin of which is unknown. Macro- and microphotos of the teleomorph of a Czech collection are available at http://www.mykologie.net/index.php/houby/podle-morfologie/perithecia/item/3035-thyronectria-abieticola (accessed 20 Aug. 2021). Like most other Thyronectria species, T. abieticola is tightly associated with Diplodia sp., indicating a fungicolous habit. The connection of the anamorph with the teleomorph has been proven by sequences obtained from conidial and ascospore isolates.

Thyronectria aurigera (Berk. & Ravenel) Jaklitsch & Voglmayr, Persoonia 33: 195 (2014). Figure 4, 5.

Fig. 4
figure 4

Thyronectria aurigera, teleomorph (a, d–q WU 44631; b, c WU 44630). a–c Stromata emerging through bark; arrows denoting pycnidia of Diplodia sp. d Transverse section through stroma and perithecium in KOH. e–h Thin section of peridium in water (e), KOH (f) and LA (g, h) with covering stromatal layer (h). i Section of stroma tissue in water. j Ascus. k Apical paraphyses. l–q Ascospores in water. Scale bars (a–c) 300 µm; (d) 50 µm; (e–q) 10 µm

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Fig. 5
figure 5

Thyronectria aurigera, anamorph on natural substrate (a–h, l) and on CMD (i–k, m) (a–h, l WU 44632; i–k, m WU 44631). a, b Sporodochia below bark; arrows denoting pycnidia of Diplodia sp. c, d Transverse section through sporodochia with superficial hymenial layer of phialides. e–h Squash mount of hymenium, showing phialides arising from globose stroma cells. i–k Phialides and pegs on CMD (after 3 d). l, m Conidia from sporodochia (l) and on CMD (m) (all in water). Scale bars (a, b) 300 µm; (c) 50 µm; (d) 10 µm; (e–m) 5 µm

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Hypostromata erumpent from bark, surrounded by bark flaps or slightly projecting above the bark, well-developed, brown in water, dark purple in 3% KOH, yellow in LA, pseudoparenchymatous of textura angularis of cells 4–18 µm diam with walls 1–1.5 μm thick. Ascomata superficial on the hypostroma, aggregated in groups of up to 30 in round or elongated clusters 0.5–2 mm long and 0.3–0.7 mm high, 200–350 µm diam, 200–300 µm high, subglobose to globose, cupulate or not when dry, brick red to red-brown, slightly darker in 3% KOH, more or less unchanged in LA, covered with an irregularly areolate, bright sulphur yellow scaly scurf. Peridium 35–50 µm thick, of two regions: outer region 20–35 μm thick, orange in water, turning purple in 3% KOH and bright yellow in LA, composed of a textura globulosa-angularis of cells 4–8 µm diam with walls 1.5 μm thick, outwardly covered by a layer of yellow amorphous crystals; inner region 10–20 μm thick, composed of a textura prismatica of elongate thin-walled, hyaline cells. Apical paraphyses numerous, branched, anastomosing, descending to the bases of asci, 2–3.5 μm wide. Asci clavate, (49–)53–70(–82) × (10–)11–14.5(–17) μm (n = 31), with 8 irregularly biseriate ascospores; apex undifferentiated. Ascospores (19.5–)22.5–27.2(–29.3) × (4.8–)5.5–6.7(–7.2) μm, l/w = (3–)3.6–4.6(–5.4) (n = 81), ellipsoidal, oblong to allantoid, curved, with broadly rounded ends, (3–)6–7-septate, hyaline, at maturity becoming yellowish-brown, smooth-walled, not to slightly constricted at the septum, not budding inside or outside the asci.

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–)1.1–1.3(–1.5) μm, l/w = (2.2–)2.3–3.1(–4.3) (n = 44), hyaline, 1-celled, straight, with 0–3 min guttules, smooth.

Distribution: North America and Europe (Austria, Czech Republic, Germany, France, Slovakia).

Ecology: On dead corticated branches of various Oleaceae; in Europe only known on Fraxinus spp., constantly associated with Diplodia sp. and possibly fungicolous.

Specimens examined: Austria, Niederösterreich, Ringelsdorf, riverine forest of the river March, 48° 34′ 13ʺ N, 16° 55′ 25ʺ E, alt. 150 m, on dead corticated branches of Fraxinus pennsylvanica, soc. Diplodia sp., 13 Mar. 2020, H. Voglmayr & I. Greilhuber (WU 44630, teleomorph); Niederösterreich, Bruck an der Leitha, Wolfsthal, Donauauen, 48° 08′ 12ʺ N, 17°01′48"E, alt. 138 m, on dead corticated branches of Fraxinus excelsior, 20 Feb. 2021, H. Voglmayr & I. Greilhuber (WU 43964); Oberösterreich, St. Willibald, Aichet, Au, 48° 21′ 13ʺ N, 13° 41′ 02ʺ E, alt. 400 m, on dead corticated branches of Fraxinus excelsior, soc. Diplodia sp., 14 Dec. 2019, H. Voglmayr (WU 44631, holomorph; culture ex teleomorph THAU); Wien, Donaustadt, Lobau, at the Donau-Oder Kanal near Uferhaus, 48° 10′ 31ʺ N, 16° 31′ 42ʺ E, alt. 155 m, on dead corticated branches of Fraxinus excelsior, soc. Diplodia sp., 14 Mar. 2020, H. Voglmayr & I. Greilhuber (WU 44632, holomorph; culture ex anamorph THAU1).

Notes: Until recently, this species was known only from North America; however, a French collection made in 2001 (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.

Thyronectria citri (C.M. Tian & Q. Yang) Voglmayr, comb. nov.

MycoBank: MB 841960.

Basionym. Neothyronectria citri C.M. Tian & Q. Yang, in Yang, Chen, Jiang & Tian, MycoKeys 56: 56 (2019).

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.

Thyronectria sophorae (Crous & Thangavel) Voglmayr, comb. nov.

MycoBank: MB 841959.

Basionym. Neothyronectria sophorae Crous & Thangavel, in Crous et al., Persoonia 37: 329 (2016).

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.

Thyronectria ulmi Polhorský, Halasů & Voglmayr, sp. nov. Figure 6, 7.

Fig. 6
figure 6

Thyronectria ulmi, teleomorph (BRA CR33224). a–c Stromata emerging through bark. d Transverse section through stroma and perithecium. e Peridium and covering stromatal layer. f–h Mature ascospores in water (f), KOH (g) and LA (h). i Mature and submature asci. j Thin section of peridium. k Thin section of stroma (left) and peridium (right). l Pseudostroma tissue with periderm fragments (all vital in water except where noted). Scale bars (a) 2 cm; (b) 3 mm; (c) 500 µm; (d) 100 μm; (e, i, k) 20 μm; (f–h, j, l) 10 μm

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Fig. 7
figure 7

Thyronectria ulmi, anamorph (BRA CR33224). a Pycnidia exuding tendrils of subhyaline conidial masses. b Transverse section through pycnidium. c Pycnidial wall. d Conidiophores and phialides lining the pycnidium. e Conidia. f Germinating ascospore on PDA, producing conidia. g Colony with scattered pycnidia at the edges (PDA, RT, 4 w). h Pycnidia with conidial masses in vitro. i Conidia in vitro, some budding. j–l Intercalary phialides with conidiogenous pegs on hyphae in vitro (all vital in water). Scale bars (a) 200 µm; (b) 100 μm; (c) 20 µm; (d–f, i–l) 10 μm; (h) 500 µm

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MycoBank: MB 841958.

Etymology: named after its hosts, Ulmus spp.

Stromata immersed to erumpent from bark, surrounded by bark flaps or projecting above the bark; inner tissue consisting of loosely to densely interwoven, (2.2–)3.1–4.6(–6.0) μm wide (n = 40) thin-walled hyphae, in places appearing more cellular, hyaline to subhyaline; covered by a 27–60 μm thick layer of orange-red to dark brown, thick-walled, angular, (2.8–)4–7(–10.4) μm wide cells encrusted in yellow-green amorphous scurf. Scurf consisting of yellow, minute, irregular particles and druses of crystallic appearance, not staining in Cresyl blue, without change in LA, Melzer or 60% aqueous ethanol, in KOH rapidly and entirely dissolving and releasing small amount of yellow pigment. Ascomata embedded in stromatic tissue, scattered or aggregated in groups of 2–20(–40) in rounded or elongated clusters, 0.8–2(–4) mm long, 0.6–1(–2.5) mm wide (n = 25) and 0.8–1.2 mm high, producing dark brown to black spore masses, subglobose to ovoid, not becoming cupulate upon drying, (270–)300–480(–510) μm diam in surface view (n = 10), 330–540 μm high in section, mostly covered by stromatal tissue, subostiolar, exposed regions covered by greenish yellow scurf. Peridium orange-red, dark red, brown, 39–54(–60) μm (n = 12) wide at the sides, consisting of a thin, up to 20 μm thick, inner layer of compressed hyaline, thin-walled cells of, (8.4–)11.8–23.6(–32.2) × (2.5–)3–4.4(–5.3) μm and a pigmented outer layer of compressed, thick-walled cells of (4.0–)5.5–12(–15.7) × (2.6–)3–4.7(–6) μm (n = 40), orange-red in KOH, (yellowish) orange in LA; cells around the ostiole small and more isodiametric. without a distinct pH-dependent colour change in KOH and LA. Ostiolar region 50–250 μm diam in surface view, concealed by the scurf when young, later black, smooth. Ostiole periphysate, 20–50 μm wide in section. Periphyses pointed, 15–30(–52) μm long, 1.2–2.2 μm wide, embedded in gelatinous matrix, projecting into the ostiole and slightly downward. Apical paraphyses numerous, indistinct in KOH, embedded in a slime matrix when young, reaching down to the base of the asci, richly branched and anastomosing, 1.8–4.3 μm wide. Asci clavate, (120–)138–170(–175) × (18–)22–26(–30) μm (n = 25), with variably long stipe with croziers and undifferentiated apex, containing (2–4–)8 obliquely uniseriate or biseriate ascospores. Ascospores broadly ellipsoid to cylindrical, rarely clavate, straight or curved, assymetrical, (20–)23–29(–33.5) × (9.3–)11.5–15(–16.2) μm, l/w = (1.4–)1.7–2.4(–3.1) (n = 80), muriform, eudistoseptate, with 5–8 (rarely 9) transverse and 1–3 longitudinal, less commonly oblique septa, hyaline and often more oblong when immature, turning olive green, yellow–brown or brown at maturity, smooth, not budding in the ascus, mature spores vividly green in KOH, wine red in LA.

Anamorph on the natural host: Pycnidia either associated with perithecia on a common stroma or separate, solitary or in small groups, ellipsoid, vertically elongated, 0.3–0.4 mm diam, distinguishable from ascomata only when wet, when producing subhyaline conidial masses. Peridium yellow-orange to red-brown, 26–53 μm wide, of thick-walled, ± isodiametric cells (2.8–)4.3–8(–11) μm diam; the interior densely lined with occasionally branching conidiophores, (9.5–)15.2–29(–36) × (1–)1.4–2(–2.4) μm. Phialides terminal, solitary, long cylindrical, slightly conical, (4.1–)7–10(–11.1) × (1.4–)1.6–2(–1.9) μm. Conidia oblong-cylindrical, (2.5–)2.8–3.8(–4.3) × 1.0–1.3(–1.4) μm, l/w = (2–)2.4–3.2(–3.6) (n = 40), hyaline, 1-celled, straight to slightly curved, eguttulate or with minute guttules, smooth.

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(–5.1) (n = 90), straight to curved, unicellular, hyaline, smooth, with few minute, often subterminal guttules, budding.

Distribution: Central Europe (Austria, Czech Republic, Slovakia).

Ecology: On dead corticated trunks and branches of Ulmus spp.; associated with effete conidiomata of Diplodia sp. and possibly fungicolous.

Type: Slovakia, Bratislava, Bratislava – Karlova Ves, Sihoť, 48° 08′ 58.0ʺ N 17° 01′ 20.1ʺ E, alt. 147 m, on recently fallen Ulmus laevis tree, stromata on corticated branches and twigs, associated with Diplodia sp., 10 Jun. 2020, A. Polhorský (BRA CR33224 holotype, WU 44633 isotype, holomorph; ex-holotype culture ex teleomorph CBS 147674 = TCH1).

Other material studied (all associated with Diplodia sp.): Austria, Niederösterreich, Engelhartstetten, Nationalpark Donauauen, 48° 09′ 40.2ʺ N, 16° 55′ 24.2ʺ E, alt. 140 m, on fallen branch of Ulmus laevis, 6 Mar. 2021, H. Voglmayr & I. Greilhuber (WU 44634, teleomorph; culture TCH2); Marchegg, WWF-Auenreservat Marchauen, 48° 17′ 01.5ʺ N, 16° 53′ 50ʺ E alt. 140 m, on corticated dead branches of a standing Ulmus laevis tree, 22 May 2021, H. Voglmayr & I. Greilhuber (WU 44635, teleomorph); ibid, same host, 13 Nov. 2021, Voglmayr & I. Greilhuber (WU 44947, holomorph); Ringelsdorf, riverine forest of the river March, 48° 34′ 17ʺ N, 16° 55′ 17ʺ E, alt. 150 m, on corticated dead branch of a standing Ulmus laevis tree, 13 Mar. 2021, H. Voglmayr & I. Greilhuber (WU 44636, teleomorph; culture TCH4); Stockerau, Stockerauer Au, riverine forest along the Göllersbach, 48° 22′ 46ʺ N, 16° 11′ 37ʺ E, alt. 170 m, on dead cut corticated branches of Ulmus laevis, 2 May 2021, H. Voglmayr & T. Kirisits (WU 44637, anamorph; culture TCH5). Czech Republic, Morava, Olomouc, Černovírské slatiniště forest, 49° 37′ 32.5ʺ N, 17° 15′ 53.7ʺ E, alt. 210 m, on corticated fallen branch of Ulmus laevis, 27 Oct. 2020, V. Halasů (unpreserved, holomorph); ibid., on the same branch, 9 Dec. 2020, V. Halasů (BRNM 828,876, holomorph); ibid., 49° 37′ 02.5ʺ N 17° 16′ 15.4ʺ E, alt. 210 m, fallen crown of a young Ulmus minor, on corticated branches, 9 Dec. 2020, V. Halasů (PRM 955819, holomorph). ibid., same place and piece of substrate, 25 Feb. 2021, V. Halasů (WU 44638, holomorph; ex anamorph culture TCH3). Slovakia, Bratislava, Bratislava – Karlova Ves, Sihoť, 48° 08′ 58.0ʺ N 17° 01′ 20.1ʺ E, alt. 147 m, recently fallen Ulmus laevis tree, on corticated branches and twigs, 4 Mar. 2020, A. Polhorský (BRA CR33231, HR B006924, holomorph).

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.

Thyronectria zanthoxyli (Peck) Ellis & Everh. [as 'xanthoxyli'], N. Amer. Pyren. (Newfield): 92 (1892). Figure 8.

Fig. 8
figure 8

Thyronectria zanthoxyli (WU 44639). a–e Stromata emerging through bark; in d, e with effete conidiomata of Diplodia sp. (white arrows). f Ascus. g–l Ascospores. m, n conidiophores from effuse conidiation in pure culture, showing pegs, phialides and conidia (CMD, RT, 4 d) (all vital in water). Scale bars (a) 1 mm; (b, c) 500 µm; (d, e) 200 μm; (f–n) 10 μm

Full size image

Specimens examined. Belgium, Prov. Antwerpen, Meerhout, 51° 8′ N, 5° 5′ E, on dead branch of Sorbus aucuparia, 12 Dec. 2019, C. van Steenwinkel (WU 44639, culture NP12). USA, Ohio, Columbus, Ohio State University, 39° 59′ 58.2ʺ N 83° 00′ 56.2ʺ W, on dead branches of Ulmus sp., 6 Nov. 2018, D. Grootmyers (WU 44640, teleomorph; culture NP11).

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.

Additional new host records

Thyronectria cucurbitula (Tode) Jaklitsch & Voglmayr, Persoonia 33: 201 (2014).

Specimen examined. Czech Republic, Morava, Olomouc, Černovírské slatiniště forest, near a small lake (former sand and gravel pit), 49° 37′ 9.26ʺ N, 17° 16′ 2ʺ E, alt. 210 m, on bark of twigs of a fallen partly decorticated crown of Pinus strobus, 2 May 2021, V. Halasů (WU 44641, teleomorph; culture THST1).

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).

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) that was published without sequence data needs confirmation.

Thyronectria rhodochlora (Mont.) Seeler, J. Arnold Arbor. 21: 455 (1940).

Specimens examined. Austria, Oberösterreich, Raab, Rotes Kreuz, Rothmayrberg, 48° 22′ 07ʺ N, 13° 39′ 57ʺ E, alt. 430 m, on dead fallen corticated branch of Acer pseudoplatanus, 23 Aug. 2020, H. Voglmayr (WU 44642, teleomorph; culture NP13). Czech Republic, Morava, Olomouc, Černovírské slatiniště forest, 49° 37′ 9.96ʺ N, 17° 15′ 51.12ʺ E, alt. 210 m, on dead twigs in a fallen crown of Fraxinus excelsior, 13 Nov. 2020, V. Halasů (PRM 955820, teleomorph).

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).

Specimen examined. Czech Republic, Moravia, Olomouc, Botanic garden of Palacký University, 49° 35′ 14ʺ N, 17° 15′ 07ʺ E, alt. 215 m, on dead twigs of Hedera colchica, 24 Mar. 2021, V. Halasů (WU 44643, teleomorph). Spain, Jaén, Valdepeñas de Jaén, Las chorreras, 30SVG 28,511 60,458, 37° 35′ 18.28ʺ N, 3° 48′ 35.04ʺ W, altitude 957 m, on dead branches of Bupleurum fruticosum, 1 Jul. 2019, S. Tello S.T.01071902 (WU 44644, teleomorph; culture THYB).

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.

Key to species of Thyronectria (adapted from the key in Jaklitsch and Voglmayr 2014)

Notes: The key is primarily based on teleomorph characters. Therefore, Thyronectria sophorae could not be included in the key, as only the pycnidial anamorph is known. Some species (e.g. from the Thyronectria cucurbitula species complex) can reliably only be identified by DNA sequence data. Unless otherwise noted, ascomata occur on dead host bark.

1. Ascospores aseptate, hyaline, allantoid to short-cylindrical...................... 2

Ascospores septate, hyaline or distinctly coloured........... 4

2. On dead bark of Populus; ascospores 3.5–5.5(–6) × 0.9–1.2(–1.4) μm......................... T. zangii

On other hosts (dead leaves of Agave, Yucca).....................3

3. On Agave; ascospores (3.3–)4.3–5.5(–7.3) × (0.9–)1.2–1.6(–2) μm.............. T. concentrica

On Yucca; ascospores (4.5–)5.5–7.3(–9.6) × (1.1–)1.4–1.8(–2.3) μm........... T. yuccae

4. Ascospores at maturity distinctly green to brown, muriform, distoseptate, not budding............. 5

Ascospores hyaline to yellowish or rosy, variously euseptate............ 10

5. Ascospores on average distinctly longer than 22 µm............ 6

Ascospores on average distinctly shorter than 22 µm.......... 8

6. On Ulmus; transverse septa of ascospores equal, ± regular, evenly spaced; ascospores turning wine red in lactic acid, without distinct hyaline sheath in KOH when immature.............. 7

Not on Ulmus; transverse septa of ascospores unequal, with first-formed 1–3 transverse septa of ascospores distinctly more pronounced than the others; ascospores irregularly muriform-septate, not turning wine red in lactic acid, 22–28(–30) × 9–13 µm, with distinct hyaline sheath in KOH when immature............... T. giennensis

7. On Ulmus americana in North America; ascospores (24–)29–34.5(–37) × (10–)12.5–15(–17) μm................................. T. chrysogramma

On Ulmus spp. in Europe; ascospores (20–)23–29(–33.5) × (9.5–)11.5–15(–16) μm..... T. ulmi

8. On Fabaceae in Southern Europe; ascospores (13–)15–20(–25.5) × (7.5–)9–11(–13) μm................. T. roseovirens

On other hosts................ 9

9. Ascospores mostly oblong, 14–22 × 6.5–9.5 μm, often curved, with 1 longitudinal septum, cells containing one large guttule when fresh T. asturiensis

Ascospores ellipsoid to subglobose, 17–22 × 10–13 µm, straight, with 1–3 longitudinal septa, cells finely multiguttulate when fresh............ T. pistaciae

10. Ascospores in nature typically not budding................................................. 11

Ascospores in nature budding in or outside asci.................................................... 27

11. Ascospores 1- to multiseptate but lacking longitudinal septa...................... 12

Ascospores muriform....................................................................................... 18

12. Ascospores (1–)6–7-septate, smooth............................................................ 13

Ascospores 1-septate, smooth or striate............................................................... 14

13. Perithecia red to red-brown; ascospores ellipsoid, oblong to allantoid, with broadly rounded ends, (15–)17–21(–25) × (4.5–)5.0–6.5(–7.3) μm; on deciduous trees (mainly Oleaceae) in Europe and North America............................................. T. aurigera

Perithecia dark brown to black; ascospores ellipsoid to fusiform, with narrowly rounded to subacute ends; (12–)18.5–26.4(–32.5) × (4.3–)6.2–8.5(–10) μm; on Berberis in East Asia (China)......................... T. berberidis

14. Ascospores striate, ellipsoid to fusiform, (13–)14–17(–18.5) × (4.5–)5.3–6.7(–7.3) μm; only known from Argentina....................... T. pseudomissouriensis

Ascospores smooth........................................... 15

15. Perithecia greenish to brownish black; ascospores (8.5–)10–13.5 × 5–7 μm; on Abies ... T. abieticola

Perithecia red to red-brown; on other hosts.......................................................... 16

16. Ascospores ellipsoid to fusiform, not constricted at the central septum, (9–)10–12(–13.5) × (3.3–)4.0–5.0(–5.7) μm; on Citrus, Gelsemium and Ilex.......... T. rubicarpa

Ascospores ellipsoid to fusiform, slightly constricted at the central septum; on Hedera, Bupleurum or Ilex.........17

17. On Ilex; ascospores ellipsoid, (9–)11–13.5(–15.5) × (4.0–)5.5–6.8(–7.5) μm; conidia in culture (5.5–)6.5–9.5(–12.5) × (2.0–)2.3–3.0(–3.3) µm...................... T. ilicicola

On Hedera or Bupleurum; ascospores ellipsoid to fusiform, (8–)10.5–13(–14.5) × (3.7–)5.0–6.5(–8.0) μm; conidia in culture (5.2–)6.0–11.0(–13.5) × (1.0–)1.5–2.5(–3.0) μm................ T. sinopica

18. Perithecia typically superficial on or partly immersed in a hypostroma...... 19

Perithecia surrounded by stromatic tissue............................. 23

19. Perithecia partly immersed in a well-developed hypostroma; reddish grey, yellowish brown, olive brown or dark umber brown to almost black................ 20

Perithecia superficial on a hypostroma; scarlet to red-brown.................................. 22

20. Perithecia typically aggregated in large groups of up to 200; ascospores with 1–3 transverse septa, subglobose to ellipsoid, straight, with broadly rounded ends, (9.7–)10–12.5(–15) × (4.8–)6.0–7.5(–10.2) μm; on Fabaceae; in nature pycnidia forming tubercular to cerebriform masses; conidia ellipsoid to oblong, (1.7–)2.3–3.0(–3.5) × (1.0–)1.3–2.0(–2.5) μm........................ T. austroamericana

Perithecia aggregated in groups up to 40; ascospores with (3–)4–10 transverse septa, ellipsoid to oblong-fusiform, commonly curved, with narrowly rounded ends; pycnidia in nature unknown....................... 21

21. Ascospores ellipsoid to subfusiform, with (3–)4–6 transverse septa, (10–)11–20(–21) × 5–7 µm; only known from China....................................... T. orientalis

Ascospores ellipsoid to oblong-fusiform, with (5–)7(− 10) transverse septa, (14.3–)18–24(− 28.4) × (4.8–)6.3–7.7(− 9.2) μm; on Caragana in Europe......................... T. caraganae

22. Ascospores of two sizes: micro-ascospores allantoid to short-cylindrical, (21–)25–30(–32.5) × (8.2–)9.5–12(–13) μm, macro-ascospores cylindrical, (37–)39–47(–49.5) × (10–)10.5–12(–13) μm; on Carya in North America ... T. missouriensis

Ascospores uniform, oblong to ellipsoid, 17–25 × 6.5–8.5 μm; on Ribes T. berolinensis

23. Saffron to sienna stroma surrounding perithecia immersed in bark, scarcely protruding, with red–orange apex; asci long and slender; on Lonicera involucrata and Symphoricarpos in the USA.................................................................. T. lonicerae

Perithecia immersed in yellowish stroma erumpent from bark; ascospores yellowish to rosy at maturity; asci clavate.................... 24

24. Asci four-spored, ascospores ellipsoid, (17–)18–21(–23.5) × 8–9(–10) μm, on Citrus in East Asia (China)...................... T. citri

Asci eight-spored; on other hosts in Europe and North America.............................. 25

25. Ascospores ellipsoid or oblong, (15–)18–25(–37) × (7–)9–12(–16) µm; chiefly on Acer campestre, but also other trees in Europe T. rhodochlora

Ascospores oblong and often curved, averaging < 9 μm wide................................. 26

26. Ascospores (13–)16–21(–23) μm long; mean colony diameter 14 mm on PDA after 7 days at 25 °C; chiefly on Rhus, but also other trees and shrubs..... T. virens

Ascospores (18–)19–24(–26.5) μm long, distinctly curved; mean colony diameter > 67 mm on PDA after 7 days at 25 °C; chiefly on Zanthoxylum, but also other trees and shrubs.................. T. zanthoxyli

27. Ascospores budding outside asci in perithecia........................ 28

Ascospores budding inside asci.......... 29

28. Ascospores 1-septate, (8.7–)10–12.5(–13.5) × (3.7–)4.5–6.0(–6.8) μm; in culture conidia 1-celled, ellipsoid, fusiform or allantoid, (5–)7–10(–11.5) × (1.8–)2.0–2.8(–3.3) μm; on Castanopsis in Japan................................. T. okinawensis

Ascospores muriform, mostly with 7 transverse septa and 1 longitudinal septum, 17–25 × 6.5–8.5 μm; in culture conidia swollen, ellipsoid, oblong-allantoid, (0–)1(–2)-septate, (9–)10–14(–20) × (2.2–)3.3–4.7(–5.5) μm; on Ribes............ T. berolinensis

29. Ascospores 1-septate................................... 30

Ascospores multiseptate or muriform............. 32

30. Perithecia dark to blackish brown; ascospores ellipsoid to broadly fusiform, 6–10 × 2–3 µm, on Eleutherococcus in China................ T. atrobrunnea

Perithecia red to red-brown; ascospores larger..................................................... 31

31. Ascospores ellipsoid to fusiform, (8–)9–11(–13) × (3.2–)4–5.5(–6.5) μm; on Ilex aquifolium; in Europe ... T. aquifolii

Ascospores cylindrical or fusiform, (8.3–)10–13(–15) × (2.2–)2.8–4(–5.3) μm; on Corylus avellana and other deciduous trees and shrubs.......................................... T. coryli

32. Ascospores filiform, transversely multiseptate............................................ 33

Ascospores muriform....................................................................................... 37

33. Ascospores 8–15-septate, hyaline, (26–)31–44(–49) × (1.3–)2.3–4.0(–4.7) μm; on Quercus ilex ssp. rotundifolia............................................... T. quercicola

Ascospores 8–44-septate, long-filiform, 22–75 μm long; on conifers....................... 34

34. On Abies; ascomatal surface scaly; ascospores 8–31-septate, (22–)29–45(–60) × (1.5–)2.0–3.2(–4.0) μm............................................................... T. rosellinii

On Pinus; ascomatal surface generally scurfy (species reliably only distinguishable by DNA sequence data)................................................................................................ 35

35. Ascospores 11–27-septate, (28–)33–63(–70) × 2.5–3.5 µm; only known from China ... T. sinensis

Ascospores with more septa ... 36

36. Mostly on Pinus subgenus Pinus; ascospores 15–39-septate, (33–)43–65(–75) × (2.3–)2.7–3.5(–3.7) μm; confirmed by DNA data only from Europe.......... T. cucurbitula

On Pinus subgenus Strobus; ascospores 12–44-septate, (22–)33–52(–64) × (2.0–)2.2–3.2(–4.0) μm; confirmed by DNA data only from North America............................ T. strobi

37. Ascospores disarticulating; part-ascospores subglobose to ellipsoid, (7.7–)8.7–12(–13.5) × (5.0–)6.5–8.5(–9.0) μm; on Platanus occidentalis and Ulmus americana; in USA........................................................................... T. chlorinella

Ascospores not disarticulating........................................................................... 38

38. Ascospores subglobose to ellipsoid, (5.0–)5.5–7.5(–9.5) × (4.0–)4.5–6.5(–8.5) μm; on Fabaceae........................ T. sphaerospora

Ascospores differently shaped........................................................................... 39

39. Ascospores distinctly clavate............................................................................... 40

Ascospores ellipsoid, oblong-clavate, cylindrical, fusoid, fusiform or vermiform ... 41

40. Ascospores broadly clavate with longitudinal septa not restricted to the upper (wider) part, (16–)18–23(–36.5) × (4.3–)4.8–6.2(–7.0) μm; ascomata collapsing; on Ribes in North America............................................................ T. clavatispora

Ascospores narrowly clavate with longitudinal septa only in the upper (wider) part, (21–)25–33(–38) × (4.0–)5.0–6.2(–7.2) µm; ascomata not collapsing; on Berberis cretica, B. hispanica, B. cf. lycium in Greece, Morocco, Spain and Pakistan......... T. caudata

41. Perithecia entirely black; ascospores fusiform, oblong, vermiform or clavate; (12.5–)17.0–24.5(–28.5) × (3.5–)4.0–5.2(–6.5) µm; on Tamarix in Europe ... T. obscura

Perithecia at least partly with shades of red.......................................................... 42

42. Ascospores fusoid or oblong to oblong-clavate, averaging > 5 μm wide; on Berberis............. 43

Ascospores ellipsoid, fusiform, cylindrical to vermiform, averaging < 5 μm wide; on conifers................................ 44

43. Perithecia orange, red to red-brown, often with a darker apex; asci (94–)108–143(–160) × (17–)18.7–26.5(–30) μm; ascospores mostly fusoid with narrowly rounded to subacute ends, (13.5–)18.5–25.5(–30.0) × (4.8–)5.5–7.2(–8.2) µm...................................................................................................... T. lamyi

Upper half of perithecia blackish brown, gradually turning red-brown towards the base; asci (57–)67–99(–118) × (9–)12–21(–26) μm; ascospores mostly oblong to oblong clavate with broadly rounded ends, (15–)19–26 (–33.5) × (3.5–)4.5–6.3(–9) μm......................................... T. berberidicola (nom. inval.)

44. On Picea; perithecial wall at the apex of three regions; ascospores with 7–25 transverse septa, cylindrical to vermiform, (15.5–)20–30(–36) × (2.8–)3.2–4.2(–4.5) μm; in culture conidia long-cylindrical to allantoid, (7.5–)9–11(–12.3) × (1.3–)1.5–2.0 μm........... T. boothii

On Abies or Pinus; perithecial wall at the apex of two regions; ascospores averaging > 4.5 μm wide........................................................................................... 45

45. On Abies; ascospores ellipsoid to fusiform with 5–9 transverse septa, (16–)20–24(–28.5) × (3–)4–5.5(–7) μm; in culture conidiophores not abundant; conidia subglobose to ellipsoid, (6.0–)6.5–7.2(–9.0) × (2.2–)2.5–3.3(–3.5) μm................................... T. balsamea

On Pinus; ascospores ellipsoid, fusiform to vermiform with 5–15 transverse septa, (14–)18–28(–46.5) × (3.2–)4.3–5.3(–7) μm; in culture conidiophores abundant; conidia oblong, slightly swollen at both ends, (5.5–)7–11(–13) × (1.7–)2.0–2.7(–3.0) μm.............. T. pinicola

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).

We here also report a new sporodochial anamorph on natural substrate for T. aurigera. This is remarkable, as Thyronectria (syn. Pleonectria) species usually have pycnidial anamorphs (Hirooka et al. 2012). However, also T. concentrica, previously known as Allantonectria miltina, has a sporodochial anamorph (Voglmayr et al. 2016). As T. aurigera and T. concentrica are located at basal positions in the phylogenetic analyses (Fig. 1), sporodochial anamorphs may be considered ancestral for the genus, and it is an additional argument for the inclusion of the genus Allantonectria in Thyronectria as advocated by Voglmayr et al. (2016). However, by reporting effuse sporulation on natural substrates for T. roseovirens, Jaklitsch and Voglmayr (2014) already pointed out that pycnidial anamorphs are not diagnostic for the genus Thyronectria.

Our multigene phylogenies also resolved the position of the genus Neothyronectria recently described by Crous et al. (2016). The results show that the genus Neothyronectria is not only embedded within Thyronectria but also closely related to the type of the genus, T. rhodochlora (Fig. 1). A close relationship is also supported by the teleomorph morphology of N. citri (Yang et al. 2019), which fully agrees with the T. rhodochlora group in ascomatal and ascospore characters. This corroborates that Neothyronectria is synonymous with Thyronectria, into which its two species are therefore newly combined here.

We also provide new country and host reports for other Thyronectria species confirmed by sequence data. Thyronectria abieticola, a recently described species from France (Lechat et al. 2018) is newly reported for Austria and Slovakia. Likewise, until recently, in Europe, T. aurigera, a primarily North American species, has been only known from France (Hirooka et al. 2012), and the current records from Fraxinus spp. are new for Austria. Concluded from several recent reports from Central Europe (e.g. Czech Republic, Germany, Slovakia), this species may have recently been favoured in the course of ash dieback disease, which has substantially increased the availability of suitable host substrates.

Additional new host and distribution records confirmed by sequence data include T. rhodochlora from Acer pseudoplatanus in Austria and Fraxinus excelsior in the Czech Republic, T. sinopica from Hedera colchica in the Czech Republic and Bupleurum fruticosum in Spain, and T. zanthoxyli from 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, suggesting that T. cucurbitula is not confined to pine species from subgen. Pinus but also occurs on subgen. Strobus. Confirmed records of T. strobi are currently only known from North America. Therefore, additional sequence data are required to reveal the species identity of the European (Hirooka et al. 2012) and Chinese (Zeng and Zhuang 2017) records of T. strobi.