Introduction

During a study of corticolous ascomycetes of Mallorca, the second author made several collections of dothideomycetes with hysteriform to apothecial ascomata embedded in host tissue and lacking an excipulum, which showed a character combination of branched, septate, apically swollen paraphyses with dark brown incrustation, saccate, bitunicate asci, and large, brown, asymmetric, one- or three-septate ascospores with an excentric euseptum and eventually two additional distosepta with large pores, each hemispore part being surrounded by a gelatinous sheath. These characters resembled the recently described genus Stigmatodiscus (Voglmayr et al. 2016, 2017), but the Mallorcan collections did not match any described species. Therefore, they were isolated it in pure culture; DNA sequence data of nuc18S-ITS-28S rDNA, rpb2, tef1, and tub2 were generated for phylogenetic analyses; and detailed morphological examinations were conducted. As a result, three new species of Stigmatodiscus were revealed, which are here described and illustrated.

Materials and methods

Morphological observations

Stereomicroscopy illustrations were captured either with a Keyence VHX-6000 system or with a Nikon SMZ 1500 stereomicroscope equipped with a Nikon DS-U2 digital camera. For certain images of ascomata, the stacking software Zerene Stacker version 1.04 (Zerene Systems LLC, Richland, WA, USA) was used. Hand sections of ascomata and conidiomata were made using a razor blade and mounted in water or 3% KOH on a microscope slide, gently torn apart with a preparation needle when necessary and covered with a cover slip. Slides were examined and photographed using a Zeiss Axio Imager.A1 (Zeiss, Jena, Germany) microscope equipped with a Zeiss Axiocam 506 colour digital camera. Measurements were done with the Keyence VHX-6000, NIS-Elements D v.3.0 or Zeiss ZEN Blue Edition software packages and are reported as maxima and minima in parentheses and the range representing the mean plus and minus the standard deviation of a number of measurements given in parentheses. The specimens were deposited in the fungarium of the University of Vienna (WU).

Pure culture isolation

Mature ascomata on corticated twigs were horizontally cut using a sterile razor blade, the apothecia separated from the surrounding host tissue, transferred to a sterile drop of water on a microscope slide, torn apart with a forceps to release the ascospores from asci, which were pipetted on a 2% malt extract agar (MEA) plate supplemented with 200 mg/l penicillin G and streptomycin sulphate (Sigma-Aldrich, St. Louis, MO). Germinated ascospores were then transferred to 2% MEA or 2% corn meal agar (CMA, Sigma-Aldrich) supplemented with 2% w/v dextrose (CMD) plates, which were sealed with laboratory film and incubated at 16 or 22 °C. Cultures were deposited at the Westerdijk Fungal Biodiversity Centre, Utrecht, The Netherlands (CBS culture collection).

DNA extraction, PCR and sequencing

Growth of liquid cultures and extraction of genomic DNA was done according to Voglmayr and Jaklitsch (2011), using the DNeasy Plant Mini Kit (QIAgen GmbH, Hilden, Germany). The following sequence regions were used for identification and phylogenetic analyses: the complete nucITS region and D1 and D2 domains of nuc28S rDNA region (ITS-LSU) were amplified using the primers V9G (de Hoog and Gerrits van den Ende 1998) and LR5 (Vilgalys and Hester 1990). The nuc18S rDNA region (SSU) was amplified with primers SL1 (Landvik et al. 1997) and NS24mod (Voglmayr and Jaklitsch 2011). A ca 1.2 kb fragment of the RNA polymerase II subunit 2 (rpb2) gene was amplified using the primers fRPB2-5f and fRPB2-7cr (Liu et al. 1999). A ca 1.3–1.7 kb fragment of translation elongation factor 1-α (tef1) gene was amplified with the primers EF1-728F (Carbone and Kohn 1999) and TEF1-LLErev (Jaklitsch et al. 2005) or EF1-2218R (Rehner and Buckley 2005), and a ca 0.8 kb fragment of the beta tubulin (tub2) gene with primers T1 (O’Donnell and Cigelnik 1997) or T1HV and BtHV2r (Voglmayr et al. 2016). PCR products were purified using an enzymatic PCR cleanup (Werle et al. 1994) as described in Voglmayr and Jaklitsch (2008). DNA was cycle-sequenced using the ABI PRISM Big Dye Terminator Cycle Sequencing Ready Reaction Kit v. 3.1 (Applied Biosystems,Warrington) and the PCR primers; in addition, the following primers were used: ITS-LSU region: ITS4 (White et al. 1990), LR2R-A (Voglmayr et al. 2012) and LR3 (Vilgalys and Hester 1990); SSU: NS1088 (Kauff and Lutzoni 2002). Sequencing was performed on an automated DNA sequencer (ABI 3730xl Genetic Analyzer, Applied Biosystems).

Phylogenetic analyses

To reveal the phylogenetic position of the new isolates produced in the present study, a matrix of aligned nucleotide sequences from the four different phylogenetic markers (SSU-ITS-LSU, rpb2, tef1 and tub2) was produced. GenBank sequences of four taxa (Anisomeridium ubianum and Megalotremis verrucosa from Monoblastiales, Dyfrolomyces rhizophorae from Dyfrolomycetales and Palawania thailandense from Palawaniaceae) were selected as outgroup according to Voglmayr et al. (2017) and the results of BLAST searches. Sequences were aligned with the server version of MAFFT (www.ebi.ac.uk/Tools/mafft) and subsequently checked and refined using BioEdit version v. 7.0.9.0 (Hall 1999). For alignment of rpb2, the alignment was translated into a protein matrix and the gap positions corrected according to the codons. The combined sequence matrix contained 6723 nucleotide positions (1770 from SSU, 1514 from ITS-LSU, 1167 from rpb2, 1417 from tef1, 855 from tub2). GenBank accession numbers of the sequences included in the phylogenetic analyses are given in Table 1.

Table 1 Isolates and GenBank accession numbers of sequences used in the phylogenetic analyses; those in bold were isolated/sequenced in the present study
Full size table

Maximum likelihood (ML) analyses were performed with RAxML (Stamatakis 2006) as implemented in raxmlGUI 1.3 (Silvestro and Michalak 2012), using the ML + rapid bootstrap setting and the GTRGAMMA substitution model with 1000 bootstrap replicates. The matrix was partitioned for the individual gene regions, and substitution model parameters were calculated separately for them.

Maximum parsimony (MP) analyses were performed with PAUP v. 4.0a161 (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 NO. Bootstrap analysis with 1000 replicates was performed in the same way, but using 5 rounds of random sequence addition and subsequent TBR branch swapping during each bootstrap replicate.

Bootstrap support below 70% was considered low, between 70 and 90% medium/moderate and above 90% high.

Results

Molecular phylogeny

For S. pinicola, no tef1 could be obtained. Of the 6723 nucleotide positions, 1016 were parsimony informative (64 from SSU, 310 from ITS-LSU, 258 from rpb2, 231 from tef1 and 153 from tub2). The parsimony analyses revealed 27 MP trees 2398 steps long, one of which is shown as phylogram in Fig. 1. Tree backbone of the 27 MP trees was identical, except for minor differences within S. enigmaticus. The best tree revealed by RAxML (-ln = 20,909.518) was fully compatible with the MP strict consensus tree.

Fig. 1
figure 1

Phylogram showing one of 27 MP trees 2398 steps long obtained from an MP analysis of the combined multigene matrix of nucSSU-ITS-LSU rDNA, rpb2, tef1 and tub2 from Stigmatodiscus. MP and ML bootstrap values above 50% are given at first and second position, respectively, above the branches. The isolates of the new species described in the present study are formatted in bold

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In the MP and ML analyses, the Stigmatodiscales were highly supported, and all Stigmatodiscus species for which more than one accession was sequenced received maximum support. Within Stigmatodiscus, the newly described Stigmatodiscus oculatus formed a highly supported clade with Asterodiscus tamaricis, and this clade was revealed as sister group to the other Stigmatodiscus species with high support. The newly described S. labiatus clustered with S. pruni with low (59% MP) to medium (72% ML) support, and the newly described S. pinicola was placed as sister species to the S. pruni-S. labiatus clade without support. The clade containing S. enigmaticus, S. labiatus, S. pinicola and S. pruni received maximum (MP) or high (96% ML) support, but within this clade, the sister group relationship of S. enigmaticus to the S. pinicola-S. pruni-S. labiatus subclade was unsupported.

Culture characteristics

Culture images of the three new Stigmatodiscus species grown on CMD are shown in Fig. 2. Detailed culture descriptions are given under the respective species.

Fig. 2
figure 2

Stigmatodiscus cultures on CMD after 42 days at 16 °C; a–c in face view, d–f reverse. a, d S. labiatus; b, e S. oculatus; c, f S. pinicola. Sources: a, d AP6516; b AP231016; c, f AP21916b; e AP311216

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Taxonomy

Stigmatodiscus labiatus Voglmayr & Pintos, sp. nov. Figs. 3, 4.

Fig. 3
figure 3

Stigmatodiscus labiatus, sexual morph. a–h Ascomata erumpent from bark in face view. i, j Vertical sections of ascomata embedded in bark. k–m Asci. n Apically inflated septate paraphyses, covered by an dark brown amorphous incrustation. o–u Vital ascospores. All in water. Sources: a–h, l, o–u WU 39973 (holotype); i–k, m, n WU 39980. Scale bars: a, b, e 500 μm; c, d, f–h 200 μm; i, j 50 μm; k–u 10 μm

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Fig. 4
figure 4

Stigmatodiscus labiatus, asexual morph. a, b Conidiomata (pycnidia) immersed in periderm in vertical section. c–e Conidiophores with densely aggregated phialides. f, g Phialides. h–p Falcate to semicircular conidia. All in water, except a, c–g, p in 3% KOH. Sources: a, c–g, p WU 39980; b, h–o WU 39973 (holotype). Scale bars: a, b 20 μm; c–g 10 μm; h–p 5 μm

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MycoBank MB 827487.

Etymology: Referring to the lip-shaped ascomata.

Ascomata hysteriform, scattered, rarely gregarious or confluent, corticolous, erumpent through the periderm, in face view (275–)380–1000(–1610) μm long, (145–)200–350(–570) μm wide (n = 51), with sides consisting of usually two, rarely three black lips (peridium), (45–)90–170(–200) μm wide (n = 50), with a narrow central slit, usually closed when dry and not exposing the blackish elongated disc. Bark tissues not visibly altered, no black line visible in bark or wood. Peridium coriaceous, pseudoparenchymatous, black, up to 130 μm thick at the apex, 17–31 μm thick at the sides, almost absent to 10 μm thick at the base, composed of small angular to rounded, dark brown, thick-walled cells 4–8 μm diam. Hamathecium composed of hyaline, septate, filiform branched paraphyses 1.7–3 μm wide, embedded in a tough hymenial gel, simple, not anastomosing, 82–130 μm long, longer than asci, swollen at their apices up to 4 μm and incrusted with dark brown granules forming an epithecium, neither staining blue in Lugol nor in Melzer’s reagent after pre-treatment with 3% KOH. Asci (67–)78–103(–108) × (41–)43–59(–71) μm (n = 18), bitunicate, broadly ellipsoid to globose, almost sessile, with a distinct apical chamber, thick-walled, typically containing 8 irregularly bi- to triseriate ascospores, very stable, fissitunicate dehiscence not observed. Ascospores (34.5–)38–43(–47.5) × (13.8–)15.5–17.5(–19.3) μm, l/w = (2.2–)2.3–2.6(–2.8) (n = 84), brown, asymmetric, broadly fusiform, straight, 1-septate, strongly constricted at the septum, each hemispore surrounded by a separate gelatinous sheath quickly dissolving in water, upper cell slightly broader, with broadly rounded ends and distinctly constricted in the middle with a ring-like thickening inside; wall finely verruculose, brown, the contents granular, usually with a large and several smaller guttules per cell.

Conidiomata on the natural substrate associated with ascomata, visible as minute black dots 80–160 μm in diam, immersed, peridermal, pycnidial, unilocular, of circular shape, opening with a central ostiole, 100–200 μm diam, marginal wall thin, ca. 10 μm, composed of subhyaline to light brown cells, wall around ostiole ca. 22–50 μm thick, composed of dark brown cells. Conidiophores short, branched up to three times. Condiogenous cells phialidic, cylindrical, (8.5–)9.0–12.3(–15.5) × (1.5–)1.7–2.3(–2.7) μm (n = 50). Conidia (14–)17–20(–22) × (0.9–)1.1–1.4(–1.7) μm (n = 36), hyaline, falcate to semicircular, aseptate.

Cultures slow-growing, with uneven lobed margins, colony on CMD reaching 42 mm diam after 42 days at 16 °C, first whitish, turning medium to dark red brown, with sparse lighter brown aerial mycelium in the centre, reverse zonate, dark brown in the centre, with medium and dark red brown concentric zones towards the margins, entire culture black after 6 months. No conidiomata seen in pure culture.

Habitat: on dead corticated braches of Quercus and Rhamnus alaternus.

Distribution: only known from Mallorca (Spain).

Holotype: Spain, Mallorca, Es Capdellà, Finca Son Marti, on corticated dead branches of Quercus sp., 6 May 2016, A. Pintos AP6516 (WU 39973), ex holotype culture CBS 144700.

Additional specimens examined: Spain, Mallorca, Calvià, Finca Pública Es Galatzó, on corticated dead branches of Quercus coccifera, 14 Dec. 2016, A. Pintos AP141216 (WU 39980); Esporlas, on dead corticated branches of Rhamnus alaternus, A. Pintos, 20 Aug. 2018 AP20818.

Notes: The hysteriform ascomata and 1-septate ascospores of S. labiatus are similar to the closely related S. pruni, but the latter has distinctly smaller ascospores (26–35 × 11–14 μm vs. 35–48 × 14–19 μm in S. labiatus), and it occurs on a different host, Prunus spinosa. In addition, the mature ascospore cells of S. labiatus are more distinctly constricted in the middle and have a ring-like thickening inside the wall.

Stigmatodiscus oculatus Voglmayr & Pintos, sp. nov. Fig. 5.

Fig. 5
figure 5

Stigmatodiscus oculatus, sexual morph. a–h Ascomata erumpent from bark in face view. i, j Vertical sections of ascomata embedded in bark. k, l Asci. m Apically inflated septate paraphyses, covered by a dark brown amorphous incrustation. n Ascoma margin in transverse section. o–v Ascospores (o immature, p–v mature; o–q vital r–v dead). All in water, except k–n, u, v in 3% KOH. Sources: a, c, e, k–n, u, v WU 39977; b, d, f–i, r–t WU 39974 (holotype); j, o–q WU 39975. Scale bars: a, b 500 μm; c–h 200 μm; i, j 50 μm; k–v 10 μm

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MycoBank MB 827488.

Etymology: Referring to its eye-shaped ascomata.

Ascomata hysteriform, scattered to gregarious, corticolous, erumpent through the periderm, commonly arranged in parallel to the branch axis, in face view (160–)280–510(–880) μm long, (110–)180–300(–380) μm wide (n = 96), with sides consisting of usually two, rarely three black lips (peridium) not in mutual contact, with a slit-like to almost circular central opening exposing the blackish elongated to broadly oval disc. Bark tissues not visibly altered, no black line visible in bark or wood. Peridium coriaceous, pseudoparenchymatous, dark brown to black, up to 120 μm thick at the apex, 38–55 μm thick at the sides, almost absent to 20 μm thick at the base, composed of small angular to rounded cells 3–10 μm diam. Hamathecium composed of hyaline, septate, filiform branched paraphyses 1.7–2.5 μm wide, embedded in tough hymenial gel, simple, not anastomosing, 82–123 μm long, longer than asci, swollen at their apices up to 5.5 μm and incrusted with dark brown granules forming an epithecium, neither staining blue in Lugol nor in Melzer’s reagent after pre-treatment with 3% KOH. Asci 72–84(–90) × (29–)35–49 μm (n = 10), bitunicate, clavate to pyriform, almost sessile, with a distinct apical chamber, thick-walled, typically containing 8 irregularly bi- to triseriate ascospores, very stable, fissitunicate dehiscence not observed. Ascospores (25.5–)27.5–31(–33) × (9.5–)10.5–12.0(–12.5) μm, l/w = (2.3–)2.5–2.7(–2.9) (n = 63), brown, asymmetric, broadly fusiform, straight, first 1-septate, developing 2 additional distosepta and becoming 3-septate with age, strongly constricted at the primary septum, weakly at secondary septa, secondary septa with large pores, each hemispore surrounded by a separate gelatinous sheath quickly dissolving in water, upper hemispore slightly broader, with broadly rounded ends; wall finely verruculose, brown, the contents granular, often with a large and several smaller guttules per cell.

Conidiomata on the natural substrate and in pure culture not observed.

Cultures slow-growing, with uneven margins, colony on CMD reaching 58 mm diam after 42 days at 16 °C, first whitish, soon turning dark olive brown, with abundant woolly surface mycelium, reverse dark brown to black.

Habitat: on dead corticated braches of various Mediterranean trees and shrubs.

Distribution: only known from Mallorca (Spain).

Holotype: Spain, Mallorca, Campos, Sa Ràpita, on corticated dead branches of Olea europaea, 16 Nov. 2016, A. Pintos AP161116 (WU 39974), ex holotype culture CBS 144701.

Additional specimens examined: Spain, Mallorca, Puig de Ros, on dead corticated branches of Populus canadensis, 10 Aug. 2016, A. Pintos AP10816 (WU 39975); Calvià, Playa Portals Vells, on dead corticated branches of Cistus albidus, 23 Oct. 2016, A. Pintos AP231016B (WU 39976); Campos, Sa Ràpita, on dead corticated branches of Pistacia lentiscus, 17 Nov. 2016, AP171116 (WU 39977), Calvià, Portals Vells, on dead corticated branches of Globularia alypum, 31 Dec. 2016, AP311216 and AP311216A (WU 39978).

Notes: Stigmatodiscus oculatus is evidently polyphagous as it has been found on corticated twigs of various shrubs and trees. Within the Stigmatodiscus species with four-celled ascospores, it is well distinct by ascospores smaller than 33 × 12.5 μm.

Stigmatodiscus pinicola Voglmayr & Pintos, sp. nov. Figs. 6 and 7.

Fig. 6
figure 6

Stigmatodiscus pinicola, sexual morph (WU 39979, holotype). a–j Ascomata erumpent from bark in face view. k Vertical section of ascoma. l Ascoma margin in transverse section. m, n Asci. o Apically inflated septate paraphyses, covered by an dark brown amorphous incrustation. p–v Ascospores (p–u vital, v dead). All in water, except l, o, v in 3% KOH. Scale bars: a 500 μm; b–k 100 μm; m, n 20 μm, l, o–v 10 μm

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Fig. 7
figure 7

Stigmatodiscus pinicola, asexual morph (WU 39979, holotype). a–c Conidiomata (pycnidia) immersed in periderm in vertical section; in c through ostiole. d–h Conidiophores with densely aggregated phialides. i–v Falcate to semicircular conidia. All in water, except b, c in 3% KOH. Scale bars: a–c 20 μm; d–h 10 μm; i–v 5 μm

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MycoBank MB 827489.

Etymology: Referring to its growth on Pinus.

Ascomata hysteriform to apothecioid, scattered, corticolous, initially covered by bark, erumpent through the periderm, in face view (205–)255–410(–600) μm long, (145–)180–285(–375) μm wide (n = 30), with sides consisting of usually 2–3 black lips (peridium) not in mutual contact, with a slit-like to circular central opening exposing the blackish elongated to circular disc. Bark tissues not visibly altered, no black line visible in bark or wood. Peridium coriaceous, pseudoparenchymatous, dark brown, up to 70 μm thick at the apex, 15–25 μm thick at the sides, almost absent to 20(–30) μm thick at the base, composed of small angular to rounded brown cells 3–9 μm diam. Hamathecium composed of hyaline, septate, filiform branched paraphyses 1.5–3.5 μm wide, embedded in tough hymenial gel, simple, not anastomosing, ca. 150–220 μm long, longer than asci, swollen at their apices up to 7 μm and incrusted with dark brown granules forming an epithecium, neither staining blue in Lugol nor in Melzer’s reagent after pre-treatment with 3% KOH. Asci (104–)111–135(–138) × 47–54(–57) μm (n = 10), bitunicate, broadly fusiform to ellipsoid, almost sessile, with a distinct apical chamber, thick-walled, typically containing 8 irregularly bi- to triseriate ascospores, very stable, fissitunicate dehiscence not observed. Ascospores (40.5–)43.5–50(–52.5) × (13.5–)14.5–16.8(–18.0) μm, l/w = (2.6–)2.8–3.2(–3.5) (n = 45), brown, asymmetric, fusiform, straight, first 1-septate, developing 2 additional distosepta and becoming 3-septate with age, strongly constricted at the primary septum, weakly at secondary septa, secondary septa with large pores, each hemispore surrounded by a separate gelatinous sheath, upper hemispore slightly broader, with subacute to rounded ends, end cells lighter brown at maturity; wall finely verruculose, brown, the contents granular, often with a large and several smaller guttules per cell.

Conidiomata on the natural substrate associated with ascomata, visible as minute black dots 110–200 μm in diam, immersed, peridermal, pycnidial, clypeate, unilocular, of circular shape, opening with a central ostiole, 140–230 μm diam, 130–210 μm high, marginal wall ca. 14–25 μm thick, of light brown cells, wall around ostiole ca. 30–60 μm thick, composed of brown cells. Ostiole dark brown, ca. 20–25 μm wide. Conidiophores short, branched up to two times. Condiogenous cells phialidic, cylindrical, (9.5–)12.0–17.2(–21.2) × (1.9–)2.2–2.9(–3.0) μm (n = 30). Conidia (18–)19–25(–28) × 1–1.6 μm (n = 30), hyaline, falcate to semicircular, aseptate.

Cultures slow-growing, with uneven margins, colony on CMD reaching 49 mm diam after 42 days at 16 °C, first whitish, then turning black, with sparse grey aerial mycelium, reverse black.

Habitat: on dead corticated braches of Pinus halepensis.

Distribution: only known from Mallorca (Spain).

Holotype: Spain, Mallorca, Es Capdellà, Castell Son Claret, on corticated dead branches of Pinus halepensis, 21 Sep. 2016, A. Pintos AP21916B (WU 39979), ex holotype culture CBS 144702.

Notes: Stigmatodiscus pinicola is well characterised by the small apothecioid-hysteriform ascomata with usually circular outline and indistinct lips and by its host, Pinus halepensis. Stigmatodiscus enigmaticus and S. tamaricis also have four-celled ascospores of similar size but have different hosts; in addition, S. enigmaticus differs by larger ascomata (0.4–1.5 vs. 0.2–0.4(–0.6) mm) which are surrounded by bark flaps, and S. tamaricis by ascospores long remaining (sub)hyaline and by paraphyses with emerald to deep blue amorphous incrustation (Voglmayr et al. 2016).

Stigmatodiscus tamaricis (Voglmayr, Gardiennet & Jaklitsch) Voglmayr & Jaklitsch, comb. nov.

MycoBank MB 827490.

Basionym: Asterodiscus tamaricis Voglmayr, Gardiennet & Jaklitsch, Fungal Diversity 80: 276 (2016).

Specimen examined: Spain, Mallorca, Calvià, Magalluf, on corticated dead branches of Tamarix sp., 9 Sep. 2016, A. Pintos AP9916A.

Notes: With the addition of Stigmatodiscus oculatus, Asterodiscus tamaricis becomes phylogenetically embedded within Stigmatodiscus and is therefore transferred to the latter. Stigmatodiscus tamaricis is widely distributed on Tamarix spp. in Central and Southern Europe, and the specimen cited above is the second record of the species for Mallorca; the first Mallorcan record was recently published in Siquier et al. (2018).

Key to the species of Stigmatodiscus

  1. 1.

    Ascospores at maturity with a primary septum, only very rarely developing two additional distosepta, brown; ascomata distinctly hysteriform........................................2

    Ascospores at maturity with a primary septum and two additional distosepta, hyaline or brown; ascomata apothecioid or hysteriform................................................3

  2. 2.

    Ascospores (26.5–)29–32.5(–34.5) × (10.8–)11.5–12.7(–13.8) μm; on Prunus spinosa ................................S. pruni

    Ascospores (34.5–)38–43(–47.5) × (13.8–)15.5–17.5(–19.3) μm; on Mediterranean Quercus spp......................................................................S. labiatus

  3. 3.

    Mature ascospores in vital asci hyaline to light brown, becoming dark brown after ejection, (33.5–)40–45(−49) × (12.8–)14.3–16.5(−17.7) μm; ascomata apothecioid, circular; paraphyses tips covered by an olivaceous, emerald to deep blue amorphous incrustation; on Tamarix spp. ...................................................S. tamaricis

    Mature ascospores brown; paraphyses tips covered by a dark brown amorphous incrustation; on other hosts..........................................................................................4

  4. 4.

    Ascospores (25.5–)27.5–31(–33) × (9.5–)10.5–12.0(–12.5) μm; ascomata hysteriform; polyphagous.................................................................. S. oculatus

    Ascospores larger than 40 × 13 μm, ascomata mostly circular, apothecioid ........................................................5

  5. 5.

    Ascomata 0.4–1.5 mm diam, surrounded by irregular bark flaps; ascospores (46–)54–64 (−73) × (16.5–)20.0–24.3(−32.5) μm; on Acer spp., Carpinus orientalis..................................................... S. enigmaticus

    Ascomata 0.2–0.4(–0.6) mm diam, not surrounded by bark flaps; ascospores (40.5–)43.5–50(–52.5) × (13.5–)14.5–16.8(–18.0); on Pinus halepensis..........................................................S. pinicola

Discussion

Voglmayr et al. (2016) established the two genera Asterodiscus and Stigmatodiscus within the new family and order Stigmatodiscaceae and Stigmatodiscales, respectively, primarily based on differences in ascomatal shape and hyaline vs. brown ascospores. The morphological boundaries and phylogenetic differences between the two genera were considered distinct enough for establishing two genera. This concept was already challenged by Voglmayr et al. (2017), who described S. pruni, another new species with brown but two-celled ascospores, which differed substantially from the generic type, S. enigmaticus, by distinctly hysteriform ascomata.

The results of the current phylogenies necessitate a re-evaluation of the genus Asterodiscus, because it forms a highly supported clade with Stigmatodiscus oculatus. Whereas the ascospore shape and septation of S. oculatus are similar to A. tamaricis, the brown ascospores are indicative of Stigmatodiscus. Therefore, if the genus Asterodiscus were maintained, this would necessitate an emendation of the genus. However, considering the morphology of the new species described since the study of Voglmayr et al. (2016), the morphological differences between Asterodiscus and Stigmatodiscus seem insufficient to maintain them as distinct genera, which are therefore here synonymised.

None of the newly described species produced asexual morphs in pure culture; however, in S. labiatus and S. pinicola, an asexual morph was found tightly associated with the sexual morphs on the natural substrate. As conidia did not germinate on MEA or CMD, the connection with the sexual morphs could not be experimentally proven. However, we are confident that the associated asexual morphs belong to the respective species, as the morphology of their conidiomata as well as their conidial ontogeny, size, and shape fully match the asexual morph of S. enigmaticus, which was documented from natural substrate as well as pure cultures originating from ascospores (Voglmayr et al. 2016), proving the connection.

It is astonishing that within a small area, three new species of Stigmatodiscus could be found. It remains so far unclear whether the new species are endemic to Mallorca, or whether they co-occur with their widely distributed hosts in other regions the Mediterranean. This once again demonstrates that in the Mediterranean, the species diversity of corticolous ascomycetes is very incompletely studied, and that many species still await description (e.g. Voglmayr and Jaklitsch 2011; Jaklitsch and Voglmayr 2011, 2014; Voglmayr et al. 2016; Jaklitsch et al. 2014, 2015, 2018a, b; Galán et al. 2015; Checa et al. 2015). Considering that the Mediterranean is amongst the main biodiversity hotspots of the world (Myers et al. 2000), additional species of Stigmatodiscus are likely to be detected in this species-rich area.