Three new species of Stigmatodiscus from Mallorca (Spain)

During a survey on corticolous Dothideomycetes in Mallorca, several collections with ascomata, asci, and ascospores matching the genus Stigmatodiscus (Stigmatodiscales, Dothideomycetes) were revealed, which did not fit any described species. Therefore, these collections were cultured and sequenced, and a multigene matrix of four loci (nuc18S-ITS-28S rDNA, rpb2, tef1 and tub2) was produced. Based on the results of the phylogenetic analyses of this matrix and of morphological investigations, three new species (Stigmatodiscus labiatus, S. oculatus, and S. pinicola) are described and illustrated, Asterodiscus is synonymised with Stigmatodiscus and the new combination S. tamaricis is proposed. A key to all currently known species of Stigmatodiscus is provided.


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(Voglmayr et al. , 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.

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).

Section Editor: Marc Stadler
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).

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.
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.

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.  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.
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. 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.

H a b i t a t : o n d e a d c o r t i c a t e d b r a c h e s o f
Stigmatodiscus oculatus Voglmayr & Pintos, sp. nov. Fig. 5.
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.
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 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 fourcelled 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 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
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 reevaluation of the genus Asterodiscus, because it forms a highly supported clade with Stigmatodiscus oculatus. Whereas t he 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;Voglmayr 2011, 2014;Voglmayr et al. 2016;Jaklitsch et al. , 2015Jaklitsch et al. , 2018aGalán 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.
Funding Information Open access funding provided by University of Vienna.
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