Fungal Diversity (2016) 80:271–284 DOI 10.1007/s13225-016-0356-y Asterodiscus and Stigmatodiscus, two new apothecial dothideomycete genera and the new order Stigmatodiscales Hermann Voglmayr1 & Alain Gardiennet 2 & Walter M. Jaklitsch1,3 Received: 25 August 2015 /Accepted: 11 January 2016 /Published online: 2 February 2016 # The Author(s) 2016. This article is published with open access at Springerlink.com Abstract During a survey on corticolous Dothideomycetes, Keywords Ascomycota . Dothideomycetes . New species . several collections with ascospores matching the genera New genus . New family . New order . Phylogenetic analysis . Asteromassaria and Stigmatomassaria (Pleomassariaceae, Taxonomy Pleosporales) were revealed from dead corticated twigs of Acer, Carpinus and Tamarix. Closer morphological examina- tion showed that their ascomata were apothecial, with a Introduction hamathecium consisting of septate, branched paraphyses, which are apically swollen at maturity. Several collections During a survey on corticolous ascomycetes in Istria, Croatia were cultured and sequenced, and a Blast search of their nuc in September 2010, we collected a dothideomycetous species 28S rDNA sequences revealed dothideomycetous affiliation, from dead corticated twigs of Carpinus orientalis which but without a close match to a specific family or order. showed ascospores with a striking resemblance to Phylogenetic analyses of a multigene matrix containing a rep- Stigmatomassaria pupula (Pleomassariaceae, Pleosporales; resentative selection of Dothideomycetes from four genes (nuc see Barr 1982,underSplanchnonema pupula). They were first 18S rDNA, nuc 28S rDNA, rpb2andtef1) revealed placement considered to represent poorly developed, aberrant ascomata within Dothideomycetes but without a supported familial or of Stigmatomassaria, but detailed morphological examination ordinal affiliation. Based on the phylogenetic analyses and showed that the ascomata were clearly apothecial, with morphological investigations, the new genera Asterodiscus ascomata opening at maturity and having septate paraphyses and Stigmatodiscus, with the two new species A. tamaricis with apical swellings at their free ends. Sequence data obtain- and S. enigmaticus, are described and illustrated, and placed ed from cultures of ascospores left no doubt that they do not in the new family Stigmatodiscaceae and new order belong to Stigmatomassaria or even Pleosporales. NCBI Stigmatodiscales. Blast searches confirmed dothideomycetous affinities, but without indication of a closer relative. Subsequently, we col- lected and cultured the fungus several times from Acer monspessulanum, A. campestre and A. sempervirens from var- * Hermann Voglmayr ious European countries. [email protected] In summer 2013, we found another dothideomycetous spe- cies on corticated dead twigs of Tamarix spp. with ascospores matching the genus Asteromassaria (Pleomassariaceae, 1 Division of Systematic and Evolutionary Botany, Department of Botany and Biodiversity Research, University of Vienna, Rennweg Pleosporales;seeBarr1982), but again with apothecial 14, A-1030 Vienna, Austria ascomata having apically swollen paraphyses. Sequence data 2 14 rue Roulette, F-21260 Véronnes, France obtained from pure cultures revealed a close phylogenetic relationship to the Stigmatomassaria-like apothecial 3 Institute of Forest Entomology, Forest Pathology and Forest Protection, Department of Forest and Soil Sciences, dothideomycete. BOKU-University of Natural Resources and Life Sciences, These fungi could not be identified upon consultation of Hasenauerstraße 38, A-1190 Vienna, Austria the taxonomic literature, and no genera or families offered 272 Fungal Diversity (2016) 80:271–284 themselves for an appropriate taxonomic placement. gene was amplified using the primer pair fRPB2-5f and Therefore, they are described in the new genera Asterodiscus fRPB2-7cr (Liu et al. 1999). A ca 1.3 kb fragment of transla- and Stigmatodiscus, which are placed in a new family and tion elongation factor 1-α (tef1) gene was amplified with the order within Dothideomycetes, according to the results of primers EF1728F (Carbone and Kohn 1999)and multigene phylogenetic analyses and detailed morphological TEF1LLErev (Jaklitsch et al. 2005). The latter fragment in- investigations. cludes the fourth and the fifth intron and a significant portion of the last large exon. A ca. 0.8 kb fragment of the β-tubulin (tub2) gene was amplified with primers T1HV (5′ Materials and methods CANMATGCGYGAGATYGTAYGT 3′)andBtHV2r(5′ CATCATRCGRTCNGGGAACTC 3′), which were newly de- Morphological observations veloped for Dothideomycetes. T1HV is a modified version of primer T1 (O’Donnell and Cigelnik 1997), whereas BtHV2r is Hand sections of ascomata were made using a razor blade and upstream from T222 (O’Donnell and Cigelnik 1997); the mounted in water on a microscope slide, gently torn apart with primers were developed from genome sequences of represen- a preparation needle when necessary and covered with a cover tative Dothideomycetes downloaded from the Joint Genome slip. Slides were examined and photographed using a Zeiss Institute (JGI, http://jgi.doe.gov/). PCR products were purified Axio Imager.A1 (Zeiss, Jena, Germany) microscope equipped using an enzymatic PCR cleanup (Werle et al. 1994)asde- with a Zeiss AxioCam ICc3 or Axiocam 506 colour digital scribed in Voglmayr and Jaklitsch (2008). DNA was cycle- camera. Measurements are reported as maxima and minima in sequenced using the ABI PRISM Big Dye Terminator Cycle parentheses and the mean plus and minus the standard devia- Sequencing Ready Reaction Kit v. 3.1 (Applied Biosystems, tion given in parentheses. For falcate conidia, the shortest Warrington) and the PCR primers; in addition, the following distance between both ends (i.e. diameter) was measured. primers were used: ITS-28S region: ITS4 (White et al. 1990), The specimens were deposited in the fungarium of the LR3 (Vilgalys and Hester 1990); SSU: NS1088 (Kauff and University of Vienna (WU). Lutzoni 2002). Sequencing was performed on an automated DNA sequencer (ABI 3730xl Genetic Analyzer, Applied Pure culture isolation Biosystems). Mature ascomata on corticated twigs were horizontally cut Phylogenetic analyses using a sterile razor blade, the apothecia separated from the surrounding host tissue, transferred in a sterile drop of water To reveal the phylogenetic position of the two new genera, a on a microscope slide, torn apart with a forceps to release the matrix of aligned nucleotide sequences from four different ascospores from asci and pipetted on a 2 % malt extract agar phylogenetic markers (18S, 28S, rpb2andtef1), and 219 rep- (MEA) plate supplemented with 200 mg/L penicillin G and resentatives of Dothideomycetes and Eurotiomycetes,includ- streptomycin sulphate (Sigma-Aldrich, St. Louis, MO). ing two species from Lecanoromycetes as outgroup, was pro- Germinated ascospores were then transferred to 2 % MEA duced. The matrix of Boehm et al. (2015) was downloaded plates, which were sealed with laboratory film and incubated from TreeBASE (www.treebase.org; submission No. S at room temperature. Cultures were deposited at CBS-KNAW 16,151) and served as the matrix basis after removal of a Fungal Biodiversity Centre, Utrecht, The Netherlands (CBS). few sequences. Sequences of six additional taxa were downloaded from the GenBank nucleotide database. These, DNA extraction, PCR and sequencing together with the newly generated sequences, were manually aligned to the matrix of Boehm et al. (2015), or realigned with Growth of liquid cultures and extraction of genomic DNAwas the server version of MAFFT (www.ebi.ac.uk/Tools/mafft). done according to Voglmayr and Jaklitsch (2011), either using The resulting alignments were subsequently checked and the modified CTAB extraction protocol of Riethmüller et al. refined using BioEdit version v. 7.0.4.1 (Hall 1999). For align- (2002) or the DNeasy Plant Mini Kit (QIAgen GmbH, Hilden, ment of rpb2, first the alignment of Boehm et al. (2015)was Germany). The following loci were used for identification and translated into a protein matrix which was then re-aligned with phylogenetic analyses: The complete ITS region and D1 and the new protein sequences using MAFFT, and the respective D2 domains of 28S nuc rDNA region (ITS-28S) were ampli- new DNA sequences were then manually aligned to the fied using the primers V9G (de Hoog and de and Gerrits van original rpb2 nucleotide matrix using the protein alignment den Ende 1998) and LR5 (Vilgalys and Hester 1990). The 18S as reference. Prior to phylogenetic analyses, the approach of nuc rDNA region was amplified with primers SL1 (Landvik et Wiens (1998) was applied to test for significant levels of lo- al. 1997) and NS24mod (Voglmayr and Jaklitsch 2011). A ca calized incongruence among the four genes, using the level of 1.1 kb fragment of the RNA polymerase II subunit 2 (rpb2) bootstrap support (Sung et al. 2007). For this, the 80 % Fungal Diversity (2016) 80:271–284 273 maximum likelihood (ML) bootstrap consensus trees for each individual partition were compared using the same parameters 2 as for the combined analysis given below, but using 100 fast tub bootstrap replicates. No significant topological conflicts were observed between the bootstrap trees of 18S, 28S, rpb2and tef1, indicating the absence of significant incongruence and 2 rpb combinability of the matrices (Wiens 1998). The resulting combined sequence matrix contained 5721 nucleotide posi- tions from four genes