Mycol Progress DOI 10.1007/s11557-017-1275-3 ORIGINAL ARTICLE Mycoceros antennatissimus gen. et sp. nov.: a mitosporic fungus capturing pollen grains D. Magyar1 & Z. Merényi2 & O. Udvardy1 & D. Kajtor-Apatini1 & P. Körmöczi3 & A. Fülöp4 & Z. Bratek2 & L. Kredics3 Received: 22 December 2016 /Revised: 18 January 2017 /Accepted: 27 January 2017 # German Mycological Society and Springer-Verlag Berlin Heidelberg 2017 Abstract Mycoceros antennatissimus gen. et sp. nov. is de- Keywords Hyphomycetes . Orbiliomycetes . Retiarius . scribed and illustrated from pollen grains deposited on the Pollen degradation . Molecular phylogeny . Direct PCR bark of Elaeagnus angustifolia and Platanus × acerifolia in Hungary. This fungus is shown to capture pollen grains by its three-dimensional shape. It clearly shows seasonality and Introduction appears to be rare. The following factors determine its ecolog- ical niche: (1) the availability of fresh Pinaceae pollen grains In 2007, a fungus was found by the first author on Pinaceae deposited from the air on the bark of a nearby standing angio- pollen grains deposited in the bark fissures of Russian olive trees sperm tree with (2) water-retaining spongious bark, and (3) (Elaeagnus angustifolia L.) near (5 m away from) a Picea abies rainy weather. Conidia are mainly dispersed by stemflow rain- (L.) H. Karst. tree. Free conidia of this fungus were also observed water, while they hardly become airborne. Direct polymerase when Alnus, Carpinus and Fagus twigs were washed (Révay chain reaction (PCR) from single conidia made it possible to and Gönczöl 2011,fig.17–18.). In 2009, the sporulating colonies perform molecular phylogenetic investigation in order to clar- of this fungus were found again on Pinaceae pollen grains de- ify its taxonomic relationship within the Ascomycota. posited in the bark fissures of a plane tree (Platanus × acerifolia (Aiton.) Willd.) near (14 m away from) a Picea abies tree. The fungus was repeatedly observed every year on this tree. In 2010, Section Editor: Marc Stadler the fungus was found on Pinaceae pollen grains deposited under the bark of a yew (Taxus baccata L.) ca. 500 m from the above- This article is part of the BSpecial Issue on ascomycete systematics in honour of Richard P. Korf who died in August 2016^. mentioned plane tree, between three Picea trees (at distances of 1.5, 1.5 and 3 m, respectively). Electronic supplementary material The online version of this article (doi:10.1007/s11557-017-1275-3) contains supplementary material, Pollen parasitism is known in many fungi, including hypho- which is available to authorized users. mycetes, Chytridiomycota and basidiomycetes (Goldstein 1960; Chou and Preece 1968;Warren1972; Hutchison and Barron * D. Magyar 1997; Huang et al. 1999; Rodrigues Marques et al. 2013). [email protected] Nutrients leached from pollen grains stimulate spore germina- tion, even in plant-pathogenic fungi, which was described as ‘ ’ 1 National Public Health Center, BudapestAlbert F. s. 2–6, 1437, the pollen effect (Fokkema 1968; 1971a, b). Especially in en- Hungary vironments with low nitrogen and phosphorus, utilisation of nu- 2 Department of Plant Physiology and Molecular Plant Biology, trients derived from pollen grains is substantial to allow fungi to Eötvös Loránd University, BudapestPázmány P. s. 1/c, 1117, complete decomposition, e.g. on litter (Stark 1972)orinoligo- Hungary trophic lakes (Wurzbacher et al. 2014). Hyphomycete species 3 Department of Microbiology, Faculty of Science and Informatics, being saprotrophs on pollen grains are common, but specialised University of Szeged, SzegedKözép fasor 52, 6726, Hungary parasites of them are rare. The present fungus is specialised to 4 Hungarian Meteorological Service, BudapestKitaibel P. s.1, 1024, capture pollen grains and is described in a new genus because of Hungary its unique morphological and molecular features. Mycol Progress Materials and methods Q water (17.75 μL); DreamTaq polymerase (Fermentas) (5 unit/μL, 0.25 μL). Thermocycling was carried out under the Small bark pieces were collected from adult, living Elaeagnus following conditions: 94 °C for 5.5 min, 40 cycles of 94 °C for angustifolia (N47.513685, E19.010648) and Platanus × acerifolia 18 s, annealing at 51 °C for 30 s, 72 °C for 45 s and a final (N47.476308, E19.091952) trees, at a height of 1.5 m above extension at 72 °C for 7 min. The ABI Prism BigDye™ ground, adjacent to spruce (Picea abies) during spruce pollen Terminator Cycle Sequencing Ready Reaction Kit v3.1 season (April–May) in Hungary. The samples were carried to the (Applied Biosystems) was applied for sequencing. Capillary laboratory in sterile polyethylene bags. During microscopic inves- gel electrophoresis was accomplished by an ABI PRISM 3100 tigations, deposited pollen grains were recovered from the bark Genetic Analyzer (Applied Biosystems), according to the in- using a wironit needle and a tape-lift method (with MACbond B structions of the manufacturer (Biomi Ltd.). The electrophero- 1200 pressure-sensitive acrylic strips, MACtac Europe S.A., grams were visually verified using FinchTV 1.4.0 (Geospiza, Brussels). The tape-lift method was described previously in detail Inc.; Seattle, WA, USA; http://www.geospiza.com). The (Magyar 2008) and successfully used to explore fungal popula- GenBank accession numbers of the sequences from the new tions inhabiting bark fissures. Digital photomicrographs were tak- species (herb no. BP105172) obtained in this work are: MD3: en with an Olympus BX-51 microscope at 600× magnification. KT186372 (ITS), MD4: KT186373 (ITS), MD5: KT186370 Fungal structures were mounted on glass slides with lactic acid in (LSU) and MD6: KT186371 (LSU). methylene blue for microscopic examination. The type material is deposited in the Hungarian Natural History Museum, Budapest Phylogenetic analysis (BP). Other materials are deposited in the first author’s collection (K.M.D.). A BLAST search (Altschul et al. 1997)oftheGenBank Nucleotide Database (National Center for Biotechnology DNA extraction and amplification Information, http://www.ncbi.nlm.nih.gov) was performed for each sequence obtained in this study and the most closely related For monospore DNA isolation, conidia were removed from the taxa were involved in the subsequent phylogenetic analysis. In substrate with the above-mentioned pressure-sensitive tape, and addition, sequences of known, morphologically similar pollen- then the tape was gently pressed to the surface of a pre-dried 2% capturing fungi [type materials of Retiarius bovicornutus (IMI Malt Extract Agar (MEA) medium. A 1 × 1-cm block was cut 223460) and R. superficiaris (IMI 223459)] were also included out of the MEA with the conidia and placed onto a microscopic (Magyar et al. in preparation). The sequences most similar to our slide. The surface of the agar block was scanned for free and clear LSU sequence, and some sequences selected from the articles of conidia under 400× magnification. The conidia were transferred Li et al. (2005), Spatafora et al. (2006) and Prieto and Wedin to 20 μL Milli-Q water in a polymerase chain reaction (PCR) (2013), were aligned with the E-INS-I algorithm MAFFT tube using a wironit needle under 200× magnification. One co- (Katoh and Toh 2008). Phylogenetic analysis was conducted with nidium was used to amplify a DNA region from the samples with both the ITS and LSU regions, but the ambiguously aligned sites the direct PCR method including a pre-PCR treatment step like mainly from the divergent ITS region were excluded using trimAl the ‘freeze–thaw method’ (Bärlocher et al. 2010)inorderto (Capella-Gutiérrez et al. 2009). Phylogenetic analysis under the disengage the DNA. The phylogenetic analysis was based on maximum-likelihood (ML) criterion was conducted with the soft- two regions of the nuclear ribosomal RNA gene cluster. The ware RAxML-VI-HPC (Stamatakis 2006;Stamatakisetal.2008) internal transcribed spacer (ITS) and the large subunit (LSU) on the CIPRES Science Gateway v. 3.1 (Miller et al. 2010). The were amplified with the primers ITS1F/ITS7 (Gardes and suitable substitution matrix for Bayesian analyses was selected Bruns 1993; Bertini et al. 1999) and LROR/LR5 (Vilgalys and with MrMODELTEST v2.3 (Nylander 2004). With regard to the Hester 1990), respectively. Because we had only conidia avail- Akaike Information Criterion (AIC), the best-fit likelihood models able, each sequence was derived from a single conidium and were found to be the GTR + I + G. Phylogenetic reconstructions numbered as MD3–MD6. The pre-PCR treatment was as fol- performed by MrBayes 3.2.6 (Huelsenbeck and Ronquist 2001) lows: PCR tubes with conidia were put into liquid nitrogen ran in four chains with 10,000,000 generations on CIPRES (around −195 °C, for 10 s), thereafter for 20 s into the wells of Science Gateway v. 3.1 (Miller et al. 2010). The average standard a PCR machine (Little Genius TC-25/H, Bioier) and heated to deviation of split frequencies was 0.0026. Every 500th generation 95 °C. This procedure was repeated 8–10 times, followed by a was sampled and the first 10% of the trees were discarded as burn- termination step at 95 °C for 3 min and centrifugation (10,000 g, in. A maximum clade credibility (MCC) tree was estimated from 1 min). The PCR master mix was added into these PCR tubes. the combined posterior sample of trees using TreeAnnotator v1.6.1 PCR was performed in a final volume of 50 μL, the components (http://beast.bio.ed.ac.uk/TreeAnnotator). The phylogenetic trees were as follows: 10× DreamTaq Green Buffer (Fermentas) with were visualised with FigTree v. 1.3.1. (Rambaut 2009)andcom- 20 mM MgCl2 (5.0 μL); dNTPmix (Fermentas) (2 mM each, pared with each other in the statistical software R (version 3.0.2; R 5.0 μL); primers for ITS or LSU (0.01 mM each, 1.0 μL), Milli- Core Team 2016) using the dendextend package (Galili 2015).