© 2019 W. Szafer Institute of Botany Polish Academy of Sciences Plant and Fungal Systematics 64(1): 81–90, 2019 ISSN 2544-7459 (print) DOI: 10.2478/pfs-2019-0009 ISSN 2657-5000 (online) New species of Capronia (Herpotrichiellaceae, Ascomycota) from Patagonian forests, Argentina Romina Magalí Sánchez1*, Andrew Nicholas Miller2 & María Virginia Bianchinotti1 Abstract. Three new species belonging to Capronia are described from plants native to the Article info Andean Patagonian forests, Argentina. The first record ofC. chlorospora in South America is Received: 2 Apr. 2019 also reported. The identity of the three new species is based on detailed morpho-anatomical Revision received: 17 Jun. 2019 observations as well as analyses of ITS and LSU nuclear rDNA. A key to the Capronia Accepted: 25 Jun. 2019 species present in Argentina is provided. Published: 30 Jul. 2019 Key words: ITS, LSU, phylogenetics, systematics, three new species Associate Editor Adam Flakus Introduction Capronia is an ascomycete genus with medically impor- of C. chlorospora in the Southern Hemisphere. A key to tant asexual morphs in the Exophiala-Ramichloridi- the Capronia species present in Argentina is provided. um-Rhinocladiella complex, known as “black yeast”, which is considered polyphyletic (Untereiner et al. 2011; Materials and methods Teixeira et al. 2017). It is characterized by small, dark and usually setose ascomata, with periphysate ostioles, the The samples were collected in Andean Patagonian forests absence of interascal filaments, bitunicate, 8- to polyspo- where native species of Nothofagus along with Cupres- rus asci, and septate, hyaline to dark-colored ascospores saceae, Proteaceae, ferns and mosses prevail. Four parks (Barr 1987; Réblová 1996). A majority of the species are were included in this survey: Parque Provincial Lago saprobic or hypersaprobic (Untereiner 2000) but about Baggilt (Chubut), Parque Nacional Lanín (Neuquén), Par- twenty species are reported to grow obligatory on lichens que Nacional Los Alerces (Chubut) and Parque Nacional (Etayo & García Sancho 2008; Etayo et al. 2013; Flakus Nahuel Huapi (Río Negro). Leaves, small branches and & Kukwa 2012; Halici et al. 2010; Tsurykau & Etayo bark showing fungal growth when observed under a 10× 2017; Zhurbenko 2012; Zhurbenko et al. 2016). About loupe were placed in paper bags and transported to the eighty species of Capronia are described and, in spite laboratory. The samples were dried at room temperature of them being common and ubiquitous, their diminutive and deposited in the Bahía Blanca Biology Herbarium ascomata, which are seldom abundant on the substrate, (BBB). For microscopy, sections were cut freehand under makes them difficult to recognize. a Leica EZ4 stereomicroscope and mounted in tap water Through our work on the biodiversity of ascomycetes and 5% KOH with phloxine or Melzer’s reagent. A Leica on trees native to the Andean Patagonian forests in Argen- DM2000 microscope fitted with a Samsung NV10 digital tina, three new species of Capronia along with C. chlo- camera was used to capture images of micromorphology. rospora were found growing on dead wood and bark. All At least 10 measurements were taken for each structure three new species of Capronia are described, illustrated mounted in tap water. Averages for ascospores are given and compared morphologically and phylogenetically to in parentheses. Material was mounted in calcofluor 0.5% other known species in the genus. This is the first report for examination of the hymenium under a Nikon Eclipse 80i fluorescence microscope with a Nikon DXM 1200F camera system and a Leica DM2000 with a Leica EC3 1 Centro de Recursos Naturales Renovables de la Zona Semiárida (CERZOS), Universidad Nacional del Sur-CONICET, Camino La camera system. Single- and multiple-ascospore isolates Carrindanga Km7; Departamento de Biología, Bioquímica y Farmacia, were attempted after 1–5 months on three different agar Universidad Nacional del Sur (UNS), San Juan 670, Bahía Blanca, media, including 2% malt extract agar (MEA), oatmeal Argentina agar (OA) and potato dextrose agar (PDA) at three dif- 2 University of Illinois Urbana-Champaign, Illinois Natural History Survey, 1816 South Oak Street, Champaign, Illinois 61820-6970, USA ferent temperatures (10°C, 15°C, room temperature) and * Corresponding author e-mail: [email protected]. light conditions (white light, fluorescent light, white This work is licensed under the Creative Commons BY-NC-ND 4.0 License Authenticated | [email protected] Download Date | 7/22/19 1:42 PM 82 Plant and Fungal Systematics 64(1): 81–90, 2019 Figure 1. Phylogenetic tree (ln = 4183.8) of Herpotrichiellaceae taxa, based on the D1–D2 region of LSU nrDNA sequences constructed using the PhyML maximum likelihood method implemented in Seaview 4.7 with 1000 bootstrap replications. Bootstrap values ≥ 70% from the PhyML and RAxML analyses are shown above or below branches. Thickened branches indicate Bayesian posterior probabilities ≥ 95%. The three new species of Capronia are bolded. Source and GenBank accession numbers are given after taxon names. Authenticated | [email protected] Download Date | 7/22/19 1:42 PM R. M. Sánchez et al.: New species of Capronia (Herpotrichiellaceae, Ascomycota) from Patagonian forests, Argentina 83 fluorescent light alternating with periods of UV light). generated from the remaining 7500 trees using PAUP* All attempts to obtain axenic cultures failed due to lack 4.0b10 (Swofford 2002). Clades with BPP ≥ 95% were of growth, most likely due to the length of time between considered significant and strongly supported (Alfaro collection and isolation, so we extracted fungal DNA et al. 2003; Larget & Simon 1999). Source and GenBank directly from the ascomata. accession numbers for all taxa included in the LSU anal- Extraction, amplification and sequencing of DNA fol- yses are shown after taxa names in Figure 1. Based on lowed Promputtha and Miller (2010). Briefly, DNA was the LSU tree results, alignments of ITS sequences were extracted directly from ~30 ascomata using an E.Z.N.A.® attempted for the most closely related taxa in each of the Microelute Genomic DNA kit (Omega Bio-tek). The two subclades containing the three proposed new species. complete internal transcribed spacer (ITS) region and the However, taxon sampling for this locus was so poor for first 600 bp of the 5’ end of 28S nuclear ribosomal large these taxa that phylogenetic analyses were abandoned. subunit (LSU) including the D1 and D2 domains were ITS sequences for Capronia austrocedri (MH809169), amplified separately using puReTaq™ Ready-To-Go PCR Capronia capucina (MH809170) and Capronia rubigi- Beads (Amersham Biosciences Corp.) according to the nosa (MH809171) were deposited in GenBank. manufacturer’s instructions, with primer sets ITS1F-ITS4 and LROR-LR3, respectively (Gardes & Bruns 1993; Results Rehner & Samuels 1995; Vilgalys & Hester 1990; White et al. 1990). PCR products were purified using a Wizard® The final LSU alignment consisted of 610 bp after removal SV Gel and PCR Clean-Up System (Promega), and tem- of missing data and ambiguous regions. The ML tree gen- plate DNA was used in 10 μL sequencing reactions with erated with PhyML based on this LSU dataset is shown in BigDye® Terminator v3.1 (Applied Biosystems), using the Figure 1. A second ML analysis using RAxML produced same primers as in PCR. Sequences were generated on an a similar tree with no significant differences in topology Applied Biosystems 3730XL high throughput capillary from the PhyML tree (BV from RAxML shown in Fig. 1). sequencer at the W.M. Keck Center at the University Overall, the relationships among taxa were similar to those of Illinois Urbana-Champaign. Consensus ITS and LSU shown in previous trees based on LSU data (Crous et al. sequences were assembled with Sequencher 5.4 (Gene 2007; Feng et al. 2014; Seyedmousavi et al. 2014; Teix- Codes Corp.). eira et al. 2017; Untereiner & Naveau 1999; Untereiner The LSU dataset comprising 103 taxa was assembled et al. 2008). Capronia capucina and C. rubiginosa formed with sequences from the three new species along with a weakly supported clade that occurred in a highly sup- sequences from previous studies (Abliz et al. 2004; Crous ported clade containing Cladophialophora chaetospira et al. 2007; De Hoog et al. 2011; Feng et al. 2014; Gue- and Chaetothyriales sp. ITS sequence divergence between idan et al. 2007; Hamada & Abe 2010; James et al. 2006; Capronia capucina and C. rubiginosa was 29 bp (5.1%) Marincowitz et al. 2008; Rakeman et al. 2005; Teixeira out of 573 bp after ignoring a large ~350 bp intron in the et al. 2017; Untereiner & Naveau 1999; Untereiner et al. 5’ end of C. rubiginosa. Capronia austrocedri occurred 2008; Vitale et al. 2002; Vu et al. 2019) using the MUS- in an unsupported clade containing Herpotrichiellaceae CLE® multiple alignment program as implemented in sp., Thysanorea papuana and Veronaea japonica. Sequencher 5.4. The alignment was rooted with Placocar- pus schaereri and Verrucula inconnexaria. Ambiguously Taxonomy aligned regions were removed from the final alignment using Gblocks (Castresana 2000; Talavera & Castresana Capronia austrocedri R.M. Sánchez, A.N. Mill. & Bian- 2007), employing the less stringent parameters. The chin., sp. nov. (Figs 2, 4) general time reversible (GTR) model (Rodríguez et al. MycoBank MB 827442 1990) was determined as the best-fit model of evolution Diagnosis: similar to Capronia mansonii but phylogenetically by jModeltest (Darriba et al. 2012; Guindon & Gascuel very distant and distinguished by its wider ascomata and asci. 2003) after evaluating 1624 possible evolutionary models Type: Argentina, Chubut, Parque Nacional Los Alerces, and implementing the Akaike information criterion (AIC) 42°46′22.2″S, 71°43′54.94″W, 551 m, on decorticated branches (Posada & Buckley 2004). A maximum likelihood (ML) of Austrocedrus chilensis, 24 Oct 2006, M. V. Bianchinotti analysis with 1000 bootstrap replicates was performed & R. M. Sánchez (BBB 204, holotype designated here!). using PhyML as implemented in Seaview 4.7 (Gouy et al. 2010), with all parameters optimized and the GTR model.