Fungal Diversity (2011) 49:225–233 DOI 10.1007/s13225-011-0097-x The phylogenetic position of the lichenicolous ascomycete Capronia peltigerae Wendy A. Untereiner & Cécile Gueidan & Mary-Jane Orr & Paul Diederich Received: 16 December 2010 /Accepted: 23 February 2011 /Published online: 8 March 2011 # Kevin D. Hyde 2011 Abstract The genus Capronia includes a number of Introduction lichenicolous (lichen-inhabiting) species, none of which have previously been characterized in vitro or considered in Capronia Sacc. (Herpotrichiellaceae, Chaetothyriales) molecular phylogenetic studies. We cultured Capronia encompasses ascomycetes characterized by their very peltigerae from Peltigera rufescens and report here the small, typically setose ascomata, aparaphysate centra, growth of this species on a variety of media and its fissitunicate asci, and septate, hyaline or pigmented phylogenetic position based on the analyses of nuclear ascospores (Munk 1957; Müller et al. 1987; Barr 1991). ribosomal RNA, mitochondrial ribosomal RNA, and RNA Species of Capronia produce dark, slow growing colonies polymerase II (RPB1) gene sequences. This species differs in vitro and conidial anamorphs that belong to the genera from the majority of Capronia studied in axenic culture in Cladophialophora Borelli, Exophiala J.W. Carmichael, lacking a conidial anamorph. Phylogenetic analyses posi- Phialophora Medlar, and Rhinocladiella Nannf. (Schol- tion C. peltigerae outside the Herpotrichiellaceae within a Schwarz 1968; Samuels and Müller 1978; Müller et al. robustly supported basal lineage of the Chaetothyriales 1987; Untereiner 1995, 1997; Untereiner et al. 1995; Okada composed primarily of melanized, rock-inhabiting anamor- et al. 1998; Untereiner and Naveau 1999). These anamorph phic fungi. Our results demonstrate that Capronia,as genera comprise a group of fungi known as the black yeasts circumscribed currently, is polyphyletic, but they do not and include species responsible for important opportunistic resolve the relationship of C. peltigerae with members of infections of vertebrates (de Hoog et al. 2000). the Chaetothyriaceae. The majority of the nearly 60 species of Capronia described to date occur on rotting wood or bark and the Keywords Ascomycota . Chaetothyriaceae . decaying stems and leaves of herbaceous plants (Untereiner Herpotrichiellaceae . Lichenicolous fungi . Molecular 2000). Plant-associated Capronia are the most frequently phylogenetics cultured members of the genus, and are therefore best represented in molecular phylogenies. Fungicolous Capronia are studied less frequently. For example, of the nine species of Capronia reported exclusively from the W. A. Untereiner (*) : M.-J. Orr fruit-bodies of other fungi, only five (C. fungicola, C. Department of Biology, Brandon University, nigerimma (R.R. Bloxam) M.E. Barr, C. dactylotricha Brandon( MB R7A 6A9, Canada Unter. et al., C. parasitica, and C. spinifera (Ellis & Everh.) e-mail: [email protected] E. Müller et al.) are known in pure culture. The phyloge- C. Gueidan netic positions of lichenicolous representatives of the genus Botany Department, Natural History Museum, have not yet been investigated. Thirteen members of the London SW7 5BD, UK genus are reported to grow obligately on lichens (Etayo and Sancho 2008; Halici et al. 2010) but none of these species P. Diederich Musée national d’histoire naturelle, have been cultivated in vitro or included in molecular L-2160 Luxembourg, Luxembourg, Germany phylogenetic studies. 226 Fungal Diversity (2011) 49:225–233 To explore this latter problem, we studied Capronia on a rotary shaker at 100 rpm. Mycelia were collected by peltigerae (= Trichosphaeria peltigerae Fuckel) the first centrifugation and stored at −20°C until lyophilized. Total lichenicolous species assigned to the genus (Hawksworth nucleic acids were isolated from ground, lyophilized 1980; Eriksson and Hawksworth 1987). Capronia peltigerae cultures and purified following Lee and Taylor (1990)or is widely distributed geographically. It was described by using a modified protocol from Zolan and Pukkila (1986) Fuckel (1874) from the thalli of Peltigera canina (L.) Willd. as detailed in Gueidan et al. (2007). Protocols used for in Switzerland and has since been recorded on species of sequencing the large (nucLSU) and small (nucSSU) Peltigera Willd. in Alaska and in a number of northern, subunits of the nuclear ribosomal RNA gene and the largest central, and southern European countries (Martínez and subunit of the RNA polymerase II (RPB1) are described in Hafellner 1998; Diederich and Sérusiaux 2000;Kocourková Gueidan et al. 2007. Those used to sequence the internal 2000; Zhurbenko and Laursen 2003; Alstrup 2004; transcribed spacer (ITS) of the nuclear ribosomal RNA Zhurbenko 2004; Schiefelbein and Rätzel 2005;Diederich gene and the small subunit of the mitochondrial ribosomal et al. 2006; Suija et al. 2009;Candanetal.2010). We RNA gene (mitSSU) followed Untereiner and Naveau obtained mass- and single-ascospore cultures of C. (1999) and Zoller et al. (1999), respectively. Primers used peltigerae from freshly collected thalli of Peltigera rufescens in the amplification and sequencing of the loci used in this (Weiss) Humb. and characterized its growth on a variety of study included (ITS) WITS3 (Untereiner et al. 1995), media. We also investigated the position of this species WITS2, WNS9 (Untereiner and Naveau 1999), LR1 within the Ascomycota and its relationship to other Capronia (Vilgalys and Hester 1990), (mitSSU) mrSSU1, mrSSU3R based on the analyses of nuclear ribosomal RNA, mitochon- (Zoller et al. 1999), (nucLSU) LR0R (Rehner and Samuels drial ribosomal RNA, and RNA polymerase II (RPB1) gene 1994), LR3R, LR5, LR7 (Vilgalys and Hester 1990), sequences. (nucSSU) nssu131, nssu634, nssu1088R (Kauff and Lutzoni 2002), NS22, NS24 (Gargas and Taylor 1992), SR7R (Spatafora et al. 1995), (RPB1) RPB1-AF, RPB1- Materials and methods G2R (B. Hall, unpublished), RPB1-6R1asc, and RPB1- DF1asc (Hofstetter et al. 2007). Fungal strains and cultural studies Taxon sampling and molecular data Mass-ascospore (UAMH 11090) and single-ascospore (UAMH 11091) isolates of Capronia peltigerae were To investigate the phylogenetic placement of Capronia obtained by streaking the contents of mature ascomata on peltigerae, a total of 57 taxa were sampled across the Modified Leonian’s agar (MLA) (Malloch 1981) containing Chaetothyriales (Table 1) representing the major phyloge- 2.5% agar, chlortetracycline (50 μg/mL), and streptomycin netic groups in this order (Gueidan et al. 2008). Two species sulfate (50 μg/mL). Germinating ascospores were sub- of Verrucariales (Placocarpus schaereri and Verrucula cultured to MLA and Potato Carrot agar (PCA) (Gams et al. inconnexaria)wereusedasanoutgroup. 1987) containing 1.5% agar. Cultures were maintained on these media at room temperature (20–21 C). Alignments and phylogenetic analyses For comparative purposes, mass- and single-ascospore cultures were grown on MLA, 2% malt extract agar (MEA), Sequences were assembled and edited using Sequencher oatmeal agar (OA) (Tuite 1969), and filtered oatmeal agar 4.2.2 (Gene Codes Corporation, Ann Arbor, MI). A (CBSOA) (Gams et al. 1987). Plates were inoculated in manual alignment was performed using MacClade 4.06 triplicatewith3x3mmsquarescutfromtheactivelygrowing (Maddison and Maddison 2003). Ambiguous regions edges of colonies on MLA and incubated at room temperature. (sensuLutzonietal.2000) and introns were delimited Colony diameter was measured and descriptions of colony manually and excluded from the alignment. Phylogenetic morphology made at 7-day intervals for 28 days. Colour relationships and confidence were inferred using a Bayes- descriptions are based on Kornerup and Wanscher (1978). ian approach based on a combined nucLSU-nucSSU- Microscopic examinations of cultures and specimens mitSSU-RPB1 dataset. Additional support values were were made from preparations mounted in distilled water. estimated using a Maximum Likelihood approach with RAxML-VI-HPC (Stamatakis et al. 2005, 2008). These DNA extraction, amplification and sequencing analyses were run on the Cipres portal (http://www.phylo. org/). As a first step, the congruence between gene regions The mass-ascospore isolate (UAMH 11090) of Capronia was tested using a 70% reciprocal bootstrap criterion peltigerae used for sequencing was grown in 50 mL (Mason-Gamer and Kellogg 1996). For each gene region, Modified Leonian’s broth (MLA lacking agar) for 5–7d a non-parametric bootstrap analysis was done with Fungal Diversity (2011) 49:225–233 227 Table 1 Sources and accession numbers of the isolates examined in this study Taxon Source a GenBank Accession Numbers nucLSU nucSSU mitSSU RPB1 Capronia fungicola (Samuels & E. Müller) Unter. CBS 614.96 FJ358224 FJ358292 FJ225722 FJ358356 C. munkii Unter. CBS 615.96 EF413604 EF413603 FJ225723 EF413605 C. parasitica (Ellis & Everhart) E. Müller et al. CBS 123.88 FJ358225 FJ358293 FJ225724 FJ358357 C. peltigerae (Fuckel) D. Hawksw. UAMH 11090 HQ613813 HQ613815 HQ613814 HQ625027 C. pilosella (P. Karsten) E. Müller et al. MUCL 39967 DQ823099 DQ823106 FJ225725 DQ840554 C. semiimmersa (Cand. & Sulmont) Unter. & F.A. Naveau MUCL 40572 FJ358226 FJ358294 FJ225726 FJ358358 Capronia sp. WUC15ss1 WUC 15ss1 FJ358227 FJ358295 FJ225727 FJ358359 Capronia sp. WUC26 WUC 26 FJ358228 FJ358296 FJ225728 FJ358360 Capronia sp. WUC102 WUC 102 FJ358229 FJ358297 FJ225729 FJ358361 Capronia sp. WUC236 WUC 236 FJ358230 FJ358298 FJ225730 FJ358362 Capronia sp. WUC315