The Lichenologist 41(3): 213–221 (2009) © 2009 British Lichen Society doi:10.1017/S0024282909008342 Printed in the United Kingdom A new species of Petractis (Ostropales s. lat., lichenized Ascomycota) from Wales Alan ORANGE Abstract: Petractis nodispora is described as a new species, characterized by an endolithic thallus with Trentepohlia as photobiont, immersed apothecia, 3-septate, halonate ascospores, and very distinctive multicellular conidia. Nuclear ribosomal SSU and LSU sequences show that it is closely related to P. luetkemuelleri, but its relationship to the type species of Petractis, P. clausa, is unclear. Key words: conidia, lichen forming fungus, limestone, nuclear ribosomal DNA Introduction Thelotremataceae clade (Diploschistes, Chroo- Petractis, as currently understood, is a small discus, Phaeographina and Graphina) and 4, a genus of calcicolous lichens with endolithic Trapeliaceae clade. Petractis hypoleuca and P. or pseudoendolithic thalli, pale, immersed thelotrematella were united with the genus apothecia, septate to muriform ascospores, Gyalecta within the Gyalectaceae. The rela- and with Trentepohlia or Scytonema as photo- tionships of several taxa, including Petractis biont. Veˇzda (1965) accepted five species, clausa (Hoffm.) Krempelh. (the type of the four of them newly transferred by him from genus) and P. luetkemuelleri (Zahlbr.) Veˇzda Gyalecta. In addition to these, Clauzade & remained unresolved. Because of the close Roux (1985) also accepted P. crozalsii (B. de relationships between the taxa, the authors Lesd.) Clauz. & Cl. Roux. Owing to uncer- recommended that Gyalectales and Ostropales tainties in the circumscription of the genus, it should be combined, under the latter name. is not possible to give precise morphological Lumbsch et al. (2004), using nuclear LSU distinctions from other genera of gyalectoid and mitochondrial SSU ribosomal DNA lichens at present. sequences to investigate supraordinal phylo- Kauff & Lutzoni (2002) used the SSU and genetic relationships in Lecanoromycetes, LSU ribosomal RNA genes to investigate distinguished a Gyalectales + Ostropales + the phylogenetic relationships of Gyalectales Graphidales clade as sister to the remainder of and Ostropales. Analyses confirmed the close Lecanoromycetes. The Gyalectales were relationship of the Gyalectales with ostro- paraphyletic in the analysis, but the mono- palean and graphidalean fungi, but four phyly of this order could not be rejected with monophyletic entities were distinguished: the data set used. The authors suggested that 1, a clade with Gyalecta, Petractis hypoleuca more data were needed before the relation- (Ach.) Veˇzda and P. thelotrematella (Bagl.) ship of the orders within this group could be Veˇzda; 2, a Coenogonium clade (including resolved. They noted that the results of Kauff the former Dimerella, now treated as a syno- & Lutzoni (2002) could support a more tra- nym of Coenogonium); 3, a Graphidaceae- ditional view (the three orders remaining separate) if Petractis clausa, P. luetkmuelleri and Ocellularia (their relationships shown with low support in the trees of Kauff & A. Orange: Department of Biodiversity and Sys- tematic Biology, National Museum of Wales, Lutzoni) were treated as belonging to Gya- Cathays Park, Cardiff CF10 3NP, UK. Email: lectales. Wedin et al. (2005) using combined [email protected] nuclear LSU rDNA and mitochondrial SSU Downloaded from https://www.cambridge.org/core. University of Athens, on 24 Sep 2021 at 14:52:59, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0024282909008342 214 THE LICHENOLOGIST Vol. 41 rDNA sequences, found Ostropales (s. str., ambiguously aligned regions (identified “by eye”) were represented by Absconditella, Conotrema, excluded from the analysis. Neobelonia and Stictis) to be sister to a Gya- Sequences for members of Ostropales sensu lato, Baeomycetales and two outgroup sequences, used in lectales + Graphidales clade; Graphidales was phylogenetic analyses by Kauff & Lutzoni (2002), were nested within Gyalectales, but the monophyly downloaded from GenBank (Table 1) and aligned with of Gyalectales could not be rejected. the newly obtained nuclear SSU rDNA and nuclear In 2006, a specimen of an unidentified LSU rDNA sequences. The sequences were analysed in two sets: 1, taxa for which both SSU and LSU sequences Petractis species was collected on the coast were available and 2, taxa for which only the LSU was of South Wales. In 2008 more abundant available. SSU and LSU sequences were concatenated material was collected from a second site before analysis. The ITS1-5.8S-ITS2 regions (available 7 km distant from the first; this enabled a only for two newly prepared sequences) were excluded better assessment of the morphological fea- from the analyses. Phylogenetic relationships and sup- port values were investigated using a Bayesian approach. tures of the taxon, and the extraction of DNA Additional support values were obtained using Maxi- for sequencing. The species is described here mum Parsimony bootstrapping. The Bayesian analysis as new. employed the GTR+I+G model, selected using the Akaike Information Criterion (AIC) in MrModeltest 2.2 (Nylander 2004). Using MrBayes 3.1.2 (Huelsenbeck & Ronquist 2005), two analyses of two parellel runs were Materials and Methods carried out for 100 000 generations (SSU + LSU data set) or 5 000 000 generations (LSU data set), with trees Sections were cut by hand and examined in water, 5% sampled every 100 generations. Stationarity was consid- KOH, and a 0·5% IKI solution (I2 0·5 g, KI 1 g, water ered to have been reached when the average standard 100 ml). Some sections were decalcified in c. 5% HCl deviation of split frequencies dropped to <0·01, and the before examination. Conidia were cleared in Lugol’s values for the Potential Scale Reduction Factor were Iodine. Indian Ink was used to detect gelatinous close to 1. A burnin sample of 250 and 12 500 trees ascospore sheaths. were discarded from each run, respectively. Maximum DNA was extracted from thallus tissue of freshly Parsimony bootstrapping was carried out using MEGA collected specimens, using the Qiagen DNeasy Plant version 4.0 (Tamura et al. 2007). The Maximum Mini Kit; the manufacturer’s instructions were followed Parsimony tree was obtained using the Close- except that warm water was used for the final elution. Neighbour-Interchange (CNI) algorithm, in which the PCR amplification was carried out using PuReTaq initial trees were obtained with the random addition of Ready-To-Go PCR Beads (Amersham Biosciences). sequences (10 replicates). The bootstrap consensus tree For one specimen, the nuclear small subunit ribosomal was inferred from 2000 bootstrap replicates. Gaps were DNA (SSU), the two internal transcribed spacer regions treated as missing data. Support values of R95% pos- and the 5.8S region (ITS1-5.8S-ITS2), and the 5' end terior probabilities and R70% MP bootstrapping were of the nuclear large subunit ribosomal DNA (LSU) were regarded as significant. amplified, and in a second specimen only the ITS-5.8S- ITS2 region was amplified. The following primers were used: nssu97A, nssu1088R (Kauff & Lutzoni 2002), NS24 (Gargas & Taylor 1992), ITS1F (Gardes & Results and Discussion Bruns 1993), LR3, LR7 (Vilgalys & Hester 1990) and nu-LSU-155-5' (Döring et al. 2000). The PCR thermal After exclusion of ambiguously aligned cycling parameters were: initial denaturation for 5 min at regions, the alignment of dataset 1 (SSU + 94°C, followed by 5 cycles of 30 s at 94°C, 30 s at 55°C, LSU) contained 350 parsimony-informative and 1 min at 72°C, then 30 cycles of 30 s at 94°C, 30 s at 52°C and 1 min at 72°C. PCR products were visualized characters. The tree resulting from the on agarose gels stained with ethidium bromide, and Bayesian analysis is shown in Fig. 1. After purified using the Sigma GenElute PCR Clean-Up Kit. exclusion of ambiguously aligned regions Sequencing was performed by The Sequencing Service and insertions, the alignment of dataset 2 (College of Life Sciences, University of Dundee, (LSU only) contained 262 parsimony- www.dnaseq.co.uk) using Applied Biosystems Big-Dye Ver 3.1 chemistry on an Applied Biosystems model 3730 informative characters. The tree resulting automated capillary DNA sequencer. from the Bayesian analysis is shown in Fig. 2. Sequences were assembled and edited using In both trees, three well-supported clades DNAstar Lasergene software (http://www.dnastar.com/ were recovered: 1, a clade containing only products/lasergene.php). Alignment was carried out using BioEdit (http://www.mbio.ncsu.edu/BioEdit/ Gyalecta species (but with only 57% MPb bioedit.html); ClustalW was used to create an initial support in Fig. 1), including two species pre- alignment, which was edited manually. Insertions and viously placed in Petractis by Veˇzda (1965) Downloaded from https://www.cambridge.org/core. University of Athens, on 24 Sep 2021 at 14:52:59, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0024282909008342 2009 Petractis nodispora—Orange 215 F. 1. Phylogenetic relationships among 13 species of Ostropales s. lat. and 3 species of Baeomycetales, based on a Bayesian analysis of combined nuclear SSU and LSU sequences. The tree was rooted using a species of Acarospora (Acarosporomycetidae). The two support values associated with each branch are posterior probabilities and maximum parsimony bootstrap values, respectively. Branches in bold indicate a support of PP R95% and MPb R70%. If a node of the Bayesian tree was not recovered by MP bootstrapping, the MPb value is replaced by a dash. (G. hypoleuca and G. thelotremella); 2, a Coe- responds to Ostropales in the broad sense nogonium clade; 3, a clade with Chroococcus, of Kauff & Lutzoni; Baeomyces, Placopsis Diploschistes, Graphina and Phaeographina. and Trapeliopsis belong to Baeomycetales These clades were also recovered in the (Lumbsch et al. 2007). A similar position for analysis of Kauf & Lutzoni (2002), where they P. clausa is shown in Fig. 2, but with low are designated as 1, Gyalectaceae,2,Coenogo- support.