A New Agonimia from Europe with a Flabelliform Thallus
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The Lichenologist 44(1): 55–66 (2012) © British Lichen Society, 2011 doi:10.1017/S0024282911000600 A new Agonimia from Europe with a flabelliform thallus Beata GUZOW-KRZEMIN´ SKA, Josef P. HALDA and Paweł CZARNOTA Abstract: Agonimia flabelliformis sp. nov. (Verrucariaceae, Ascomycota) is described as a new species from the Czech Republic, Germany and Great Britain. Except for the distinctive, flabelliform to minutely coralloid thallus the species mostly resembles A. allobata. It differs from other related species of Agonimia in the absence of cortical papillae and in ascospore size. The distinctness of the new species and its placement within the genus Agonimia is supported by analyses of mitochondrial small subunit ribosomal DNA sequences from several samples of the taxon, and from many other representatives of Verrucariales including newly sequenced A. repleta and A. vouauxii. Additionally, ITS rDNA sequence data supports the distinction of A. flabelliformis from A. allobata. However, A. allobata was found to be highly variable and relationships, as well as the monophyly of taxa within Agonimia, are still unresolved and need further investigation. Key words: ITS rDNA, lichenized fungi, lichens, mtSSU rDNA, new species, phylogeny, taxonomy Introduction rocks and various types of soil, and some of The genus Agonimia Zahlbr. is one of the few them are found on plant debris. genera in the Verrucariaceae characterized by The genus Agonimia comprises 13 taxa; dark pigmented perithecia with multilayered eleven species, and two forms [Index walls, lack of an involucrellum, and colour- Fungorum, 2010, except for A. papillata (see less muriform ascospores; some species of Kashiwadani 2008; Muggia et al. 2009)]. the genus share a ± squamulose thallus and The currently accepted generitype, A. tris- minute cortical papillae, rarely present in ticula (Nyl.) Zahlbr., was transferred from representatives of other genera, such as Pso- Verrucaria at the beginning of the 20th cen- roglaena Müll. Arg. (Orange 2009). Other tury (Zahlbruckner 1909). At the same time perithecial features, diagnostic for all Verru- he described another member of the genus, cariaceae, include hamathecium composed Agonimia latzeli Zahlbr., later reduced by only of well-developed periphyses, and thin- Servı´t (1936) to the forma level as A. tristicula walled asci (e.g. Gueidan et al. 2007; Smith f. latzeli (Zahlbr.) Servı´t. The last author also et al. 2009). Most members of the genus described another form of A. tristicula, A. t.f. grow in shaded, moist places as epiphytes or pseudopallens Servı´t. After these works by epibryophytes on the base of trees, on roots, Servı´t, there were no new discoveries or taxonomic innovations within the genus until Coppins & James (1978) described A. octospora from Ireland. The genus began to B. Guzow-Krzemin´ka: Department of Molecular receive more attention from the 1990s when Biology, Faculty of Biology, University of Gdan´sk, Kładki 24, PL-80-822 Gdan´sk, Poland. Email: several species from other genera in the Ver- [email protected] rucariaceae (e.g. Amphoroblastia, Flakea, Ver- J. P. Halda: Muzeum a galerie Orlických hor, Jiráskova rucaria and Polyblastia) were transferred to 2, CZ-516 01 Rychnov n. Kn., Czech Republic. Agonimia (Coppins et al. 1992; Aptroot et al. P. Czarnota: Faculty of Biology and Agriculture, Department of Agroecology and Landscape Architec- 1997; Sérusiaux et. al. 1999). Also, Ago- ture, University of Rzeszów, C´ wiklin´skiej 2, PL-35-601 nimiella pacifica Harada, reported from Japan Rzeszów, Poland. (Harada 1993), was newly combined by 56 THE LICHENOLOGIST Vol. 44 Diederich into Agonimia (Aptroot et al. The aims of the present work were: 1) 1997). Based on the morphological similarity description of the new species within the of the squamulose thallus, Veˇzda (1997) genus Agonimia based on phenetic and mol- even transferred Phaeophyscia opuntiella ecular evidence; 2) investigation of its phylo- (Buschardt & Poelt) Hafellner, formerly genetic placement within Verrucariales based in the Physciaceae, into the genus Agonimia, on analysis of newly sequenced members although its ascocarps are unknown. The of the genus, focusing mainly on its relation- most recent addition to the genus is another ship to the morphologically most similar East Asian species, Agonimia koreana A. allobata. Kashiw. & K. H. Moon (Kashiwadani 2008). Recent molecular studies of the Verrucari- Materials and Methods aceae, including several species of Agonimia, showed, however, that the genus is not Taxon sampling monophyletic and some phenetic features, Newly generated mtSSU rDNA and nuclear ITS so far attributed to Agonimia, may be pre- rDNA sequences of Agonimia spp., including six and five sent also in other genera (Muggia et al. sequences, respectively, from A. flabelliformis, and an 2009, 2010). Agonimia papillata (O. E. ITS rDNA sequence from Verrucaria viridigrana,aswell as sequences of many representatives of Verrucariales Erikss.) Diederich & Aptroot forms its own obtained from GenBank, were used for the analysis. well supported clade, not related to Ago- Detailed descriptions of the newly sequenced materials nimia s. str. and should be treated rather as with their GenBank accession numbers are presented in a separate monotypic genus Flakea (Muggia Table 1, and GenBank accession numbers of sequences et al. 2009), as by Eriksson (1992). Other downloaded from GenBank are given in Figures 1 & 2 after the species names. work of Muggia et al. (2010), on phylo- genetic placement of some members of DNA extraction, PCR amplification and DNA Verrucariales, suggests that Flakea may sequencing be related to Agonimia repleta Czarnota & Fragments of perithecia were used for total genomic Coppins, which surprisingly is separated DNA extraction using a modified CTAB method from other Agonimia spp. analyzed. Agonimia according to Guzow-Krzemin´ska&We˛grzyn (2000). repleta was described as an Agonimia, based DNA was resuspended in sterile distilled water. PCR on the papillate subsquamulose thallus, amplifications were performed using Mastercycler (Eppendorf). PCR reactions of 50 l volume were pre- multi-layered melanized perithecial wall and pared using 5 lof10×Taq polymerase reaction buffer, muriform ascospores also found in the type 4 lofMgCl2,1unitofTaq polymerase (Fermentas), of the genus (Czarnota & Coppins 2000), 0·2 mM of each of the four dNTP’s, 0·5 M of each but in the light of the molecular studies primer and 10–50 ng of DNA. mentioned above it might represent a separ- Fragments of mitochondrial SSU rDNA were ampli- fied with mrSSU1 and mrSSU3R primers (Zoller et al. ate genus. These studies suggest that we still 1999) and the following conditions were used for PCR: need more molecular data to determine initial denaturation at 95°C for 5 min followed by 6 the correct placement of the species within cycles at 95°C for 1 min, 62°C for 1 min and 72°C for Verrucariaceae. 1 min 45 s, and then 30 cycles with the modified anneal- ing step at 56°C for 1 min; followed by a final elongation The new species presented here seems to step at 72°C for 10 min. ITS1, 5.8S and ITS2 regions belong to the genus Agonimia based on peri- were amplified with ITS1F (Gardes & Bruns 1993) and thecial anatomy and ecology, despite the fla- ITS4 primers (White et al. 1990). The following con- bellate to coralloid thallus and non-papillate ditions were used for PCR: initial denaturation at 95°C cortex, not previously known from the genus. for 5 min followed by 35 cycles at 95°C for 40 s, 54°C for 45 s and 72°C for 1 min; followed by a final elongation To support our taxonomic hypothesis step at 72°C for 10 min. PCR products were resolved on mtSSU rDNA and ITS rDNA analyses were agarose gels in order to determine DNA fragment performed, including new sequences ob- lengths. Then the residual oligonucleotides and dNTP’s tained from A. allobata, A. flabelliformis, A. were eliminated from the 20 l of PCR products using 10 units of Exonuclease I (Fermentas) and 2 units repleta, A. vouauxii and Verrucaria viridi- of Shrimp Alkaline Phosphatase (Fermentas), starting grana, and sequences of other Verrucariaceae with the incubation at 37°C for 35 min and followed that are available in GenBank. by enzyme inactivation at 80°C for 20 min. DNA 2012 Agonimia flabelliformis—Guzow-Krzemin´ska et al. 57 sequencing was performed using Macrogen (Korea) ser- scribed A. flabelliformis, as well as two of A. vice (www.macrogen.com). For sequencing the same vouauxii that had not previously been se- primers as for DNA amplification were employed. The newly determined sequences (Table 1) were compared quenced. Additionally, four new mtSSU to the sequences available in GenBank using a BLAST rDNA and five ITS rDNA sequences of search (Altschul et al. 1990) in order to confirm their A. allobata were generated. Moreover, we identity. also obtained sequences from A. repleta and Verrucaria viridigrana. Sequence alignment and phylogenetic analysis The newly sequenced mtSSU rDNA genes The new sequences were aligned with sequences of (Table 1) were aligned with 33 sequences selected representatives of the genus Agonimia and other obtained from GenBank (for list of taxa and taxa from Verrucariales obtained from GenBank, using their accession numbers see Fig. 1). In total, ClustalX software (Thompson et al. 1997) (with the following parameters: gap opening = 15; gap extension = 46 mtSSU rDNA sequences were analyzed. 6·66). Then the pre-alignment was manually optimized After exclusion of ambiguous regions, the using Seaview software (Galtier et al. 1996). Portions of final alignment consisted of 555 sites, of the alignment with ambiguous positions that might not which 236 were found to be variable. Among have been homologous were eliminated from the data- them, 143 characters were parsimony- set. The phylogenetic analyses were performed using informative. PAUP* 4.0b10 (Swofford 2001) with maximum parsi- A parsimony analysis using PAUP soft- mony (MP) method as optimality criterion.