Molecular studies of photobionts of selected lichens from the coastal vegetation of Brazil

FEMS Microbiology Ecology 54 (2005) 381–390 www.fems-microbiology.org Molecular studies of photobionts of selected lichens from the coastal vegetation of Brazil a Centro de Ciências Médicas e Farmacêuticas, Universidade Estadual do Oeste do Paraná – UNIOESTE, CEP 85819-110, Cascavel, PR, Brazil b Departamento de Bioquı́mica, Universidade Federal do Paraná, CP 19.046, CEP 81.531-990, Curitiba, PR, Brazil c Institute of Plant Physiology, University of Salzburg, Hellbrunner Strasse 34, A-5020 Salzburg, Austria d Institute of Plant Sciences, Karl-Franzens-University, Graz, Holteigasse 6, A-8010 Graz, Austria Received 14 November 2004; received in revised form 16 February 2005; accepted 4 May 2005 First published online 13 June 2005 Abstract A light microscopic and molecular analysis of photobionts in Ramalina and Cladonia from coastal habitats of Brazil is presented. A Bayesian phylogenetic analysis of ITS rDNA sequences suggests a Trebouxia lineage which is preferentially tropical in geographic distribution. This highly diverse clade also includes the morphological similar species Trebouxia higginsiae and galapagensis. Within the predominantly tropical clade of Trebouxia we distinguish several subclades, three of which are represented in our samples of Ramalina species. Since sexuality has not been recognized in coccal lichenised photobionts until recently, we cannot apply a biological species concept, but when compared with the sequence diversity between known species we conclude that several new species need to be described in this clade. The mutually exclusive presence of other Trebouxia lineages in temperate samples of Ramalina suggests an evolution towards higher selectivity in this genus. A strictly tropical lineage is not conspicuous in the photobionts of the genus Asterochloris sampled from Cladonia so far. Ó 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. Keywords: Photobionts; Lichen; Trebouxia; Asterochloris; Ramalina; Cladonia; ITS rDNA 1. Introduction Compared with the mycobiont partner of lichens, still little is known about the geographic distribution and ecology of the associated photobionts. Historically, this can be explained by the general difficulty to recognize the species in the lichen thallus. Previous identifications therefore included the isolation and axenic culture of algal symbionts. With the use of molecular tools, the interest in this symbiotic partner has increased. Friedl and * Corresponding author. Tel.: +55 41 3611655; fax: +55 41 2662042. E-mail address: (M. Iacomini). Rokitta [1] showed a clear heterogeneity of trebouxioid photobionts. Species with a chloroplast closely appressed to the cell wall at certain stages and an indistinct pyrenoid, containing regular thylakoids, were found to be distantly related to the core Trebouxia cluster. These data agree with the findings of Tschermak-Woess [2] who splitted Trebouxia in two subgenera Trebouxia and Eleutherococcus. Piercey-Normore and DePriest [3] compared the sequences of several photobionts of Cladoniineae, including the subgenus Eleutherococcus sensu Tschermak-Woess and the isolated photobionts from Anzina carneonivea, i.e. Asterochloris phycobiontica [4]. They found 93% of similarity on their ITS sequences, suggesting that all these photobionts belong 0168-6496/$22.00 Ó 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.femsec.2005.05.003 Lucimara M.C. Cordeiro a, Rodrigo A. Reis b, Leonardo M. Cruz b, Elfriede Stocker-Wörgötter c, MartinGrube d, Marcello Iacomini b,* 382 L.M.C. Cordeiro et al. / FEMS Microbiology Ecology 54 (2005) 381–390 the mangrove does not seem to be much different from that in other tropical rain forest close to the coast [23]. In the present paper, we describe the phylogenetic position (ITS rDNA sequence comparison) and light microscopic characters of photobionts from some tropical lichens belonging to the families Ramalinaceae and Cladoniaceae, from different coastal environments of Brazil. 2. Material and methods 2.1. Lichen material Samples utilized in this study were collected in different environments of Brazil: Restinga: Ramalina sprengelii 68; Ramalina gracilis 63; Ramalina peruviana 67 – 25°32 0 0500 S/48°20 0 3000 W; sea level (0 m); Ilha do Mel, Pontal do Paraná – PR, Brazil (February/2004). Ramalina sorediosa 59; Ramalina anceps 70 – 25°19 0 5200 S/ 48°25 0 1000 W; 8 m; Ilha Rasa, Guaraquecaba – PR, Brazil (February/2004). R. gracilis, Ramalina complanata – 27°35 0 4800 S/48°32 0 5700 W, 3 m, Campeche Beach, Santa Catarina Island – SC, Brazil (August/2001). Cladina confusa – 25°32 0 0500 S/48°20 0 3000 W; on sandy soil, sea level, Ilha do Mel, Pontal do Paraná – PR, Brazil (August/ 2001). Mixed rain forest: R. peruviana 58 – 25°26 0 1500 S/ 49°03 0 4500 W; 940 m; Piraquara – PR, Brazil (February/ 2004). Mangrove: Ramalina sorediosa 60 – 25°19 0 5100 S/ 48°27 0 4000 W; sea level; Guaraquecaba – PR, Brazil (February/2004). R. anceps 78 (6) 25°19 0 3100 S/48°25 0 4900 W; sea level; Guaraquecaba – PR, Brazil (February/2004). Ramalina dendroides 79 (8) 25°19 0 0400 S/48°26 0 4100 W; sea level; Guaraquecaba – PR, Brazil (February/2004). Recovery area of Atlantic rain forest: R. peruviana 81 (5) 25°14 0 3600 S/48°29 0 3800 W; 40 m; Guaraquecaba – PR, Brazil (February/2004). Caatinga: Cladonia verticillaris – 7°26 0 S/34°56 0 W; Alhandra – PB, Brazil. Atlantic rain forest – Savanna (contact zone): Cladonia crinita; Cladonia fissidens – 20°05 0 S/43°29 0 W; Sandy Soil, Monastério do Caraça – MG, Brazil. Some samples from non-tropical environments were included: Cladonia perforata, Eric Mendes Biological Station, Florida, USA (subtropical; gift from Rebecca Yahr). Ramalina fraxinea FB30 and Ramalina farinaceae FB22: Slovenia, 1997. All the voucher specimens are placed in the UPCB Herbarium (UFPR-Curitiba, Brazil). 2.2. DNA extraction, PCR and sequencing Total DNA was extracted according to a modified CTAB method [24]. DNA-extracts were used for PCRamplification of the ITS regions including the 5.8S gene to the same genus Asterochloris. Phylogenetic studies of ITS rDNA from Trebouxia allowed the assignment of photobionts to certain clades [5,6] or focused on selectivity/specificity of fungi for photobionts [7]. These studies show that there are clear differences in photobiont selectivity in lichens. Only two species of Trebouxia were observed in some foliose Physciaceae by Dahlkild et al. [8], while in Parmelia and in the lichen genus Umbilicaria from Antarctica, several photobionts were found in different thalli of a single species [6,9]. There are few studies dealing with the diversity of photobionts of particular habitats. Beck et al. [5,10] indicated that a green algal species can generally be selected from a more diverse ‘‘pool of locally available algae’’. A similar situation has been found in lichens that are assoc (...truncated)