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Microbial Cargo: Do Bacteria on Symbiotic Propagules Reinforce the Microbiome of Lichens?

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Microbial Cargo: Do Bacteria on Symbiotic Propagules Reinforce the Microbiome of Lichens? bs_bs_banner Environmental Microbiology (2014) 16(12), 3743–3752 doi:10.1111/1462-2920.12658 Microbial cargo: do bacteria on symbiotic propagules reinforce the microbiome of lichens? Ines Aline Aschenbrenner,1,2 Introduction Massimiliano Cardinale,1,2 Gabriele Berg1 and Lichens are known as ecologically diversified symbioses Martin Grube2* that colonize almost all climate zones and occur on a wide 1Institute of Environmental Biotechnology, Graz range of substrates (Nash, 2008). In the symbiotic stage, University of Technology, Petersgasse 12, 8010 Graz, the microscopic fungi (mycobionts) produce macroscopic Austria. thalli of characteristic appearance. They provide a long- 2Institute of Plant Sciences, University of Graz, living habitat for the photoautotrophic partners (alga Holteigasse 6, 8010 Graz, Austria. and/or cyanobacterium), which are sheltered between peripheral fungal layers (Honegger, 2012). Despite a Summary general ubiquity of lichens, individual species of lichen- forming fungi may be highly specialized for their ecolo- According to recent research, bacteria contribute gical niches. One prominent example for ecological as recurrent associates to the lichen symbiosis. specialization is the lung lichen, a conspicuous foliose Yet, the variation of the microbiomes within species species adapted to old-growth forests and highly sensitive and across geographically separated populations to air pollution (Scheidegger, 1995; Scheidegger and remained largely elusive. As a quite common disper- Werth, 2009). This species is among the most intensely sal mode, lichens evolved vertical transmission studied lichens because of its importance as indicator of both fungal and algal partners in specifically species (Rose, 1992). Detailed studies have assessed designed mitotic propagules. Bacteria, if co- chemical, ecological and ecophysiological aspects in lung transmitted with these symbiotic propagules, could lichens (e.g. McEvoy et al., 2007; Bidussi et al., 2013), as contribute to a geographical structure of lichen- well as in population genetics, phylogeography and repro- associated microbiomes. ductive biology (e.g., Jüriado et al., 2011; Widmer et al., The lung lichen was sampled from three localities in 2012). The lung lichen represents a tripartite lichen as it eastern Austria to analyse their associated bacterial hosts additionally to the mycobiont (Lobaria pulmonaria) communities by bar-coded pyrosequencing, network two distinct photobionts internally. The green-algae analysis and fluorescence in situ hybridization. For Dictyochloropsis reticulata is found beneath more than the first time, bacteria were documented to colonize 90% of the lichen surface and strains of cyanobacterium symbiotic propagules of lichens developed for Nostoc are locally restricted to gall-like colonies, in short-distance transmission of the symbionts. The so-called internal cephalodia (Myllys et al., 2007). These propagules share the overall bacterial community Nostoc strains are generally captured from the surface to structure with the thalli at class level, except for fila- become gradually integrated into the lichen thallus, where mentous Cyanobacteria (Nostocophycideae), and they primarily contribute to the symbiotic system by nitro- with Alphaproteobacteria as predominant group. All gen fixation (Jordan, 1970; Cornejo and Scheidegger, three sampling sites share a core fraction of the 2013). microbiome. Bacterial communities of lichen thalli Although the lung lichen is among the fastest growing from the same sampling site showed higher similarity lichens, it is only fertile under rare optimal habitat condi- than those of distant populations. This variation and tions. Apothecia are usually only found on older and the potential co-dispersal of a microbiome fraction sometimes even senescing thallus lobes. On the other with structures of the host organism contribute new hand, mitotic propagules for simultaneous dispersal of aspects to the ‘everything is everywhere’ hypothesis. both symbionts (fungus and alga), a common propagation strategy of many lichens, are almost generally present in this species. Their function as dispersal units has also Received 14 April, 2014; revised 30 September, 2014; accepted 1 October, 2014. *For correspondence. E-mail martin.grube been shown experimentally (Scheidegger, 1995), and a @uni-graz.at; Tel. (+43) 316 380 5655; Fax (+43) 316 380 9880. recent microsatellite analysis revealed co-dispersal of © 2014 Society for Applied Microbiology and John Wiley & Sons Ltd 3744 I. A. Aschenbrenner, M. Cardinale, G. Berg and M. Grube both symbionts as one of the key factors shaping the assume that this process could reinforce the composition genetic structure of this species (Werth and Scheidegger, of the lichen-associated microbiome and contribute to its 2012). The symbiotic propagules of the lung lichen can be geographical structure. We studied this possibility by called isidioid soredia, which are developed at margins microcopy to assess bacterial colonization on the and ridges of the upper thallus surfaces as dust-like par- propagules and by analyses of pyrosequencing data and ticles comprising a few algal and fungal cells, as typical for community fingerprinting patterns. soredia structures. Before the propagules detach, they normally develop a peripheral cortex to resemble isidia Results (stratified propagules). These symbiotic propagules of the lung lichen are much larger and heavier than the sexual First insights into the bacterial communities on the fungal spores of this species. Therefore, the propagules lichen thalli and symbiotic propagules appear to be less suitable for long-distance dispersal, Single strand conformation analysis (SSCP) of 16S rRNA compared with the much smaller meiotically produced gene fragments obtained from three sampling sites fungal ascospores. Previous work underlined that symbi- (Tamischbachgraben, St. Oswald/Eibiswald, Johnsbach otic dispersal with isidioid soredia is rather local in this in Styria, Austria) revealed generally high numbers of species (Walser, 2004). More recently, Dal Grande and bands (> 20) in samples of both thalli and vegetative colleagues (2012) provided a quantitative assessment of propagules (isidioid soredia). The entire thalli were char- both propagation strategies and showed that vertical acterized by several additional strong bands, which were transmission decreases significantly within short distance not present in the samples of the symbiotic propagules (approximately cut in half over distances of 10 m). (Fig. S2). Long-living lichen thalli provide various niches for asso- ciated microorganisms, of which colonizing fungi have Microbial community composition of lichen thalli and been well studied (Lawrey and Diederich, 2003; Werth symbiotic propagules et al., 2013). Even though earlier culture-dependent search for microorganisms revealed the presence of A comparison of the lichen-associated bacterial commu- lichen-associated bacteria (Grube and Berg, 2009), the nity compositions on both thallus and isidioid soredia by molecular characterization has only just started in the last amplicon sequencing showed that the lichen propagules few years. According to fluorescence in situ hybridization shared most of the dominant taxa (at class level) with and confocal laser scanning microscopy (FISH-CLSM), entire thalli except for the filamentous Nostocophycideae bacteria colonize lichens in a biofilm-like manner (Cyanobacteria), which were only present on thalli (Cardinale et al., 2008). The composition of these com- (Fig. 1A). Alphaproteobacteria were the predominant munities is host specific (Grube et al., 2009; Bates et al., taxon on both lichen parts [on average: soredia 33%, 2011), with Alphaproteobacteria as the prominent bacte- standard deviation (SD) 6.7%; thalli 26%, SD 4.7%] fol- rial class on many lichens (e.g. Grube et al., 2009; lowed by Sphingobacteria (on average: soredia 23%, SD Schneider et al., 2011). Other lineages may also be 8.9%; thalli 14%, SD 2.8%). The relative abundance of detected at considerable relative abundances, includ- Betaproteobacteria, Gammaproteobacteria and Delta- ing Acidobacteria, Actinobacteria, Betaproteobacteria, proteobacteria as well as Actinobacteria on thalli was Spartobacteria and Sphingobacteria, among others at least twofold higher compared with that on the (Bjelland et al., 2010; Mushegian et al., 2011; Grube propagules, and in contrast, the relative abundance of et al., 2012; Hodkinson et al., 2012). These surface- Spartobacteria was twofold higher on the propagules than colonizing bacteria of lichens are supposed to play a on thalli (Fig. 1A). The lichen-associated communities on beneficial role for lichens by contributing to the lichen thalli and symbiotic propagules were high in alpha diver- metabolism with release of nitrogen compounds like sity and richness indices (Table S1). amino acids, vitamins and phytohormones, or by More than 89% of all sequences were shared by both solubilization of phosphate (Liba et al., 2006; Grube et al., thalli and propagules and represented 28% of all 2009; 2014; Schneider et al., 2011). observed operational taxonomic units (OTUs) within this Despite new insights into the structure and function of data set (Fig. S3A). Only 9% of the sequences were lichen-associated bacteria, little is known so far about the unique for the thalli and 2% for the propagules. The major- intraspecific variation of microbiome composition, and ity of the OTUs (49%) occurred exclusively on lichen thalli; also, how lichens
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