Abstract
The current literature reveals that the intrathalline coexistence of multiple microalgal taxa in lichens is more common than previously thought, and additional complexity is supported by the coexistence of bacteria and basidiomycete yeasts in lichen thalli. This replaces the old paradigm that lichen symbiosis occurs between a fungus and a single photobiont. The lichen Ramalina farinacea has proven to be a suitable model to study the multiplicity of microalgae in lichen thalli due to the constant coexistence of Trebouxia sp. TR9 and T. jamesii in long-distance populations. To date, studies involving phycobiont diversity within entire thalli are based on Sanger sequencing, but this method seems to underestimate the diversity. Here, we aim to analyze both the microalgal diversity and its community structure in a single thallus of the lichen R. farinacea by applying a 454 pyrosequencing approach coupled with a careful ad hoc-performed protocol for lichen sample processing prior to DNA extraction. To ascertain the reliability of the pyrosequencing results and the applied bioinformatics pipeline results, the thalli were divided into three sections (apical, middle and basal zones), and a mock community sample was used. The developed methodology allowed 40448 filtered algal reads to be obtained from a single lichen thallus, which encompassed 31 OTUs representative of different microalgae genera. In addition to corroborating the coexistence of the two Trebouxia sp. TR9 and T. jamesii taxa in the same thallus, this study showed a much higher microalgal diversity associated with the lichen. Along the thallus ramifications, we also detected variations in phycobiont distribution that might correlate with different microenvironmental conditions. These results highlight R. farinacea as a suitable material for studying microalgal diversity and further strengthen the concept of lichens as multispecies microecosystems. Future analyses will be relevant to ecophysiological and evolutionary studies to understand the roles of the multiple photobionts in lichen symbioses.
Highlights
Lichens have been traditionally described as symbiotic organisms resulting from the close morpho-physiological interaction between a heterotroph and at least one photosynthetic partner, which can be green microalga and/or cyanobacteria [1]
Additional complexity was reported inside a single lichen thallus by the intrathalline coexistence of multiple microalgae [2,3,4,5,6,7], bacteria and yeasts [8,9], overthrowing the old paradigm that lichens considered to be mutualistic associations between one lichenized fungus and a single photobiont
high-throughput sequencing (HTS) approaches are providing unprecedented amounts of information to assess species diversity at diverse taxonomic levels and in diverse ecological settings. These techniques have been progressively applied in lichenological studies throughout the past few years; some authors have noted how the sample storage, the selection of primers and the application of different DNA extraction protocols can influence the final perception of species diversity and its patterns [18,47]
Summary
Lichens have been traditionally described as symbiotic organisms (holobionts) resulting from the close morpho-physiological interaction between a heterotroph (mycobiont) and at least one photosynthetic (photobiont) partner, which can be green microalga (phycobiont) and/or cyanobacteria (cyanobiont) [1]. Previous studies performed on the lichen Ramalina farinacea (L.) Ach. showed the recurrent co-occurrence of at least two phycobionts TR1 = T. jamesii) inside the thalli using microscopic techniques, culture isolations of both phycobionts and molecular characterization with different genetic markers [10,11]. The coexistence of these two phycobionts was verified in several populations of R. farinacea from the Iberian Peninsula, the Canary Islands and North America [3,11]. Several studies have demonstrated that these two phycobionts respond differently to abiotic stress [3,12,13,14]; their coexistence would be advantageous for the whole lichen under extreme environmental conditions and would represent a common, basic phenomenon in ecologically adapted lichens
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