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Unexpected associated microalgal diversity in the lichen Ramalina farinacea is uncovered by pyrosequencing analyses

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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.

No MeSH data available.


Scheme of Real Time-PCR (RT-PCR) strategies.For the preparation of the algal DNA, two strategies were followed: A a ‘reamplification strategy’ for the HW, MW, A, M, B, A+M+B and Random treatments and B a ‘non-reamplification strategy’ for samples of the individual T. jamesii, T. asymmetrica and Trebouxia sp. TR9 cultures, the mock community, and the A+M+B non-ream. See the text for more details. CT: cycle threshold.
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pone.0175091.g002: Scheme of Real Time-PCR (RT-PCR) strategies.For the preparation of the algal DNA, two strategies were followed: A a ‘reamplification strategy’ for the HW, MW, A, M, B, A+M+B and Random treatments and B a ‘non-reamplification strategy’ for samples of the individual T. jamesii, T. asymmetrica and Trebouxia sp. TR9 cultures, the mock community, and the A+M+B non-ream. See the text for more details. CT: cycle threshold.

Mentions: The preparation of the algal DNA for pyrosequencing followed two strategies (Fig 2). We performed a ‘reamplification strategy’ for the HW, MW, A, M, B, A+M+B and Random treatments (Fig 2A) and a ‘non-reamplification strategy’ (‘non-ream’) for samples of the individual T. jamesii, T. asymmetrica and Trebouxia sp. TR9 cultures and the mock community and for the A+M+B treatment, which were distinguished as ‘A+M+B non-ream’ (Fig 2B).


Unexpected associated microalgal diversity in the lichen Ramalina farinacea is uncovered by pyrosequencing analyses
Scheme of Real Time-PCR (RT-PCR) strategies.For the preparation of the algal DNA, two strategies were followed: A a ‘reamplification strategy’ for the HW, MW, A, M, B, A+M+B and Random treatments and B a ‘non-reamplification strategy’ for samples of the individual T. jamesii, T. asymmetrica and Trebouxia sp. TR9 cultures, the mock community, and the A+M+B non-ream. See the text for more details. CT: cycle threshold.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5392050&req=5

pone.0175091.g002: Scheme of Real Time-PCR (RT-PCR) strategies.For the preparation of the algal DNA, two strategies were followed: A a ‘reamplification strategy’ for the HW, MW, A, M, B, A+M+B and Random treatments and B a ‘non-reamplification strategy’ for samples of the individual T. jamesii, T. asymmetrica and Trebouxia sp. TR9 cultures, the mock community, and the A+M+B non-ream. See the text for more details. CT: cycle threshold.
Mentions: The preparation of the algal DNA for pyrosequencing followed two strategies (Fig 2). We performed a ‘reamplification strategy’ for the HW, MW, A, M, B, A+M+B and Random treatments (Fig 2A) and a ‘non-reamplification strategy’ (‘non-ream’) for samples of the individual T. jamesii, T. asymmetrica and Trebouxia sp. TR9 cultures and the mock community and for the A+M+B treatment, which were distinguished as ‘A+M+B non-ream’ (Fig 2B).

View Article: PubMed Central - PubMed

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.

No MeSH data available.