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

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.

Molecular and morphological diversity of Trebouxia microalgae in sphaerothallioid Circinaria spp. lichens1

“…Despite the 454pyrosequencing limitations described in Moya et al. (low‐coverage samples, the region selected, etc. ), this approach represents a powerful and complementary approach to traditional Sanger sequencing, and helped us to shed light on microalgal coexistence.…”

“…Reads were processed as described in Moya et al. () and clustered based on S 99‐L 0.9 criteria. The consensus sequences of the OTUs were identified using the BLAST tool in the GenBank database (Altschul et al.…”

“…, Moya et al. ). Lichen specimens analyzed by Sanger sequencing showing high variability in their phycobiont preference, or coexistence (double peaks in the electrophoretograms) should be studied using different techniques.…”

The complexity of symbiotic interactions influences the ecological amplitude of the host: A case study inStereocaulon(lichenized Ascomycota)

“…However, as the rbcL marker generally shows lower resolution than the ITS rDNA, we did not further analyse it here. Although previously overlooked, the co‐occurrence of several phycobionts in individual lichen thalli (i.e., algal plurality) is a relative common phenomenon (Bačkor et al., ; Moya, Molins, Martinez‐Alberola, Muggia, & Barreno, ; Muggia, Baloch, Stabentheiner, Grube, & Wedin, ; Muggia et al., ; Onuț‐Brännström et al., ; Park et al., ; Voytsekhovich & Beck, ). We also obtained evidence for algal plurality in several Stereocaulon samples, which strengthens the potential of this lichen genus as a suitable model for high‐throughput sequencing studies.…”

Environment and host identity structure communities of green algal symbionts in lichens

“…Although a few studies have previously reported this phenomenon (e.g. del Campo et al ., ; Dal Grande et al ., ; Moya et al ., ), lichen thalli are still predominantly considered as pair‐wise interactions between a lichen‐forming fungus and a single alga. Our results suggest that this view may oversimplify the complexity of lichen thalli.…”