A multi-tool approach to assess microalgal diversity in lichens: isolation, Sanger sequencing, HTS and ultrastructural correlations

Abstract

Lichen thalli represent the most conspicuous examples of fungal-algal interactions. Studies that describe phycobiont diversity within entire thalli are based mainly on Sanger sequencing. In some lichen species, this technique could underestimate the intrathalline coexistence of multiple microalgae. In this study different multi-tool approaches were applied to two lichen taxa, Circinaria hispida and Flavoparmelia soredians, to detect algal coexistence. Here, we combined Sanger sequencing, a specific polymerase chain reaction (PCR) primer, 454-pyrosequencing, phycobiont isolation and ultrastructural characterization. Furthermore, we compared pyrenoid ultrastructural features of lichenized phycobionts with microalgae isolated in culture. An improved methodology was used to isolate and propagate phycobionts which, in combination with fast genetic identification, resulted in a considerable reduction in time and cost to complete the process. This isolation method, coupled with a specific PCR primer, allowed for the detection of coexisting algae in C. hispida (four Trebouxia lineages). 454-pyrosequencing detected only a fraction of such diversity, while Sanger sequencing identified only the primary phycobiont. Ultrastructural features of the isolated algae were observed by transmission electron microscopy; the maintenance of the pyrenoid characteristics suggested the existence of different Trebouxia lineages. In F. soredians a single Trebouxia lineage was identified using all these approaches.In cases of lichens with algal coexistence, a combination of different molecular and ultrastructural approaches may be required to reveal the underlying algal diversity within a single thallus. The approach proposed in this study provides information about the relationship between molecular and ultrastructural data, and represents an improvement in the delimitation of taxonomic features which is needed to recognize intrathalline Trebouxia diversity.

Myrmecia israeliensis as the primary symbiotic microalga in squamulose lichens growing in European and Canary Island terricolous communities

“…Recent literature (Casano et al 2011;Molins et al 2013;Catalá et al 2015;Moya et al 2015;Molins et al 2017) proved that TEM observations should be previously mainly known as free living.…”

“…The second combination included the newly designed primer specific to green algae zeleny_F2 (5'-TTC TTA GTT GGT GGG TTG CC-3', located at the end of 18S rDNA) + the universal primer ITS4 (White et al 1990), respectively. In the IB_CI samples, PCR reactions and Sanger sequencing were performed as described in Molins et al (2017). In the N_CE samples, PCRs were performed in 20 µl using MyTaq™ DNA polymerase (Bioline, London, UK) containing: 4 µm of buffer, 0,3 µm zeleny_F2/ITS4 primers, 0,2 µm MyTaq polymerase and 1 μl of template DNA.Sterile Milli-Q water was used to bring to volume.…”

“…Fractured thalli were attached to the holder, coated with palladium/gold and viewed with a Hitachi (S4800). For TEM, the cells were fixed and dehydrated as described in Molins et al (2017). Samples were embedded in Spurr's resin according to the manufacturer's instructions.…”

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

“…PCR reactions were performed as described in Molins et al. () (Table in the Supporting Information).…”

“…The majority of molecular studies in sphaerothallioid lichens are focused on mycobiont analyses, but phycobionts have been mostly ignored and are poorly known (Molins et al. ). Moreover, these sphaerothallioid lichen species are interesting candidates to analyze the phycobiont diversity due to the different morphology and growth forms which occur under diverse ecological settings.…”