From strains to communities: an omics approach to study the diversity and adaptation of Antarctic cyanobacteria

Ice-free areas cover less than 0.5 % of the Antarctic, yet they host most of the terrestrial biodiversity of the whole continent. These terrestrial habitats are characterized by truncated and simple food webs consisting almost uniquely of microbial communities. Cyanobacteria are among the main primary producers in these habitats where they are often regarded as ‘ecosystem engineers. Despite the important ecological role cyanobacteria play, our understanding of the factors shaping their diversity and community structure, as well as their adaptions to cope with the extreme abiotic conditions in the Antarctic remains limited. The main aims of this Ph.D. were to (1) characterize the community structure and distribution of bacteria and micro-eukaryotes in a variety of substrates from different nunataks and valleys of the Sør Rondane Mountains region (East Antarctica); (2) identify keystone taxa in these food-webs and the main abiotic factors structuring these communities (Chapter 2); (3) provide a detailed understanding of the cyanobacterial diversity and community structure in these regions (Chapter 3); (4) identify the main physiological and genetic adaptation mechanisms that a pair of species of Nostoc (cyanobacteria) employ under desiccation – rehydration events which frequently occur the terrestrial environments of the Antarctic (Chapter 4). To achieve these goals, a multidisciplinary approach was applied combining ecophysiological and several omics analyses. In Chapter 2, the composition of bacterial and micro-eukaryotic communities was examined using high-throughput sequencing of parts of the 16S rRNA and 18S rRNA genes in 105 soil samples across 9 sites within a 100 km radius in the Sør Rondane Mountains (East Antarctica), differing in substrate type and associated physical and chemical conditions. In moraine soils, Actinomycetota and Cercozoa were among the most abundant bacterial and eukaryotic phyla, whereas on gneiss, granite and marble bedrock, Cyanobacteriota and Metazoa were the among the dominant bacterial and eukaryotic phyla. A distinct differentiation was observed within the Cyanobacteriota phylum at a higher taxonomic resolution depending on bedrock type, with granite being dominated by the Nostocaceae family and marble by the Chroococcidiopsaceae family. Surprisingly, metazoans reached a relatively high abundance in the eukaryotic dataset even in samples from the most arid sites, such as moraines in Austkampane and Widerøefjellet. Overall, this study showed that different substrate types support distinct microbial communities in inland Antarctic nunataks and valleys, and that lithological diversity is a major determinant of terrestrial microbiome biodiversity. In Chapter 3, the cyanobacterial community structures of the same sampled sites as in Chapter 2 were further investigated based on the use of cyanobacterial-specific primers targeting the V3-V4 region of the 16S rRNA gene in a high-throughput sequencing approach. The latest cyanobacterial taxonomic insights used in the recently released CyanoSeq database were applied to these sequences. Ordination analyses revealed that these different substrates harbour distinct cyanobacterial communities. Differences in community structure were significantly related to differences in pH, concentration of TN and N-NO3-, and electrical conductivity. The granitic soils of the Pingvinane nunataks hosted the highest richness followed by those from the granitic Utsteinen ridge. Granitic substrates were characterized by high TN concentrations and a neutral pH and hosted a variety of filamentous Cyanobacteria, such as Leptolyngbyaceae, Gomontiellaceae, Microcoleaceae, Oculatellaceae, Nostocaceae, but also unicellular taxa (Cyanothecaceae). The moraine samples from Austkampane and Widerøefjellet were dominated by Microcoleaceae and Gomontiellaceae, respectively. They were characterized by a high dry weight and low TN, and the diversity and abundance of cyanobacteria was low. The taxonomic richness was also low in soils from the alkaline marble bedrock and the gneiss bedrock from the Perlebandet nunatak, which were both characterized by high TN. Marble bedrock was dominated by Alitarella sp. (Chroococcidiopsaceae), whereas gneiss by Cyanothece sp. (Cyanothecaceae). Overall, our data show that different substrate types host distinct cyanobacterial communities. Cyanobacteria need to deal frequently with desiccation and rehydration events in Antarctic terrestrial and lacustrine habitats as well as during dispersal between habitats. In Chapter 4, the response to short-term desiccation exposure and rehydration of a terrestrial and freshwater strain belonging to the same Nostoc species was investigated. To achieve this, the pigments and osmolytes (trehalose and sucrose) concentrations as well as the photosynthetic efficiency were measured in combination to a metatranscriptomic (RNA-seq) analysis of the two Nostoc strains after 3h of desiccation, followed by rehydration after 10 min, 24h and up to 72h. Both strains reacted to dehydration by accumulating sucrose, whereas trehalose was present in lower concentrations. Only the freshwater strain showed a recovery in chlorophyll a content after 72h of rehydration. However, the photosynthetic efficiency remained below the original levels measured before desiccation in both strains. Transcriptomics profiles showed that both strains protected their cells during dehydration by inducing stress-related genes, such as those for the production of carotenoids, trehalose synthase and nitrogen fixation-related genes. The terrestrial strain responded with the up-regulation of a higher number of genes compared to the freshwater strain. The photosynthetic metabolism, however, slowly decreased after 24h of rehydration as evidenced by the down-regulation of genes related to phycobilisomes and photosystems, whilst psbA2 gene, encoding the D1 protein synthase necessary for PSII, was found to be significantly up-regulated under desiccation in the terrestrial ULC180, but not in the freshwater ULC008. These results suggest habitat specific adaptions to environmental stress in cyanobacteria, and provide novel insights into the strategies related to survival and adaptation of non-model Antarctic Nostoc strains to desiccation and rehydration events during the first 3 days experienced in extreme environments. In the final chapter the main findings of this Ph.D. thesis were discussed and a number of future research perspectives are formulated. These include the use of shotgun metagenomics over amplicon sequencing or the use of more specific primers in bacterial and micro-eukaryotic surveys, the characterization of biotic interactions and the integration of non-model organisms in experiments concerning their abiotic tolerance. Overall, the importance of integrating both ecophysiology and ‘omics’ disciplines is highlighted throughout this Ph.D. thesis, with the aim of maximizing our understanding about the ecologic role of microbial communities inhabiting the extreme Antarctic ice-free areas.