The underestimated fraction: diversity, challenges and novel insights into unicellular cyanobionts of lichens.

Lichens are remarkable and classic examples of symbiotic organisms that have fascinated scientists for centuries. Yet, it has only been for a couple of decades that significant advances have focused on the diversity of their green algal and/or cyanobacterial photobionts. Cyanolichens, which contain cyanobacteria as their photosynthetic partner, include up to 10% of all known lichens and, as such, studies on their cyanobionts are much rarer compared to their green algal counterparts. For the unicellular cyanobionts, i.e. cyanobacteria that do not form filaments, these studies are even scarcer. Nonetheless, these currently include at least 10 different genera in the cosmopolitan lichen order Lichinales. An international consortium (International Network of CyanoBionts; INCb) will tackle this lack of knowledge. In this article, we discuss the status of current unicellular cyanobiont research, compare the taxonomic resolution of photobionts from cyanolichens with those of green algal lichens (chlorolichens), and give a roadmap of research on how to recondition the underestimated fraction of symbiotic unicellular cyanobacteria in lichens.

A direct PCR approach with low-biomass insert opens new horizons for molecular sciences on cryptogam communities.

Molecular sequence data have transformed research on cryptogams (e.g., lichens, microalgae, fungi, and symbionts thereof) but methods are still strongly hampered by the small size and intermingled growth of the target organisms, poor cultivability and detrimental effects of their secondary metabolites. Here, we aim to showcase examples on which a modified direct PCR approach for diverse aspects of molecular work on environmental samples concerning biocrusts, biofilms, and cryptogams gives new options for the research community. Unlike traditional approaches, this methodology only requires biomass equivalent to colonies and fragments of 0.2 mm in diameter, which can be picked directly from the environmental sample, and includes a quick DNA lysis followed by a standardized PCR cycle that allows co-cycling of various organisms/target regions in the same run. We demonstrate that this modified method can (i) amplify the most widely used taxonomic gene regions and those used for applied and environmental sciences from single colonies and filaments of free-living cyanobacteria, bryophytes, fungi, and lichens, including their mycobionts, chlorobionts, and cyanobionts from both isolates and in situ material during co-cycling; (ii) act as a tool to confirm that the dominant lichen photobiont was isolated from the original sample; and (iii) optionally remove inhibitory secondary lichen substances. Our results represent examples which highlight the method's potential for future applications covering mycology, phycology, biocrusts, and lichenology, in particular.IMPORTANCECyanobacteria, green algae, lichens, and other cryptogams play crucial roles in complex microbial systems such as biological soil crusts of arid biomes or biofilms in caves. Molecular investigations on environmental samples or isolates of these microorganisms are often hampered by their dense aggregation, small size, or metabolism products which complicate DNA extraction and subsequent PCRs. Our work presents various examples of how a direct DNA extraction and PCR method relying on low biomass inserts can overcome these common problems and discusses additional applications of the workflow including adaptations.

Roccellinastrum, Cenozosia and Heterodermia: Ecology and phylogeny of fog lichens and their photobionts from the coastal Atacama Desert.

Some deserts on Earth such as the Namib or the Atacama are influenced by fog which can lead to the formation of local fog oases - unique environments hosting a great diversity of specialized plants and lichens. Lichens of the genera Ramalina, Niebla or Heterodermia have taxonomically been investigated from fog oases around the globe but not from the Atacama Desert, one of the oldest and driest deserts. Conditioned by its topography and the presence of orographic fog, the National Park Pan de Azúcar in the Atacama Desert is considered to be such a lichen hotspot. Applying multi-gen loci involving phylogenetic analyses combined with intense morphological and chemical characterization, we determined the taxonomic position of five of the most abundant epiphytic lichens of this area. We evaluated Roccellinastrumspongoideum and Heterodermiafollmannii which were both described from the area but also finally showed that the genus Cenozosia is the endemic sister genus to Ramalina, Vermilacinia, Namibialina and Niebla. As a result, we have described the species Heterodermiaadunca, C.cava and C.excorticata as new lichen species. This work provides a comprehensive dataset for common fog lichen genera of the Coastal Range of the Atacama Desert that can be used as a baseline for monitoring programs and environmental health assessments.

Water availability shapes edaphic and lithic cyanobacterial communities in the Atacama Desert.

In the Atacama Desert, cyanobacteria grow on various substrates such as soils (edaphic) and quartz or granitoid stones (lithic). Both edaphic and lithic cyanobacterial communities have been described but no comparison between both communities of the same locality has yet been undertaken. In the present study, we compared both cyanobacterial communities along a precipitation gradient ranging from the arid National Park Pan de Azúcar (PA), which resembles a large fog oasis in the Atacama Desert extending to the semiarid Santa Gracia Natural Reserve (SG) further south, as well as along a precipitation gradient within PA. Various microscopic techniques, as well as culturing and partial 16S rRNA sequencing, were applied to identify 21 cyanobacterial species; the diversity was found to decline as precipitation levels decreased. Additionally, under increasing xeric stress, lithic community species composition showed higher divergence from the surrounding edaphic community, resulting in indigenous hypolithic and chasmoendolithic cyanobacterial communities. We conclude that rain and fog water, respectively, cause contrasting trends regarding cyanobacterial species richness in the edaphic and lithic microhabitats.

Ecophysiology and phylogeny of new terricolous and epiphytic chlorolichens in a fog oasis of the Atacama Desert.

The Atacama Desert is one of the driest and probably oldest deserts on Earth where only a few extremophile organisms are able to survive. This study investigated two terricolous and two epiphytic lichens from the fog oasis "Las Lomitas" within the National Park Pan de Azúcar which represents a refugium for a few vascular desert plants and many lichens that can thrive on fog and dew alone. Ecophysiological measurements and climate records were combined with molecular data of the mycobiont, their green algal photobionts and lichenicolous fungi to gain information about the ecology of lichens within the fog oasis. Phylogenetic and morphological investigations led to the identification and description of the new lichen species Acarospora conafii sp. nov. as well as the lichenicolous fungi that accompanied them and revealed the trebouxioid character of all lichen photobionts. Their photosynthetic responses were compared during natural scenarios such as reactivation by high air humidity and in situ fog events to elucidate the activation strategies of this lichen community. Epiphytic lichens showed photosynthetic activity that was rapidly induced by fog and high relative air humidity whereas terricolous lichens were only activated by fog.

Strong in combination: Polyphasic approach enhances arguments for cold-assigned cyanobacterial endemism.

Cyanobacteria of biological soil crusts (BSCs) represent an important part of circumpolar and Alpine ecosystems, serve as indicators for ecological condition and climate change, and function as ecosystem engineers by soil stabilization or carbon and nitrogen input. The characterization of cyanobacteria from both polar regions remains extremely important to understand geographic distribution patterns and community compositions. This study is the first of its kind revealing the efficiency of combining denaturing gradient gel electrophoresis (DGGE), light microscopy and culture-based 16S rRNA gene sequencing, applied to polar and Alpine cyanobacteria dominated BSCs. This study aimed to show the living proportion of cyanobacteria as an extension to previously published meta-transcriptome data of the same study sites. Molecular fingerprints showed a distinct clustering of cyanobacterial communities with a close relationship between Arctic and Alpine populations, which differed from those found in Antarctica. Species richness and diversity supported these results, which were also confirmed by microscopic investigations of living cyanobacteria from the BSCs. Isolate-based sequencing corroborated these trends as cold biome clades were assigned, which included a potentially new Arctic clade of Oculatella. Thus, our results contribute to the debate regarding biogeography of cyanobacteria of cold biomes.