Peltigera frigida Lichens and Their Substrates Reduce the Influence of Forest Cover Change on Phosphate Solubilizing Bacteria.

Phosphorus (P) is one of the most critical macronutrients in forest ecosystems. More than 70 years ago, some Chilean Patagonian temperate forests suffered wildfires and the subsequent afforestation with foreign tree species such as pines. Since soil P turnover is interlinked with the tree cover, this could influence soil P content and bioavailability. Next to soil microorganisms, which are key players in P transformation processes, a vital component of Patagonian temperate forest are lichens, which represent microbial hotspots for bacterial diversity. In the present study, we explored the impact of forest cover on the abundance of phosphate solubilizing bacteria (PSB) from three microenvironments of the forest floor: Peltigera frigida lichen thallus, their underlying substrates, and the forest soil without lichen cover. We expected that the abundance of PSB in the forest soil would be strongly affected by the tree cover composition since the aboveground vegetation influences the edaphic properties; but, as P. frigida has a specific bacterial community, lichens would mitigate this impact. Our study includes five sites representing a gradient in tree cover types, from a mature forest dominated by the native species Nothofagus pumilio, to native second-growth forests with a gradual increase in the presence of Pinus contorta in the last sites. In each site, we measured edaphic parameters, P fractions, and the bacterial potential to solubilize phosphate by quantifying five specific marker genes by qPCR. The results show higher soluble P, labile mineral P, and organic matter in the soils of the sites with a higher abundance of P. contorta, while most of the molecular markers were less abundant in the soils of these sites. Contrarily, the abundance of the molecular markers in lichens and substrates was less affected by the tree cover type. Therefore, the bacterial potential to solubilize phosphate is more affected by the edaphic factors and tree cover type in soils than in substrates and thalli of P. frigida lichens. Altogether, these results indicate that the microenvironments of lichens and their substrates could act as an environmental buffer reducing the influence of forest cover composition on bacteria involved in P turnover.

The Bacterial Community of the Foliose Macro-lichen Peltigera frigida Is More than a Mere Extension of the Microbiota of the Subjacent Substrate.

Lichens host highly diverse microbial communities, with bacteria being one of the most explored groups in terms of their diversity and functioning. These bacteria could partly originate from symbiotic propagules developed by many lichens and, perhaps more commonly and depending on environmental conditions, from different sources of the surroundings. Using the narrowly distributed species Peltigera frigida as an object of study, we propose that bacterial communities in these lichens are different from those in their subjacent substrates, even if some taxa might be shared. Ten terricolous P. frigida lichens and their substrates were sampled from forested sites in the Coyhaique National Reserve, located in an understudied region in Chile. The mycobiont identity was confirmed using partial 28S and ITS sequences. Besides, 16S fragments revealed that mycobionts were associated with the same cyanobacterial haplotype. From both lichens and substrates, Illumina 16S amplicon sequencing was performed using primers that exclude cyanobacteria. In lichens, Proteobacteria was the most abundant phylum (37%), whereas soil substrates were dominated by Acidobacteriota (39%). At lower taxonomic levels, several bacterial groups differed in relative abundance among P. frigida lichens and their substrates, some of them being highly abundant in lichens but almost absent in substrates, like Sphingomonas (8% vs 0.2%), and others enriched in lichens, as an unassigned genus of Chitinophagaceae (10% vs 2%). These results reinforce the idea that lichens would carry some components of their microbiome when propagating, but they also could acquire part of their bacterial community from the substrates.

Carbon Consumption Patterns of Microbial Communities Associated with Peltigera Lichens from a Chilean Temperate Forest.

Lichens are a symbiotic association between a fungus and a green alga or a cyanobacterium, or both. They can grow in practically any terrestrial environment and play crucial roles in ecosystems, such as assisting in soil formation and degrading soil organic matter. In their thalli, they can host a wide diversity of non-photoautotrophic microorganisms, including bacteria, which play important functions and are considered key components of the lichens. In this work, using the BioLog® EcoPlate system, we studied the consumption kinetics of different carbon-sources by microbial communities associated with the thallus and the substrate of Peltigera lichens growing in a Chilean temperate rain forest dominated by Nothofagus pumilio. Based on the similarity of the consumption of 31 carbon-sources, three groups were formed. Among them, one group clustered the microbial metabolic profiles of almost all the substrates from one of the sampling sites, which exhibited the highest levels of consumption of the carbon-sources, and another group gathered the microbial metabolic profiles from the lichen thalli with the most abundant mycobiont haplotypes. These results suggest that the lichen thallus has a higher impact on the metabolism of its microbiome than on the microbial community of its substrate, with the latter being more diverse in terms of the metabolized sources and whose activity level is probably related to the availability of soil nutrients. However, although significant differences were detected in the microbial consumption of several carbon-sources when comparing the lichen thallus and the underlying substrate, d-mannitol, l-asparagine, and l-serine were intensively metabolized by both communities, suggesting that they share some microbial groups. Likewise, some communities showed high consumption of 2-hydroxybenzoic acid, d-galacturonic acid, and itaconic acid; these could serve as suitable sources of microorganisms as bioresources of novel bioactive compounds with biotechnological applications.

Substrates of Peltigera Lichens as a Potential Source of Cyanobionts.

Photobiont availability is one of the main factors determining the success of the lichenization process. Although multiple sources of photobionts have been proposed, there is no substantial evidence confirming that the substrates on which lichens grow are one of them. In this work, we obtained cyanobacterial 16S ribosomal RNA gene sequences from the substrates underlying 186 terricolous Peltigera cyanolichens from localities in Southern Chile and maritime Antarctica and compared them with the sequences of the cyanobionts of these lichens, in order to determine if cyanobacteria potentially available for lichenization were present in the substrates. A phylogenetic analysis of the sequences showed that Nostoc phylotypes dominated the cyanobacterial communities of the substrates in all sites. Among them, an overlap was observed between the phylotypes of the lichen cyanobionts and those of the cyanobacteria present in their substrates, suggesting that they could be a possible source of lichen photobionts. Also, in most cases, higher Nostoc diversity was observed in the lichens than in the substrates from each site. A better understanding of cyanobacterial diversity in lichen substrates and their relatives in the lichens would bring insights into mycobiont selection and the distribution patterns of lichens, providing a background for hypothesis testing and theory development for future studies of the lichenization process.

Intrinsic factors of Peltigera lichens influence the structure of the associated soil bacterial microbiota.

Definition of lichens has evolved from bi(tri)partite associations to multi-species symbioses, where bacteria would play essential roles. Besides, although soil bacterial communities are known to be affected by edaphic factors, when lichens grow upon them these could become less preponderant. We hypothesized that the structure of both the lichen microbiota and the microbiota in the soil underneath lichens is shaped by lichen intrinsic and extrinsic factors. In this work, intrinsic factors corresponded to mycobiont and cyanobiont identities of Peltigera lichens, metabolite diversity and phenoloxidase activity and extrinsic factors involved the site of the forest where lichens grow. Likewise, the genetic and metabolic structure of the lichen and soil bacterial communities were analyzed by fingerprinting. Among the results, metabolite diversity was inversely related to the genetic structure of bacterial communities of lichens and soils, highlighting the far-reaching effect of these substances; while phenoloxidase activity was inversely related to the metabolic structure only of the lichen bacterial microbiota, presuming a more limited effect of the products of these enzymes. Soil bacterial microbiota was different depending on the site and, strikingly, according to the cyanobiont present in the lichen over them, which could indicate an influence of the photobiont metabolism on the availability of soil nutrients.

Phylogenetic Diversity of Peltigera Cyanolichens and Their Photobionts in Southern Chile and Antarctica.

The lichen genus Peltigera has been mainly revised in the Northern Hemisphere, with most species being recorded in Europe and North America. This study assessed the phylogenetic diversity of the mycobionts and cyanobionts of Peltigera cyanolichens collected in Southern Chile and Antarctica, areas in which lichens are extremely diverse but poorly studied. The operational taxonomic units (OTUs) of each symbiont were defined by analyzing the genetic diversity of the LSU and SSU rDNA of the mycobionts and cyanobionts, respectively, and a phylogenetic approach was used to relate these OTUs with sequences previously reported for Peltigera and Nostoc. Among the 186 samples collected, 8 Peltigera and 15 Nostoc OTUs were recognized, corresponding to sections Peltigera, Horizontales, and Polydactylon, in the case of the mycobionts, and to the Nostoc clade II, in the case of the cyanobionts. Since some of the OTUs recognized in this study had not previously been described in these areas, our results suggest that the diversity of Peltigera reported to date in the regions studied using traditional morphological surveys has underestimated the true diversity present; therefore, further explorations of these areas are recommended.