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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.

This study analyses the interactions among crustose and lichenicolous lichens growing on gypsum biocrusts. The selected community was composed of Acarospora nodulosa , Acarospora placodiiformis , Diploschistes diacapsis , Rhizocarpon malenconianum and Diplotomma rivas-martinezii . These species represent an optimal system for investigating the strategies used to share phycobionts because Acarospora spp . are parasites of D. diacapsis during their first growth stages, while in mature stages, they can develop independently. R. malenconianum is an obligate lichenicolous lichen on D. diacapsis , and D. rivas-martinezii occurs physically close to D. diacapsis . Microalgal diversity was studied by Sanger sequencing and 454-pyrosequencing of the nrITS region, and the microalgae were characterized ultrastructurally. Mycobionts were studied by performing phylogenetic analyses. Mineralogical and macro- and micro-element patterns were analysed to evaluate their influence on the microalgal pool available in the substrate. The intrathalline coexistence of various microalgal lineages was confirmed in all mycobionts. D. diacapsis was confirmed as an algal donor, and the associated lichenicolous lichens acquired their phycobionts in two ways: maintenance of the hosts’ microalgae and algal switching. Fe and Sr were the most abundant microelements in the substrates but no significant relationship was found with the microalgal diversity. The range of associated phycobionts are influenced by thallus morphology.

Trebouxiophyceae (Chlorophyta) is a species-rich class of green algae with a remarkable morphological and ecological diversity. Currently, there are a few completely sequenced mitochondrial genomes (mtDNA) from diverse Trebouxiophyceae but none from lichen symbionts. Here, we report the mitochondrial genome sequence of Trebouxia sp. TR9 as the first complete mtDNA sequence available for a lichen-symbiont microalga. A comparative study of the mitochondrial genome of Trebouxia sp. TR9 with other chlorophytes showed important organizational changes, even between closely related taxa. The most remarkable change is the enlargement of the genome in certain Trebouxiophyceae, which is principally due to larger intergenic spacers and seems to be related to a high number of large tandem repeats. Another noticeable change is the presence of a relatively large number of group II introns interrupting a variety of tRNA genes in a single group of Trebouxiophyceae, which includes Trebouxiales and Prasiolales. In addition, a fairly well-resolved phylogeny of Trebouxiophyceae, along with other Chlorophyta lineages, was obtained based on a set of seven well-conserved mitochondrial genes.

Completely sequenced plastomes provide a valuable source of information about the duplication, loss, and transfer events of chloroplast genes and phylogenetic data for resolving relationships among major groups of plants. Moreover, they can also be useful for exploiting chloroplast genetic engineering technology. Ericales account for approximately six per cent of eudicot diversity with 11,545 species from which only three complete plastome sequences are currently available. With the aim of increasing the number of ericalean complete plastome sequences, and to open new perspectives in understanding Mediterranean plant adaptations, a genomic study on the basis of the complete chloroplast genome sequencing of Arbutus unedo and an updated phylogenomic analysis of Asteridae was implemented. The chloroplast genome of A. unedo shows extensive rearrangements but a medium size (150,897 nt) in comparison to most of angiosperms. A number of remarkable distinct features characterize the plastome of A. unedo: five-fold dismissing of the SSC region in relation to most angiosperms; complete loss or pseudogenization of a number of essential genes; duplication of the ndhH-D operon and its location within the two IRs; presence of large tandem repeats located near highly re-arranged regions and pseudogenes. All these features outline the primary evolutionary split between Ericaceae and other ericalean families. The newly sequenced plastome of A. unedo with the available asterid sequences allowed the resolution of some uncertainties in previous phylogenies of Asteridae.

Two microalgal species, Trebouxia jamesii and Trebouxia sp. TR9, were detected as the main photobionts coexisting in the thalli of the lichen Ramalina farinacea. Trebouxia sp. TR9 emerged as a new taxon in lichen symbioses and was successfully isolated and propagated in in vitro culture and thoroughly investigated. Several years of research have confirmed the taxon Trebouxia sp. TR9 to be a model/reference organism for studying mycobiont–photobiont association patterns in lichen symbioses. Trebouxia sp. TR9 is the first symbiotic, lichen-forming microalga for which an exhaustive characterization of cellular ultrastructure, physiological traits, genetic and genomic diversity is available. The cellular ultrastructure was studied by light, electron and confocal microscopy; physiological traits were studied as responses to different abiotic stresses. The genetic diversity was previously analyzed at both the nuclear and organelle levels by using chloroplast, mitochondrial, and nuclear genome data, and a multiplicity of phylogenetic analyses were carried out to study its intraspecific diversity at a biogeographical level and its specificity association patterns with the mycobiont. Here, Trebouxia sp. TR9 is formally described by applying an integrative taxonomic approach and is presented to science as Trebouxia lynnae, in honor of Lynn Margulis, who was the primary modern proponent for the significance of symbiosis in evolution. The complete set of analyses that were carried out for its characterization is provided.

Lichens, self-supporting mutualistic associations between a fungal partner and one or more photosynthetic partners, also harbor non-photosynthetic bacteria. The diversity and contribution of these bacteria to the functioning of lichen symbiosis have recently begun to be studied, often by culture-independent techniques due to difficulties in their isolation and culture. However, culturing as yet unculturable lichenic bacteria is critical to unravel their potential functional roles in lichen symbiogenesis, to explore and exploit their biotechnological potential and for the description of new taxa. Our objective was to improve the recovery of lichen associated bacteria by developing novel isolation and culture approaches, initially using the lichen Pseudevernia furfuracea. We evaluated the effect of newly developed media enriched with novel lichen extracts, as well as the influence of thalli washing time and different disinfection and processing protocols of thalli. The developed methodology included: i) the use of lichen enriched media to mimic lichen nutrients, supplemented with the fungicide natamycin; ii) an extended washing of thalli to increase the recovery of ectolichenic bacteria, thus allowing the disinfection of thalli to be discarded, hence enhancing endolichenic bacteria recovery; and iii) the use of an antioxidant buffer to prevent or reduce oxidative stress during thalli disruption. The optimized methodology allowed significant increases in the number and diversity of culturable bacteria associated with P. furfuracea, and it was also successfully applied to the lichens Ramalina farinacea and Parmotrema pseudotinctorum. Furthermore, we provide, for the first time, data on the abundance of culturable ecto- and endolichenic bacteria that naturally colonize P. furfuracea, R. farinacea and P. pseudotinctorum, some of which were only able to grow on lichen enriched media. This innovative methodology is also applicable to other microorganisms inhabiting these and other lichen species.

Lichen phycobiomes have recently emerged as a source of biodiversity and new species of microalgae. Although in the genus Diplosphaera free-living microalgae are common, numerous strains belonging to this genus have frequently been recognized or isolated from lichen thalli. In this study, a comprehensive analysis of the strain Diplosphaera sp. ASUV135, isolated from a lichen thallus, has been carried out using an integrative taxonomic approach. The SSU and nuclear-encoding ITS rDNA, as well as the chloroplast rbcL gene, were sequenced and analyzed to ascertain its taxonomic position and phylogenetic relationships within the genus Diplosphaera. This strain was also analyzed by light, confocal and transmission microscopy for morphological and ultrastructural characterization. The phenotypic plasticity in this strain was also confirmed by changes in its morphology under different growth conditions, as well as those of modulated Chlorophyll a fluorescence emissions, to understand its photosynthetic functioning. Our results pointed out that this strain represents a new taxon within the genus Diplosphaera (Prasiola group), described here as Diplosphaera elongate sp. nova. This study also provides tools for future research on organisms with high phenotypic plasticity by using molecular, morphological, ultrastructural and physiological approaches.

Buellia zoharyi is a crustose placodioid lichen, usually occurring on biocrusts of semiarid ecosystems in circum-Mediterranean/Macaronesian areas. In previous work, we found that this lichenized fungus was flexible in its phycobiont choice in the Canary Islands. Here we test whether geography and habitat influence phycobiont diversity in populations of this lichen from the Iberian Peninsula and Balearic Islands using Sanger and high throughput sequencing (HTS). Additionally, three thallus section categories (central, middle and periphery) were analyzed to explore diversity of microalgal communities in each part. We found that B. zoharyi populations hosted at least three different Trebouxia spp., and this lichen can associate with distinct phycobiont strains in different habitats and geographic regions. This study also revealed that the Trebouxia composition of this lichen showed significant differences when comparing the Iberian Peninsula with the Balearics thalli. No support for differences in microalgal communities was found among thallus sections; however, several thalli showed different predominant Trebouxia spp. at each section. This result corroborate that thallus parts selected for DNA extraction in metabarcoding analyses are key to not bias the total phycobiont diversity detected. This study highlights that inclusion of HTS analysis is crucial to understand lichen symbiotic microalgal diversity.

Ramalina farinacea is a widely distributed epiphytic lichen from the Macaronesian archipelagos to Mediterranean and Boreal Europe. Previous studies have indicated a specific association between R. farinacea and Trebouxia microalgae species. Here, we examined the symbiotic interactions in this lichen and its closest allies (the so-called 'R. farinacea group') across ten biogeographic subregions, spanning diverse macroclimates, analyzing the climatic niche of the primary phycobionts, and discussing the specificity of these associations across the studied area. The most common phycobionts in the 'R. farinacea group' were T. jamesii and T. lynnae, which showed a preference for continentality and insularity, respectively. The Canarian endemic R. alisiosae associated exclusively with T. lynnae, while the other Ramalina mycobionts interacted with both microalgae. The two phycobionts exhibited extensive niche overlap in an area encompassing Mediterranean, temperate Europe, and Macaronesian localities. However, T. jamesii occurred in more diverse climate types, whereas T. lynnae preferred warmer and more humid climates, often close to the sea, which could be related to its tolerance to salinity. With the geographical perspective gained in this study, it was possible to show how the association with different phycobionts may shape the ecological adaptation of lichen symbioses.

The lichenized green microalga Trebouxia lynnae Barreno has been recently described and is considered a model organism for studying lichen chlorobionts. Its cellular ultrastructure has already been studied in detail by light, electron, and confocal microscopy, and its nuclear, chloroplast and mitochondrial genomes have been sequenced and annotated. Here, we investigated in detail the ultrastructure of in vitro grown cultures of T. lynnae observed by Low Temperature Scanning Electron Microscopy (LTSEM) applying a protocol with minimum intervention over the biological samples. This methodology allowed for the discovery of ultrastructural features previously unseen in Trebouxiophyceae microalgae. In addition, original Transmission Electron Microscopy (TEM) images of T. lynnae were reinterpreted based on the new information provided by LTSEM. The nucleolar vacuole, dictyosomes, and endoplasmic reticulum were investigated and reported for the first time in T. lynnae and most likely in other Trebouxia lineages.