Origin and evolution of chloroplast group I introns in lichen algae.

The history of group I introns is characterized by repeated horizontal transfers, even among phylogenetically distant species. The symbiogenetic thalli of lichens are good candidates for the horizontal transfer of genetic material among distantly related organisms, such as fungi and green algae. The main goal of this study was to determine whether there were different trends in intron distribution and properties among Chlorophyte algae based on their phylogenetic relationships and living conditions. Therefore, we investigated the occurrence, distribution and properties of group I introns within the chloroplast LSU rDNA in 87 Chlorophyte algae including lichen and free-living Trebouxiophyceae compared to free-living non-Trebouxiophyceae species. Overall, our findings showed that there was high diversity of group I introns and homing endonucleases (HEs) between Trebouxiophyceae and non-Trebouxiophyceae Chlorophyte algae, with divergence in their distribution patterns, frequencies and properties. However, the differences between lichen Trebouxiophyceae and free-living Trebouxiophyceae were smaller. An exception was the cL2449 intron, which was closely related to ω elements in yeasts. Such introns seem to occur more frequently in lichen Trebouxiophyceae compared to free-living Trebouxiophyceae. Our data suggest that lichenization and maintenance of lichen symbiosis for millions of years of evolution may have facilitated horizontal transfers of specific introns/HEs between symbionts. The data also suggest that sequencing of more chloroplast genes harboring group I introns in diverse algal groups may help us to understand the group I intron/HE transmission process within these organisms.

The genetic structure of the cosmopolitan three-partner lichen Ramalina farinacea evidences the concerted diversification of symbionts.

The epiphytic lichen Ramalina farinacea is distributed throughout the northern hemisphere in which the same two algal Trebouxia species (provisionally named TR1 and TR9) coexist in every thallus. Ramalina farinacea symbionts were characterized based on the two fungal nuclear loci (nrITS and rpb2) along with the primary and secondary structures of nrITS from each Trebouxia species in the Iberian Peninsula and Canary Islands. The results indicated a noticeable genetic differentiation between mycobionts from these two geographic areas and also suggested concerted changes in the three partners of a lichen symbiosis toward two clearly distinguishable 'holobiont' lineages. Modeling of ITS2 RNA secondary structures suggested their temperature sensitivity in TR1 but not in TR9, which was consistent with the observed superior physiological performance of TR9 phycobionts under relatively high temperatures. Both TR1 and TR9 phycobionts have been also found in a variety of taxonomically distinct lichens with a preferably Mediterranean distribution, being TR1 much more widespread than TR9. Our observations support a model in which ecological diversification and speciation of lichen symbionts in different habitats could include a transient phase consisting of associations with more than one photobiont in individual thalli. Such diversification is likely to be promoted by different physiological backgrounds.

Different strategies to achieve Pb-tolerance by the two Trebouxia algae coexisting in the lichen Ramalina farinacea.

Lichen thalli are permeable to airborne substances, including heavy metals, which are harmful to cell metabolism. Ramalina farinacea shows a moderate tolerance to Pb. This lichen comprises two Trebouxia phycobionts, provisionally referred to as TR1 and TR9, with distinct physiological responses to acute oxidative stress. Thus, there is a more severe decay in photosynthesis and photosynthetic pigments in TR1 than in TR9. Similarly, under oxidative stress, antioxidant enzymes and HSP70 protein decrease in TR1 but increase in TR9. Since Pb toxicity is associated with increased ROS formation, we hypothesized greater Pb tolerance in this phycobiont. Accordingly, the aim of the present study was to characterize the physiological differences in the responses of TR1 and TR9 to Pb exposure. Liquid cultures of isolated phycobionts were incubated for 7 days in the presence of Pb(NO3)2. Thereafter, extracellular and intracellular Pb accumulation, photosynthetic pigments, and photosynthesis (as modulated chlorophyll fluorescence) were analyzed along with the antioxidant enzymes glutathione reductase (GR), superoxide dismutase (SOD), ascorbate peroxidase (APx), and catalase (CAT), and the stress-related protein HSP70. Pb uptake increased with the amount of supplied Pb in both algae. However, while significantly more metal was immobilized extracellularly by TR9, the amount of intracellular Pb accumulation was three times higher in TR1. In neither of the phycobionts were significant effects on photosynthetic pigments or photosynthetic electron transport observed. While under control conditions GR, SOD, and APx levels were significantly higher in TR1 than in TR9, only in the latter were these enzymes induced by Pb. This resulted in quantitatively similar antioxidant activities in the two algae when exposed to Pb. In conclusion, the phycobionts of R. farinacea make use of two different strategies against stress, in which the integration of distinct anatomical and physiological features affords similar levels of Pb tolerance.

Two Trebouxia algae with different physiological performances are ever-present in lichen thalli of Ramalina farinacea. Coexistence versus competition?

Ramalina farinacea is an epiphytic fruticose lichen that is relatively abundant in areas with Mediterranean, subtropical or temperate climates. Little is known about photobiont diversity in different lichen populations. The present study examines the phycobiont composition of several geographically distant populations of R. farinacea from the Iberian Peninsula, Canary Islands and California as well as the physiological performance of isolated phycobionts. Based on anatomical observations and molecular analyses, the coexistence of two different taxa of Trebouxia (working names, TR1 and TR9) was determined within each thallus of R. farinacea in all of the analysed populations. Examination of the effects of temperature and light on growth and photosynthesis indicated a superior performance of TR9 under relatively high temperatures and irradiances while TR1 thrived at moderate temperature and irradiance. Ramalina farinacea thalli apparently represent a specific and selective form of symbiotic association involving the same two Trebouxia phycobionts. Strict preservation of this pattern of algal coexistence is likely favoured by the different and probably complementary ecophysiological responses of each phycobiont, thus facilitating the proliferation of this lichen in a wide range of habitats and geographic areas.

Oxidative stress induces distinct physiological responses in the two Trebouxia phycobionts of the lichen Ramalina farinacea.

BACKGROUND AND AIMS: Most lichens form associations with Trebouxia phycobionts and some of them simultaneously include genetically different algal lineages. In other symbiotic systems involving algae (e.g. reef corals), the relative abundances of different endosymbiotic algal clades may change over time. This process seems to provide a mechanism allowing the organism to respond to environmental stress. A similar mechanism may operate in lichens with more than one algal lineage, likewise protecting them against environmental stresses. Here, the physiological responses to oxidative stress of two distinct Trebouxia phycobionts (provisionally named TR1 and TR9) that coexist within the lichen Ramalina farinacea were analysed. METHODS: Isolated phycobionts were exposed to oxidative stress through the reactive oxygen species propagator cumene hydroperoxide (CuHP). Photosynthetic pigments and proteins, photosynthesis (through modulated chlorophyll fluorescence), the antioxidant enzymes superoxide dismutase (SOD) and glutathione reductase (GR), and the stress-related protein HSP70 were analysed. KEY RESULTS: Photosynthetic performance was severely impaired by CuHP in phycobionts, as indicated by decreases in the maximal PSII photochemical efficiency (F(v)/F(m)), the quantum efficiency of PSII (Φ(PSII)) and the non-photochemical dissipation of energy (NPQ). However, the CuHP-dependent decay in photosynthesis was significantly more severe in TR1, which also showed a lower NPQ and a reduced ability to preserve chlorophyll a, carotenoids and D1 protein. Additionally, differences were observed in the capacities of the two phycobionts to modulate antioxidant activities and HPS70 levels when exposed to oxidative stress. In TR1, CuHP significantly diminished HSP70 and GR but did not change SOD activities. In contrast, in TR9 the levels of both antioxidant enzymes and those of HSP70 increased in response to CuHP. CONCLUSIONS: The better physiological performance of TR9 under oxidative conditions may reflect its greater capacity to undertake key metabolic adjustments, including increased non-photochemical quenching, higher antioxidant protection and the induction of repair mechanisms.