The chloroplast genome of the lichen-symbiont microalga Trebouxia sp. Tr9 (Trebouxiophyceae, Chlorophyta) shows short inverted repeats with a single gene and loss of the rps4 gene, which is encoded by the nucleus.

The Trebouxiophyceae is the class of Chlorophyta algae from which the highest number of chloroplast genome (cpDNA) sequences has been obtained. Several species in this class participate in symbioses with fungi to form lichens. However, no cpDNA has been obtained from any Trebouxia lichen-symbiont microalgae, which are present in approximately half of all lichens. Here, we report the sequence of the completely assembled cpDNA from Trebouxia sp. TR9 and a comparative study with other Trebouxio-phyceae. The organization of the chloroplast genome of Trebouxia sp. TR9 has certain features that are unusual in the Trebouxiophyceae and other green algae. The most remarkable characteristics are the presence of long intergenic spacers, a quadripartite structure with short inverted repeated sequences (IRs), and the loss of the rps4 gene. The presence of long intergenic spacers accounts for a larger cpDNA size in comparison to other closely related Trebouxiophyceae. The IRs, which were thought to be lost in the Trebouxiales, are distinct from most of cpDNAs since they lack the rRNA operon and uniquely includes the rbcL gene. The functional transfer of the rps4 gene to the nuclear genome has been confirmed by sequencing and examination of the gene architecture, which includes three spliceosomal introns as well as the verification of the presence of the corresponding transcript. This is the first documented transfer of the rps4 gene from the chloroplast to the nucleus among Viridiplantae. Additionally, a fairly well-resolved phylogenetic reconstruction, including Trebouxia sp. TR9 along with other Trebouxiophyceae, was obtained based on a set of conserved chloroplast genes.

Dynamic evolution of mitochondrial genomes in Trebouxiophyceae, including the first completely assembled mtDNA from a lichen-symbiont microalga (Trebouxia sp. TR9).

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.

Characterization of the responses to saline stress in the symbiotic green microalga Trebouxia sp. TR9.

MAIN CONCLUSION: For the first time we provide a study on the physiological, ultrastructural and molecular effects of salt stress on a terrestrial symbiotic green microalga, Trebouxia sp. TR9. Although tolerance to saline conditions has been thoroughly studied in plants and, to an extent, free-living microalgae, scientific data regarding salt stress on symbiotic lichen microalgae is scarce to non-existent. Since lichen phycobionts are capable of enduring harsh, restrictive and rapidly changing environments, it is interesting to study the metabolic machinery operating under these extreme conditions. We aim to determine the effects of prolonged exposure to high salt concentrations on the symbiotic phycobiont Trebouxia sp. TR9, isolated from the lichen Ramalina farinacea. Our results suggest that, when this alga is confronted with extreme saline conditions, the cellular structures are affected to an extent, with limited chlorophyll content loss and photosynthetic activity remaining after 72 h of exposure to 5 M NaCl. Furthermore, this organism displays a rather different molecular response compared to land plants and free-living halophile microalgae, with no noticeable increase in ABA levels and ABA-related gene expression until the external NaCl concentration is raised to 3 M NaCl. Despite this, the ABA transduction pathway seems functional, since the ABA-related genes tested are responsive to exogenous ABA. These observations could suggest that this symbiotic green alga may have developed alternative molecular pathways to cope with highly saline environments.

Unexpected associated microalgal diversity in the lichen Ramalina farinacea is uncovered by pyrosequencing analyses.

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.

Balanced gene losses, duplications and intensive rearrangements led to an unusual regularly sized genome in Arbutus unedo chloroplasts.

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.

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.