Intracellular bound chlorophyll residues identify 1 Gyr-old fossils as eukaryotic algae.

The acquisition of photosynthesis is a fundamental step in the evolution of eukaryotes. However, few phototrophic organisms are unambiguously recognized in the Precambrian record. The in situ detection of metabolic byproducts in individual microfossils is the key for the direct identification of their metabolisms. Here, we report a new integrative methodology using synchrotron-based X-ray fluorescence and absorption. We evidence bound nickel-geoporphyrins moieties in low-grade metamorphic rocks, preserved in situ within cells of a ~1 Gyr-old multicellular eukaryote, Arctacellularia tetragonala. We identify these moieties as chlorophyll derivatives, indicating that A. tetragonala was a phototrophic eukaryote, one of the first unambiguous algae. This new approach, applicable to overmature rocks, creates a strong new proxy to understand the evolution of phototrophy and diversification of early ecosystems.

Gaia Pigino: Inside the cell.

Gaia Pigino studies the molecular mechanisms and principles of self-organization in cilia using 3D cryo-EM.

Centrosome structure and biogenesis: Variations on a theme?

Centrosomes are composed of two orthogonally arranged centrioles surrounded by an electron-dense matrix called the pericentriolar material (PCM). Centrioles are cylinders with diameters of ~250 nm, are several hundred nanometres in length and consist of 9-fold symmetrically arranged microtubules (MT). In dividing animal cells, centrosomes act as the principal MT-organising centres and they also organise actin, which tunes cytoplasmic MT nucleation. In some specialised cells, the centrosome acquires additional critical structures and converts into the base of a cilium with diverse functions including signalling and motility. These structures are found in most eukaryotes and are essential for development and homoeostasis at both cellular and organism levels. The ultrastructure of centrosomes and their derived organelles have been known for more than half a century. However, recent advances in a number of techniques have revealed the high-resolution structures (at Å-to-nm scale resolution) of centrioles and have begun to uncover the molecular principles underlying their properties, including: protein components; structural elements; and biogenesis in various model organisms. This review covers advances in our understanding of the features and processes that are critical for the biogenesis of the evolutionarily conserved structures of the centrosomes. Furthermore, it discusses how variations of these aspects can generate diversity in centrosome structure and function among different species and even between cell types within a multicellular organism.

Detrimental effect of UV-B radiation on growth, photosynthetic pigments, metabolites and ultrastructure of some cyanobacteria and freshwater chlorophyta.

BACKGROUND: Global warming directly influencing ozone layer depletion, which eventually is increasing ultraviolet radiation penetration having far-reaching impacts on living biota. This particularly influences the primary producer microalgae which are the basic unit of food webs in the aquatic habitats. Therefore, it is necessary to concentrate the research at this micro-level to understand the harmful impact of increased UV-B radiation ever before. Consequently, the present attempt aimed to focus on the influence of UV-B on growth criteria, photosynthetic pigments, some metabolites, and ultrastructure of the freshwater cyanobacteria, Planktothrix cryptovaginata (Microcoleaceae), Nostoc carneum (Nostocaceae), Microcystis aeruginosa (Microcystaceae), the Chlorophyte Scenedesmus acutus (Scenedesmaceae), and the marine Cyanobacterium Microcystis (Microcystaceae). METHODS: The cultures of investigated algae were subjected directly to different duration periods (1, 3, 5, and 7 h) of artificial UV-B in addition to unirradiated control culture and allowed to grow for 10 days, after which the algal samples were analyzed for growth, photosynthetic activities, primary metabolities and cellular ultrastructure. RESULTS: A remarkable inhibitory influence of UV-B was observed on growth criteria (measured as optical density and dry weight) and photosynthetic pigments of P. cryptovaginata, N. carneum, M. aeruginosa, S. acutus, and marine Microcystis. Where increasing the exposure time of UV-B was accompanied by increased inhibition. The variation in carbohydrate and protein contents under UV stress was based on the exposure periods and the algal species. The variation in algal ultrastructure by UV-B stress was noticed by an Electron Microscope. Cells damage and lysis, cell wall and cell membrane ruptured and release of intracellular substances, loss of cell inclusion, plasmolysis and necrosis, or apoptosis of the algal cells were observed by exposure to 7 h of UV-B. CONCLUSION: Exposure to UV-B has a marked harmful impact on the growth, pigments, and metabolic activity, as well as the cellular ultrastructure of some cyanobacteria and chlorophytes.

Biomineralization Capacities of Chlorodendrophyceae: Correlation Between Chloroplast Morphology and the Distribution of Micropearls in the Cell.

Several species of the genus Tetraselmis (Chlorodendrophyceae, Chlorophyta) were recently discovered to possess unsuspected biomineralization capacities: they produce multiple intracellular inclusions of amorphous calcium carbonate (ACC), called micropearls. Early light-microscopists had spotted rows of refractive granules in some species, although without identifying their mineral nature. Scanning electron microscope (SEM) observations showed that the distribution of the micropearls in the cell forms a pattern, which appears to be characteristic for a given species. The present study shows that this pattern correlates with the shape of the chloroplast, which differs between Tetraselmis species, because micropearls align themselves along the incisions between chloroplast lobes. This was observed both by SEM and in live cells by light microscopy (LM) using Nomarski differential interference contrast. Additionally, molecular phylogenetic analyses, of rbcL and ITS2 gene sequences from diverse strains of Chlorodendrophyceae, corroborated the morphological observations by identifying two groups among nominal Tetraselmis spp. that differ in chloroplast morphology, micropearl arrangement, and ITS2 RNA secondary structure.

Formally described species woefully underrepresent phylogenetic diversity in the common lichen photobiont genus Trebouxia (Trebouxiophyceae, Chlorophyta): An impetus for developing an integrated taxonomy.

Lichens provide valuable systems for studying symbiotic interactions. In lichens, these interactions are frequently described in terms of availability, selectivity and specificity of the mycobionts and photobionts towards one another. The lichen-forming, green algal genus Trebouxia Puymaly is among the most widespread photobiont, associating with a broad range of lichen-forming fungi. To date, 29 species have been described, but studies consistently indicate that the vast majority of species-level lineages still lack formal description, and new, previously unrecognized lineages are frequently reported. To reappraise the diversity and the evolutionary relationships of species-level lineages in Trebouxia, we assembled DNA sequence data from over 1600 specimens, compiled from a range of sequences from previously published studies, axenic algal cultures, and lichens collected from poorly sampled regions. From these samples, we selected representatives of the currently known genetic diversity in the lichenized Trebouxia and inferred a phylogeny from multi-locus sequence data (ITS, rbcL, cox2). We demonstrate that the current formally described species woefully underrepresent overall species-level diversity in this important lichen-forming algal genus. We anticipate that an integrative taxonomic approach, incorporating morphological and physiological data from axenic cultures with genetic data, will be required to establish a robust, comprehensive taxonomy for Trebouxia. The data presented here provide an important impetus and reference dataset for more reliably characterizing diversity in lichenized algae and in using lichens to investigate the evolution of symbioses and holobionts.

Integrated ultrasound-assisted liquid biphasic flotation for efficient extraction of astaxanthin from Haematococcus pluvialis.

The purpose of this investigation is to evaluate the implementation of ultrasound-assisted liquid biphasic flotation (LBF) system for the recovery of natural astaxanthin from Haematococcus pluvialis microalgae. Various operating conditions of ultrasound-assisted LBF systems such as the position of ultrasound horn, mode of ultrasonication (pulse and continuous), amplitude of ultrasonication, air flowrate, duration of air flotation, and mass of H. pluvialis microalgae were evaluated. The effect of ultrasonication on the cellular morphology of microalgae was also assessed using microscopic analysis. Under the optimized operating conditions of UALBF, the maximum recovery yield, extraction efficiency, and partition coefficient of astaxanthin were 95.08 ± 3.02%, 99.74 ± 0.05%, and 185.09 ± 4.78, respectively. In addition, the successful scale-up operation of ultrasound-assisted LBF system verified the practicability of this integrated approach for an effective extraction of natural astaxanthin.

Dinoflagellates with relic endosymbiont nuclei as models for elucidating organellogenesis.

Nucleomorphs are relic endosymbiont nuclei so far found only in two algal groups, cryptophytes and chlorarachniophytes, which have been studied to model the evolutionary process of integrating an endosymbiont alga into a host-governed plastid (organellogenesis). However, past studies suggest that DNA transfer from the endosymbiont to host nuclei had already ceased in both cryptophytes and chlorarachniophytes, implying that the organellogenesis at the genetic level has been completed in the two systems. Moreover, we have yet to pinpoint the closest free-living relative of the endosymbiotic alga engulfed by the ancestral chlorarachniophyte or cryptophyte, making it difficult to infer how organellogenesis altered the endosymbiont genome. To counter the above issues, we need novel nucleomorph-bearing algae, in which endosymbiont-to-host DNA transfer is on-going and for which endosymbiont/plastid origins can be inferred at a fine taxonomic scale. Here, we report two previously undescribed dinoflagellates, strains MGD and TGD, with green algal endosymbionts enclosing plastids as well as relic nuclei (nucleomorphs). We provide evidence for the presence of DNA in the two nucleomorphs and the transfer of endosymbiont genes to the host (dinoflagellate) genomes. Furthermore, DNA transfer between the host and endosymbiont nuclei was found to be in progress in both the MGD and TGD systems. Phylogenetic analyses successfully resolved the origins of the endosymbionts at the genus level. With the combined evidence, we conclude that the host-endosymbiont integration in MGD/TGD is less advanced than that in cryptophytes/chrorarachniophytes, and propose the two dinoflagellates as models for elucidating organellogenesis.

Four New Members of Foliicolous Green Algae Within the Watanabea Clade (Trebouxiophyceae, Chlorophyta) from China.

Members of the Watanabea clade of Trebouxiophyceae are genetically diverse and widely distributed in all kinds of habitats, especially in most terrestrial habitats. Ten new strains of terrestrial algae isolated from the tropical rainforest in China, and four published strains were investigated in this study. Morphological observation and molecular phylogenetic analyses based on the 18S, ITS, rbcL, and tufA genes were used to identify the new strains. Four previously described species were reinvestigated to supplement molecular data and autospores' morphological photographs. The phylogenetic analyses based on 18S only, the concatenated dataset of 18S and ITS, as well as the concatenated dataset of rbcL and tufA, showed the same phylogenetic positions and relationships of these new strains. According to the phylogenetic analysis and morphological comparisons results, we described these 10 strains as four new members within the Watanabea clade, Polulichloris yunnanensis sp. nov., Polulichloris ovale sp. nov., Massjukichlorella orientale sp. nov., and Massjukichlorella minus sp. nov., and two known species, Massjukichlorella epiphytica, and Mysteriochloris nanningensis. Additionally, we provide strong evidence proving that Phyllosiphon, Mysteriochloris, Polulichloris, and Desertella all reproduce through unequal sized autospores.

Lipid Droplets Mediate Salt Stress Tolerance in Parachlorella kessleri.

Microalgae are known to respond to salinity stress via mechanisms that include accumulation of compatible solutes and synthesis of antioxidants. Here, we describe a salinity-tolerance mechanism mediated by lipid droplets (LDs). In the alga Parachlorella kessleri grown under salt-stress conditions, we observed significant increases in cell size and LD content. LDs that were closely grouped along the plasma membrane shrank as the plasma membrane expanded, and some LDs were engulfed by vacuoles. Transcriptome analysis showed that genes encoding lysophospholipid acyltransferases (LPLATs) and phospholipase A2 were significantly up-regulated following salt stress. Diacylglycerol kinase and LPLAT were identified in the proteome of salt-induced LDs, alongside vesicle trafficking and plastidial proteins and histone H2B. Analysis of fatty acid composition revealed an enrichment of C18:1 and C18:2 at the expense of C18:3 in response to salt stress. Pulse-chase experiments further suggested that variations of fatty acid composition were associated with LDs. Acetate stimulation research further confirmed a positive role of LDs in cell growth under salt stress. These results suggest that LDs play important roles in salt-stress tolerance, through harboring proteins, participating in cytoplasmic component recycling, and providing materials and enzymes for membrane modification and expansion.

Metabolomic foundation for differential responses of lipid metabolism to nitrogen and phosphorus deprivation in an arachidonic acid-producing green microalga.

The green oleaginous microalga Lobosphaera incisa accumulates storage lipids triacylglycerols (TAG) enriched in the long-chain polyunsaturated fatty acid arachidonic acid under nitrogen (N) deprivation. In contrast, under phosphorous (P) deprivation, the production of the monounsaturated oleic acid prevails. We compared physiological responses, ultrastructural, and metabolic consequences of L. incisa acclimation to N and P deficiency to provide novel insights into the key determinants of ARA accumulation. Differential responses to nutrient deprivation on growth performance, carbon-to-nitrogen stoichiometry, membrane lipid composition and TAG accumulation were demonstrated. Ultrastructural analyses suggested a dynamic role for vacuoles in sustaining cell homeostasis under conditions of different nutrient availability and their involvement in autophagy in L. incisa. Paralleling ARA-rich TAG accumulation in lipid droplets, N deprivation triggered intensive chloroplast dismantling and promoted catabolic processes. Metabolome analysis revealed depletion of amino acids and pyrimidines, and repression of numerous biosynthetic hubs to favour TAG biosynthesis under N deprivation. Under P deprivation, despite the relatively low growth penalties, the presence of the endogenous P reserves and the characteristic lipid remodelling, metabolic signatures of energy deficiency were revealed. Metabolome adjustments to P deprivation included depletion in ATP and phosphorylated nucleotides, increased levels of TCA-cycle intermediates and osmoprotectants. We conclude that characteristic cellular and metabolome adjustments tailor the adaptive responses of L. incisa to N and P deprivation modulating its LC-PUFA production.

Long-Chain Polyunsaturated Fatty Acids in the Green Microalga Lobosphaera incisa Contribute to Tolerance to Abiotic Stresses.

Lobosphaera incisa is a green microalga that accumulates high levels of the valuable omega-6 long-chain polyunsaturated fatty acids (LC-PUFA) arachidonic acid (ARA, 20:4n-6) in triacylglycerols (TAG) under nitrogen (N) starvation. LC-PUFA accumulation is a rare trait in photosynthetic microalgae with insufficiently understood physiological significance. In this study, RNAi was attempted, for the first time in L. incisa, to produce knockdown lines for the Δ5 desaturase gene. Two lines, termed modified lines, which were isolated during screening for transgenic events, demonstrated alterations in their LC-PUFA profile, ARA-biosynthesis gene expression and lipid class distribution. In line M5-78, which appeared to carry a mutation in the Δ6 elongase gene, LC-PUFA were substituted by 18:3n-6 in all glycerolipids. Line M2-35, for which the exact genetic background has not been established, displayed a dramatic reduction in 20:4n-6, concomitant with an augmented proportion of 18:1n-9, in particular in the extraplastidial membrane lipids and TAG. The physiological responses of the modified lines to stressful conditions were compared with the wild type and the Δ5 desaturase mutant. In the N-replete cells of modified lines, the frequency of lipid droplets was reduced, while a number of starch grains increased, suggesting altered partitioning of assimilated carbon into reserve products. Furthermore, both lines exhibited reduced ability to accumulate TAG under N deprivation and recover from N starvation. Both lines demonstrated lower photosynthetic pigment contents, impairments in photosynthesis under a range of stressful conditions, and less efficient functioning of photoprotection under optimal conditions. Possible implications of fatty acids modifications in the stress response of L. incisa are addressed.

Transcriptional and physiological responses of Dunaliella salina to cadmium reveals time-dependent turnover of ribosome, photosystem, and ROS-scavenging pathways.

Effects on short-term (6 h) and long-term (96 h) exposure to cadmium (Cd) at 0.1, 0.5 and 2.5 mg/L in microalga Dunaliella salina were assessed using both physiological end points and gene expression analysis. Different physiological responses between the short-term and long-term exposures were observed. Upon 6 h after Cd exposure, lipid peroxidation and cell ultrastructure remained unchanged, while contents of chlorophyll a, chlorophyll b, carotenoids were increased at 0.5 and 2.5 mg/L Cd. Contrarily, 96 h after Cd exposure, lipid peroxidation levels were increased, while pigments content was decreased, and damaged cell ultrastructure was apparent at 2.5 mg/L Cd. Activities of antioxidant enzymes (APX, SOD, GST, GPX, and GR) changed differently both at 6 h and 96 h after Cd exposure. Upon 6 h after Cd exposure, SOD and GST activity increased at all three doses, GR and GPX activity increased at 0.5 mg/L Cd while APX activity increased at 0.1 mg/L Cd. Contrarily, 96 h after Cd exposure, activities of all the antioxidant enzymes increased both at 0.1 and 0.5 mg/L Cd; but there was a decrease in SOD and GR activity in D. salina exposed to 2.5 mg/L Cd. RNA-seq and qRT-PCR analyses indicated that genes involved in ROS-scavenge, photosystem, and ribosome functions were differentially expressed. The most significantly enriched function was the ribosome, in which more than 30 ribosome genes were up-regulated at 6 h but down-regulated at 96 h after Cd exposure at 2.5 mg/L. Our study indicated for the first time that genes encoding ribosomal proteins are the primary target for Cd in microalgae, which allowed gaining new insights into temporal dynamics of toxicity and adaptive response pathways in microalgae exposed to metals.

Correlative 3D x-ray fluorescence and ptychographic tomography of frozen-hydrated green algae.

Accurate knowledge of elemental distributions within biological organisms is critical for understanding their cellular roles. The ability to couple this knowledge with overall cellular architecture in three dimensions (3D) deepens our understanding of cellular chemistry. Using a whole, frozen-hydrated Chlamydomonas reinhardtii cell as an example, we report the development of 3D correlative microscopy through a combination of simultaneous cryogenic x-ray ptychography and x-ray fluorescence microscopy. By taking advantage of a recently developed tomographic reconstruction algorithm, termed GENeralized Fourier Iterative REconstruction (GENFIRE), we produce high-quality 3D maps of the unlabeled alga's cellular ultrastructure and elemental distributions within the cell. We demonstrate GENFIRE's ability to outperform conventional tomography algorithms and to further improve the reconstruction quality by refining the experimentally intended tomographic angles. As this method continues to advance with brighter coherent light sources and more efficient data handling, we expect correlative 3D x-ray fluorescence and ptychographic tomography to be a powerful tool for probing a wide range of frozen-hydrated biological specimens, ranging from small prokaryotes such as bacteria, algae, and parasites to large eukaryotes such as mammalian cells, with applications that include understanding cellular responses to environmental stimuli and cell-to-cell interactions.

Non-invasive magnetic resonance imaging of oils in Botryococcus braunii green algae: Chemical shift selective and diffusion-weighted imaging.

Botryococcus braunii is an oleaginous green algae with the distinctive property of accumulating high quantities of hydrocarbons per dry weight in its colonies. Large variation in colony structure exists, yet its implications and influence of oil distribution and diffusion dynamics are not known and could not be answered due to lack of suitable in vivo methods. This publication seeks to further the understanding on oil dynamics, by investigating naturally relevant large (700-1500µm) and extra-large (1500-2500µm) sized colonies of Botryococcus braunii (race B, strain Showa) in vivo, using a comprehensive approach of chemical shift selective imaging, chemical shift imaging and spin echo diffusion measurements at high magnetic field (17.6T). Hydrocarbon distribution in large colonies was found to be localised in two concentric oil layers with different thickness and concentration. Extra-large colonies were highly unstructured and oil was spread throughout colonies, but with large local variations. Interestingly, fluid channels were observed in extra-large colonies. Diffusion-weighted MRI revealed a strong correlation between colony heterogeneity, oil distribution, and diffusion dynamics in different parts of Botryococcus colonies. Differences between large and extra-large colonies were characterised by using T2 weighted MRI along with relaxation measurements. Our result, therefore, provides first non-invasive MRI means to obtain spatial information on oil distribution and diffusion dynamics in Botryococcus braunii colonies.

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.

Subdiffraction-resolution live-cell imaging for visualizing thylakoid membranes.

The chloroplast is the chlorophyll-containing organelle that produces energy through photosynthesis. Within the chloroplast is an intricate network of thylakoid membranes containing photosynthetic membrane proteins that mediate electron transport and generate chemical energy. Historically, electron microscopy (EM) has been a powerful tool for visualizing the macromolecular structure and organization of thylakoid membranes. However, an understanding of thylakoid membrane dynamics remains elusive because EM requires fixation and sectioning. To improve our knowledge of thylakoid membrane dynamics we need to consider at least two issues: (i) the live-cell imaging conditions needed to visualize active processes in vivo; and (ii) the spatial resolution required to differentiate the characteristics of thylakoid membranes. Here, we utilize three-dimensional structured illumination microscopy (3D-SIM) to explore the optimal imaging conditions for investigating the dynamics of thylakoid membranes in living plant and algal cells. We show that 3D-SIM is capable of examining broad characteristics of thylakoid structures in chloroplasts of the vascular plant Arabidopsis thaliana and distinguishing the structural differences between wild-type and mutant strains. Using 3D-SIM, we also visualize thylakoid organization in whole cells of the green alga Chlamydomonas reinhardtii. These data reveal that high light intensity changes thylakoid membrane structure in C. reinhardtii. Moreover, we observed the green alga Chromochloris zofingiensis and the moss Physcomitrella patens to show the applicability of 3D-SIM. This study demonstrates that 3D-SIM is a promising approach for studying the dynamics of thylakoid membranes in photoautotrophic organisms during photoacclimation processes.

Relation between water status and desiccation-affected genes in the lichen photobiont Trebouxia gelatinosa.

The relation between water status and expression profiles of desiccation -related genes has been studied in the desiccation tolerant (DT) aeroterrestrial green microalga Trebouxia gelatinosa, a common lichen photobiont. Algal colonies were desiccated in controlled conditions and during desiccation water content (WC) and water potential (Ψ) were measured to find the turgor loss point (Ψtlp). Quantitative real-time PCR was performed to measure the expression of ten genes related to photosynthesis, antioxidant defense, expansins, heat shock proteins (HSPs), and desiccation related proteins in algal colonies collected during desiccation when still at full turgor (WC > 6 g H2O g-1 dry weight), immediately before and after Ψtlp (-4 MPa; WC ∼ 1 g H2O g-1 dry weight) and before and after complete desiccation (WC < 0.01 g H2O g-1 dry weight), quantifying the HSP70 protein levels by immunodetection. Our analysis showed that the expression of eight out of ten genes changed immediately before and after Ψtlp. Interestingly, the expression of five out of ten genes changed also before complete desiccation, i.e. between 0.2 and 0.01 g H2O g-1 dry weight. However, the HSP70 protein levels were not affected by changes in water status. The study provides new evidences of the link between the loss of turgor and the expression of genes related to the desiccation tolerance of T. gelatinosa, suggesting the former as a signal triggering inducible mechanisms.

Molecular and morphological diversity of Trebouxia microalgae in sphaerothallioid Circinaria spp. lichens1.

Three vagrant (Circinaria hispida, Circinaria gyrosa, and Circinaria sp. 'paramerae') and one crustose (semi-vagrant, Circinaria sp. 'oromediterranea') lichens growing in very continental areas in the Iberian Peninsula were selected to study the phycobiont diversity. Mycobiont identification was checked using nrITS DNA barcoding: Circinaria sp. 'oromediterranea' and Circinaria sp. 'paramerae' formed a new clade. Phycobiont diversity was analyzed in 50 thalli of Circinaria spp. using nrITS DNA and LSU rDNA, with microalgae coexistence being found in all the species analyzed by Sanger sequencing. The survey of phycobiont diversity showed up to four different Trebouxia spp. as the primary phycobiont in 20 thalli of C. hispida, in comparison with the remaining Circinaria spp., where only one Trebouxia was the primary microalga. In lichen species showing coexistence, some complementary approaches are needed (454 pyrosequencing and/or ultrastructural analyses). Five specimens were selected for high-throughput screening (HTS) analyses: 22 Trebouxia OTUs were detected, 10 of them not previously known. TEM analyses showed three different cell morphotypes (Trebouxia sp. OTU A12, OTU S51, and T. cretacea) whose ultrastructure is described here in detail for the first time. HTS revealed a different microalgae pool in each species studied, and we cannot assume a specific pattern between these pools and the ecological and/or morphological characteristics. The mechanisms involved in the selection of the primary phycobiont and the other microalgae by the mycobiont are unknown, and require complex experimental designs. The systematics of the genus Circinaria is not yet well resolved, and more analyses are needed to establish a precise delimitation of the species.

A model suite of green algae within the Scenedesmaceae for investigating contrasting desiccation tolerance and morphology.

Microscopic green algae inhabiting desert microbiotic crusts are remarkably diverse phylogenetically, and many desert lineages have independently evolved from aquatic ancestors. Here we worked with five desert and aquatic species within the family Scenedesmaceae to examine mechanisms that underlie desiccation tolerance and release of unicellular versus multicellular progeny. Live cell staining and time-lapse confocal imaging coupled with transmission electron microscopy established that the desert and aquatic species all divide by multiple (rather than binary) fission, although progeny were unicellular in three species and multicellular (joined in a sheet-like coenobium) in two. During division, Golgi complexes were localized near nuclei, and all species exhibited dynamic rotation of the daughter cell mass within the mother cell wall at cytokinesis. Differential desiccation tolerance across the five species, assessed from photosynthetic efficiency during desiccation/rehydration cycles, was accompanied by differential accumulation of intracellular reactive oxygen species (ROS) detected using a dye sensitive to intracellular ROS. Further comparative investigation will aim to understand the genetic, ultrastructural and physiological characteristics supporting unicellular versus multicellular coenobial morphology, and the ability of representatives in the Scenedesmaceae to colonize ecologically diverse, even extreme, habitats.