The underestimated fraction: diversity, challenges and novel insights into unicellular cyanobionts of lichens.

Lichens are remarkable and classic examples of symbiotic organisms that have fascinated scientists for centuries. Yet, it has only been for a couple of decades that significant advances have focused on the diversity of their green algal and/or cyanobacterial photobionts. Cyanolichens, which contain cyanobacteria as their photosynthetic partner, include up to 10% of all known lichens and, as such, studies on their cyanobionts are much rarer compared to their green algal counterparts. For the unicellular cyanobionts, i.e. cyanobacteria that do not form filaments, these studies are even scarcer. Nonetheless, these currently include at least 10 different genera in the cosmopolitan lichen order Lichinales. An international consortium (International Network of CyanoBionts; INCb) will tackle this lack of knowledge. In this article, we discuss the status of current unicellular cyanobiont research, compare the taxonomic resolution of photobionts from cyanolichens with those of green algal lichens (chlorolichens), and give a roadmap of research on how to recondition the underestimated fraction of symbiotic unicellular cyanobacteria in lichens.

Quantifying gliding forces of filamentous cyanobacteria by self-buckling.

Filamentous cyanobacteria are one of the oldest and today still most abundant lifeforms on earth, with manifold implications in ecology and economics. Their flexible filaments, often several hundred cells long, exhibit gliding motility in contact with solid surfaces. The underlying force generating mechanism is not yet understood. Here, we demonstrate that propulsion forces and friction coefficients are strongly coupled in the gliding motility of filamentous cyanobacteria. We directly measure their bending moduli using micropipette force sensors, and quantify propulsion and friction forces by analyzing their self-buckling behavior, complemented with analytical theory and simulations. The results indicate that slime extrusion unlikely generates the gliding forces, but support adhesion-based hypotheses, similar to the better-studied single-celled myxobacteria. The critical self-buckling lengths align well with the peaks of natural length distributions, indicating the importance of self-buckling for the organization of their collective in natural and artificial settings.

Genomes of multicellular algal sisters to land plants illuminate signaling network evolution.

Zygnematophyceae are the algal sisters of land plants. Here we sequenced four genomes of filamentous Zygnematophyceae, including chromosome-scale assemblies for three strains of Zygnema circumcarinatum. We inferred traits in the ancestor of Zygnematophyceae and land plants that might have ushered in the conquest of land by plants: expanded genes for signaling cascades, environmental response, and multicellular growth. Zygnematophyceae and land plants share all the major enzymes for cell wall synthesis and remodifications, and gene gains shaped this toolkit. Co-expression network analyses uncover gene cohorts that unite environmental signaling with multicellular developmental programs. Our data shed light on a molecular chassis that balances environmental response and growth modulation across more than 600 million years of streptophyte evolution.

Environmental gradients reveal stress hubs pre-dating plant terrestrialization.

Plant terrestrialization brought forth the land plants (embryophytes). Embryophytes account for most of the biomass on land and evolved from streptophyte algae in a singular event. Recent advances have unravelled the first full genomes of the closest algal relatives of land plants; among the first such species was Mesotaenium endlicherianum. Here we used fine-combed RNA sequencing in tandem with a photophysiological assessment on Mesotaenium exposed to a continuous range of temperature and light cues. Our data establish a grid of 42 different conditions, resulting in 128 transcriptomes and ~1.5 Tbp (~9.9 billion reads) of data to study the combinatory effects of stress response using clustering along gradients. Mesotaenium shares with land plants major hubs in genetic networks underpinning stress response and acclimation. Our data suggest that lipid droplet formation and plastid and cell wall-derived signals have denominated molecular programmes since more than 600 million years of streptophyte evolution-before plants made their first steps on land.

A microplate-based bioassay for toxicity testing using the large benthic algal species Closterium ehrenbergii.

Pollution of water bodies by metals has long been studied but still remains a threat to healthy ecosystems. While most ecotoxicological studies on algae are performed with planktonic standard species such as Raphidocelis subcapitata, benthic algae may depict the majority of the algal flora in rivers and streams. These species encounter different exposure scenarios to pollutants as they are sedentary and not carried away by the current. This particular way of life leads to an integration of toxic effects over time. Therefore, in this study, the effects of six metals on the large unicellular benthic species Closterium ehrenbergii were examined. A miniaturized bioassay with low cell densities of 10-15 cells/mL using microplates was developed. Through chemical analysis, metal complexing properties in the culture medium were demonstrated, that could lead to an underestimation of metal toxicity. Thus, the medium was modified by excluding EDTA and TRIS. The toxicity of the six metals ranked by EC50 values in descending order, was as follows: Cu (5.5 µg/L) > Ag (9.2 µg/L) > Cd (18 µg/L) > Ni (260 µg/L) > Cr (990 µg/L) > Zn (1200 µg/L). In addition, toxic effects on the cell morphology were visualized. Based on a literature review, C. ehrenbergii was shown to be partly more sensitive than R. subcapitata which suggests that it can be a useful addition to ecotoxicological risk assessment.

Chromosome-level genomes of multicellular algal sisters to land plants illuminate signaling network evolution.

The filamentous and unicellular algae of the class Zygnematophyceae are the closest algal relatives of land plants. Inferring the properties of the last common ancestor shared by these algae and land plants allows us to identify decisive traits that enabled the conquest of land by plants. We sequenced four genomes of filamentous Zygnematophyceae (three strains of Zygnema circumcarinatum and one strain of Z. cylindricum) and generated chromosome-scale assemblies for all strains of the emerging model system Z. circumcarinatum. Comparative genomic analyses reveal expanded genes for signaling cascades, environmental response, and intracellular trafficking that we associate with multicellularity. Gene family analyses suggest that Zygnematophyceae share all the major enzymes with land plants for cell wall polysaccharide synthesis, degradation, and modifications; most of the enzymes for cell wall innovations, especially for polysaccharide backbone synthesis, were gained more than 700 million years ago. In Zygnematophyceae, these enzyme families expanded, forming co-expressed modules. Transcriptomic profiling of over 19 growth conditions combined with co-expression network analyses uncover cohorts of genes that unite environmental signaling with multicellular developmental programs. Our data shed light on a molecular chassis that balances environmental response and growth modulation across more than 600 million years of streptophyte evolution.

The plastomes of Hyalomonas oviformis and Hyalogonium fusiforme evolved dissimilar architectures after the loss of photosynthesis.

The loss of photosynthesis in land plants and algae is typically associated with parasitism but can also occur in free-living species, including chlamydomonadalean green algae. The plastid genomes (ptDNAs) of colorless chlamydomonadaleans are surprisingly diverse in architecture, including highly expanded forms (Polytoma uvella and Leontynka pallida) as well as outright genome loss (Polytomella species). Here, we explore the ptDNAs of Hyalomonas (Hm.) oviformis (SAG 62-27; formerly known as Polytoma oviforme) and Hyalogonium (Hg.) fusiforme (SAG 62-1c), each representing independent losses of photosynthesis within the Chlamydophyceae. The Hm. oviformis ptDNA is moderately sized (132 kb) with a reduced gene complement (but still encoding the ATPase subunits) and is in fact smaller than that of its photosynthetic relative Hyalomonas chlamydogama SAG 11-48b (198.3 kb). The Hg. fusiforme plastome, however, is the largest yet observed in nonphotosynthetic plants or algae (~463 kb) and has a coding repertoire that is almost identical to that of its photosynthetic relatives in the genus Chlorogonium. Furthermore, the ptDNA of Hg. fusiforme shows no clear evidence of pseudogenization, which is consistent with our analyses showing that Hg. fusiforme is the nonphotosynthetic lineage of most recent origin among known colorless Chlamydophyceae. Together, these new ptDNAs clearly show that, in contrast to parasitic algae, plastid genome compaction is not an obligatory route following the loss of photosynthesis in free-living algae, and that certain chlamydomonadalean algae have a remarkable propensity for genomic expansion, which can persist regardless of the trophic strategy.

Submergence of the filamentous Zygnematophyceae Mougeotia induces differential gene expression patterns associated with core metabolism and photosynthesis.

The streptophyte algal class Zygnematophyceae is the closest algal sister lineage to land plants. In nature, Zygnematophyceae can grow in both terrestrial and freshwater habitats and how they do this is an important unanswered question. Here, we studied what happens to the zygnematophyceaen alga Mougeotia sp., which usually occurs in permanent and temporary freshwater bodies, when it is shifted to liquid growth conditions after growth on a solid substrate. Using global differential gene expression profiling, we identified changes in the core metabolism of the organism interlinked with photosynthesis; the latter went hand in hand with measurable impact on the photophysiology as assessed via pulse amplitude modulation (PAM) fluorometry. Our data reveal a pronounced change in the overall physiology of the alga after submergence and pinpoint candidate genes that play a role. These results provide insight into the importance of photophysiological readjustment when filamentous Zygnematophyceae transition between terrestrial and aquatic habitats.

Comparative genome analysis of test algal strain NIVA-CHL1 (Raphidocelis subcapitata) maintained in microalgal culture collections worldwide.

Raphidocelis subcapitata is one of the most frequently used species for algal growth inhibition tests. Accordingly, many microalgal culture collections worldwide maintain R. subcapitata for distribution to users. All R. subcapitata strains maintained in these collections are derived from the same cultured strain, NIVA-CHL1. However, considering that 61 years have passed since this strain was isolated, we suspected that NIVA-CHL1 in culture collections might have acquired various mutations. In this study, we compared the genome sequences among NIVA-CHL1 from 8 microalgal culture collections and one laboratory in Japan to evaluate the presence of mutations. We found single-nucleotide polymorphisms or indels at 19,576 to 28,212 sites per strain in comparison with the genome sequence of R. subcapitata NIES-35, maintained at the National Institute for Environmental Studies, Tsukuba, Japan. These mutations were detected not only in non-coding but also in coding regions; some of the latter mutations may affect protein function. In growth inhibition test with 3,5-dichlorophenol, EC50 values varied 2.6-fold among the 9 strains. In the ATCC 22662-2 and CCAP 278/4 strains, we also detected a mutation in the gene encoding small-conductance mechanosensitive ion channel, which may lead to protein truncation and loss of function. Growth inhibition test with sodium chloride suggested that osmotic regulation has changed in ATCC 22662-2 and CCAP 278/4 in comparison with NIES-35.

The Draft Genome of Coelastrum proboscideum (Sphaeropleales, Chlorophyta).

Coelastrum proboscideum Bohlin, 1896 (Sphaeropleales, Scenedesmaceae, Chlorophyta) is a coenobial species with cosmopolitan distribution in diverse freshwater habitats. Coelastrum spp. are widely tested for biotechnological applications such as carotenoid and lipid production, and in bioremediation of wastewater. Here, we report the draft genome of C. proboscideum var. dilatatum strain SAG 217-2. The final assembly comprised 125,935,854 bp with over 8357 scaffolds. The whole-genome data is publicly available in the Nucleotide Sequence Archive (CNSA) of China National GeneBank (CNGB) (https://db.cngb.org/cnsa/) under the accession number CNA0014153.

The Draft Genome of Hariotina reticulata (Sphaeropleales, Chlorophyta) Provides Insight into the Evolution of Scenedesmaceae.

Hariotina reticulata P. A. Dangeard 1889 (Sphaeropleales, Chlorophyta) is a common member of the summer phytoplankton of meso- to highly eutrophic water bodies with a worldwide distribution. Here, we report the draft whole-genome shotgun sequencing of H. reticulata strain SAG 8.81. The final assembly comprises 107,596,510bp with over 15,219 scaffolds (>100bp). This whole-genome project is publicly available in the CNSA (https://db.cngb.org/cnsa/) of CNGBdb under the accession number CNP0000705.

The Draft Genome of the Small, Spineless Green Alga Desmodesmus costato-granulatus (Sphaeropleales, Chlorophyta).

Desmodesmus costato-granulatus (Skuja) Hegewald 2000 (Sphaeropleales, Chlorophyta) is a small, spineless green alga that is abundant in the freshwater phytoplankton of oligo- to eutrophic waters worldwide. It has a high lipid content and is considered for sustainable production of diverse compounds, including biofuels. Here, we report the draft whole-genome shotgun sequencing of D. costato-granulatus strain SAG 18.81. The final assembly comprises 48,879,637bp with over 4,141 scaffolds. This whole-genome project is publicly available in the CNSA (https://db.cngb.org/cnsa/) of CNGBdb under the accession number CNP0000701.

ATP Content and Cell Viability as Indicators for Cryostress Across the Diversity of Life.

In many natural environments, organisms get exposed to low temperature and/or to strong temperature shifts. Also, standard preservation protocols for live cells or tissues involve ultradeep freezing in or above liquid nitrogen (-196°C or -150°C, respectively). To which extent these conditions cause cold- or cryostress has rarely been investigated systematically. Using ATP content as an indicator of the physiological state of cells, we found that representatives of bacteria, fungi, algae, plant tissue, as well as plant and human cell lines exhibited similar responses during freezing and thawing. Compared to optimum growth conditions, the cellular ATP content of most model organisms decreased significantly upon treatment with cryoprotectant and cooling to up to -196°C. After thawing and a longer period of regeneration, the initial ATP content was restored or even exceeded the initial ATP levels. To assess the implications of cellular ATP concentration for the physiology of cryostress, cell viability was determined in parallel using independent approaches. A significantly positive correlation of ATP content and viability was detected only in the cryosensitive algae Chlamydomonas reinhardtii SAG 11-32b and Chlorella variabilis NC64A, and in plant cell lines of Solanum tuberosum. When comparing mesophilic with psychrophilic bacteria of the same genera, and cryosensitive with cryotolerant algae, ATP levels of actively growing cells were generally higher in the psychrophilic and cryotolerant representatives. During exposure to ultralow temperatures, however, psychrophilic and cryotolerant species showed a decline in ATP content similar to their mesophilic or cryosensitive counterparts. Nevertheless, psychrophilic and cryotolerant species attained better culturability after freezing. Cellular ATP concentrations and viability measurements thus monitor different features of live cells during their exposure to ultralow temperatures and cryostress.

Contrasting biodiversity-ecosystem functioning relationships in phylogenetic and functional diversity.

It is well known that ecosystem functioning is positively influenced by biodiversity. Most biodiversity-ecosystem functioning experiments have measured biodiversity based on species richness or phylogenetic relationships. However, theoretical and empirical evidence suggests that ecosystem functioning should be more closely related to functional diversity than to species richness. We applied different metrics of biodiversity in an artificial biodiversity-ecosystem functioning experiment using 64 species of green microalgae in combinations of two to 16 species. We found that phylogenetic and functional diversity were positively correlated with biomass overyield, driven by their strong correlation with species richness. At low species richness, no significant correlation between overyield and functional and phylogenetic diversity was found. However, at high species richness (16 species), we found a positive relationship of overyield with functional diversity and a negative relationship with phylogenetic diversity. We show that negative phylogenetic diversity-ecosystem functioning relationships can result from interspecific growth inhibition. The opposing performances of facilitation (functional diversity) and inhibition (phylogenetic diversity) we observed at the 16 species level suggest that phylogenetic diversity is not always a good proxy for functional diversity and that results from experiments with low species numbers may underestimate negative species interactions.

Mixotrophy in the terrestrial green alga Apatococcus lobatus (Trebouxiophyceae, Chlorophyta).

The green microalga Apatococcus lobatus is widely distributed in terrestrial habitats throughout many climatic zones. It dominates green biofilms on natural and artificial substrata in temperate latitudes and is regarded as a key genus of obligate terrestrial consortia. Until now, its isolation, cultivation and application as a terrestrial model organism has been hampered by slow growth rates and low growth capacities. A mixotrophic culturing approach clearly enhanced the accumulation of biomass, thereby permitting the future application of A. lobatus in different types of bio-assays necessary for material and biofilm research. The ability of A. lobatus to grow mixotrophically is assumed as a competitive advantage in terrestrial habitats.

Biodiversity effects on ecosystem functioning change along environmental stress gradients.

Positive relationship between biodiversity and ecosystem functioning has been observed in many studies, but how this relationship is affected by environmental stress is largely unknown. To explore this influence, we measured the biomass of microalgae grown in microcosms along two stress gradients, heat and salinity, and compared our results with 13 published case studies that measured biodiversity-ecosystem functioning relationships under varying environmental conditions. We found that positive effects of biodiversity on ecosystem functioning decreased with increasing stress intensity in absolute terms. However, in relative terms, increasing stress had a stronger negative effect on low-diversity communities. This shows that more diverse biotic communities are functionally less susceptible to environmental stress, emphasises the need to maintain high levels of biodiversity as an insurance against impacts of changing environmental conditions and sets the stage for exploring the mechanisms underlying biodiversity effects in stressed ecosystems.

Encapsulation dehydration colligative cryoprotective strategies and amplified fragment length polymorphism markers to verify the identity and genetic stability of euglenoids following cryopreservation.

An encapsulation/dehydration procedure was developed for Euglena gracilis Klebs as a 'model alga' to examine various cryoprotective regimes combined with controlled rate cooling to cryopreserve other Euglenoid taxa. Cryoprotective variables were optimised to enable reproducible growth following a combination of alginate encapsulation, sucrose osmotic dehydration, air desiccation, methanol treatment, cooling to -40 degrees C and plunging into liquid nitrogen (LN). Amplified Fragment Length Polymorphism (AFLP) analysis was adapted to: (i) verify algal identity by discriminating between different Euglenoids and (ii) examine the genetic stability of algal cultures prior to various stages of cryoprotective treatments and following exposure to LN. AFLPs were highly reproducible (> 99%) as reliable diagnostic markers, where a single DNA fragment change accounted for -0.4% of the detectable variation in an AFLP pattern. AFLP changes were detected in cryoprotective treatments following LN exposure. Successive stages of the dehydration and desiccation treatments did not accumulate AFLP changes indicating these are random events.

Assessing genetic stability of a range of terrestrial microalgae after cryopreservation using amplified fragment length polymorphism (AFLP).

Cryopreservation is the long-term, indefinite storage of living biological resources at ultralow temperatures. It is almost universally assumed that cryogenic storage supports genetic and phenotypic stability of organisms. However, certain components of the cryopreservation process, particularly some cryoprotective additives (CPAs) and free radical mediated cryoinjury, may potentially cause genetic alterations. Genetic integrity in cryopreserved microalgae was assessed using a very sensitive molecular fingerprinting technique, AFLP, on 28 terrestrial microalgal strains. In about half of all investigated strains the AFLP fingerprints revealed, with high levels of reproducibility, clearly detectable genomic differences after cryopreservation employing a widely used standard two-step cooling protocol. Differences ranged from a single fragment position to multiple fragment changes and were compared to differences found between wild-type and UV-light- or radioisotope-induced mutants of Parachlorella kessleri. The basis of the changes are discussed in terms of their reversibility, as may be the case if they are attributed to DNA methylation and/or whether they are true mutations that may potentially manifest in the phenotype. The possibility that cryopreservation selects for genotypically different subpopulations of microalgae is also considered.