Testing for terrestrial and freshwater microalgae productivity under elevated CO2 conditions and nutrient limitation.

BACKGROUND: Microalgae CO2 fixation results in the production of biomass rich in high-valuable products, such as fatty acids and carotenoids. Enhanced productivity of valuable compounds can be achieved through the microalgae's ability to capture CO2 efficiently from sources of high CO2 contents, but it depends on the species. Culture collections of microalgae offer a wide variety of defined strains. However, an inadequate understanding of which groups of microalgae and from which habitats they originate offer high productivity under increased CO2 concentrations hampers exploiting microalgae as a sustainable source in the bioeconomy. RESULTS: A large variety of 81 defined algal strains, including new green algal isolates from various terrestrial environments, were studied for their growth under atmospheres with CO2 levels of 5-25% in air. They were from a pool of 200 strains that had been pre-selected for phylogenetic diversity and high productivity under ambient CO2. Green algae from terrestrial environments exhibited enhanced growth up to 25% CO2. In contrast, in unicellular red algae and stramenopile algae, which originated through the endosymbiotic uptake of a red algal cell, growth at CO2 concentrations above 5% was suppressed. While terrestrial stramenopile algae generally tolerated such CO2 concentrations, their counterparts from marine phytoplankton did not. The tests of four new strains in liquid culture revealed enhanced biomass and chlorophyll production under elevated CO2 levels. The 15% CO2 aeration increased their total carotenoid and fatty acid contents, which were further stimulated when combined with the starvation of macronutrients, i.e., less with phosphate and more with nitrogen-depleted culture media. CONCLUSION: Green algae originating from terrestrial environments, Chlorophyceae and Trebouxiophyceae, exhibit enhanced productivity of carotenoids and fatty acids under elevated CO2 concentrations. This ability supports the economic and sustainable production of valuable compounds from these microalgae using inexpensive sources of high CO2 concentrations, such as industrial exhaust fumes.

Global Biodiversity Patterns of the Photobionts Associated with the Genus Cladonia (Lecanorales, Ascomycota).

The diversity of lichen photobionts is not fully known. We studied here the diversity of the photobionts associated with Cladonia, a sub-cosmopolitan genus ecologically important, whose photobionts belong to the green algae genus Asterochloris. The genetic diversity of Asterochloris was screened by using the ITS rDNA and actin type I regions in 223 specimens and 135 species of Cladonia collected all over the world. These data, added to those available in GenBank, were compiled in a dataset of altogether 545 Asterochloris sequences occurring in 172 species of Cladonia. A high diversity of Asterochloris associated with Cladonia was found. The commonest photobiont lineages associated with this genus are A. glomerata, A. italiana, and A. mediterranea. Analyses of partitioned variation were carried out in order to elucidate the relative influence on the photobiont genetic variation of the following factors: mycobiont identity, geographic distribution, climate, and mycobiont phylogeny. The mycobiont identity and climate were found to be the main drivers for the genetic variation of Asterochloris. The geographical distribution of the different Asterochloris lineages was described. Some lineages showed a clear dominance in one or several climatic regions. In addition, the specificity and the selectivity were studied for 18 species of Cladonia. Potentially specialist and generalist species of Cladonia were identified. A correlation was found between the sexual reproduction frequency of the host and the frequency of certain Asterochloris OTUs. Some Asterochloris lineages co-occur with higher frequency than randomly expected in the Cladonia species.

A Molecular Analysis of Microalgae from Around the Globe to Revise Raphidonema (Trebouxiophyceae, Chlorophyta).

We isolated five microalgal strains from alpine snow near Vancouver, Canada, which display morphological features suggestive of the genera Koliella and Raphidonema. Due to variations in cell size and shape, we could not make a clear delimitation based on morphology. We proceeded to a molecular analysis and included 22 strains from the CCCryo culture collection, previously identified as members of four closely related genera: Raphidonema, Koliella, Stichococcus, and Pseudochlorella. For greater taxonomic context in our phylogenetic analysis, we also obtained authentic strains for the type species of Koliella and Pseudochlorella, but were unable to find one for Raphidonema. To examine generic boundaries, we did a phylogenetic analysis on the rbcL gene for all strains, establishing distinct lineages. Our novel isolates fell within Raphidonema, and so we analyzed the ITS2 gene of all Raphidonema strains to delimit species. To support species delimitations, we did a Compensatory Base Change analysis using the secondary structure of the ITS2 gene to assist in aligning the sequence. We also computed a maximum likelihood phylogenetic tree to examine species clades of Raphidonema. We assigned epitypes for two Raphidonema species based on the best morphological match to strains in the ITS2 clades. We then amended their diagnoses so they can be more reliably identified using DNA sequence data. We also propose two new species, R. catena and R. monicae, that formed their own species clades according to our ITS2 analysis.

Cophylogenetic patterns in algal symbionts correlate with repeated symbiont switches during diversification and geographic expansion of lichen-forming fungi in the genus Sticta (Ascomycota, Peltigeraceae).

Species in the fungal genus Sticta form symbiotic associations primarily with either green algae or cyanobacteria, but tripartite associations or photosymbiodemes involving both types of photobionts occur in some species. Sticta is known to associate with green algae in the genus Symbiochloris. However, previous studies have shown that algae from other genera, such as Heveochlorella, may also be suitable partners for Sticta. We examined the diversity of green algal partners in the genus Sticta and assessed the patterns of association between the host fungus and its algal symbiont. We used multi-locus sequence data from multiple individuals collected in Australia, Cuba, Madagascar, Mauritius, New Zealand, Reunion and South America to infer phylogenies for fungal and algal partners and performed tests of congruence to assess coevolution between the partners. In addition, event-based methods were implemented to examine which cophylogenetic processes have led to the observed association patterns in Sticta and its green algal symbionts. Our results show that in addition to Symbiochloris, Sticta associates with green algae from the genera Chloroidium, Coccomyxa, Elliptochloris and Heveochlorella, the latter being the most common algal symbiont associated with Sticta in this study. Geography plays a strong role in shaping fungal-algal association patterns in Sticta as mycobionts associate with different algal lineages in different geographic locations. While fungal and algal phylogenies were mostly congruent, event-based methods did not find any evidence for cospeciation between the partners. Instead, the association patterns observed in Sticta and associated algae, were largely explained by other cophylogenetic events such as host-switches, losses of symbiont and failure of the symbiont to diverge with its host. Our results also show that tripartite associations with green algae evolved multiple times in Sticta.

Fatty Acid Content and Profile of the Novel Strain of Coccomyxa elongata (Trebouxiophyceae, Chlorophyta) Cultivated at Reduced Nitrogen and Phosphorus Concentrations.

A novel freshwater strain of Coccomyxa elongata (MZ-Ch64) was isolated from the Zaporizhia region, Ukraine. The identification was based on the phylogenetic analysis of SSU rDNA gene and ITS1-5.8S rDNA-ITS2 region and predicted secondary structure of the ITS2. Phylogenetic analysis placed this strain in the Coccomyxa group, within the class Trebouxiophyceae. The novel strain MZ-Ch64 formed a strongly supported lineage closest with C. elongata. The MZ-Ch64 strain differed from the morphological description of the species by the size of vegetative cells and absence of small mucilaginous caps at one end of the cell. A number of experiments with different concentrations of phosphate and nitrate were conducted to evaluate changes in the resulting fatty acid profiles and biomass productivity. The fatty acid profile and total fatty acids varied significantly under different nutrient deficiencies. The dominant fatty acid during cultivation on standard BBM medium, as well as in phosphorus-depleted conditions, was oleic acid (to 48.0%-54.6% of total fatty acids). Absence of nitrogen alone, and absence of both nitrogen and phosphorus, led to an increase of palmitic acid (to 24.7%-25.6%), cis-7-hexadecenoic acid (to 14.8%) and α-linolenic acid (to 9.1%-10.1%) in comparison with the control sample. The greatest variation was found for oleic acid (31.9%-54.6%). Thus, this strain can be considered as a potential producer of oleic acid or cis-7-hexadecenoic and α-linolenic acids for biotechnological applications.

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.

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.

Molecular and morphological delimitation and generic classification of the family Oocystaceae (Trebouxiophyceae, Chlorophyta).

The family Oocystaceae (Chlorophyta) is a group of morphologically and ultrastructurally distinct green algae that constitute a well-supported clade in the class Trebouxiophyceae. Despite the family's clear delimitation, which is based on specific cell wall features, only a few members of the Oocystaceae have been examined using data other than morphological. In previous studies of Trebouxiophyceae, after the establishment of molecular phylogeny, the taxonomic status of the family was called into question. The genus Oocystis proved to be paraphyletic and some species were excluded from Oocystaceae, while a few other species were newly redefined as members of this family. We investigated 54 strains assigned to the Oocystaceae using morphological, ultrastructural and molecular data (SSU rRNA and rbcL genes) to clarify the monophyly of and diversity within Oocystaceae. Oonephris obesa and Nephrocytium agardhianum clustered within the Chlorophyceae and thus are no longer members of the Oocystaceae. On the other hand, we transferred the coenobial Willea vilhelmii to the Oocystaceae. Our findings combined with those of previous studies resulted in the most robust definition of the family to date. The division of the family into three subfamilies and five morphological clades was suggested. Taxonomical adjustments in the genera Neglectella, Oocystidium, Oocystis, and Ooplanctella were established based on congruent molecular and morphological data. We expect further taxonomical changes in the genera Crucigeniella, Eremosphaera, Franceia, Lagerheimia, Oocystis, and Willea in the future.

New features of desiccation tolerance in the lichen photobiont Trebouxia gelatinosa are revealed by a transcriptomic approach.

Trebouxia is the most common lichen-forming genus of aero-terrestrial green algae and all its species are desiccation tolerant (DT). The molecular bases of this remarkable adaptation are, however, still largely unknown. We applied a transcriptomic approach to a common member of the genus, T. gelatinosa, to investigate the alteration of gene expression occurring after dehydration and subsequent rehydration in comparison to cells kept constantly hydrated. We sequenced, de novo assembled and annotated the transcriptome of axenically cultured T. gelatinosa by using Illumina sequencing technology. We tracked the expression profiles of over 13,000 protein-coding transcripts. During the dehydration/rehydration cycle c. 92 % of the total protein-coding transcripts displayed a stable expression, suggesting that the desiccation tolerance of T. gelatinosa mostly relies on constitutive mechanisms. Dehydration and rehydration affected mainly the gene expression for components of the photosynthetic apparatus, the ROS-scavenging system, Heat Shock Proteins, aquaporins, expansins, and desiccation related proteins (DRPs), which are highly diversified in T. gelatinosa, whereas Late Embryogenesis Abundant Proteins were not affected. Only some of these phenomena were previously observed in other DT green algae, bryophytes and resurrection plants, other traits being distinctive of T. gelatinosa, and perhaps related to its symbiotic lifestyle. Finally, the phylogenetic inference extended to DRPs of other chlorophytes, embryophytes and bacteria clearly pointed out that DRPs of chlorophytes are not orthologous to those of embryophytes: some of them were likely acquired through horizontal gene transfer from extremophile bacteria which live in symbiosis within the lichen thallus.

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 of green algae covering artificial hard substrate surfaces in a suburban environment: a case study using molecular approaches.

In Middle European suburban environments green algae often cover open surfaces of artificial hard substrates. Microscopy reveals the Apatococcus/Desmococcus morphotype predominant over smaller coccoid forms. Adverse conditions such as limited water availability connected with high PAR and UV irradiance may narrow the algal diversity to a few specialists in these subaerial habitats. We used rRNA gene cloning/sequencing from both DNA extracts of the biofilms without culturing as well as cultures, for the unambiguous determination of the algal composition and to assess the algal diversity more comprehensively. The culture independent approach revealed mainly just two genera (Apatococcus, Trebouxia) for all study sites and five molecular operational taxonomic units (OTUs) for a particular study site, which based on microscopic observation was the one with the highest morphological diversity. The culture approach, however, revealed seven additional OTUs from five genera (Chloroidium, Coccomyxa, Coenochloris, Pabia, Klebsormidium) and an unidentified trebouxiophyte lineage for that same site; only two OTUs were shared by both approaches. Two OTUs or species were recovered for which references have been isolated only from Antarctica so far. However, the internal transcribed spacer (ITS) sequence differences among them supported they are representing distinct populations of the same species. Within Apatococcus five clearly distinct groups of ITS sequences, each putatively representing a distinct species, were recovered with three or four such ITS types co-occurring at the same study site. Except for the streptophyte Klebsormidium only members of Trebouxiophyceae were detected suggesting these algae may be particularly well-adapted to subaerial habitats.

Meiotic genes and sexual reproduction in the green algal class Trebouxiophyceae (Chlorophyta).

Sexual reproduction is widespread in eukaryotes and is well documented in chlorophytan green algae. In this lineage, however, the Trebouxiophyceae represent a striking exception: in contrast to its relatives Chlorophyceae and Ulvophyceae this group appears to be mostly asexual, as fertilization has been rarely observed. Assessments of sexual reproduction in the Trebouxiophyceae have been based on microscopic observation of gametes fusing. New genomic data offer now the opportunity to check for the presence of meiotic genes, which represent an indirect evidence of a sexual life cycle. Using genomic and transcriptomic data for 12 taxa spanning the phylogenetic breadth of the class, we tried to clarify whether genuine asexuality or cryptic sexuality is the most likely case for the numerous putatively asexual trebouxiophytes. On the basis of these data and a bibliographic review, we conclude that the view of trebouxiophytes as primarily asexual is incorrect. In contrast to the limited number of reports of fertilization, meiotic genes were found in all genomes and transcriptomes examined, even in species presumed asexual. In the taxa examined the totality or majority of the genes were present, Helicosporidium and Auxenochlorella being the only partial exceptions (only four genes present). The evidence of sex provided by the meiotic genes is phylogenetically widespread in the class and indicates that sexual reproduction is not associated with any particular morphological or ecological trait. On the basis of the results, we expect that the existence of the meiotic genes will be documented in all trebouxiophycean genomes that will become available in the future.

Ecological specialization in Trebouxia (Trebouxiophyceae) photobionts of Ramalina menziesii (Ramalinaceae) across six range-covering ecoregions of western North America.

• Premise of the study: Many lichens exhibit extensive ranges spanning several ecoregions. It has been hypothesized that this wide ecological amplitude is facilitated by fungal association with locally adapted photobiont strains.• Methods: We studied the identity and geographic distribution of photobionts of the widely distributed North American lichen Ramalina menziesii based on rbcL (chloroplast DNA) and nuclear ribosomal ITS DNA sequences. To test for ecological specialization, we associate photobiont genotypes with local climate and phorophyte.• Key results: Of the photobiont lineages of R. menziesii, 94% belong to a clade including Trebouxia decolorans. The remaining are related to T. jamesii. The photobionts showed (1) significant structure according to ecoregion and phorophyte species and (2) genetic associations with phorophyte species and climate.• Conclusions: Geography, climate, and ecological specialization shape genetic differentiation of lichen photobionts. One great advantage of independent dispersal of the fungus is symbiotic association with locally adapted photobiont strains.

Complete mitochondrial and chloroplast DNA sequences of the freshwater green microalga Medakamo hakoo.

We report the complete organellar genome sequences of an ultrasmall green alga, Medakamo hakoo strain M-hakoo 311, which has the smallest known nuclear genome in freshwater green algae. Medakamo hakoo has 90.8-kb chloroplast and 36.5-kb mitochondrial genomes containing 80 and 33 putative protein-coding genes, respectively. The mitochondrial genome is the smallest in the Trebouxiophyceae algae studied so far. The GC content of the nuclear genome is 73%, but those of chloroplast and mitochondrial genomes are 41% and 35%, respectively. Codon usages in the organellar genomes have a different tendency from that in the nuclear genome. The organellar genomes have unique characteristics, such as the biased encoding of mitochondrial genes on a single strand and the absence of operon structures in chloroplast ribosomal genes. Medakamo hakoo will be helpful for understanding the evolution of the organellar genome and the regulation of gene expression in chloroplasts and mitochondria.

Myrmecia, Not Asterochloris, Is the Main Photobiont of Cladonia subturgida (Cladoniaceae, Lecanoromycetes).

This study explores the diversity of photobionts associated with the Mediterranean lichen-forming fungus Cladonia subturgida. For this purpose, we sequenced the whole ITS rDNA region by Sanger using a metabarcoding method for ITS2. A total of 41 specimens from Greece, Italy, France, Portugal, and Spain were studied. Additionally, two specimens from Spain were used to generate four cultures. Our molecular studies showed that the genus Myrmecia is the main photobiont of C. subturgida throughout its geographic distribution. This result contrasts with previous studies, which indicated that the main photobiont for most Cladonia species is Asterochloris. The identity of Myrmecia was also confirmed by ultrastructural studies of photobionts within the lichen thalli and cultures. Photobiont cells showed a parietal chloroplast lacking a pyrenoid, which characterizes the species in this genus. Phylogenetic analyses indicate hidden diversity within this genus. The results of amplicon sequencing showed the presence of multiple ASVs in 58.3% of the specimens studied.

The Safety and Efficacy of 1-Monoeicosapentaenoin Isolated from the Trebouxiophyceae Micractinium on Anti-Wrinkle: A Split-Face Randomized, Double-Blind Placebo-Controlled Clinical Study.

The skin aging process is governed by intrinsic and extrinsic factors causing skin wrinkles, sagging, and loosening. The 1-monoeicosapentaenoin (1-MEST) is a component isolated from Micractinium, a genus of microalgae (Chlorophyta, Trebouxiophyceae). However, the anti-wrinkle effects of 1-MEST are not yet known. This study aimed to evaluate the anti-wrinkle effects of 1-MEST in vitro and in clinical trials. The cytotoxicity of 1-MEST was investigated in vitro using the MTS assay in human epidermal keratinocytes (HEKs). Expression of matrix metalloproteinase (MMP)-1 and MMP-9 was determined by ELISA in HEKs irradiated with UVB after treatment with 1-MEST. A split-face randomized, double-blind, placebo-controlled study was conducted to evaluate the safety and efficacy of 1-MEST. The study evaluated wrinkle parameters and visual assessment, self-efficacy and usability questionnaires, and adverse events. The study showed that the 1-MEST was not cytotoxic in HEKs, suppressed MMP-1 secretion and MMP-9 protein expression in HEKs irradiated with UVB. The wrinkle parameters and mean visual assessment score were significantly decreased in the test group after 12 weeks and differed from the control group. There were no significant differences in efficacy and usability. Adverse effects were also not observed. The 1-MEST showed anti-wrinkle properties to slow down or prevent skin aging.

Evaluation of the effects of input variables on the growth of two microalgae classes during wastewater treatment.

Wastewater treatment carried out by microalgae is usually affected by the type of algal strain and the combination of cultivation parameters provided during the process. Every microalga strain has a different tolerance level towards cultivation parameters, including temperature, pH, light intensity, CO2 content, initial inoculum level, pretreatment method, reactor type and nutrient concentration in wastewater. Therefore, it is vital to supply the right combination of cultivation parameters to increase the wastewater treatment efficiency and biomass productivity of different microalgae classes. In the current investigation, the decision tree was used to analyse the dataset of class Trebouxiophyceae and Chlorophyceae. Various combinations of cultivation parameters were determined to enhance their performance in wastewater treatment. Nine combinations of cultivation parameters leading to high biomass production and eleven combinations each for high nitrogen removal efficiency and high phosphorus removal efficiency for class Trebouxiophyceae were detected by decision tree models. Similarly, eleven combinations for high biomass production, nine for high nitrogen removal efficiency, and eight for high phosphorus removal efficiency were detected for class Chlorophyceae. The results obtained through decision tree analysis can provide the optimum conditions of cultivation parameters, saving time in designing new experiments for treating wastewater at a large scale.

Mitochondrial genome of the Antarctic microalga Micractinium simplicissimum KSF0127 (Chlorellaceae, Trebouxiophyceae).

We report the first mitochondrial genome of the Antarctic microalga Micractinium simplicissimum KSF0127. The circular mitochondrial genome was 67,923 bp in length and contained 45 protein-coding genes, one ribosomal RNA gene, and 60 transfer RNA genes. The phylogenetic tree was constructed with eight previously reported mitogenome sequences and showed the phylogenetic position of M. simplicissimum KSF0127 within the Chlorellaceae family.

Thorsmoerkia curvula gen. et spec. nov. (Trebouxiophyceae, Chlorophyta), a semi-terrestrial microalga from Iceland exhibits high levels of unsaturated fatty acids.

A terrestrial green alga was isolated at Iceland, and the strain (SAG 2627) was described for its morphology and phylogenetic position and tested for biotechnological capabilities. Cells had a distinctly curved, crescent shape with conical poles and a single parietal chloroplast. Phylogenetic analyses of 18S rDNA and rbcL markers placed the strain into the Trebouxiophyceae (Chlorophyta). The alga turned out to belong to an independent lineage without an obvious sister group within the Trebouxiophyceae. Based on morphological and phylogenetic data, the strain was described as a new genus and species, Thorsmoerkia curvula gen. et sp. nov. Biomass was generated in column reactors and subsequently screened for promising metabolites. Growth was optimized by pH-regulated, episodic CO2 supplement during the logarithmic growth-phase, and half of the biomass was thereafter exposed to nitrogen and phosphate depletion. The biomass yield reached up to 53.5 mg L-1 day-1. Fatty acid (FA) production peaked at 24 mg L-1 day-1 and up to 83% of all FAs were unsaturated. At the end of the log phase, approximately 45% of dry mass were lipids, including eicosapentaenoic acid. Carotenoid production reached up to 2.94 mg L-1 day-1 but it was halted during the stress phase. The N-linked glycans of glycoproteins were assessed to reveal chemotaxonomic patterns. The study demonstrated that new microalgae can be found at Iceland, potentially suitable for applied purposes. The advantage of T. curvula is its robustness and that significant amounts of lipids are already accumulated during log phase, making a subsequent stress exposure dispensable.

Turnover of Lecanoroid Mycobionts and Their Trebouxia Photobionts Along an Elevation Gradient in Bolivia Highlights the Role of Environment in Structuring the Lichen Symbiosis.

Shifts in climate along elevation gradients structure mycobiont-photobiont associations in lichens. We obtained mycobiont (lecanoroid Lecanoraceae) and photobiont (Trebouxia alga) DNA sequences from 89 lichen thalli collected in Bolivia from a ca. 4,700 m elevation gradient encompassing diverse natural communities and environmental conditions. The molecular dataset included six mycobiont loci (ITS, nrLSU, mtSSU, RPB1, RPB2, and MCM7) and two photobiont loci (ITS, rbcL); we designed new primers to amplify Lecanoraceae RPB1 and RPB2 with a nested PCR approach. Mycobionts belonged to Lecanora s.lat., Bryonora, Myriolecis, Protoparmeliopsis, the "Lecanora" polytropa group, and the "L." saligna group. All of these clades except for Lecanora s.lat. occurred only at high elevation. No single species of Lecanoraceae was present along the entire elevation gradient, and individual clades were restricted to a subset of the gradient. Most Lecanoraceae samples represent species which have not previously been sequenced. Trebouxia clade C, which has not previously been recorded in association with species of Lecanoraceae, predominates at low- to mid-elevation sites. Photobionts from Trebouxia clade I occur at the upper extent of mid-elevation forest and at some open, high-elevation sites, while Trebouxia clades A and S dominate open habitats at high elevation. We did not find Trebouxia clade D. Several putative new species were found in Trebouxia clades A, C, and I. These included one putative species in clade A associated with Myriolecis species growing on limestone at high elevation and a novel lineage sister to the rest of clade C associated with Lecanora on bark in low-elevation grassland. Three different kinds of photobiont switching were observed, with certain mycobiont species associating with Trebouxia from different major clades, species within a major clade, or haplotypes within a species. Lecanoraceae mycobionts and Trebouxia photobionts exhibit species turnover along the elevation gradient, but with each partner having a different elevation threshold at which the community shifts completely. A phylogenetically defined sampling of a single diverse family of lichen-forming fungi may be sufficient to document regional patterns of Trebouxia diversity and distribution.