Identification of Indicator Genes for Agar Accumulation in Gracilariopsis lemaneiformis (Rhodophyta).

Agar, as a seaweed polysaccharide mainly extracted from Gracilariopsis lemaneiformis, has been commercially applied in multiple fields. To investigate factors indicating the agar accumulation in G. lemaneiformis, the agar content, soluble polysaccharides content, and expression level of 11 genes involved in the agar biosynthesis were analysed under 4 treatments, namely salinity, temperature, and nitrogen and phosphorus concentrations. The salinity exerted the greatest impact on the agar content. Both high (40‱) and low (10‱, 20‱) salinity promoted agar accumulation in G. lemaneiformis by 4.06%, 2.59%, and 3.00%, respectively. The content of agar as a colloidal polysaccharide was more stable than the soluble polysaccharide content under the treatments. No significant correlation was noted between the two polysaccharides, and between the change in the agar content and the relative growth rate of the algae. The expression of all 11 genes was affected by the 4 treatments. Furthermore, in the cultivar 981 with high agar content (21.30 ± 0.95%) compared to that (16.23 ± 1.59%) of the wild diploid, the transcriptional level of 9 genes related to agar biosynthesis was upregulated. Comprehensive analysis of the correlation between agar accumulation and transcriptional level of genes related to agar biosynthesis in different cultivation conditions and different species of G. lemaneiformis, the change in the relative expression level of glucose-6-phosphate isomerase II (gpiII), mannose-6-phosphate isomerase (mpi), mannose-1-phosphate guanylyltransferase (mpg), and galactosyltransferase II (gatII) genes was highly correlated with the relative agar accumulation. This study lays a basis for selecting high-yield agar strains, as well as for targeted breeding, by using gene editing tools in the future.

Physiological, Transcriptome, and Metabolome Analyses Reveal the Tolerance to Cu Toxicity in Red Macroalgae Gracilariopsis lemaneiformis.

Heavy metal copper (Cu) will inevitably impact the marine macroalgae Gracilariopsis lemaneiformis (G. lemaneiformis), which is a culture of economic importance along China's coastline. In this study, the detoxification mechanism of Cu stress on G. lemaneiformis was revealed by assessing physiological indicators in conjunction with transcriptome and metabolome analyses at 1 d after Cu stress. Our findings revealed that 25 µM Cu stimulated ROS synthesis and led to the enzymatic oxidation of arachidonic acid residues. This process subsequently impeded G. lemaneiformis growth by suppressing photosynthesis, nitrogen metabolism, protein synthesis, etc. The entry of Cu ions into the algae was facilitated by ZIPs and IRT transporters, presenting as Cu2+. Furthermore, there was an up-regulation of Cu efflux transporters HMA5 and ABC family transporters to achieve compartmentation to mitigate the toxicity. The results revealed that G. lemaneiformis elevated the antioxidant enzyme superoxide dismutase and ascorbate-glutathione cycle to maintain ROS homeostasis. Additionally, metabolites such as flavonoids, 3-O-methylgallic acid, 3-hydroxy-4-keto-gama-carotene, and eicosapentaenoic acid were up-regulated compared with the control, indicating that they might play roles in response to Cu stress. In summary, this study offers a comprehensive insight into the detoxification mechanisms driving the responses of G. lemaneiformis to Cu exposure.

Diverse fates of ancient horizontal gene transfers in extremophilic red algae.

Horizontal genetic transfer (HGT) is a common phenomenon in eukaryotic genomes. However, the mechanisms by which HGT-derived genes persist and integrate into other pathways remain unclear. This topic is of significant interest because, over time, the stressors that initially favoured the fixation of HGT may diminish or disappear. Despite this, the foreign genes may continue to exist if they become part of a broader stress response or other pathways. The conventional model suggests that the acquisition of HGT equates to adaptation. However, this model may evolve into more complex interactions between gene products, a concept we refer to as the 'Integrated HGT Model' (IHM). To explore this concept further, we studied specialized HGT-derived genes that encode heavy metal detoxification functions. The recruitment of these genes into other pathways could provide clear examples of IHM. In our study, we exposed two anciently diverged species of polyextremophilic red algae from the Galdieria genus to arsenic and mercury stress in laboratory cultures. We then analysed the transcriptome data using differential and coexpression analysis. Our findings revealed that mercury detoxification follows a 'one gene-one function' model, resulting in an indivisible response. In contrast, the arsH gene in the arsenite response pathway demonstrated a complex pattern of duplication, divergence and potential neofunctionalization, consistent with the IHM. Our research sheds light on the fate and integration of ancient HGTs, providing a novel perspective on the ecology of extremophiles.

In situ decrease in rhodolith growth associated with Arctic climate change.

Rhodoliths built by crustose coralline algae (CCA) are ecosystem engineers of global importance. In the Arctic photic zone, their three-dimensional growth emulates the habitat complexity of coral reefs but with a far slower growth rate, growing at micrometers per year rather than millimeters. While climate change is known to exert various impacts on the CCA's calcite skeleton, including geochemical and structural alterations, field observations of net growth over decade-long timescales are lacking. Here, we use a temporally explicit model to show that rising ocean temperatures over nearly 100 years were associated with reduced rhodolith growth at different depths in the Arctic. Over the past 90 years, the median growth rate was 85 µm year-1 but each °C increase in summer seawater temperature decreased growth by a mean of 8.9 µm (95% confidence intervals = 1.32-16.60 µm °C-1, p < .05). The decrease was expressed for rhodolith occurrences in 11 and 27 m water depth but not at 46 m, also having the shortest time series (1991-2015). Although increasing temperatures can spur plant growth, we suggest anthropogenic climate change has either exceeded the population thermal optimum for these CCA, or synergistic effects of warming, ocean acidification, and/or increasing turbidity impair rhodolith growth. Rhodoliths built by calcitic CCA are important habitat providers worldwide, so decreased growth would lead to yet another facet of anthropogenic habitat loss.

Unveiling the Role of Nano-Formulated Red Algae Extract in Cancer Management.

Cancer is one of the major causes of death, and its negative impact continues to rise globally. Chemotherapy, which is the most common therapy, has several limitations due to its tremendous side effects. Therefore, developing an alternate therapeutic agent with high biocompatibility is indeed needed. The anti-oxidative effects and bioactivities of several different crude extracts of marine algae have been evaluated both in vitro and in vivo. In the present study, we synthesized the aqueous extract (HA) from the marine algae Amphiroa anceps, and then, a liposome was formulated for that extract (NHA). The extracts were characterized using different photophysical tools like dynamic light scattering, UV-visible spectroscopy, FTIR, scanning electron microscopy, and GC-MS analysis. The SEM image revealed a size range of 112-185 nm for NHA and the GC-MS results showed the presence of octadecanoic acid and n-Hexadecanoic acid in the majority. The anticancer activity was studied using A549 cells, and the NHA inhibited the cancer cells dose-dependently, with the highest killing of 92% at 100 µg/mL. The in vivo studies in the zebrafish model showed that neither the HA nor NHA of Amphiroa anceps showed any teratogenic effect. The outcome of our study showed that NHA can be a potential drug candidate for inhibiting cancer with good biocompatibility up to a dose of 100 µg/mL.

Study of Dispersed Repeats in the Cyanidioschyzon merolae Genome.

In this study, we applied the iterative procedure (IP) method to search for families of highly diverged dispersed repeats in the genome of Cyanidioschyzon merolae, which contains over 16 million bases. The algorithm included the construction of position weight matrices (PWMs) for repeat families and the identification of more dispersed repeats based on the PWMs using dynamic programming. The results showed that the C. merolae genome contained 20 repeat families comprising a total of 33,938 dispersed repeats, which is significantly more than has been previously found using other methods. The repeats varied in length from 108 to 600 bp (522.54 bp in average) and occupied more than 72% of the C. merolae genome, whereas previously identified repeats, including tandem repeats, have been shown to constitute only about 28%. The high genomic content of dispersed repeats and their location in the coding regions suggest a significant role in the regulation of the functional activity of the genome.

A 70% Ethanol Neorhodomela munita Extract Attenuates RANKL-Induced Osteoclast Activation and H2O2-Induced Osteoblast Apoptosis In Vitro.

The rapid aging of the population worldwide presents a significant social and economic challenge, particularly due to osteoporotic fractures, primarily resulting from an imbalance between osteoclast-mediated bone resorption and osteoblast-mediated bone formation. While conventional therapies offer benefits, they also present limitations and a range of adverse effects. This study explores the protective impact of Neorhodomela munita ethanol extract (EN) on osteoporosis by modulating critical pathways in osteoclastogenesis and apoptosis. Raw264.7 cells and Saos-2 cells were used for in vitro osteoclast and osteoblast models, respectively. By utilizing various in vitro methods to detect osteoclast differentiation/activation and osteoblast death, it was demonstrated that the EN's potential to inhibit RANKL induced osteoclast formation and activation by targeting the MAPKs-NFATc1/c-Fos pathway and reducing H2O2-induced cell death through the downregulation of apoptotic signals. This study highlights the potential benefits of EN for osteoporosis and suggests that EN is a promising natural alternative to traditional treatments.

Understanding Antidiabetic Potential of Oligosaccharides from Red Alga Dulse Devaleraea inkyuleei Xylan by Investigating α-Amylase and α-Glucosidase Inhibition.

In this study, the α-glucosidase (maltase-glucoamylase: MGAM) and α-amylase inhibitory properties elicited by xylooligosaccharides (XOSs) prepared from dulse xylan were analysed as a potential mechanism to control postprandial hyperglycaemia for type-2 diabetes prevention and treatment. Xylan was purified from red alga dulse powder and used for enzymatic hydrolysis using Sucrase X to produce XOSs. Fractionation of XOSs produced xylobiose (X2), ß-(1→3)-xylosyl xylobiose (DX3), xylotriose (X3), ß-(1→3)-xylosyl-xylotriose (DX4), and a dulse XOS mixture with n ≥ 4 xylose units (DXM). The different fractions exhibited moderate MGAM (IC50 = 11.41-23.44 mg/mL) and α-amylase (IC50 = 18.07-53.04 mg/mL) inhibitory activity, which was lower than that of acarbose. Kinetics studies revealed that XOSs bound to the active site of carbohydrate digestive enzymes, limiting access to the substrate by competitive inhibition. A molecular docking analysis of XOSs with MGAM and α-amylase clearly showed moderate strength of interactions, both hydrogen bonds and non-bonded contacts, at the active site of the enzymes. Overall, XOSs from dulse could prevent postprandial hyperglycaemia as functional food by a usual and continuous consumption.

Extraction, Isolation and Biological Activity of Two Glycolipids from Bangia fusco-purpurea.

In order to explore the extraction and activity of macroalge glycolipids, six macroalgae (Bangia fusco-purpurea, Gelidium amansii, Gloiopeltis furcata, Gracilariopsis lemaneiformis, Gracilaria sp. and Pyropia yezoensis) glycolipids were extracted with five different solvents firstly. Considering the yield and glycolipids concentration of extracts, Bangia fusco-purpurea, Gracilaria sp. and Pyropia yezoensis were selected from six species of marine macroalgae as the raw materials for the extraction of glycolipids. The effects of the volume score of methanol, solid-liquid ratio, extraction temperature, extraction time and ultrasonic power on the yield and glycolipids concentration of extracts of the above three macroalgae were analyzed through a series of single-factor experiments. By analyzing the antioxidant activity in vitro, moisture absorption and moisturizing activity, the extraction process of Bangia fusco-purpurea glycolipids was further optimized by response surface method to obtain suitable conditions for glycolipid extraction (solid-liquid ratio of 1:27 g/mL, extraction temperature of 48 °C, extraction time of 98 min and ultrasonic power of 450 W). Bangia fusco-purpurea extracts exhibited a certain scavenging effect on DPPH free radicals, as well as good moisture-absorption and moisture retaining activities. Two glycolipids were isolated from Bangia fusco-purpurea by liquid-liquid extraction, silica gel column chromatography and thin-layer chromatography, and they showed good scavenging activities against DPPH free radicals and total antioxidant capacity. Their scavenging activities against DPPH free radicals were about 60% at 1600 µg/mL, and total antioxidant capacity was better than that of Trolox. Among them, the moisturizing activity of a glycolipid was close to that of sorbierite and sodium alginate. These two glycolipids exhibited big application potential as food humectants and antioxidants.

Hot springs viruses at Yellowstone National Park have ancient origins and are adapted to thermophilic hosts.

Geothermal springs house unicellular red algae in the class Cyanidiophyceae that dominate the microbial biomass at these sites. Little is known about host-virus interactions in these environments. We analyzed the virus community associated with red algal mats in three neighboring habitats (creek, endolithic, soil) at Lemonade Creek, Yellowstone National Park (YNP), USA. We find that despite proximity, each habitat houses a unique collection of viruses, with the giant viruses, Megaviricetes, dominant in all three. The early branching phylogenetic position of genes encoded on metagenome assembled virus genomes (vMAGs) suggests that the YNP lineages are of ancient origin and not due to multiple invasions from mesophilic habitats. The existence of genomic footprints of adaptation to thermophily in the vMAGs is consistent with this idea. The Cyanidiophyceae at geothermal sites originated ca. 1.5 Bya and are therefore relevant to understanding biotic interactions on the early Earth.

Effect of Post-Extraction Ultrasonication on Compositional Features and Antioxidant Activities of Enzymatic/Alkaline Extracts of Palmaria palmata.

Palmaria palmata is a viable source of nutrients with bioactive properties. The present study determined the potential role of post-extraction ultrasonication on some compositional features and antioxidant properties of enzymatic/alkaline extracts of P. palmata (EAEP). No significant difference was detected in terms of protein content and recovery, as well as the amino acid composition of the extracts. The nitrogen-to-protein conversion factor of 5 was found to be too high for the seaweed and EAEP. The extracts sonicated by bath for 10 min and not sonicated showed the highest and lowest total phenolic contents (p < 0.05), respectively. The highest radical scavenging and lowest metal-chelating activities were observed for the non-sonicated sample, as evidenced by IC50 values. The extract sonicated by bath for 10 min showed the most favorable in vitro antioxidant properties since its radical scavenging was not significantly different from that of the not-sonicated sample (p > 0.05). In contrast, its metal-chelating activity was significantly higher (p < 0.05). To conclude, post-extraction ultrasonication by an ultrasonic bath for 10 min is recommended to increase phenolic content and improve the antioxidant properties of EAEP.

Hydrothermal processing of Sarcopeltis skottsbergii and study of the potential of its carrageenan for tissue engineering.

The red macroalga Sarcopeltis skottsbergii was subjected to hydrothermal processing to maximize the solubilization and recovery of carrageenan. Once isolated by ethanol precipitation, the carrageenan was further chemically (oligosaccharides composition), and structurally (TGA/DTG, DSC, HPSEC, FTIR-ATR, 1H NMR, SEM, etc.) characterized, as well as employed as source for the synthesis of hydrogels. The rheological properties of the carrageenan showed promising results as biopolymer for food applications due to the high molecular weight (500 kDa) presenting higher cell viability than 70 %. The evaluation of immune activation using ELISA test reflected a lower inflammatory response for concentrations of 0.025 % of carrageenan. Conversely, the cell viability of the synthesized hydrogels did not surpass 50 %. This work represents a considerable step forward to obtain a biopolymer from natural sources and a thorough study of their chemical, structural and biological properties.

Halogenated Cyclic Monoterpenoids with Anti-Biofouling Activity from the Okinawan Red Marine Algae Portieria Hornemannii.

The red algal genus Portieria is a prolific producer of halogenated monoterpenoids. In this study, we isolated and characterised monoterpenoids from the Okinawan red algae Portieria hornemannii. A new polyhalogenated cyclic monoterpenoid, 2(R)-chloro-1,6(S)-dibromo-3(8)(Z)-ochtoden-4(R)-ol (1), along with three known monoterpenoids, (2R,3(8)E,4S,6R)-6-bromo-2-chloro-1,4-oxido-3(8)-ochtodene (2), 1-bromo-2-chloroochtoda-3(8),5-dien-4-one (3), and 2-chloro-1-hydroxyochtoda-3(8),5-dien-4-one (4) were isolated from the methanol extract of three populations of P. hornemannii. These compounds were characterised using a combination of spectroscopic methods and chemical synthesis, and the absolute stereochemistry of compounds 1 and 2 was determined. In addition, all isolated compounds were screened for their anti-biofouling activity against the mussel Mytilus galloprovincialis, and 1 exhibited strong activity. Therefore, halogenated monoterpenoids have the potential to be used as natural anti-biofouling drugs.

Biocatalytic Conversion of Carrageenans for the Production of 3,6-Anhydro-D-galactose.

Marine biomass stands out as a sustainable resource for generating value-added chemicals. In particular, anhydrosugars derived from carrageenans exhibit a variety of biological functions, rendering them highly promising for utilization and cascading in food, cosmetic, and biotechnological applications. However, the limitation of available sulfatases to break down the complex sulfation patterns of carrageenans poses a significant limitation for the sustainable production of valuable bioproducts from red algae. In this study, we screened several carrageenolytic polysaccharide utilization loci for novel sulfatase activities to assist the efficient conversion of a variety of sulfated galactans into the target product 3,6-anhydro-D-galactose. Inspired by the carrageenolytic pathways in marine heterotrophic bacteria, we systematically combined these novel sulfatases with other carrageenolytic enzymes, facilitating the development of the first enzymatic one-pot biotransformation of ι- and κ-carrageenan to 3,6-anhdyro-D-galactose. We further showed the applicability of this enzymatic bioconversion to a broad series of hybrid carrageenans, rendering this process a promising and sustainable approach for the production of value-added biomolecules from red-algal feedstocks.

Bioaccessibility of toxic heavy metals/metalloids in edible seaweeds: Exposure and health risk assessment.

This study assesses the health risk due to heavy metals/metalloids (HMs/Ms) in edible seaweeds (Caulerpa racemosa, Kappaphycus alvarezii, and Ulva lactuca) through an in vitro bioaccessibility study. The percentage of bioabsorbed HMs/Ms in unprocessed and processed C. racemosa, U. lactuca, and K. alvarezii ranged from 3 % to 46 %, 3 % to 42 %, and 3 % to 40 %, respectively. The levels of HMs/Ms in seawater, sediment, and seaweeds were below the levels recommended by the European Commission (EC) and World Health Organization/Food and Agriculture Organization (WHO/FAO). The maximum accumulation of HMs/Ms was found during monsoons and post-monsoon seasons, and Cd, Pb, Hg, Cr, As, and Pb were predominant in all the samples. Tukey's post hoc test and t-test confirmed that thermal processing significantly reduced HMs/Ms in seaweeds. On the basis of the bioabsorption of HMs/Ms, the TTHQ values were found to be < 1, and the LCR values were within the acceptable limit (10-06 to 10-04), indicating no carcinogenic risks through seaweeds.

Complex Endosymbioses I: From Primary to Complex Plastids, Serial Endosymbiotic Events.

A considerable part of the diversity of eukaryotic phototrophs consists of algae with plastids that evolved from endosymbioses between two eukaryotes. These complex plastids are characterized by a high number of envelope membranes (more than two) and some of them contain a residual nucleus of the endosymbiotic alga called a nucleomorph. Complex plastid-bearing algae are thus chimeric cell assemblies, eukaryotic symbionts living in a eukaryotic host. In contrast, the primary plastids of the Archaeplastida (plants, green algae, red algae, and glaucophytes) possibly evolved from a single endosymbiosis with a cyanobacterium and are surrounded by two membranes. Complex plastids have been acquired several times by unrelated groups of eukaryotic heterotrophic hosts, suggesting that complex plastids are somewhat easier to obtain than primary plastids. Evidence suggests that complex plastids arose twice independently in the green lineage (euglenophytes and chlorarachniophytes) through secondary endosymbiosis, and four times in the red lineage, first through secondary endosymbiosis in cryptophytes, then by higher-order events in stramenopiles, alveolates, and haptophytes. Engulfment of primary and complex plastid-containing algae by eukaryotic hosts (secondary, tertiary, and higher-order endosymbioses) is also responsible for numerous plastid replacements in dinoflagellates. Plastid endosymbiosis is accompanied by massive gene transfer from the endosymbiont to the host nucleus and cell adaptation of both endosymbiotic partners, which is related to the trophic switch to phototrophy and loss of autonomy of the endosymbiont. Such a process is essential for the metabolic integration and division control of the endosymbiont in the host. Although photosynthesis is the main advantage of acquiring plastids, loss of photosynthesis often occurs in algae with complex plastids. This chapter summarizes the essential knowledge of the acquisition, evolution, and function of complex plastids.

Jasmonates and Ethylene Shape Floridoside Synthesis during Carposporogenesis in the Red Seaweed Grateloupia imbricata.

Floridoside is a galactosyl-glycerol compound that acts to supply UDP-galactose and functions as an organic osmolyte in response to salinity in Rhodophyta. Significantly, the UDP-galactose pool is shared for sulfated cell wall galactan synthesis, and, in turn, affected by thallus development alongside carposporogenesis induced by volatile growth regulators, such as ethylene and methyl jasmonate, in the red seaweed Grateloupia imbricata. In this study, we monitored changes in the floridoside reservoir through gene expression controlling both the galactose pool and glyceride pool under different reproductive stages of G. imbricata and we considered changing salinity conditions. Floridoside synthesis was followed by expression analysis of galactose-1-phosphate uridyltransferase (GALT) as UDP-galactose is obtained from UDP-glucose and glucose-1P, and through α-galactosidase gene expression as degradation of floridoside occurs through the cleavage of galactosyl residues. Meanwhile, glycerol 3-phosphate is connected with the galactoglyceride biosynthetic pathway by glycerol 3-phosphate dehydrogenase (G3PD), monogalactosyl diacylglyceride synthase (MGDGS), and digalactosyl diacylglyceride synthase (DGDGS). The results of our study confirm that low GALT transcripts are correlated with thalli softness to locate reproductive structures, as well as constricting the synthesis of UDP-hexoses for galactan backbone synthesis in the presence of two volatile regulators and methionine. Meanwhile, α-galactosidase modulates expression according to cystocarp maturation, and we found high transcripts in late development stages, as occurred in the presence of methyljasmonate, compared to early stages in ethylene. Regarding the acylglyceride pool, the upregulation of G3PD, MGDGS, and DGDGS gene expression in G. imbricata treated with MEJA supports lipid remodeling, as high levels of transcripts for MGDGS and DGDGS provide membrane stability during late development stages of cystocarps. Similar behavior is assumed in three naturally collected thalli development stages-namely, fertile, fertilized, and fertile-under 65 psu salinity conditions. Low transcripts for α-galactosidase and high for G3PD are reported in infertile and fertilized thalli, which is the opposite to high transcripts for α-galactosidase and low for G3PD encountered in fertile thalli within visible cystocarps compared to each of their corresponding stages in 35 psu. No significant changes are reported for MGDGS and DGDGS. It is concluded that cystocarp and thallus development stages affect galactose and glycerides pools with interwoven effects on cell wall polysaccharides.

Mycosporine-like Amino Acids in Palmaria palmata (Rhodophyta): Specific Implication of Usujirene in Photoprotection.

The effect of UV radiation on the accumulation of mycosporine-like amino acids (MAAs) and pigments was investigated on red macroalga Palmaria palmata cultivated for 21 days. The data were combined with the effect of NaNO3 to further investigate the synthesis of these nitrogenous compounds. A progressive decrease in both total MAA and pigment contents was observed, with a positive effect of nitrate supply. Usujirene was the only MAA exhibiting a significantly increasing content when exposed to UV radiation, changing from 9% to 24% of the total MAA's contribution, with no variation observed with NaNO3. This suggests a specific induction or synthesis pathway of usujirene for photoprotection, while the synthesis of other MAAs could have been limited by an insufficient amount of UV radiation and/or irradiance. The photoprotective ability of some MAAs could have been impacted by nitrogen starvation over time, resulting in a limited synthesis and/or potential use of MAAs as a nitrogen source for red macroalgae. The data confirmed the multiple effects of environmental factors on the synthesis of MAAs while providing new insights into the specific synthesis of usujirene, which could find an application in the cosmetics sector as natural sunscreen or an anti-ageing agent.

The structure of PSI-LHCI from Cyanidium caldarium provides evolutionary insights into conservation and diversity of red-lineage LHCs.

Light-harvesting complexes (LHCs) are diversified among photosynthetic organisms, and the structure of the photosystem I-LHC (PSI-LHCI) supercomplex has been shown to be variable depending on the species of organisms. However, the structural and evolutionary correlations of red-lineage LHCs are unknown. Here, we determined a 1.92-Å resolution cryoelectron microscopic structure of a PSI-LHCI supercomplex isolated from the red alga Cyanidium caldarium RK-1 (NIES-2137), which is an important taxon in the Cyanidiophyceae. We subsequently investigated the correlations of PSI-LHCIs from different organisms through structural comparisons and phylogenetic analysis. The PSI-LHCI structure obtained shows five LHCI subunits surrounding a PSI-monomer core. The five LHCIs are composed of two Lhcr1s, two Lhcr2s, and one Lhcr3. Phylogenetic analysis of LHCs bound to PSI in the red-lineage algae showed clear orthology of LHCs between C. caldarium and Cyanidioschyzon merolae, whereas no orthologous relationships were found between C. caldarium Lhcr1-3 and LHCs in other red-lineage PSI-LHCI structures. These findings provide evolutionary insights into conservation and diversity of red-lineage LHCs associated with PSI.

Rosetta gen. nov. (Chlorophyta): Resolving the identity of red snow algal rosettes.

Thick-walled rosette-like snow algae were long thought to be a life stage of various other species of snow algae. Rosette-like cells have not been cultured, but by manually isolating cells from 38 field samples in southern British Columbia, we assigned a variety of rosette morphologies to DNA sequence. Phylogenetic analysis of Rubisco large-subunit (rbcL) gene, ribosomal internal transcribed spacer 2 (ITS2) rRNA region, and 18S rRNA gene revealed that the rosette-like cells form a new clade within the phylogroup Chloromonadinia. Based on these data, we designate a new genus, Rosetta, which comprises five novel species: R. castellata, R. floranivea, R. stellaria, R. rubriterra, and R. papavera. In a survey of 762 snow samples from British Columbia, we observed R. floranivea exclusively on snow overlying high-elevation glaciers, whereas R. castellata was observed at lower elevations, near the tree line. The other three species were rarely observed. Spherical red cells enveloped in a thin translucent sac were conspecific with Rosetta, possibly a developmental stage. These results highlight the unexplored diversity among snow algae and emphasize the utility of single-cell isolation to advance the centuries-old problem of disentangling life stages and cryptic species.