Gene expression response under thermal stress in two Hawaiian corals is dominated by ploidy and genotype.

Transcriptome data are frequently used to investigate coral bleaching; however, the factors controlling gene expression in natural populations of these species are poorly understood. We studied two corals, Montipora capitata and Pocillopora acuta, that inhabit the sheltered Kane'ohe Bay, Hawai'i. M. capitata colonies in the bay are outbreeding diploids, whereas P. acuta is a mixture of clonal diploids and triploids. Populations were sampled from six reefs and subjected to either control (no stress), thermal stress, pH stress, or combined pH and thermal stress treatments. RNA-seq data were generated to test two competing hypotheses: (1) gene expression is largely independent of genotype, reflecting a shared treatment-driven response (TDE) or, (2) genotype dominates gene expression, regardless of treatment (GDE). Our results strongly support the GDE model, even under severe stress. We suggest that post-transcriptional processes (e.g., control of translation, protein turnover) modify the signal from the transcriptome, and may underlie the observed differences in coral bleaching sensitivity via the downstream proteome and metabolome.

Whole-genome duplication in an algal symbiont bolsters coral heat tolerance.

The algal endosymbiont Durusdinium trenchii enhances the resilience of coral reefs under thermal stress. D. trenchii can live freely or in endosymbiosis, and the analysis of genetic markers suggests that this species has undergone whole-genome duplication (WGD). However, the evolutionary mechanisms that underpin the thermotolerance of this species are largely unknown. Here, we present genome assemblies for two D. trenchii isolates, confirm WGD in these taxa, and examine how selection has shaped the duplicated genome regions using gene expression data. We assess how the free-living versus endosymbiotic lifestyles have contributed to the retention and divergence of duplicated genes, and how these processes have enhanced the thermotolerance of D. trenchii. Our combined results suggest that lifestyle is the driver of post-WGD evolution in D. trenchii, with the free-living phase being the most important, followed by endosymbiosis. Adaptations to both lifestyles likely enabled D. trenchii to provide enhanced thermal stress protection to the host coral.

From dusk till dawn: cell cycle progression in the red seaweed Gracilariopsis chorda (Rhodophyta).

The conserved eukaryotic functions of cell cycle genes have primarily been studied using animal/plant models and unicellular algae. Cell cycle progression and its regulatory components in red (Rhodophyta) seaweeds are poorly understood. We analyzed diurnal gene expression data to investigate the cell cycle in the red seaweed Gracilariopsis chorda. We identified cell cycle progression and transitions in G. chorda which are induced by interactions of key regulators such as E2F/DP, RBR, cyclin-dependent kinases, and cyclins from dusk to dawn. However, several typical CDK inhibitor proteins are absent in red seaweeds. Interestingly, the G1-S transition in G. chorda is controlled by delayed transcription of GINS subunit 3. We propose that the delayed S phase entry in this seaweed may have evolved to minimize DNA damage (e.g., due to UV radiation) during replication. Our results provide important insights into cell cycle-associated physiology and its molecular mechanisms in red seaweeds.

Evidence of a putative CO2 delivery system to the chromatophore in the photosynthetic amoeba Paulinella.

The photosynthetic amoeba, Paulinella provides a recent (ca. 120 Mya) example of primary plastid endosymbiosis. Given the extensive data demonstrating host lineage-driven endosymbiont integration, we analysed nuclear genome and transcriptome data to investigate mechanisms that may have evolved in Paulinella micropora KR01 (hereinafter, KR01) to maintain photosynthetic function in the novel organelle, the chromatophore. The chromatophore is of α-cyanobacterial provenance and has undergone massive gene loss due to Muller's ratchet, but still retains genes that encode the ancestral α-carboxysome and the shell carbonic anhydrase, two critical components of the biophysical CO2 concentrating mechanism (CCM) in cyanobacteria. We identified KR01 nuclear genes potentially involved in the CCM that arose via duplication and divergence and are upregulated in response to high light and downregulated under elevated CO2. We speculate that these genes may comprise a novel CO2 delivery system (i.e., a biochemical CCM) to promote the turnover of the RuBisCO carboxylation reaction and counteract photorespiration. We posit that KR01 has an inefficient photorespiratory system that cannot fully recycle the C2 product of RuBisCO oxygenation back to the Calvin-Benson cycle. Nonetheless, both these systems appear to be sufficient to allow Paulinella to persist in environments dominated by faster-growing phototrophs.

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.

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.

Massive genome reduction predates the divergence of Symbiodiniaceae dinoflagellates.

Dinoflagellates in the family Symbiodiniaceae are taxonomically diverse, predominantly symbiotic lineages that are well-known for their association with corals. The ancestor of these taxa is believed to have been free-living. The establishment of symbiosis (i.e. symbiogenesis) is hypothesized to have occurred multiple times during Symbiodiniaceae evolution, but its impact on genome evolution of these taxa is largely unknown. Among Symbiodiniaceae, the genus Effrenium is a free-living lineage that is phylogenetically positioned between two robustly supported groups of genera within which symbiotic taxa have emerged. The apparent lack of symbiogenesis in Effrenium suggests that the ancestral features of Symbiodiniaceae may have been retained in this lineage. Here, we present de novo assembled genomes (1.2-1.9 Gbp in size) and transcriptome data from three isolates of Effrenium voratum and conduct a comparative analysis that includes 16 Symbiodiniaceae taxa and the other dinoflagellates. Surprisingly, we find that genome reduction, which is often associated with a symbiotic lifestyle, predates the origin of Symbiodiniaceae. The free-living lifestyle distinguishes Effrenium from symbiotic Symbiodiniaceae vis-à-vis their longer introns, more-extensive mRNA editing, fewer (~30%) lineage-specific gene sets, and lower (~10%) level of pseudogenization. These results demonstrate how genome reduction and the adaptation to distinct lifestyles intersect to drive diversification and genome evolution of Symbiodiniaceae.

Genome-wide transcriptome analysis reveals the diversity and function of long non-coding RNAs in dinoflagellates.

Dinoflagellates are a diverse group of phytoplankton, ranging from harmful bloom-forming microalgae to photosymbionts of coral reefs. Genome-scale data from dinoflagellates reveal atypical genomic features, extensive genomic divergence, and lineage-specific innovation of gene functions. Long non-coding RNAs (lncRNAs), known to regulate gene expression in eukaryotes, are largely unexplored in dinoflagellates. Here, using high-quality genome and transcriptome data, we identified 48039 polyadenylated lncRNAs in three dinoflagellate species: the coral symbionts Cladocopium proliferum and Durusdinium trenchii, and the bloom-forming species, Prorocentrum cordatum. These lncRNAs have fewer introns and lower G+C content than protein-coding sequences; 37 768 (78.6%) are unique with respect to sequence similarity. We classified all lncRNAs based on conserved motifs (k-mers) into distinct clusters, following properties of protein-binding and/or subcellular localisation. Interestingly, 3708 (7.7%) lncRNAs are differentially expressed under heat stress, algal lifestyle, and/or growth phase, and share co-expression patterns with protein-coding genes. Based on inferred triplex interactions between lncRNA and putative promoter regions, we identified 19 460 putative gene targets for 3721 lncRNAs; 907 genes exhibit differential expression under heat stress. These results reveal, for the first time, the diversity of lncRNAs in dinoflagellates and how lncRNAs may regulate gene expression as a heat-stress response in these ecologically important microbes.

Exceptional Quantum Efficiency Powers Biomass Production in Halotolerant Algae Picochlorum sp.

The green algal genus Picochlorum is of biotechnological interest because of its robust response to multiple environmental stresses. We compared the metabolic performance of P. SE3 and P. oklahomense to diverse microbial phototrophs and observed exceptional performance of photosystem II (PSII) in light energy conversion in both Picochlorum species. The quantum yield (QY) for O2 evolution is the highest of any phototroph yet observed, 32% (20%) by P. SE3 (P. okl) when normalized to total PSII subunit PsbA (D1) protein, and 80% (75%) normalized per active PSII, respectively. Three factors contribute: (1) an efficient water oxidizing complex (WOC) with the fewest photochemical misses of any organism; (2) faster reoxidation of reduced (PQH2)B in P. SE3 than in P. okl. (period-2 Fourier amplitude); and (3) rapid reoxidation of the plastoquinol pool by downstream electron carriers (Cyt b6f/PETC) that regenerates PQ faster in P. SE3. This performance gain is achieved without significant residue changes around the QB site and thus points to a pull mechanism involving faster PQH2 reoxidation by Cyt b6f/PETC that offsets charge recombination. This high flux in P. SE3 may be explained by genomically encoded plastoquinol terminal oxidases 1 and 2, whereas P. oklahomense has neither. Our results suggest two distinct types of PSII centers exist, one specializing in linear electron flow and the other in PSII-cyclic electron flow. Several amino acids within D1 differ from those in the low-light-descended D1 sequences conserved in Viridiplantae, and more closely match those in cyanobacterial high-light D1 isoforms, including changes near tyrosine Yz and a water/proton channel near the WOC. These residue changes may contribute to the exceptional performance of Picochlorum at high-light intensities by increasing the water oxidation efficiency and the electron/proton flux through the PSII acceptors (QAQB).

Diurnal Rhythms in the Red Seaweed Gracilariopsis chorda are Characterized by Unique Regulatory Networks of Carbon Metabolism.

Cellular and physiological cycles are driven by endogenous pacemakers, the diurnal and circadian rhythms. Key functions such as cell cycle progression and cellular metabolism are under rhythmic regulation, thereby maintaining physiological homeostasis. The photoreceptors phytochrome and cryptochrome, in response to light cues, are central input pathways for physiological cycles in most photosynthetic organisms. However, among Archaeplastida, red algae are the only taxa that lack phytochromes. Current knowledge about oscillatory rhythms is primarily derived from model species such as Arabidopsis thaliana and Chlamydomonas reinhardtii in the Viridiplantae, whereas little is known about these processes in other clades of the Archaeplastida, such as the red algae (Rhodophyta). We used genome-wide expression profiling of the red seaweed Gracilariopsis chorda and identified 3,098 rhythmic genes. Here, we characterized possible cryptochrome-based regulation and photosynthetic/cytosolic carbon metabolism in this species. We found a large family of cryptochrome genes in G. chorda that display rhythmic expression over the diurnal cycle and may compensate for the lack of phytochromes in this species. The input pathway gates regulatory networks of carbon metabolism which results in a compact and efficient energy metabolism during daylight hours. The system in G. chorda is distinct from energy metabolism in most plants, which activates in the dark. The green lineage, in particular, land plants, balance water loss and CO2 capture in terrestrial environments. In contrast, red seaweeds maintain a reduced set of photoreceptors and a compact cytosolic carbon metabolism to thrive in the harsh abiotic conditions typical of intertidal zones.

Facultative lifestyle drives diversity of coral algal symbionts.

The photosynthetic symbionts of corals sustain biodiverse reefs in nutrient-poor, tropical waters. Recent genomic data illuminate the evolution of coral symbionts under genome size constraints and suggest that retention of the facultative lifestyle, widespread among these algae, confers a selective advantage when compared with a strict symbiotic existence. We posit that the coral symbiosis is analogous to a 'bioreactor' that selects winner genotypes and allows them to rise to high numbers in a sheltered habitat prior to release by the coral host. Our observations lead to a novel hypothesis, the 'stepping-stone model', which predicts that local adaptation under both the symbiotic and free-living stages, in a stepwise fashion, accelerates coral alga diversity and the origin of endemic strains and species.

Multi-omics analysis reveals the molecular response to heat stress in a "red tide" dinoflagellate.

BACKGROUND: "Red tides" are harmful algal blooms caused by dinoflagellate microalgae that accumulate toxins lethal to other organisms, including humans via consumption of contaminated seafood. These algal blooms are driven by a combination of environmental factors including nutrient enrichment, particularly in warm waters, and are increasingly frequent. The molecular, regulatory, and evolutionary mechanisms that underlie the heat stress response in these harmful bloom-forming algal species remain little understood, due in part to the limited genomic resources from dinoflagellates, complicated by the large sizes of genomes, exhibiting features atypical of eukaryotes. RESULTS: We present the de novo assembled genome (~ 4.75 Gbp with 85,849 protein-coding genes), transcriptome, proteome, and metabolome from Prorocentrum cordatum, a globally abundant, bloom-forming dinoflagellate. Using axenic algal cultures, we study the molecular mechanisms that underpin the algal response to heat stress, which is relevant to current ocean warming trends. We present the first evidence of a complementary interplay between RNA editing and exon usage that regulates the expression and functional diversity of biomolecules, reflected by reduction in photosynthesis, central metabolism, and protein synthesis. These results reveal genomic signatures and post-transcriptional regulation for the first time in a pelagic dinoflagellate. CONCLUSIONS: Our multi-omics analyses uncover the molecular response to heat stress in an important bloom-forming algal species, which is driven by complex gene structures in a large, high-G+C genome, combined with multi-level transcriptional regulation. The dynamics and interplay of molecular regulatory mechanisms may explain in part how dinoflagellates diversified to become some of the most ecologically successful organisms on Earth.

Developing model systems for dinoflagellates in the post-genomic era.

Dinoflagellates are a diverse group of eukaryotic microbes that are ubiquitous in aquatic environments. Largely photosynthetic, they encompass symbiotic, parasitic, and free-living lineages with a broad spectrum of trophism. Many free-living taxa can produce bioactive secondary metabolites such as biotoxins, some of which cause harmful algal blooms. In contrast, most symbiotic species are crucial for sustaining coral reef health. The year 2023 marked a decade since the first genome data of dinoflagellates became available. The growing genome-scale resources for these taxa are highlighting their remarkable evolutionary and genomic complexities. Here, we discuss the prospect of developing dinoflagellate models using the criteria of accessibility, tractability, resources, research support, and promise. Moving forward in the post-genomic era, we argue for the development of fit-to-purpose models that tailor to specific biological contexts, and that a one-size-fits-all model is inadequate for encapsulating the complex biology, ecology, and evolutionary history of dinoflagellates.

Peeling back the layers of coral holobiont multi-omics data.

The integration of multiple 'omics' datasets is a promising avenue for answering many important and challenging questions in biology, particularly those relating to complex ecological systems. Although multi-omics was developed using data from model organisms with significant prior knowledge and resources, its application to non-model organisms, such as coral holobionts, is less clear-cut. We explore, in the emerging rice coral model Montipora capitata, the intersection of holobiont transcriptomic, proteomic, metabolomic, and microbiome amplicon data and investigate how well they correlate under high temperature treatment. Using a typical thermal stress regime, we show that transcriptomic and proteomic data broadly capture the stress response of the coral, whereas the metabolome and microbiome datasets show patterns that likely reflect stochastic and homeostatic processes associated with each sample. These results provide a framework for interpreting multi-omics data generated from non-model systems, particularly those with complex biotic interactions among microbial partners.

Gene duplication is the primary driver of intraspecific genomic divergence in coral algal symbionts.

Dinoflagellates in the order Suessiales include the family Symbiodiniaceae, which have essential roles as photosymbionts in corals, and their cold-adapted sister group, Polarella glacialis. These diverse taxa exhibit extensive genomic divergence, although their genomes are relatively small (haploid size < 3 Gbp) when compared with most other free-living dinoflagellates. Different strains of Symbiodiniaceae form symbiosis with distinct hosts and exhibit different regimes of gene expression, but intraspecific whole-genome divergence is poorly understood. Focusing on three Symbiodiniaceae species (the free-living Effrenium voratum and the symbiotic Symbiodinium microadriaticum and Durusdinium trenchii) and the free-living outgroup P. glacialis, for which whole-genome data from multiple isolates are available, we assessed intraspecific genomic divergence with respect to sequence and structure. Our analysis, based on alignment and alignment-free methods, revealed a greater extent of intraspecific sequence divergence in Symbiodiniaceae than in P. glacialis. Our results underscore the role of gene duplication in generating functional innovation, with a greater prevalence of tandemly duplicated single-exon genes observed in the genomes of free-living species than in symbionts. These results demonstrate the remarkable intraspecific genomic divergence in dinoflagellates under the constraint of reduced genome sizes, shaped by genetic duplications and symbiogenesis events during the diversification of Symbiodiniaceae.

Ploidy Variation and Its Implications for Reproduction and Population Dynamics in Two Sympatric Hawaiian Coral Species.

Standing genetic variation is a major driver of fitness and resilience and therefore of fundamental importance for threatened species such as stony corals. We analyzed RNA-seq data generated from 132 Montipora capitata and 119 Pocillopora acuta coral colonies collected from Kane'ohe Bay, O'ahu, Hawai'i. Our goals were to determine the extent of colony genetic variation and to study reproductive strategies in these two sympatric species. Surprisingly, we found that 63% of the P. acuta colonies were triploid, with putative independent origins of the different triploid clades. These corals have spread primarily via asexual reproduction and are descended from a small number of genotypes, whose diploid ancestor invaded the bay. In contrast, all M. capitata colonies are diploid and outbreeding, with almost all colonies genetically distinct. Only two cases of asexual reproduction, likely via fragmentation, were identified in this species. We report two distinct strategies in sympatric coral species that inhabit the largest sheltered body of water in the main Hawaiian Islands. These data highlight divergence in reproductive behavior and genome biology, both of which contribute to coral resilience and persistence.

A k-mer-Based Approach for Phylogenetic Classification of Taxa in Environmental Genomic Data.

In the age of genome sequencing, whole-genome data is readily and frequently generated, leading to a wealth of new information that can be used to advance various fields of research. New approaches, such as alignment-free phylogenetic methods that utilize k-mer-based distance scoring, are becoming increasingly popular given their ability to rapidly generate phylogenetic information from whole-genome data. However, these methods have not yet been tested using environmental data, which often tends to be highly fragmented and incomplete. Here, we compare the results of one alignment-free approach (which utilizes the D2 statistic) to traditional multi-gene maximum likelihood trees in 3 algal groups that have high-quality genome data available. In addition, we simulate lower-quality, fragmented genome data using these algae to test method robustness to genome quality and completeness. Finally, we apply the alignment-free approach to environmental metagenome assembled genome data of unclassified Saccharibacteria and Trebouxiophyte algae, and single-cell amplified data from uncultured marine stramenopiles to demonstrate its utility with real datasets. We find that in all instances, the alignment-free method produces phylogenies that are comparable, and often more informative, than those created using the traditional multi-gene approach. The k-mer-based method performs well even when there are significant missing data that include marker genes traditionally used for tree reconstruction. Our results demonstrate the value of alignment-free approaches for classifying novel, often cryptic or rare, species, that may not be culturable or are difficult to access using single-cell methods, but fill important gaps in the tree of life.

Origin of minicircular mitochondrial genomes in red algae.

Eukaryotic organelle genomes are generally of conserved size and gene content within phylogenetic groups. However, significant variation in genome structure may occur. Here, we report that the Stylonematophyceae red algae contain multipartite circular mitochondrial genomes (i.e., minicircles) which encode one or two genes bounded by a specific cassette and a conserved constant region. These minicircles are visualized using fluorescence microscope and scanning electron microscope, proving the circularity. Mitochondrial gene sets are reduced in these highly divergent mitogenomes. Newly generated chromosome-level nuclear genome assembly of Rhodosorus marinus reveals that most mitochondrial ribosomal subunit genes are transferred to the nuclear genome. Hetero-concatemers that resulted from recombination between minicircles and unique gene inventory that is responsible for mitochondrial genome stability may explain how the transition from typical mitochondrial genome to minicircles occurs. Our results offer inspiration on minicircular organelle genome formation and highlight an extreme case of mitochondrial gene inventory reduction.

Building consensus around the assessment and interpretation of Symbiodiniaceae diversity.

Within microeukaryotes, genetic variation and functional variation sometimes accumulate more quickly than morphological differences. To understand the evolutionary history and ecology of such lineages, it is key to examine diversity at multiple levels of organization. In the dinoflagellate family Symbiodiniaceae, which can form endosymbioses with cnidarians (e.g., corals, octocorals, sea anemones, jellyfish), other marine invertebrates (e.g., sponges, molluscs, flatworms), and protists (e.g., foraminifera), molecular data have been used extensively over the past three decades to describe phenotypes and to make evolutionary and ecological inferences. Despite advances in Symbiodiniaceae genomics, a lack of consensus among researchers with respect to interpreting genetic data has slowed progress in the field and acted as a barrier to reconciling observations. Here, we identify key challenges regarding the assessment and interpretation of Symbiodiniaceae genetic diversity across three levels: species, populations, and communities. We summarize areas of agreement and highlight techniques and approaches that are broadly accepted. In areas where debate remains, we identify unresolved issues and discuss technologies and approaches that can help to fill knowledge gaps related to genetic and phenotypic diversity. We also discuss ways to stimulate progress, in particular by fostering a more inclusive and collaborative research community. We hope that this perspective will inspire and accelerate coral reef science by serving as a resource to those designing experiments, publishing research, and applying for funding related to Symbiodiniaceae and their symbiotic partnerships.

Algae obscura: The potential of rare species as model systems.

Model organism research has provided invaluable knowledge about foundational biological principles. However, most of these studies have focused on species that are in high abundance, easy to cultivate in the lab, and represent only a small fraction of extant biodiversity. Here, we present three examples of rare algae with unusual features that we refer to as "algae obscura." The Cyanidiophyceae (Rhodophyta), Glaucophyta, and Paulinella (rhizarian) lineages have all transitioned out of obscurity to become models for fundamental evolutionary research. Insights have been gained into the prevalence and importance of eukaryotic horizontal gene transfer, early Earth microbial community dynamics, primary plastid endosymbiosis, and the origin of Archaeplastida. By reviewing the research that has come from the exploration of these organisms, we demonstrate that underappreciated algae have the potential to help us formulate, refine, and substantiate core hypotheses and that such organisms should be considered when establishing future model systems.