Endosymbiotic ratchet accelerates divergence after organelle origin: The Paulinella model for plastid evolution: The Paulinella model for plastid evolution.

We hypothesize that as one of the most consequential events in evolution, primary endosymbiosis accelerates lineage divergence, a process we refer to as the endosymbiotic ratchet. Our proposal is supported by recent work on the photosynthetic amoeba, Paulinella, that underwent primary plastid endosymbiosis about 124 Mya. This amoeba model allows us to explore the early impacts of photosynthetic organelle (plastid) origin on the host lineage. The current data point to a central role for effective population size (Ne ) in accelerating divergence post-endosymbiosis due to limits to dispersal and reproductive isolation that reduce Ne , leading to local adaptation. We posit that isolated populations exploit different strategies and behaviors and assort themselves in non-overlapping niches to minimize competition during the early, rapid evolutionary phase of organelle integration. The endosymbiotic ratchet provides a general framework for interpreting post-endosymbiosis lineage evolution that is driven by disruptive selection and demographic and population shifts. Also see the video abstract here: https://youtu.be/gYXrFM6Zz6Q.

Retrotransposition facilitated the establishment of a primary plastid in the thecate amoeba Paulinella.

The evolution of eukaryotic life was predicated on the development of organelles such as mitochondria and plastids. During this complex process of organellogenesis, the host cell and the engulfed prokaryote became genetically codependent, with the integration of genes from the endosymbiont into the host nuclear genome and subsequent gene loss from the endosymbiont. This process required that horizontally transferred genes become active and properly regulated despite inherent differences in genetic features between donor (endosymbiont) and recipient (host). Although this genetic reorganization is considered critical for early stages of organellogenesis, we have little knowledge about the mechanisms governing this process. The photosynthetic amoeba Paulinella micropora offers a unique opportunity to study early evolutionary events associated with organellogenesis and primary endosymbiosis. This amoeba harbors a "chromatophore," a nascent photosynthetic organelle derived from a relatively recent cyanobacterial association (∼120 million years ago) that is independent of the evolution of primary plastids in plants (initiated ∼1.5 billion years ago). Analysis of the genome and transcriptome of Paulinella revealed that retrotransposition of endosymbiont-derived nuclear genes was critical for their domestication in the host. These retrocopied genes involved in photoprotection in cyanobacteria became expanded gene families and were "rewired," acquiring light-responsive regulatory elements that function in the host. The establishment of host control of endosymbiont-derived genes likely enabled the cell to withstand photo-oxidative stress generated by oxygenic photosynthesis in the nascent organelle. These results provide insights into the genetic mechanisms and evolutionary pressures that facilitated the metabolic integration of the host­endosymbiont association and sustained the evolution of a photosynthetic organelle.

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.

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.

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.

Amoeba Genome Reveals Dominant Host Contribution to Plastid Endosymbiosis.

Eukaryotic photosynthetic organelles, plastids, are the powerhouses of many aquatic and terrestrial ecosystems. The canonical plastid in algae and plants originated >1 Ga and therefore offers limited insights into the initial stages of organelle evolution. To address this issue, we focus here on the photosynthetic amoeba Paulinella micropora strain KR01 (hereafter, KR01) that underwent a more recent (∼124 Ma) primary endosymbiosis, resulting in a photosynthetic organelle termed the chromatophore. Analysis of genomic and transcriptomic data resulted in a high-quality draft assembly of size 707 Mb and 32,361 predicted gene models. A total of 291 chromatophore-targeted proteins were predicted in silico, 208 of which comprise the ancestral organelle proteome in photosynthetic Paulinella species with functions, among others, in nucleotide metabolism and oxidative stress response. Gene coexpression analysis identified networks containing known high light stress response genes as well as a variety of genes of unknown function ("dark" genes). We characterized diurnally rhythmic genes in this species and found that over 49% are dark. It was recently hypothesized that large double-stranded DNA viruses may have driven gene transfer to the nucleus in Paulinella and facilitated endosymbiosis. Our analyses do not support this idea, but rather suggest that these viruses in the KR01 and closely related P. micropora MYN1 genomes resulted from a more recent invasion.

Evidence for a robust photosystem II in the photosynthetic amoeba Paulinella.

Paulinella represents the only known case of an independent primary plastid endosymbiosis, outside Archaeplastida, that occurred c. 120 (million years ago) Ma. These photoautotrophs grow very slowly in replete culture medium with a doubling time of 6-7 d at optimal low light, and are highly sensitive to photodamage under moderate light levels. We used genomic and biophysical methods to investigate the extreme slow growth rate and light sensitivity of Paulinella, which are key to photosymbiont integration. All photosystem II (PSII) genes except psb28-2 and all cytochrome b6 f complex genes except petM and petL are present in Paulinella micropora KR01 (hereafter, KR01). Biophysical measurements of the water oxidation complex, variable chlorophyll fluorescence, and photosynthesis-irradiance curves show no obvious evidence of PSII impairment. Analysis of photoacclimation under high-light suggests that although KR01 can perform charge separation, it lacks photoprotection mechanisms present in cyanobacteria. We hypothesize that Paulinella species are restricted to low light environments because they are deficient in mitigating the formation of reactive oxygen species formed within the photosystems under peak solar intensities. The finding that many photoprotection genes have been lost or transferred to the host-genome during endosymbiont genome reduction, and may lack light-regulation, is consistent with this hypothesis.

Hypothesis: Trans-splicing Generates Evolutionary Novelty in the Photosynthetic Amoeba Paulinella.

Plastid primary endosymbiosis has occurred twice, once in the Archaeplastida ancestor and once in the Paulinella (Rhizaria) lineage. Both events precipitated massive evolutionary changes, including the recruitment and activation of genes that are horizontally acquired (HGT) and the redeployment of existing genes and pathways in novel contexts. Here we address the latter aspect in Paulinella micropora KR01 (hereafter, KR01) that has independently evolved spliced leader (SL) trans-splicing (SLTS) of nuclear-derived transcripts. We investigated the role of this process in gene regulation, novel gene origination, and endosymbiont integration. Our analysis shows that 20% of KR01 genes give rise to transcripts with at least one (but in some cases, multiple) sites of SL addition. This process, which often occurs at canonical cis-splicing acceptor sites (internal introns), results in shorter transcripts that may produce 5'-truncated proteins with novel functions. SL-truncated transcripts fall into four categories that may show: (i) altered protein localization, (ii) altered protein function, structure, or regulation, (iii) loss of valid alternative start codons, preventing translation, or (iv) multiple SL addition sites at the 5'-terminus. The SL RNA genes required for SLTS are putatively absent in the heterotrophic sister lineage of photosynthetic Paulinella species. Moreover, a high proportion of transcripts derived from genes of endosymbiotic gene transfer (EGT) and HGT origin contain SL sequences. We hypothesize that truncation of transcripts by SL addition may facilitate the generation and expression of novel gene variants and that SLTS may have enhanced the activation and fixation of foreign genes in the host genome of the photosynthetic lineages, playing a key role in primary endosymbiont integration.

Comparison of 15 dinoflagellate genomes reveals extensive sequence and structural divergence in family Symbiodiniaceae and genus Symbiodinium.

BACKGROUND: Dinoflagellates in the family Symbiodiniaceae are important photosynthetic symbionts in cnidarians (such as corals) and other coral reef organisms. Breakdown of the coral-dinoflagellate symbiosis due to environmental stress (i.e. coral bleaching) can lead to coral death and the potential collapse of reef ecosystems. However, evolution of Symbiodiniaceae genomes, and its implications for the coral, is little understood. Genome sequences of Symbiodiniaceae remain scarce due in part to their large genome sizes (1-5 Gbp) and idiosyncratic genome features. RESULTS: Here, we present de novo genome assemblies of seven members of the genus Symbiodinium, of which two are free-living, one is an opportunistic symbiont, and the remainder are mutualistic symbionts. Integrating other available data, we compare 15 dinoflagellate genomes revealing high sequence and structural divergence. Divergence among some Symbiodinium isolates is comparable to that among distinct genera of Symbiodiniaceae. We also recovered hundreds of gene families specific to each lineage, many of which encode unknown functions. An in-depth comparison between the genomes of the symbiotic Symbiodinium tridacnidorum (isolated from a coral) and the free-living Symbiodinium natans reveals a greater prevalence of transposable elements, genetic duplication, structural rearrangements, and pseudogenisation in the symbiotic species. CONCLUSIONS: Our results underscore the potential impact of lifestyle on lineage-specific gene-function innovation, genome divergence, and the diversification of Symbiodinium and Symbiodiniaceae. The divergent features we report, and their putative causes, may also apply to other microbial eukaryotes that have undergone symbiotic phases in their evolutionary history.

Why is primary endosymbiosis so rare?

Endosymbiosis is a relationship between two organisms wherein one cell resides inside the other. This affiliation, when stable and beneficial for the 'host' cell, can result in massive genetic innovation with the foremost examples being the evolution of eukaryotic organelles, the mitochondria and plastids. Despite its critical evolutionary role, there is limited knowledge about how endosymbiosis is initially established and how host-endosymbiont biology is integrated. Here, we explore this issue, using as our model the rhizarian amoeba Paulinella, which represents an independent case of primary plastid origin that occurred c. 120 million yr ago. We propose the 'chassis and engine' model that provides a theoretical framework for understanding primary plastid endosymbiosis, potentially explaining why it is so rare.

Genomes of the dinoflagellate Polarella glacialis encode tandemly repeated single-exon genes with adaptive functions.

BACKGROUND: Dinoflagellates are taxonomically diverse and ecologically important phytoplankton that are ubiquitously present in marine and freshwater environments. Mostly photosynthetic, dinoflagellates provide the basis of aquatic primary production; most taxa are free-living, while some can form symbiotic and parasitic associations with other organisms. However, knowledge of the molecular mechanisms that underpin the adaptation of these organisms to diverse ecological niches is limited by the scarce availability of genomic data, partly due to their large genome sizes estimated up to 250 Gbp. Currently available dinoflagellate genome data are restricted to Symbiodiniaceae (particularly symbionts of reef-building corals) and parasitic lineages, from taxa that have smaller genome size ranges, while genomic information from more diverse free-living species is still lacking. RESULTS: Here, we present two draft diploid genome assemblies of the free-living dinoflagellate Polarella glacialis, isolated from the Arctic and Antarctica. We found that about 68% of the genomes are composed of repetitive sequence, with long terminal repeats likely contributing to intra-species structural divergence and distinct genome sizes (3.0 and 2.7 Gbp). For each genome, guided using full-length transcriptome data, we predicted > 50,000 high-quality protein-coding genes, of which ~40% are in unidirectional gene clusters and ~25% comprise single exons. Multi-genome comparison unveiled genes specific to P. glacialis and a common, putatively bacterial origin of ice-binding domains in cold-adapted dinoflagellates. CONCLUSIONS: Our results elucidate how selection acts within the context of a complex genome structure to facilitate local adaptation. Because most dinoflagellate genes are constitutively expressed, Polarella glacialis has enhanced transcriptional responses via unidirectional, tandem duplication of single-exon genes that encode functions critical to survival in cold, low-light polar environments. These genomes provide a foundational reference for future research on dinoflagellate evolution.

Evidence That Inconsistent Gene Prediction Can Mislead Analysis of Dinoflagellate Genomes.

Comparative algal genomics often relies on predicted genes from de novo assembled genomes. However, the artifacts introduced by different gene-prediction approaches, and their impact on comparative genomic analysis remain poorly understood. Here, using available genome data from six dinoflagellate species in the Symbiodiniaceae, we identified methodological biases in the published genes that were predicted using different approaches and putative contaminant sequences in the published genome assemblies. We developed and applied a comprehensive customized workflow to predict genes from these genomes. The observed variation among predicted genes resulting from our workflow agreed with current understanding of phylogenetic relationships among these taxa, whereas the variation among the previously published genes was largely biased by the distinct approaches used in each instance. Importantly, these biases affect the inference of homologous gene families and synteny among genomes, thus impacting biological interpretation of these data. Our results demonstrate that a consistent gene-prediction approach is critical for comparative analysis of dinoflagellate genomes.

Analysis of the Draft Genome of the Red Seaweed Gracilariopsis chorda Provides Insights into Genome Size Evolution in Rhodophyta.

Red algae (Rhodophyta) underwent two phases of large-scale genome reduction during their early evolution. The red seaweeds did not attain genome sizes or gene inventories typical of other multicellular eukaryotes. We generated a high-quality 92.1 Mb draft genome assembly from the red seaweed Gracilariopsis chorda, including methylation and small (s)RNA data. We analyzed these and other Archaeplastida genomes to address three questions: 1) What is the role of repeats and transposable elements (TEs) in explaining Rhodophyta genome size variation, 2) what is the history of genome duplication and gene family expansion/reduction in these taxa, and 3) is there evidence for TE suppression in red algae? We find that the number of predicted genes in red algae is relatively small (4,803-13,125 genes), particularly when compared with land plants, with no evidence of polyploidization. Genome size variation is primarily explained by TE expansion with the red seaweeds having the largest genomes. Long terminal repeat elements and DNA repeats are the major contributors to genome size growth. About 8.3% of the G. chorda genome undergoes cytosine methylation among gene bodies, promoters, and TEs, and 71.5% of TEs contain methylated-DNA with 57% of these regions associated with sRNAs. These latter results suggest a role for TE-associated sRNAs in RNA-dependent DNA methylation to facilitate silencing. We postulate that the evolution of genome size in red algae is the result of the combined action of TE spread and the concomitant emergence of its epigenetic suppression, together with other important factors such as changes in population size.

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.

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.

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.

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.

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.

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.