Genome-powered classification of microbial eukaryotes: focus on coral algal symbionts.

Modern microbial taxonomy generally relies on the use of single marker genes or sets of concatenated genes to generate a framework for the delineation and classification of organisms at different taxonomic levels. However, given that DNA is the 'blueprint of life', and hence the ultimate arbiter of taxonomy, classification systems should attempt to use as much of the blueprint as possible to capture a comprehensive phylogenetic signal. Recent analysis of whole-genome sequences from coral reef symbionts (dinoflagellates of the family Symbiodiniaceae) and other microalgal groups has uncovered extensive divergence not recognised by current algal taxonomic approaches. In the era of 'sequence everything', we argue that whole-genome data are pivotal to guide informed taxonomic inference, particularly for microbial eukaryotes.

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.

Morphological stasis masks ecologically divergent coral species on tropical reefs.

Coral reefs are the epitome of species diversity, yet the number of described scleractinian coral species, the framework-builders of coral reefs, remains moderate by comparison. DNA sequencing studies are rapidly challenging this notion by exposing a wealth of undescribed diversity, but the evolutionary and ecological significance of this diversity remains largely unclear. Here, we present an annotated genome for one of the most ubiquitous corals in the Indo-Pacific (Pachyseris speciosa) and uncover, through a comprehensive genomic and phenotypic assessment, that it comprises morphologically indistinguishable but ecologically divergent lineages. Demographic modeling based on whole-genome resequencing indicated that morphological crypsis (across micro- and macromorphological traits) was due to ancient morphological stasis rather than recent divergence. Although the lineages occur sympatrically across shallow and mesophotic habitats, extensive genotyping using a rapid molecular assay revealed differentiation of their ecological distributions. Leveraging "common garden" conditions facilitated by the overlapping distributions, we assessed physiological and quantitative skeletal traits and demonstrated concurrent phenotypic differentiation. Lastly, spawning observations of genotyped colonies highlighted the potential role of temporal reproductive isolation in the limited admixture, with consistent genomic signatures in genes related to morphogenesis and reproduction. Overall, our findings demonstrate the presence of ecologically and phenotypically divergent coral species without substantial morphological differentiation and provide new leads into the potential mechanisms facilitating such divergence. More broadly, they indicate that our current taxonomic framework for reef-building corals may be scratching the surface of the ecologically relevant diversity on coral reefs, consequently limiting our ability to protect or restore this diversity effectively.

Genomic signatures in the coral holobiont reveal host adaptations driven by Holocene climate change and reef specific symbionts.

Genetic signatures caused by demographic and adaptive processes during past climatic shifts can inform predictions of species' responses to anthropogenic climate change. To identify these signatures in Acropora tenuis, a reef-building coral threatened by global warming, we first assembled the genome from long reads and then used shallow whole-genome resequencing of 150 colonies from the central inshore Great Barrier Reef to inform population genomic analyses. We identify population structure in the host that reflects a Pleistocene split, whereas photosymbiont differences between reefs most likely reflect contemporary (Holocene) conditions. Signatures of selection in the host were associated with genes linked to diverse processes including osmotic regulation, skeletal development, and the establishment and maintenance of symbiosis. Our results suggest that adaptation to post-glacial climate change in A. tenuis has involved selection on many genes, while differences in symbiont specificity between reefs appear to be unrelated to host population structure.

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.

Comparative transcriptomic analyses of Chromera and Symbiodiniaceae.

Reef-building corals live in a mutualistic relationship with photosynthetic algae (family Symbiodiniaceae) that usually provide most of the energy required by the coral host. This relationship is sensitive to temperature stress; as little as a 1°C increase often leads to the collapse of the association. This sensitivity has led to an interest in the potential of more stress-tolerant algae to supplement or substitute for the normal Symbiodiniaceae mutualists. In this respect, the apicomplexan-like microalga Chromera is of particular interest due to its greater temperature tolerance. We generated a de novo transcriptome for a Chromera strain isolated from a GBR coral ('GBR Chromera') and compared with those of the reference strain of Chromera ('Sydney Chromera'), and to those of Symbiodiniaceae (Fugacium kawagutii, Cladocopium goreaui and Breviolum minutum), as well as the apicomplexan parasite, Plasmodium falciparum. In contrast to the high sequence divergence amongst representatives of different genera within the family Symbiodiniaceae, the two Chromera strains featured low sequence divergence at orthologous genes, implying that they are likely to be conspecifics. Although KEGG categories provide few criteria by which true coral mutualists might be identified, they do supply a molecular rationalization that explains the ecological dominance of Cladocopium spp. amongst Indo-Pacific reef corals. The presence of HSP20 genes may contribute to the high thermal tolerance of Chromera.

A genomic view of the reef-building coral Porites lutea and its microbial symbionts.

Corals and the reef ecosystems that they support are in global decline due to increasing anthropogenic pressures such as climate change1. However, effective reef conservation strategies are hampered by a limited mechanistic understanding of coral biology and the functional roles of the diverse microbial communities that underpin coral health2,3. Here, we present an integrated genomic characterization of the coral species Porites lutea and its microbial partners. High-quality genomes were recovered from P. lutea, as well as a metagenome-assembled Cladocopium C15 (the dinoflagellate symbiont) and 52 bacterial and archaeal populations. Comparative genomic analysis revealed that many of the bacterial and archaeal genomes encode motifs that may be involved in maintaining association with the coral host and in supplying fixed carbon, B-vitamins and amino acids to their eukaryotic partners. Furthermore, mechanisms for ammonia, urea, nitrate, dimethylsulfoniopropionate and taurine transformation were identified that interlink members of the holobiont and may be important for nutrient acquisition and retention in oligotrophic waters. Our findings demonstrate the critical and diverse roles that microorganisms play within the coral holobiont and underscore the need to consider all of the components of the holobiont if we are to effectively inform reef conservation strategies.

Quantitative Modelling of the Waddington Epigenetic Landscape.

C.H. Waddington introduced the epigenetic landscape as a metaphor to represent cellular decision-making during development. Like a population of balls rolling down a rough hillside, developing cells follow specific trajectories (valleys) and eventually come to rest in one or another low-energy state that represents a mature cell type. Waddington depicted the topography of this landscape as determined by interactions among gene products, thereby connecting genotype to phenotype. In modern terms, each point on the landscape represents a state of the underlying genetic regulatory network, which in turn is described by a gene expression profile. In this chapter we demonstrate how the mathematical formalism of Hopfield networks can be used to model this epigenetic landscape. Hopfield networks are auto-associative artificial neural networks; input patterns are stored as attractors of the network and can be recalled from noisy or incomplete inputs. The resulting models capture the temporal dynamics of a gene regulatory network, yielding quantitative insight into cellular development and phenotype.

Genome Evolution of Coral Reef Symbionts as Intracellular Residents.

Coral reefs are sustained by symbioses between corals and symbiodiniacean dinoflagellates. These symbioses vary in the extent of their permanence in and specificity to the host. Although dinoflagellates are primarily free-living, Symbiodiniaceae diversified mainly as symbiotic lineages. Their genomes reveal conserved symbiosis-related gene functions and high sequence divergence. However, the evolutionary mechanisms that underpin the transition from the free-living lifestyle to symbiosis remain poorly understood. Here, we discuss the genome evolution of Symbiodiniaceae in diverse ecological niches across the broad spectrum of symbiotic associations, from free-living to putative obligate symbionts. We pose key questions regarding genome evolution vis-à-vis the transition of dinoflagellates from free-living to symbiotic and propose strategies for future research to better understand coral-dinoflagellate and other eukaryote-eukaryote symbioses.

Whole-genome sequence of the bovine blood fluke Schistosoma bovis supports interspecific hybridization with S. haematobium.

Mesenteric infection by the parasitic blood fluke Schistosoma bovis is a common veterinary problem in Africa and the Middle East and occasionally in the Mediterranean Region. The species also has the ability to form interspecific hybrids with the human parasite S. haematobium with natural hybridisation observed in West Africa, presenting possible zoonotic transmission. Additionally, this exchange of alleles between species may dramatically influence disease dynamics and parasite evolution. We have generated a 374 Mb assembly of the S. bovis genome using Illumina and PacBio-based technologies. Despite infecting different hosts and organs, the genome sequences of S. bovis and S. haematobium appeared strikingly similar with 97% sequence identity. The two species share 98% of protein-coding genes, with an average sequence identity of 97.3% at the amino acid level. Genome comparison identified large continuous parts of the genome (up to several 100 kb) showing almost 100% sequence identity between S. bovis and S. haematobium. It is unlikely that this is a result of genome conservation and provides further evidence of natural interspecific hybridization between S. bovis and S. haematobium. Our results suggest that foreign DNA obtained by interspecific hybridization was maintained in the population through multiple meiosis cycles and that hybrids were sexually reproductive, producing viable offspring. The S. bovis genome assembly forms a highly valuable resource for studying schistosome evolution and exploring genetic regions that are associated with species-specific phenotypic traits.

Alignment-free inference of hierarchical and reticulate phylogenomic relationships.

We are amidst an ongoing flood of sequence data arising from the application of high-throughput technologies, and a concomitant fundamental revision in our understanding of how genomes evolve individually and within the biosphere. Workflows for phylogenomic inference must accommodate data that are not only much larger than before, but often more error prone and perhaps misassembled, or not assembled in the first place. Moreover, genomes of microbes, viruses and plasmids evolve not only by tree-like descent with modification but also by incorporating stretches of exogenous DNA. Thus, next-generation phylogenomics must address computational scalability while rethinking the nature of orthogroups, the alignment of multiple sequences and the inference and comparison of trees. New phylogenomic workflows have begun to take shape based on so-called alignment-free (AF) approaches. Here, we review the conceptual foundations of AF phylogenetics for the hierarchical (vertical) and reticulate (lateral) components of genome evolution, focusing on methods based on k-mers. We reflect on what seems to be successful, and on where further development is needed.

Whole-genome sequence of the oriental lung fluke Paragonimus westermani.

Background: Foodborne infections caused by lung flukes of the genus Paragonimus are a significant and widespread public health problem in tropical areas. Approximately 50 Paragonimus species have been reported to infect animals and humans, but Paragonimus westermani is responsible for the bulk of human disease. Despite their medical and economic importance, no genome sequence for any Paragonimus species is available. Results: We sequenced and assembled the genome of P. westermani, which is among the largest of the known pathogen genomes with an estimated size of 1.1 Gb. A 922.8 Mb genome assembly was generated from Illumina and Pacific Biosciences (PacBio) sequence data, covering 84% of the estimated genome size. The genome has a high proportion (45%) of repeat-derived DNA, particularly of the long interspersed element and long terminal repeat subtypes, and the expansion of these elements may explain some of the large size. We predicted 12,852 protein coding genes, showing a high level of conservation with related trematode species. The majority of proteins (80%) had homologs in the human liver fluke Opisthorchis viverrini, with an average sequence identity of 64.1%. Assembly of the P. westermani mitochondrial genome from long PacBio reads resulted in a single high-quality circularized 20.6 kb contig. The contig harbored a 6.9 kb region of non-coding repetitive DNA comprised of three distinct repeat units. Our results suggest that the region is highly polymorphic in P. westermani, possibly even within single worm isolates. Conclusions: The generated assembly represents the first Paragonimus genome sequence and will facilitate future molecular studies of this important, but neglected, parasite group.

k-mer Similarity, Networks of Microbial Genomes, and Taxonomic Rank.

Microbial genomes have been shaped by parent-to-offspring (vertical) descent and lateral genetic transfer. These processes can be distinguished by alignment-based inference and comparison of phylogenetic trees for individual gene families, but this approach is not scalable to whole-genome sequences, and a tree-like structure does not adequately capture how these processes impact microbial physiology. Here we adopted alignment-free approaches based on k-mer statistics to infer phylogenomic networks involving 2,783 completely sequenced bacterial and archaeal genomes and compared the contributions of rRNA, protein-coding, and plasmid sequences to these networks. Our results show that the phylogenomic signal arising from ribosomal RNAs is strong and extends broadly across all taxa, whereas that from plasmids is strong but restricted to closely related groups, particularly Proteobacteria. However, the signal from the other chromosomal regions is restricted in breadth. We show that mean k-mer similarity can correlate with taxonomic rank. We also link the implicated k-mers to genome annotation (thus, functions) and define core k-mers (thus, core functions) in specific phyletic groups. Highly conserved functions in most phyla include amino acid metabolism and transport as well as energy production and conversion. Intracellular trafficking and secretion are the most prominent core functions among Spirochaetes, whereas energy production and conversion are not highly conserved among the largely parasitic or commensal Tenericutes. These observations suggest that differential conservation of functions relates to niche specialization and evolutionary diversification of microbes. Our results demonstrate that k-mer approaches can be used to efficiently identify phylogenomic signals and conserved core functions at the multigenome scale. IMPORTANCE Genome evolution of microbes involves parent-to-offspring descent, and lateral genetic transfer that convolutes the phylogenomic signal. This study investigated phylogenomic signals among thousands of microbial genomes based on short subsequences without using multiple-sequence alignment. The signal from ribosomal RNAs is strong across all taxa, and the signal of plasmids is strong only in closely related groups, particularly Proteobacteria. However, the signal from other chromosomal regions (∼99% of the genomes) is remarkably restricted in breadth. The similarity of subsequences is found to correlate with taxonomic rank and informs on conserved and differential core functions relative to niche specialization and evolutionary diversification of microbes. These results provide a comprehensive, alignment-free view of microbial genome evolution as a network, beyond a tree-like structure.

Core genes in diverse dinoflagellate lineages include a wealth of conserved dark genes with unknown functions.

Dinoflagellates are a diverse group of unicellular primary producers and grazers that exhibit some of the most remarkable features known among eukaryotes. These include gigabase-sized nuclear genomes, permanently condensed chromosomes and highly reduced organelle DNA. However, the genetic inventory that allows dinoflagellates to thrive in diverse ecological niches is poorly characterised. Here we systematically assess the functional capacity of 3,368,684 predicted proteins from 47 transcriptome datasets spanning eight dinoflagellate orders. We find that 1,232,023 proteins do not share significant sequence similarity to known sequences, i.e. are "dark". Of these, we consider 441,006 (13.1% of overall proteins) that are found in multiple taxa, or occur as alternative splice variants, to comprise the high-confidence dark proteins. Even with unknown function, 43.3% of these dark proteins can be annotated with conserved structural features using an exhaustive search against available data, validating their existence and importance. Furthermore, these dark proteins and their putative homologs are largely lineage-specific and recovered in multiple taxa. We also identified conserved functions in all dinoflagellates, and those specific to toxin-producing, symbiotic, and cold-adapted lineages. Our results demonstrate the remarkable divergence of gene functions in dinoflagellates, and provide a platform for investigations into the diversification of these ecologically important organisms.

Erratum: Publisher Correction: Symbiodinium genomes reveal adaptive evolution of functions related to coral-dinoflagellate symbiosis.

[This corrects the article DOI: 10.1038/s42003-018-0098-3.].

Symbiodinium genomes reveal adaptive evolution of functions related to coral-dinoflagellate symbiosis.

Symbiosis between dinoflagellates of the genus Symbiodinium and reef-building corals forms the trophic foundation of the world's coral reef ecosystems. Here we present the first draft genome of Symbiodinium goreaui (Clade C, type C1: 1.03 Gbp), one of the most ubiquitous endosymbionts associated with corals, and an improved draft genome of Symbiodinium kawagutii (Clade F, strain CS-156: 1.05 Gbp) to further elucidate genomic signatures of this symbiosis. Comparative analysis of four available Symbiodinium genomes against other dinoflagellate genomes led to the identification of 2460 nuclear gene families (containing 5% of Symbiodinium genes) that show evidence of positive selection, including genes involved in photosynthesis, transmembrane ion transport, synthesis and modification of amino acids and glycoproteins, and stress response. Further, we identify extensive sets of genes for meiosis and response to light stress. These draft genomes provide a foundational resource for advancing our understanding of Symbiodinium biology and the coral-algal symbiosis.

Deciphering the nature of the coral-Chromera association.

Since the discovery of Chromera velia as a novel coral-associated microalga, this organism has attracted interest because of its unique evolutionary position between the photosynthetic dinoflagellates and the parasitic apicomplexans. The nature of the relationship between Chromera and its coral host is controversial. Is it a mutualism, from which both participants benefit, a parasitic relationship, or a chance association? To better understand the interaction, larvae of the common Indo-Pacific reef-building coral Acropora digitifera were experimentally infected with Chromera, and the impact on the host transcriptome was assessed at 4, 12, and 48 h post-infection using Illumina RNA-Seq technology. The transcriptomic response of the coral to Chromera was complex and implies that host immunity is strongly suppressed, and both phagosome maturation and the apoptotic machinery is modified. These responses differ markedly from those described for infection with a competent strain of the coral mutualist Symbiodinium, instead resembling those of vertebrate hosts to parasites and/or pathogens such as Mycobacterium tuberculosis. Consistent with ecological studies suggesting that the association may be accidental, the transcriptional response of A. digitifera larvae leads us to conclude that Chromera could be a coral parasite, commensal, or accidental bystander, but certainly not a beneficial mutualist.

Signatures of adaptation and symbiosis in genomes and transcriptomes of Symbiodinium.

Symbiodinium is best-known as the photosynthetic symbiont of corals, but some clades are symbiotic in other organisms or include free-living forms. Identifying similarities and differences among these clades can help us understand their relationship with corals, and thereby inform on measures to manage coral reefs in a changing environment. Here, using sequences from 24 publicly available transcriptomes and genomes of Symbiodinium, we assessed 78,389 gene families in Symbiodinium clades and the immediate outgroup Polarella glacialis, and identified putative overrepresented functions in gene families that (1) distinguish Symbiodinium from other members of Order Suessiales, (2) are shared by all of the Symbiodinium clades for which we have data, and (3) based on available information, are specific to each clade. Our findings indicate that transmembrane transport, mechanisms of response to reactive oxygen species, and protection against UV radiation are functions enriched in all Symbiodinium clades but not in P. glacialis. Enrichment of these functions indicates the capability of Symbiodinium to establish and maintain symbiosis, and to respond and adapt to its environment. The observed differences in lineage-specific gene families imply extensive genetic divergence among clades. Our results provide a platform for future investigation of lineage- or clade-specific adaptation of Symbiodinium to their environment.

Evolutionary conservation of a core root microbiome across plant phyla along a tropical soil chronosequence.

Culture-independent molecular surveys of plant root microbiomes indicate that soil type generally has a stronger influence on microbial communities than host phylogeny. However, these studies have mostly focussed on model plants and crops. Here, we examine the root microbiomes of multiple plant phyla including lycopods, ferns, gymnosperms, and angiosperms across a soil chronosequence using 16S rRNA gene amplicon profiling. We confirm that soil type is the primary determinant of root-associated bacterial community composition, but also observe a significant correlation with plant phylogeny. A total of 47 bacterial genera are associated with roots relative to bulk soil microbial communities, including well-recognized plant-associated genera such as Bradyrhizobium, Rhizobium, and Burkholderia, and major uncharacterized lineages such as WPS-2, Ellin329, and FW68. We suggest that these taxa collectively constitute an evolutionarily conserved core root microbiome at this site. This lends support to the inference that a core root microbiome has evolved with terrestrial plants over their 400 million year history.Yeoh et al. study root microbiomes of different plant phyla across a tropical soil chronosequence. They confirm that soil type is the primary determinant of root-associated bacterial communities, but also observe a clear correlation with plant phylogeny and define a core root microbiome at this site.

Modeling the Attractor Landscape of Disease Progression: a Network-Based Approach.

Genome-wide regulatory networks enable cells to function, develop, and survive. Perturbation of these networks can lead to appearance of a disease phenotype. Inspired by Conrad Waddington's epigenetic landscape of cell development, we use a Hopfield network formalism to construct an attractor landscape model of disease progression based on protein- or gene-correlation networks of Parkinson's disease, glioma, and colorectal cancer. Attractors in this landscape correspond to normal and disease states of the cell. We introduce approaches to estimate the size and robustness of these attractors, and take a network-based approach to study their biological features such as the key genes and their functions associated with the attractors. Our results show that the attractor of cancer cells is wider than the attractor of normal cells, suggesting a heterogeneous nature of cancer. Perturbation analysis shows that robustness depends on characteristics of the input data (number of samples per time-point, and the fraction which converge to an attractor). We identify unique gene interactions at each stage, which reflect the temporal rewiring of the gene regulatory network (GRN) with disease progression. Our model of the attractor landscape, constructed from large-scale gene expression profiles of individual patients, captures snapshots of disease progression and identifies gene interactions specific to different stages, opening the way for development of stage-specific therapeutic strategies.