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Brown algae are a group of multicellular, heterokont algae that have convergently evolved developmental complexity that rivals that of embryophytes, animals or fungi. Early in development, brown algal zygotes establish a basal and an apical pole, which will become respectively the basal system (holdfast) and the apical system (thallus) of the adult alga. Brown algae are interesting models for understanding the establishment of cell polarity in a broad evolutionary context, because they exhibit a large diversity of life cycles, reproductive strategies and, importantly, their zygotes are produced in large quantities free of parental tissue, with symmetry breaking and asymmetric division taking place in a highly synchronous manner. This review describes the current knowledge about the establishment of the apical-basal axis in the model brown seaweeds Ectocarpus, Dictyota, Fucus and Saccharina, highlighting the advantages and specific interests of each system. Ectocarpus is a genetic model system that allows access to the molecular basis of early development and life-cycle control over apical-basal polarity. The oogamous brown alga Fucus, together with emerging comparative models Dictyota and Saccharina, emphasize the diversity of strategies of symmetry breaking in determining a cell polarity vector in brown algae. A comparison with symmetry-breaking mechanisms in land plants, animals and fungi, reveals that the one-step zygote polarisation of Fucus compares well to Saccharomyces budding and Arabidopsis stomata development, while the two-phased symmetry breaking in the Dictyota zygote compares to Schizosaccharomyces fission, the Caenorhabditis anterior-posterior zygote polarisation and Arabidopsis prolate pollen polarisation. The apical-basal patterning in Saccharina zygotes on the other hand, may be seen as analogous to that of land plants. Overall, brown algae have the potential to bring exciting new information on how a single cell gives rise to an entire complex body plan.

The spontaneous mutation rate µ is a crucial parameter to understand evolution and biodiversity. Mutation rates are highly variable across species, suggesting that µ is susceptible to selection and drift and that species life cycle and life history may impact its evolution. In particular, asexual reproduction and haploid selection are expected to affect mutation rate, but very little empirical data is available to test this expectation. Here, we sequence 30 genomes of a parent-offspring pedigree in the model brown alga Ectocarpus sp.7, and 137 genomes of an interspecific cross of the closely related brown alga Scytosiphon to have access to the spontaneous mutation rate of representative organisms of a complex multicellular eukaryotic lineage outside animals and plants, and to evaluate the potential impact of life cycle on mutation rate. Brown algae alternate between a haploid and a diploid stage, both multicellular and free living, and utilize both sexual and asexual reproduction. They are therefore excellent models to empirically test expectations of the effect of asexual reproduction and haploid selection on mutation rate evolution. We estimate that Ectocarpus has a base substitution rate of µbs = 4.07 × 10−10 per site per generation, whereas the Scytosiphon interspecific cross had µbs =1.22 × 10−9. Overall, our estimations suggest that these brown algae, despite being multicellular complex eukaryotes, have unusually low mutation rates. In Ectocarpus, effective population size (Ne) could not entirely explain the low µb. We propose that the haploid-diploid life cycle, combined with extensive asexual reproduction may be additional key drivers of mutation rate.

SummaryThe first mitotic division of the initial cell is a key event in all multicellular organisms and is usually concomitant with the establishment of major developmental axes and cell fates. The brown alga Ectocarpus has a haploid-diploid life cycle that involves the development of two multicellular and independent generations, the sporophyte and the gametophyte. Each generation deploys a distinct developmental program autonomously from an initial cell, whose first cell division sets up the future body pattern. Here, we show that mutations in the BASELESS (BAS) gene result in multiple cellular defects during the first division of the initial cell and subsequently failure to produce basal structures (rhizoids and prostrate filaments) during both generations of the life cycle. Cloning-by-sequencing revealed that BAS encodes a type B” regulatory subunit of protein phosphatase 2A, and transcriptomic analysis of early developmental stages uncovered potential effector genes involved in setting up basal cell fate in this organism. The bas mutant phenotype is very similar to that observed in the distag (dis) mutants, which lack a functional TBCCd1 protein, at both the cellular and morphological levels. The high level of similarity of the dis and bas mutant phenotypes indicate that TBCCd1 and PP2A are two critical components of the cellular machinery that regulates the division of the initial cell and mediates the establishment of basal cell fate in the developing thallus.

Type II DNA topoisomerases regulate topology by double-stranded DNA cleavage and ligation. The TopoVI family of DNA topoisomerase, first identified and biochemically characterized in Archaea, represents, with TopoVIII and mini-A, the type IIB family. TopoVI has several intriguing features in terms of function and evolution. TopoVI has been identified in some eukaryotes, and a global view is lacking to understand its evolutionary pattern. In addition, in eukaryotes, the two TopoVI subunits (TopoVIA and TopoVIB) have been duplicated and have evolved to give rise to Spo11 and TopoVIBL, forming TopoVI-like (TopoVIL), a complex essential for generating DNA breaks that initiate homologous recombination during meiosis. TopoVIL is essential for sexual reproduction. How the TopoVI subunits have evolved to ensure this meiotic function is unclear. Here, we investigated the phylogenetic conservation of TopoVI and TopoVIL. We demonstrate that BIN4 and RHL1, potentially interacting with TopoVIB, have co-evolved with TopoVI. Based on model structures, this observation supports the hypothesis for a role of TopoVI in decatenation of replicated chromatids and predicts that in eukaryotes the TopoVI catalytic complex includes BIN4 and RHL1. For TopoVIL, the phylogenetic analysis of Spo11, which is highly conserved among Eukarya, highlighted a eukaryal-specific N-terminal domain that may be important for its regulation. Conversely, TopoVIBL was poorly conserved, giving rise to ATP hydrolysis-mutated or -truncated protein variants, or was undetected in some species. This remarkable plasticity of TopoVIBL provides important information for the activity and function of TopoVIL during meiosis.

The emergence of new genes is an important driver of evolutionary novelty. Yet, we lack a conceptual and computational approach that accurately traces gene-family founder events and effectively associates them with trait innovation and major radiation events. Here, we present GenEra, a DIAMOND-fuelled gene-family founder inference framework that addresses previously raised limitations and biases of founder gene detection in genomic phylostratigraphy by accounting for homology detection failure (HDF). We demonstrate how GenEra can accelerate gene-family founder computations from several months to a few days for any query genome of interest. We analyzed 30 genomes to explore the emergence of new gene families during the major evolutionary transitions in plants, animals and fungi. The detection of highly conserved protein domains in these gene families indicates that neofunctionalization of preexisting protein domains is a richer source of gene-family founder events compared with de novo gene birth. We report vastly different patterns of gene-family founder events in animal and fungi before and after accounting for HDF. Only plants exhibit a consistent pattern of founder gene emergence after accounting for HDF, suggesting they are more likely to evolve novelty through the emergence of new genes compared to opisthokonts. Finally, we show that gene-family founder bursts are associated with the transition to multicellularity in streptophytes, the terrestrialization of land plants and the origin of angiosperms, as well as with the evolution of bilateral symmetry in animals.

International audience; A vast diversity of types of life cycles exists in nature, and several theories have been advanced to explain how this diversity has evolved and how each type of life cycle is retained over evolutionary time. Here, we exploited the diversity of life cycles and reproductive traits of the brown algae (Phaeophyceae) to test several hypotheses on the evolution of life cycles. We investigated the evolutionary dynamics of four life-history traits: life cycle, sexual system, level of gamete dimorphism and gamete parthenogenetic capacity. We assigned states to up to 77 representative species of the taxonomic diversity of the brown algal group, in a multi-gene phylogeny. We used maximum likelihood and Bayesian analyses of correlated evolution, while taking the phylogeny into account, to test for correlations between traits, and to investigate the chronological sequence of trait acquisition. Our analyses are consistent with the prediction that diploid growth evolves when sexual reproduction is preferred over asexual reproduction, possibly because it allows the complementation of deleterious mutations. We also found that haploid sex determination is ancestral in relation to diploid sex determination. However, our results could not address whether increased zygotic and diploid growth are associated with increased sexual dimorphism. Our analyses suggest that in the brown algae, isogamous species evolved from anisogamous ancestors, contrary to the commonly reported pattern where evolution proceeds from isogamy to anisogamy.

While the process of meiosis is highly conserved across eukaryotes, the sexual systems that govern life cycle phase transitions are surprisingly labile. Switches between sexual systems have profound evolutionary and ecological consequences, in particular for plants, but our understanding of the fundamental mechanisms and ultimate causes underlying these transitions is still surprisingly incomplete. We explore here the idea that brown and green algae may be interesting comparative models that can increase our understanding of relevant processes in plant reproductive biology, from evolution of gamete dimorphism, gametogenesis, sex determination and transitions in sex-determining systems.

The spontaneous mutation rate µ is a crucial parameter driving evolution and biodiversity. Mutation rates are highly variable across species, suggesting that µ is susceptible to selection and drift and that species life cycle and life history may impact its evolution. In particular, asexual reproduction and haploid selection are expected to affect mutation rate, but very little empirical data is available to test this expectation. Here, we sequence 30 genomes of a parent-offspring pedigree in the brown algae Ectocarpus to test the impact of its life cycle on mutation rate. Ectocarpus alternates between a haploid and a diploid stage, both multicellular and free living, and utilizes both sexual and asexual reproduction. Ectocarpus is therefore a unique model to empirically test expectations of the effect of asexual reproduction and haploid selection on mutation rate evolution. We estimate that Ectocarpus has a nucleotide mutation rate of µbs=4.07×10−10 per site per generation, a surprisingly low number for a multicellular complex eukaryote. Effective population size (Ne) and genome size could not explain this low mutation rate. We propose that the haploid-diploid life cycle, combined with extensive asexual reproduction may be key drivers of mutation rate.

Ectocarpus is a genus of filamentous, marine brown algae. Brown algae belong to the stramenopiles, a large supergroup of organisms that are only distantly related to animals, land plants and fungi. Brown algae are also one of only a small number of eukaryotic lineages that have evolved complex multicellularity. For many years, little information was available concerning the molecular mechanisms underlying multicellular development in the brown algae, but this situation has changed with the emergence of Ectocarpus as a model brown alga. Here we summarise some of the main questions that are being addressed and areas of study using Ectocarpus as a model organism and discuss how the genomic information, genetic tools and molecular approaches available for this organism are being employed to explore developmental questions in an evolutionary context.

Co-sexuality has evolved repeatedly from unisexual (dioicous) ancestors across a wide range of taxa. However, the molecular changes underpinning this important transition remain unknown, particularly in organisms with haploid sexual systems such as bryophytes, red algae and brown algae. Here we explore four independent events of emergence of co-sexuality from unisexual ancestors in brown algal clades to examine the nature, evolution and degree of convergence of gene expression changes that accompany the breakdown of dioicy. The amounts of male versus female phenotypic differences in dioicous species were not correlated with the extent of sex-biased gene expression, in stark contrast to what is observed in animals. Although sex-biased genes exhibited a high turnover rate during brown alga diversification, some of their predicted functions were conserved across species. Transitions to co-sexuality consistently involved adaptive gene expression shifts and rapid sequence evolution, particularly for male-biased genes. Gene expression in co-sexual species was more similar to that in females rather than males of related dioicous species, suggesting that co-sexuality may have arisen from ancestral females. Finally, extensive convergent gene expression changes, driven by selection, were associated with the transition to co-sexuality. Together, our observations provide insights on how co-sexual systems arise from ancestral, haploid UV sexual systems.

In many eukaryotes, such as dioicous mosses and many algae, sex is determined by UV sex chromosomes and is expressed during the haploid phase of the life cycle. In these species, the male and female developmental programs are initiated by the presence of the U- or V-specific regions of the sex chromosomes but, as in XY and ZW systems, sexual differentiation is largely driven by autosomal sex-biased gene expression. The mechanisms underlying the regulation of sex-biased expression of genes during sexual differentiation remain elusive. Here, we investigated the extent and nature of epigenomic changes associated with UV sexual differentiation in the brown alga Ectocarpus, a model UV system. Six histone modifications were quantified in near-isogenic lines, leading to the identification of 16 chromatin signatures across the genome. Chromatin signatures correlated with levels of gene expression and histone PTMs changes in males versus females occurred preferentially at genes involved in sex-specific pathways. Despite the absence of chromosome scale dosage compensation and the fact that UV sex chromosomes recombine across most of their length, the chromatin landscape of these chromosomes was remarkably different to that of autosomes. Hotspots of evolutionary young genes in the pseudoautosomal regions appear to drive the exceptional chromatin features of UV sex chromosomes.

• Sex-biased gene expression is considered to be an underlying cause of sexually dimorphic traits. Although the nature and degree of sex-biased expression has been well-documented in several animal and plant systems, far less is known about the evolution of sex-biased genes in more distant eukaryotic groups. • Here we investigate sex-biased gene expression in two brown algal dioecious species, Fucus serratus and Fucus vesiculosus, where male heterogamety (XX/XY) has recently emerged. • We find that in contrast to evolutionary distant plant and animal lineages, male-biased genes do not experience high turnover rates, but instead reveal remarkable conservation of bias and expression levels between the two species, suggesting their importance in sexual differentiation. Genes with consistent male-bias were enriched in functions related to gamete production, along with sperm competition and include three flagellar proteins under positive selection. • We present one of the first reports, outside of the animal kingdom, showing that male-biased genes display accelerated rates of coding sequence evolution compared to female-biased or unbiased genes. Our results imply that evolutionary forces affect male and female sex-biased genes differently on structural and regulatory levels, resulting in unique properties of differentially expressed transcripts during reproductive development in Fucus algae.

Co-sexuality has evolved repeatedly from ancestors with separate sexes across a wide range of taxa. The switch to co-sexuality is expected to involve major molecular readjustments at the level of gene expression patterns, as modified males or females will express the opposite sexual function for which their phenotypes have been optimized. However, the molecular changes underpinning this important transition remain unknown, particularly in organisms with haploid sexual systems such as bryophytes, red and brown algae. Here, we explore four independent events of emergence of co-sexuality from uni-sexual (dioicous) ancestors in brown algal clades in order to examine the nature, evolution and degree of convergence of gene expression changes that accompany the breakdown of dioicy. The amount of male versus female phenotypic differences in dioicous species were not correlated with the extent of sex-biased gene expression, in strike contrast to what is observed in animals. Although sex-biased genes exhibited a high turnover rate during brown alga diversification, their predicted functions were remarkably conserved. Transition to co-sexuality consistently involved adaptive gene expression shifts and rapid sequence evolution, particularly of male-biased genes. The gene expression profiles of co-sexual species were more similar to those of females than to males of related dioicous species, suggesting that the former may have arisen from ancestral females. Finally, we identified extensive convergent gene expression changes associated with the transition to co-sexuality, and these changes appear to be driven by selection. Together, our observations provide novel insights on how co-sexual systems arise from ancestral, haploid UV sexual systems.

International audience; Algae are photosynthetic eukaryotes whose taxonomic breadth covers a range of life histories, degrees of cellular and developmental complexity, and diverse patterns of sexual reproduction. These patterns include haploid-and diploid-phase sex determination, and isogamous mating systems, and dimorphic sexes. Despite the ubiquity of sexual reproduction in algae, their mating-type-determination and sex-determination mechanisms have been investigated in only a limited number of representatives. These include volvocine green algae, where sexual cycles and sex-determining mechanisms have shed light on the transition from mating types to sexes, and brown algae, which are a model for UV sex chromosome evolution in the context of a complex haplodiplontic life cycle. Recent advances in genomics have aided progress in understanding sexual cycles in less-studied taxa including ulvophyte, charophyte, and prasinophyte green algae, as well as in diatoms.

SummaryIn UV sexual systems, sex is determined during the haploid phase of the life cycle and males have a V chromosome whereas females have a U chromosome. Previous work in the brown algal model Ectocarpus revealed that the V chromosome has a dominant role in male sex determination and suggested that the female developmental program may occur by ‘default’, triggered in the absence of the male master sex determination gene(s). Here, we describe the identification of a genetically male giant kelp strain presenting phenotypic features typical of a female, despite lacking the U-specific region. The conversion to the female developmental program is however incomplete, because gametes of this feminised male are unable to produce the sperm-attracting pheromone lamoxirene. We identify the transcriptomic patterns underlying the male and female specific developmental programs, and reveal the faster evolutionary rates of male-biased genes compared to female-biased and unbiased genes. Moreover, we show that the phenotypic feminisation of the variant strain is associated with both feminisation and de-masculinisation of gene expression patterns. Importantly, the feminisation phenotype was associated with the dramatic downregulation of two V-specific genes including a candidate sex-determining gene on the V-specific region. Our results reveal the transcriptional changes associated with sexual differentiation in a UV system with marked sexual dimorphism, and contribute to disentangling the role of sex-linked genes and autosomal gene expression in the initiation of the male and female developmental programs. Overall, the data presented here imply that the U-specific region in the giant kelp is not required to initiate the female developmental program, but is critical to produce fully functional eggs, arguing against the idea that female is the ‘default’ sex in this species.

Co-sexuality has evolved repeatedly from unisexual (dioicous) ancestors across a wide range of taxa. However, the molecular changes underpinning this important transition remain unknown, particularly in organisms with haploid sexual systems such as bryophytes, red algae and brown algae. Here we explore four independent events of emergence of co-sexuality from unisexual ancestors in brown algal clades to examine the nature, evolution and degree of convergence of gene expression changes that accompany the breakdown of dioicy. The amounts of male versus female phenotypic differences in dioicous species were not correlated with the extent of sex-biased gene expression, in stark contrast to what is observed in animals. Although sex-biased genes exhibited a high turnover rate during brown alga diversification, some of their predicted functions were conserved across species. Transitions to co-sexuality consistently involved adaptive gene expression shifts and rapid sequence evolution, particularly for male-biased genes. Gene expression in co-sexual species was more similar to that in females rather than males of related dioicous species, suggesting that co-sexuality may have arisen from ancestral females. Finally, extensive convergent gene expression changes, driven by selection, were associated with the transition to co-sexuality. Together, our observations provide insights on how co-sexual systems arise from ancestral, haploid UV sexual systems.

• A comparative study of copper (Cu) toxicity and tolerance in three populations of Fucus serratus was conducted by examining Cu2+ effects on various physiological parameters. • Chlorophyll fluorescence, oxygen evolution, copper content, and relative growth rate of embryos and adults were measured on Cu2+-exposed material. • Algae naturally exposed to elevated total Cu concentration (CuT), were more Cu2+ resistant than those from clean sites, as indicated by higher embryo and adult growth rates and lower copper contents. The Cu2+ tolerance of F. serratus is at least partly inherited and relies partly on metal exclusion. • There were inhibitory effects of Cu2+ on oxygen exchange rates in both tolerant and non-tolerant algae. By contrast to sensitive algae, the maximum efficiency of photosystem II (Fv/Fm), maximum fluorescence (Fm) and zero fluorescence (Fo) of resistant algae were unaffected by Cu2+, whereas decreased quantum yield (ΦPSII) and increased nonphotochemical quenching (NPQ) were most pronounced in resistant algae. Inhibitory effects of Cu2+ on ΦPSII may result in the excitation energy being dissipated through xanthophyll-dependent quenching mechanisms in tolerant algae. In nontolerant algae, lower energy dissipation may result in chlorophyll degradation.

International audience; Brown algae are an important group of multicellular eukaryotes, phylogenetically distinct from both the animal and land plant lineages. Ectocarpus has emerged as a model organism to study diverse aspects of brown algal biology, but this system currently lacks an effective reverse genetics methodology to analyse the functions of selected target genes.Here, we report that mutations at specific target sites are generated following the introduction of CRISPR-Cas9 ribonucleoproteins into Ectocarpus cells, using either biolistics or microinjection as the delivery method.Individuals with mutations affecting the ADENINE PHOSPHORIBOSYL TRANSFERASE (APT) gene were isolated following treatment with 2-fluoroadenine, and this selection system was used to isolate individuals in which mutations had been introduced simultaneously at APT and at a second gene. This double mutation approach could potentially be used to isolate mutants affecting any Ectocarpus gene, providing an effective reverse genetics tool for this model organism.The availability of this tool will significantly enhance the utility of Ectocarpus as a model organism for this ecologically and economically important group of marine organisms. Moreover, the methodology described here should be readily transferable to other brown algal species.

International audience; The haploid-diploid life cycle of the filamentous brown alga Ectocarpus involves alternation between two independent and morphologically distinct multicellular generations, the sporophyte and the gametophyte. Deployment of the sporophyte developmental program requires two TALE homeodomain transcription factors OUROBOROS and SAMSARA. In addition, the sporophyte generation has been shown to secrete a diffusible factor that can induce uni-spores to switch from the gametophyte to the sporophyte developmental program. Here, we determine optimal conditions for production, storage and detection of this diffusible factor and show that it is a heat-resistant, high molecular weight molecule. Based on a combined approach involving proteomic analysis of sporophyte-conditioned medium and the use of biochemical tools to characterize arabinogalactan proteins, we present evidence that sporophyte-conditioned medium contains AGP epitopes and suggest that the diffusible factor may belong to this family of glycoproteins.

International audience; Model organisms are extensively used in research as accessible and convenient systems to study a particular area or question in biology. Traditionally, only a limited number of organisms have been studied in detail, but modern genomic tools are enabling researchers to extend beyond the set of classical model organisms to include novel species from less well-studied phylogenetic groups. This review focuses on model species for an important group of multicellular organisms, the brown algae. The development of genetic and genomic tools for the filamentous brown alga Ectocarpus has led to it emerging as a general model system for this group, but additional models, such as Fucus or Dictyota dichotoma, remain of interest for specific biological questions. In addition, Saccharina japonica has emerged as a model system to directly address applied questions related to algal aquaculture. We discuss the past, present, and future of brown algal model organisms in relation to the opportunities and challenges in brown algal research.

SummaryIn many eukaryotes, such as dioicous mosses and many algae, sex is determined by UV sex chromosomes and is expressed during the haploid phase of the life cycle. In these species, the male and female developmental programs are initiated by the presence of the U- or V-specific regions of the sex chromosomes but, as in XY and ZW systems, phenotypic differentiation is largely driven by autosomal sex-biased gene expression. The mechanisms underlying sex-biased transcription in XY, ZW or UV sexual systems currently remain elusive. Here, we set out to understand the extent and nature of epigenomic changes associated with sexual differentiation in the brown alga Ectocarpus, which has a well described UV system. Five histone modifications, H3K4me3, H3K27Ac, H3K9Ac, H3K36me3, H4K20me3, were quantified in near-isogenic male and female lines, leading to the identification of 13 different chromatin states across the Ectocarpus genome that showed different patterns of enrichment at transcribed, silent, housekeeping or narrowly-expressed genes. Chromatin states were strongly correlated with levels of gene expression indicating a relationship between the assayed marks and gene transcription. The relative proportion of each chromatin state across the genome remained stable in males and females, but a subset of genes exhibited different chromatin states in the two sexes. In particular, males and females displayed distinct patterns of histone modifications at sex-biased genes, indicating that chromatin state transitions occur preferentially at genes involved in sex-specific pathways. Finally, our results reveal a unique chromatin landscape of the U and V sex chromosomes compared to autosomes. Taken together, our observations reveal a role for histone modifications in sex determination and sexual differentiation in a UV sexual system, and suggest that the mechanisms of epigenetic regulation of genes on the UV sex chromosomes may differ from those operating on autosomal genes.

Background Brown algae evolved complex multicellularity independently of the animal and land plant lineages and are the third most developmentally complex phylogenetic group on the planet. An understanding of developmental processes in this group is expected to provide important insights into the evolutionary events necessary for the emergence of complex multicellularity. Here, we focus on mechanisms of epigenetic regulation involving post-translational modifications of histone proteins. Results A total of 47 histone post-translational modifications are identified, including a novel mark H2AZR38me1, but Ectocarpus lacks both H3K27me3 and the major polycomb complexes. ChIP-seq identifies modifications associated with transcription start sites and gene bodies of active genes and with transposons. H3K79me2 exhibits an unusual pattern, often marking large genomic regions spanning several genes. Transcription start sites of closely spaced, divergently transcribed gene pairs share a common nucleosome-depleted region and exhibit shared histone modification peaks. Overall, patterns of histone modifications are stable through the life cycle. Analysis of histone modifications at generation-biased genes identifies a correlation between the presence of specific chromatin marks and the level of gene expression. Conclusions The overview of histone post-translational modifications in the brown alga presented here will provide a foundation for future studies aimed at understanding the role of chromatin modifications in the regulation of brown algal genomes.

International audience; The brown alga Ectocarpus has a haploid-diploid life cycle that involves alternation between two multicellular generations, the sporophyte and the gametophyte. Life cycle generation is not determined by ploidy but by a genetic system that includes two different three amino acid loop extension homeodomain transcription factors called OUROBOROS and SAMSARA. In addition, sporophytes have been shown to secrete a diffusible factor into the medium that can induce gametophyte initial cells to switch from the gametophyte to the sporophyte developmental program. The protocol presented here describes how to produce sporophyte-conditioned medium containing the diffusible sporophyte-inducing factor and how to assay for activity of the factor using a meio-spore-based bioassay. The protocol, which describes how several steps of these procedures can be optimised, will represent a useful tool for future work aimed at characterising the diffusible factor and investigating its mode of action.

Hi-C has become a popular technique in recent genome assembly projects. Hi-C exploits contact frequencies between pairs of loci to bridge and order contigs in draft genomes, resulting in chromosome-level assemblies. However, application of this approach is currently hampered by a lack of robust programs that are capable of effectively treating this type of data, particularly open source programs. We developed instaGRAAL, a complete overhaul of the GRAAL program, which has adapted the latter to allow efficient assembly of large genomes. Both GRAAL, and instaGRAAL use a Markov Chain Monte Carlo algorithm to perform Hi-C scaffolding, but instaGRAAL features a number of improvements including a modular polishing approach that optionally integrates independent data. To validate the program, we used it to generate chromosome-level assemblies for two brown algae, Desmarestia herbacea and the model Ectocarpus sp., and quantified improvements compared to the initial draft for the latter. Overall, instaGRAAL is a program able to generate, using default parameters with minimal human intervention, near-complete assemblies.

Although evolutionary transitions from sexual to asexual reproduction are frequent in eukaryotes, the genetic bases of these shifts remain largely elusive. Here, we used classic quantitative trait analysis, combined with genomic and transcriptomic information to dissect the genetic basis of asexual, parthenogenetic reproduction in the brown algaEctocarpus. We found that parthenogenesis is controlled by the sex locus, together with two additional autosomal loci, highlight the key role of the sex chromosome as a major regulator of asexual reproduction. Importantly, we identify several negative effects of parthenogenesis on male fitness, but also different fitness effects between parthenogenesis and life cycle generations, supporting the idea that parthenogenesis may be under both sexual selection and generation/ploidally-antagonistic selection. Overall, our data provide the first empirical illustration, to our knowledge, of a trade-off between the haploid and diploid stages of the life cycle, where distinct parthenogenesis alleles have opposing effects on sexual and asexual reproduction and may contribute to the maintenance of genetic variation. These types of fitness trade-offs have profound evolutionary implications in natural populations and may structure life history evolution in organisms with haploid-diploid life cycles.

Brown algae are characterized by a remarkable diversity of life cycles, sexual systems, and reproductive modes, and these traits seem to be very labile across the whole group. This diversity makes them ideal models to test existing theories on the evolution of alternation between generations, and to examine correlations between life cycle and reproductive life history traits. In this study, we investigate the dynamics of trait evolution for four life-history traits: life cycle, sexual system, level of gamete dimorphism and gamete parthenogenetic capacity. We assign states to up to 70 species in a multi-gene phylogeny of brown algae, and use maximum likelihood and Bayesian analyses of correlated evolution, taking phylogeny into account, to test for correlations between life history traits and sexual systems, and to investigate the sequence of trait acquisition. Our analyses are consistent with the prediction that diploid growth may evolve because it allows the complementation of deleterious mutations, and that haploid sex determination is ancestral in relation to diploid sex determination. However, the idea that increased zygotic and diploid growth is associated with increased sexual dimorphism is not supported by our analysis. Finally, it appears that in the brown algae isogamous species evolved from anisogamous ancestors.

International audience; Sexual reproduction is a nearly universal feature of eukaryotic organisms. Meiosis appears to have had a single ancient origin, but the mechanisms underlying male or female sex determination are diverse and have emerged repeatedly and independently in the different eukaryotic groups. The brown algae are a group of multicellular photosynthetic eukaryotes that have a distinct evolutionary history compared with animals and plants, as they have been evolving independently for over 1 billion yr. Here, we review recent work using the brown alga Ectocarpus as a model organism to study haploid sex chromosomes, and highlight how the diversity of reproductive and life cycle features of the brown algae offer unique opportunities to characterize the evolutionary forces and the mechanisms underlying the evolution of sex determination.

Most eukaryotes inherit their mitochondria from only one of their parents. When there are different sexes, it is almost always the maternal mitochondria that are transmitted. Indeed, maternal uniparental inheritance has been reported for the brown alga Ectocarpus but we show in this study that different strains of Ectocarpus can exhibit different patterns of inheritance: Ectocarpus siliculosus strains showed maternal uniparental inheritance, as expected, but crosses using different Ectocarpus species 7 strains exhibited either paternal uniparental inheritance or an unusual pattern of transmission where progeny inherited either maternal or paternal mitochondria, but not both. A possible correlation between the pattern of mitochondrial inheritance and male gamete parthenogenesis was investigated. Moreover, in contrast to observations in the green lineage, we did not detect any change in the pattern of mitochondrial inheritance in mutant strains affected in life cycle progression. Finally, an analysis of field-isolated strains provided evidence of mitochondrial genome recombination in both Ectocarpus species.

Three amino acid loop extension homeodomain transcription factors (TALE HD TFs) act as life cycle regulators in green algae and land plants. In mosses these regulators are required for the deployment of the sporophyte developmental program. We demonstrate that mutations in either of two TALE HD TF genes, OUROBOROS or SAMSARA, in the brown alga Ectocarpus result in conversion of the sporophyte generation into a gametophyte. The OUROBOROS and SAMSARA proteins heterodimerise in a similar manner to TALE HD TF life cycle regulators in the green lineage. These observations demonstrate that TALE-HD-TF-based life cycle regulation systems have an extremely ancient origin, and that these systems have been independently recruited to regulate sporophyte developmental programs in at least two different complex multicellular eukaryotic supergroups, Archaeplastida and Chromalveolata.

International audience; Three types of sex chromosome system exist in nature: diploid XY and ZW systems and haploid UV systems. For many years, research has focused exclusively on XY and ZW systems, leaving UV chromosomes and haploid sex determination largely neglected. Here, we perform a detailed analysis of DNA sequence neutral diversity levels across the U and V sex chromosomes of the model brown alga Ectocarpus using a large population dataset. We show that the U and V non-recombining regions of the sex chromosomes (SDR) exhibit about half as much neutral diversity as the autosomes. This difference is consistent with the reduced effective population size of these regions compared with the rest of the genome, suggesting that the influence of additional factors such as background selection or selective sweeps is minimal. The pseudoautosomal region (PAR) of this UV system, in contrast, exhibited surprisingly high neutral diversity and there were several indications that genes in this region may be under balancing selection. The PAR of Ectocarpus is known to exhibit unusual genomic features and our results lay the foundation for further work aimed at understanding whether, and to what extent, these structural features underlie the high level of genetic diversity. Overall, this study fills a gap between available information on genetic diversity in XY/ZW systems and UV systems and significantly contributes to advancing our knowledge of the evolution of UV sex chromosomes.

BackgroundSexual life cycles in eukaryotes involve a cyclic alternation between haploid and diploid phases. While most animals possess a diploid life cycle, plants and algae alternate between multicellular haploid (gametophyte) and diploid (sporophyte) generations. In many algae, gametophytes and sporophytes are independent and free living, and may present dramatic phenotypic differences. The same shared genome can therefore be subject to different, even conflicting, selection pressures in each of the life cycle generations. Here, we have analysed the nature and extent of genome-wide generation-biased gene expression in four species of brown algae with contrasting levels of dimorphism between life cycle generations, in order to assess the potential role of generation-specific selection in shaping patterns of gene expression and divergence.ResultsWe show that the proportion of the transcriptome that is generation-biased is associated with the level of phenotypic dimorphism between the life cycle stages. Importantly, our data reveals a remarkably high turnover rate for life-cycle-related gene sets across the brown algae and highlights the importance not only of co-option of regulatory programs from one generation to the other but also a key role for newly emerged, lineage-specific genes in the evolution of the gametophyte and sporophyte developmental programs in this major eukaryotic group. Moreover, we show that generation-biased genes display distinct evolutionary modes, with gametophyte-biased genes evolving rapidly at the coding sequence level whereas sporophyte-biased genes exhibit changes in their patterns of expression.ConclusionOur analysis uncovers the characteristics, expression patterns and evolution of generation-biased genes and underline the selective forces that shape this previously underappreciated source of phenotypic diversity.

The evolutionary stability of haploid-diploid life cycles is still controversial. Mathematical models indicate that niche differences between ploidy phases may be a necessary condition for the evolution and maintenance of these life cycles. Nevertheless, experimental support for this prediction remains elusive. In the present work, we explored this hypothesis in natural populations of the brown alga Ectocarpus. Consistent with the life cycle described in culture, Ectocarpus crouaniorum in NW France and E. siliculosus in SW Italy exhibited an alternation between haploid gametophytes and diploid sporophytes. Our field data invalidated, however, the long-standing view of an isomorphic alternation of generations. Gametophytes and sporophytes displayed marked differences in size and, conforming to theoretical predictions, occupied different spatiotemporal niches. Gametophytes were found almost exclusively on the alga Scytosiphon lomentaria during spring whereas sporophytes were present year-round on abiotic substrata. Paradoxically, E. siliculosus in NW France exhibited similar habitat usage despite the absence of alternation of ploidy phases. Diploid sporophytes grew both epilithically and epiphytically, and this mainly asexual population gained the same ecological advantage postulated for haploid-diploid populations. Consequently, an ecological interpretation of the niche differences between haploid and diploid individuals does not seem to satisfactorily explain the evolution of the Ectocarpus life cycle.

Brown algae are one of the most developmentally complex groups within the eukaryotes. As in many land plants and animals, their main body axis is established early in development, when the initial cell gives rise to two daughter cells that have apical and basal identities, equivalent to shoot and root identities in land plants, respectively. We show here that mutations in the

International audience

International audience; The sporophyte generation of the brown alga Ectocarpus sp. exhibits an unusual pattern of development compared to the majority of brown algae. The first cell division is symmetrical and the apical/basal axis is established late in development. In the IMMEDIATE UPRIGHT mutant the initial cell undergoes an asymmetric division to immediately establish the apical/basal axis. We provide evidence which suggests that this phenotype corresponds to the ancestral state of the sporophyte. The IMM gene encodes a protein of unknown function, containing a repeated motif also found in the EsV-1-7 gene of the Ectocarpus virus. Brown algae possess large families of EsV-1-7 domain genes but these genes are rare in other stramenopiles suggesting that the expansion of this family may have been linked with the emergence of multicellular complexity. EsV-1-7 domain genes have a patchy distribution across eukaryotic supergroups and occur in several viral genomes, suggesting possible horizontal transfer during eukaryote evolution.

International audience; The genome of the filamentous brown alga Ectocarpus was the first to be completely sequenced from within the brown algal group and has served as a key reference genome both for this lineage and for the stramenopiles. We present a complete structural and functional reannotation of the Ectocarpus genome. The large-scale assembly of the Ectocarpus genome was significantly improved and genome-wide gene re-annotation using extensive RNA-seq data improved the structure of 11 108 existing protein-coding genes and added 2030 new loci. A genome-wide analysis of splicing isoforms identified an average of 1.6 transcripts per locus. A large number of previously undescribed noncoding genes were identified and annotated, including 717 loci that produce long noncoding RNAs. Conservation of lncRNAs between Ectocarpus and another brown alga, the kelp Saccharina japonica, suggests that at least a proportion of these loci serve a function. Finally, a large collection of single nucleotide polymorphism-based markers was developed for genetic analyses. These resources are available through an updated and improved genome database.This study significantly improves the utility of the Ectocarpus genome as a high-quality reference for the study of many important aspects of brown algal biology and as a reference for genomic analyses across the stramenopiles.

Algae are (mostly) photosynthetic eukaryotes that occupy multiple branches of the tree of life, and are vital for planet function and health. This review highlights a transformative period in studies of the evolution and functioning of this extraordinary group of organisms and their potential for novel applications, wrought by high- throughput 'omic' and reverse genetic methods. It covers the origin and diversification of algal groups, explores advances in understanding the link between phenotype and genotype, considers algal sex determination, and reviews progress in understanding the roots of algal multicellularity. Experimental evolution studies to determine how algae evolve in changing environments are highlighted, as is their potential as production platforms for compounds of commercial interest such as biofuel precursors, nutraceuticals, or therapeutics., This manuscript is an outcome of a symposium hosted in June 2016 by The Royal Society entitled ‘Into the genome: advances in the world of algal genomics’, at Chicheley Hall, Buckinghamshire, UK. The symposium organizers, J.B. and D.B., are grateful to the Royal Society for supporting this event. We thank Dr. Doris Gangl for designing Figure 2. The University of Dundee is a registered Scottish charity, No. 015096.