Olivier Godfroy

Min Zheng

Haiqin Yao

Agnes Henschen

Akira F Peters

Delphine Scornet

Sebastien Colin

Paolo Ronchi

Katharina Hipp

Chikako Nagasato

Taizo Motomura

J Mark Cock
email: cock@sb-roscoff.fr

Susana M Coelho
email: susana.coelho@tuebingen.mpg.de

The baseless mutant links protein phosphatase 2A with basal cell identity in the brown alga Ectocarpus

The 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.

Introduction

In most animals and plants key events during the first cell division lead to the establishment of one or more major body axes, providing the foundation for the deployment of the subsequent steps of multicellular developmental programs (reviewed in [START_REF] Radoeva | Evolution, Initiation, and Diversity in Early Plant Embryogenesis[END_REF][START_REF] Rose | Polarity establishment, asymmetric division and segregation of fate determinants in early C. elegans embryos[END_REF]. In animals, partitioning defective (PAR) proteins play a key role in establishing the anterior/posterior body axis, whilst a number of pathways are involved in establishing the dorsoventral axis (e.g. [START_REF] Mongera | Mechanics of Anteroposterior Axis Formation in Vertebrates[END_REF]. In the land plant Arabidopsis, both the apical/basal axis and apical/basal cell identities are established at the time of the first cell division, and genetic analysis has identified two main pathways involved in this process (reviewed in [START_REF] Bayer | Early plant embryogenesis-dark ages or dark matter?[END_REF][START_REF] Ueda | New cues for body axis formation in plant embryos[END_REF]. The first pathway involves SHORT SUSPENSOR (an interleukin-1 receptor-associated kinase/Pelle-like kinase), YODA (a MAP kinase kinase), MPK3 and MPK6 (MAP kinases) and downstream transcription factors, which may include WRKY2 and GROUNDED/RKD4. This pathway may also be influenced maternally through secreted peptide factors such as EMBRYO SURROUNDING FACTOR1 and CLV3/ESR-RELATED8. The second pathway involves auxin and consists of PIN-FORMED 7 (auxin efflux regulator), MONOPTEROS/AUXIN RESPONSE FACTOR 5 (transcription factor) and BODENLOS/INDOLE-3-ACETIC-ACID 12 (auxin response inhibitor). The establishment of zygote polarity is a prerequisite of the asymmetrical first cell division. This process involves movement of the nucleus and other organelles, enlargement of the vacuole, and reorganization of microtubules [START_REF] Kimata | Cytoskeleton dynamics control the first asymmetric cell division in Arabidopsis zygote[END_REF][START_REF] Ueda | New cues for body axis formation in plant embryos[END_REF].

The mechanisms underlying the onset of early development from an initial cell in multicellular plants and animals are relatively well understood, but research has lagged behind for the third most complex group of multicellular eukaryotes, the brown algae. The brown algae offer an interesting contrast to animals and plants because of their phylogenetic position and the fact that they evolved complex multicellularity independently from those groups. Moreover, in brown algae, the two generations are independent and often very distinct morphologically. The same genome, therefore, regulates the set-up of two independent and distinct developmental programs from two different types of initial cells, opening interesting questions about the molecular control of alternation of generations [START_REF] Arun | Convergent recruitment of TALE homeodomain life cycle regulators to direct sporophyte development in land plants and brown algae[END_REF][START_REF] Cock | Evolution and regulation of complex life cycles: a brown algal perspective[END_REF][START_REF] Coelho | Complex life cycles of multicellular eukaryotes: New approaches based on the use of model organisms[END_REF][START_REF] Coelho | OUROBOROS is a master regulator of the gametophyte to sporophyte life cycle transition in the brown alga Ectocarpus[END_REF]. Furthermore, gametophytes and sporophytes of brown algae develop from single cells outside the parent organism, indicating that they likely establish polarity in a cell-autonomous manner, without the involvement of factors delivered from the parental tissues, simplifying the study of polarity, axis establishment and pattern formation.

Investigations using the brown alga Fucus have shown that asymmetrical first cell division is driven by apical-basal polarity established within the zygotic cell [START_REF] Brownlee | Polarity determination in Fucus: from zygote to multicellular embryo[END_REF][START_REF] Goodner | Fucus Embryogenesis: A Model to Study the Establishment of Polarity[END_REF]; reviewed in [START_REF] Bogaert | Polarization of brown algal zygotes[END_REF]. The two daughter cells of this first division divide to produce the apical and basal systems of the alga, the thallus and the rhizoid, respectively [START_REF] Brownlee | Polarity determination in Fucus: from zygote to multicellular embryo[END_REF]. Studies using Fucus zygotes have underlined the role of Ca 2+ asymmetries, mRNA distribution and position-dependent information from the cell wall (involving an unknown diffusible apoplastic factor) in the determination of the fate of the basal and apical systems [START_REF] Berger | Cell fate determination by the cell wall in early fucus development[END_REF][START_REF] Bogaert | Polarization of brown algal zygotes[END_REF][START_REF] Bouget | Localization of Actin mRNA during the Establishment of Cell Polarity and Early Cell Divisions in Fucus Embryos[END_REF][START_REF] Brownlee | Polarity determination in Fucus: from zygote to multicellular embryo[END_REF].

More recently, Ectocarpus has emerged as a suitable model to investigate the molecular mechanisms underlying initial cell division and cell fate determination in the brown algae [START_REF] Bogaert | Polarization of brown algal zygotes[END_REF]. Advantages of this model include the availability of a range of genetic and genomic tools [START_REF] Avia | High-density genetic map and identification of QTLs for responses to temperature and salinity stresses in the model brown alga Ectocarpus[END_REF][START_REF] Badis | Targeted CRISPR-Cas9-based gene knockouts in the model brown alga Ectocarpus[END_REF][START_REF] Bourdareau | Histone modifications during the life cycle of the brown alga Ectocarpus[END_REF][START_REF] Cock | Rapid Evolution of microRNA Loci in the Brown Algae[END_REF][START_REF] Coelho | Ectocarpus: an evo-devo model for the brown algae[END_REF][START_REF] Cormier | Re-annotation, improved large-scale assembly and establishment of a catalogue of noncoding loci for the genome of the model brown alga Ectocarpus[END_REF][START_REF] Umen | Algal sex determination and the evolution of anisogamy[END_REF] but also, importantly, the regularity of the first cell division that characterises the early stages of development of both the gametophyte and sporophyte generations. In this organism, the gametophyte generation exhibits an asymmetrical initial cell division that produces a basal rhizoid cell and an apical cell, the latter further dividing to form the apical system of upright filaments. The upright filaments bear the reproductive structures (plurilocular gametangia, which produce the gametes by mitosis). In contrast, the initial cell of the sporophyte generation undergoes a symmetrical initial cell division to produce two daughter cells with similar fates, both being components of the basal system [START_REF] Godfroy | DISTAG/TBCCd1 Is Required for Basal Cell Fate Determination in Ectocarpus[END_REF][START_REF] Peters | Life-cycle-generation-specific developmental processes are modified in the immediate upright mutant of the brown alga Ectocarpus siliculosus[END_REF](Figure 1). The apical system of the sporophyte (upright filaments) are produced later, after an extensive system of basal filaments has been established. Reproductive structures (unilocular and plurilocular sporangia) are produced on the upright filaments.

Earlier work identified an Ectocarpus mutant, distag (dis), with abnormal cellular features during the first cell division, and that was unable to develop basal systems (rhizoids in the gametophyte, prostrate filaments in the sporophyte) [START_REF] Godfroy | DISTAG/TBCCd1 Is Required for Basal Cell Fate Determination in Ectocarpus[END_REF]. dis mutant alleles exhibit a strong phenotype in the initial cell, with disordered microtubule networks, larger cell size, altered Golgi structure and mispositioned nuclei and centrioles [START_REF] Godfroy | DISTAG/TBCCd1 Is Required for Basal Cell Fate Determination in Ectocarpus[END_REF]. The cell division plane, however, is normal and the cellular defects are only observed during the division of the initial cell, suggesting that DIS function may be specific to this cell. DIS encodes a Tubulin Binding Cofactor C (TBCC) domain protein of the TBCCd1 class, with conserved roles in animal and plants [START_REF] André | The tubulin cofactor C family member TBCCD1 orchestrates cytoskeletal filament formation[END_REF][START_REF] Feldman | ASQ2 encodes a TBCC-like protein required for motherdaughter centriole linkage and mitotic spindle orientation[END_REF].

Here, we report the identification of the BASELESS (BAS) locus in Ectocarpus. bas mutants exhibit phenotypes that closely resemble those of dis mutants, including an atypical initial cell division that leads to failure to deploy a basal system in the adult organism, abnormal cellular features such as disorganised microtubule cytoskeleton, loss of bipolar germination and more extensive Golgi apparatus compared with wild-type cells. These phenotypic features are underlie by important modifications in patterns of gene expression during very early stages of development. BAS encodes a protein phosphatase 2A regulatory subunit type B" with EF-hand domains. Together, our results are consistent with BAS being involved in a pathway that plays a key role in initial cell division and basal cell fate determination during both the gametophyte and sporophyte generations of the Ectocarpus life cycle. [START_REF] Coelho | Ectocarpus: an evo-devo model for the brown algae[END_REF][START_REF] Coelho | Ectocarpus: an evo-devo model for the brown algae[END_REF]. Male and female gametophytes produce male and female gametes, respectively, which are released into the seawater from plurilocular gametangia (pg). Gamete fusion produces a zygote which will initiate the (diploid) sporophyte generation. However, gametes that do not meet a gamete of the opposite sex settle and lose their flagella, and they may still function as initial cells of the sporophyte generation because they can develop parthenogenically into a (haploid) partheno-sporophyte. Note that there is no visible (morphological) difference between haploid and diploid sporophytes. Meiosis occurs in the sporophyte (in the unilocular sporangia, U), producing several hundred haploid meio-spores. These meio-spores are released into the seawater and develop into male or female gametophytes. Partheno-sporophytes and diploid sporophytes may also be maintained by production of mitospores in plurilocular sporangia (ps): released mito-spores recycle the partheno-sporophyte generation (circular arrow in the scheme).

Results

baseless mutants lack a basal system during both the sporophyte and gametophyte generations During the Ectocarpus gametophyte generation, the two cells derived from the division of the initial cell develop as two germ tubes, and establish a rhizoid (a basal, root-like organ) and an upright filamentous thallus (an apical, shoot-like organ) (Figure 2A,2B). The first cell division of the sporophyte generation, in contrast, produces two daughter cells with similar morphology and equivalent cell fates (Figure 2C). These two cells then divide to produce a prostrate filament, which branches to establish the basal system and the apical system differentiates later in A screen of a large population of individuals mutagenised by ultraviolet (UV) irradiation identified two mutant strains (Ec800 and Ec801; Table S1) that failed to develop any of the basal structures normally observed during either the gametophyte or the sporophyte generation of wild-type strains (Figure 2). The screen used gametes, which in absence of fertilisation develop into partheno-sporophytes, being thus initial cells of the sporophyte generation (Figure 1) [START_REF] Coelho | OUROBOROS is a master regulator of the gametophyte to sporophyte life cycle transition in the brown alga Ectocarpus[END_REF][START_REF] Godfroy | Genome-wide comparison of ultraviolet and ethyl methanesulphonate mutagenesis methods for the brown alga Ectocarpus[END_REF][START_REF] Godfroy | DISTAG/TBCCd1 Is Required for Basal Cell Fate Determination in Ectocarpus[END_REF][START_REF] Peters | Life-cycle-generation-specific developmental processes are modified in the immediate upright mutant of the brown alga Ectocarpus siliculosus[END_REF]. Initial cells of Ec800 and Ec801 gametophytes immediately developed as apical upright filament cells and no rhizoid cells were produced. Similarly, during the sporophyte generation, neither mutant strain produced the network of prostrate basal filaments typical of the wild-type sporophyte and, instead, the first divisions of the initial cell directly produced an upright filament (Figure 2).

In wild-type Ectocarpus, secondary rhizoids, which are analogous to the adventitious roots produced from the stems of some land plants [START_REF] Atkinson | Branching out in roots: uncovering form, function, and regulation[END_REF], are produced from apical upright filament cells at a late stage of development (Figure 2E) [START_REF] Peters | Life-cycle-generation-specific developmental processes are modified in the immediate upright mutant of the brown alga Ectocarpus siliculosus[END_REF]. The Ec800 and Ec801 mutants did not produce secondary rhizoids (Figure 2, Figure S1). Hence, production of all basal, attachment structures, both primary and secondary, was abolished in these mutants. Taking into account these phenotypes, the Ec800 and Ec801 mutants were named baseless-1 (bas-1) and baseless-2 (bas-2) respectively.

The establishment of reproductive structures on apical systems in both the gametophyte and sporophyte generations was unaffected in the bas-1 mutant, which was fully fertile after three weeks in culture (Figure 2F-2S). In the bas-2 mutant, the formation of the plurilocular sporangia (which contain mito-spores) was unaffected whereas unilocular sporangia (where meiosis takes place) aborted and no functional meio-spores were produced, preventing generation of gametophytes (Figure 2T).

bas mutants exhibit reduced bipolar germination compared with wild-type strains

In wild-type Ectocarpus, the majority of the initial cells (91%) exhibited a bipolar pattern of germination, with two germ tubes emerging from opposite poles of the initial cell (Figure 2U; [START_REF] Peters | Life-cycle-generation-specific developmental processes are modified in the immediate upright mutant of the brown alga Ectocarpus siliculosus[END_REF]. In contrast, only 21% of the initial cells of bas-1 partheno-sporophytes exhibited this bipolar pattern of germination, the remaining 79% undergoing unipolar germination (Figure 2U). A proportion of the bas-1 partheno-sporophytes that exhibited a bipolar germination pattern produced one or more enlarged and abnormally shaped cells at the extremity where the second germ tube would normally emerge, possibly corresponding to an aborted germ tube (Figure 2O). Similar phenotypes were observed for bas-2 parthenosporophytes (Table S2).

Disorganisation of the microtubule cytoskeleton in bas mutant initial cells Mutations at the DIS locus strongly affect the organisation of the microtubule cytoskeleton [START_REF] Godfroy | DISTAG/TBCCd1 Is Required for Basal Cell Fate Determination in Ectocarpus[END_REF]. Because of the similarity between the morphological phenotypes of bas and dis mutants, we investigated the distribution of the microtubule network during early development of bas mutants compared with wild-type germlings (Figure 3A-B). The microtubule cytoskeleton was markedly disorganised in the bas mutants, with supernumerary microtubule filaments and a disordered network (Figure 3C). This microtubule phenotype is reminiscent of that of the dis mutant [START_REF] Godfroy | DISTAG/TBCCd1 Is Required for Basal Cell Fate Determination in Ectocarpus[END_REF]. Also, similarly to the dis mutant, we did not detect any abnormalities in the positioning of the cell division plane during early development; all bas initial cells produced a cell division plane perpendicular to the growing axis (Figure 2U).

Figure 3. The organization of the microtubule cytoskeleton is affected in bas mutant germinating cells. A) Confocal maximum z-projections showing representative cells of wild type, bas-1, and bas-2 partheno-sporophytes at several stages of early development (24h, 2-5 days). Microtubules (MT) were immune-stained with an anti-tubulin antibody (green). Nuclear DNA was counterstained with DAPI (mauve). Microtubule (MT) bundles were wavy and more abundant in both bas-1 and bas-2 mutant cells compared with the wild-type during the germination of the initial cell. B) Cartoons summarising the stages shown in A) in wild-type and bas mutants. C) Number of microtubule bundles during germination in wild-type (WT), bas-1 and bas-2 mutants at 2 days and 5 days after germination of the initial cell of the sporophyte generation.

Ultrastructural analysis of bas initial cells

Transmission electron microscopy (TEM) and Focused Ion Beam-Scanning Electron Microscopy (FIB-SEM) were used to further characterise the cellular architecture of bas mutants. We focused on the early development of the sporophyte generation, i.e., when unfused gametes had started developing into partheno-sporophytes (2-5 cells), which is the stage when conspicuous morphological differences were observed (Figure 2). This is also the stage when dis mutants exhibit altered subcellular phenotypes, including significantly more abundant cisternae and more fragmented Golgi compared with wild-type cells [START_REF] Godfroy | DISTAG/TBCCd1 Is Required for Basal Cell Fate Determination in Ectocarpus[END_REF].

Morphometric analyses of the subcellular structures in bas mutant and wild-type indicated that the Golgi apparatus were about twice as numerous in bas as in wild-type at the same developmental stage, but this difference was not significant (Figure 4E; Table S3). We also noticed that the number of cisternae per Golgi tended to be slightly higher in bas (Figure 4F, Table S3). Therefore, bas exhibited a Golgi defect, but this defect appeared to be less conspicuous than that of dis mutants. We did not observe any other organellar defects in bas cells, such as abnormal structure or position of the nucleus, centrioles, mitochondria or chloroplasts. Moreover, no visible defect, in particular at the Golgi, could be observed in the gametes prior to their release from the plurilocular gametangia (Figure 4G). This indicates that bas cellular defects are detectable only once the initial cell initiates germination.

Genetic analysis of the BAS gene

A male bas-1 gametophyte (Ec800) was crossed with a wild-type female gametophyte (strain Ec25; (Table S1 andFigure S2). The resulting sporophyte (Ec805) exhibited a wild-type pattern of development, indicating that the bas-1 mutation was recessive. A segregating family of 38 partheno-sporophyte individuals derived from this cross consisted of 16 and 22 phenotypically wild-type and mutant individuals, respectively, consistent with a 1:1 segregation ratio and Mendelian inheritance of a single-locus recessive mutation (Chi-square test with Yates correction = 0.4767, p-value = 0.4899; Table S5). The bas-1 mutation segregated with the phenotype in the 38 individuals used for the phenotype segregation analysis (Table S4).

bas-1 and bas-2 resemble dis mutants but are unaffected in the DIS gene

The phenotypes of bas-1 and bas-2 strongly resembled that of the dis mutant [START_REF] Godfroy | DISTAG/TBCCd1 Is Required for Basal Cell Fate Determination in Ectocarpus[END_REF]. The dis mutant also fails to produce any basal structures, during both the sporophyte and gametophyte generations, and lacks secondary rhizoids, again during both generations. Sporophytes resulting from crosses either between the bas-1 strain Ec800 and strains carrying either the dis-1 or the dis-2 allele had wild-type phenotypes (Figure S3 and Table S1), indicating complementation, and therefore that the DIS gene was not mutated in the bas-1 mutants. gametangia in bas-1 and WT, filled with gametes. Note that no difference in the ultrastructure could be observed in the mutant compared with the wild-type samples prior to the release of the gametes from the plurilocular gametangia. Chl: chloroplast, G: Golgi; N: nucleus.

Figure 4. Sub-cellular architecture of wild-type and bas (bas-2) germinating cells. 3D visualization with FIB-SEM (focused ion beam scanning electron microscopy) of representative wild-type (A-B) and bas-2 mutant (C-D) developing sporophytes (2 cell stage), with Golgi (cyan) and nucleus (violet) highlighted. The scale bar represents 200 nm. E) Number of cisternae per Golgi apparatus in wild-type and bas-2 developing sporophytes (2 cell stage). F) Number of Golgi apparatus per cell in wild-type and bas-2 developing sporophytes (2 cell stage). G) Plurilocular

BAS encodes a protein phosphatase 2A type B" regulatory subunit Genome resequencing identified a candidate locus on chromosome 21 for the location of the bas-1 and bas-2 mutations (Figure 5). Whole genome resequencing (WGS) was carried out for the Ec800 (bas-1) and Ec801 (bas-2) mutant strains and the data compared to the wild-type Ectocarpus sp. strain Ec32 reference genome. More than 41,000 putative variants were detected for each strain. Those variants were compared to a list of 567,532 variants called during the analysis of 14 other mutant lines that showed a range of different phenotypes (Table S5) and shared variants were eliminated. This approach allowed the identification of 827 and 769 variants that were unique to the Ec800 and Ec801 mutants, respectively. Quality filtering of those variants (see methods for details) resulted in 118 and 67 putative mutations for the Ec800 and Ec801 strains, respectively, corresponding to one mutation every 1.7 to 3.0 Mb of genome. Of these 185 putative mutations, 26 and 15 were in coding regions (CDS) (22%) in Ec800 and Ec801, respectively. Only one gene (locusID: Ec-21_001770) contained a CDS mutation in both strains. A single nucleotide transition from T to C, at position 2,806,985 was identified in the bas-1 mutant (strain Ec800) and a G to A transition at position 2,807,321 in the bas-2 mutant (strain Ec801) (Figure 5). The bas-1 mutation segregated with the phenotype in the 38 individuals used for the phenotype segregation analysis (Table S6).

The Ec-21_001770 gene encodes a protein of 646 amino-acids similar to protein phosphatase 2A regulatory subunit type B" proteins. This polypeptide contains three predicted functional domains: a disordered region between positions 50 to 185 and two EF-hand domains at positions 280 to 370 and 380 to 550. The bas-1 mutation affects the first EF-hand, replacing a positively charged lysine residue with a negatively charged glutamic acid (K302E). This modification of electric charge may disrupt domain folding and/or function at least for the first EF-hand. It is possible that the bas-1 mutation leads to the production of a protein that is partially active. The bas-2 mutant carries a non-sense mutation that creates a premature stop codon at position 190 of the protein. This mutation is predicted to result in the production of a truncated protein that lacks both EF-hand domains (Figure 5A).

BAS is predicted to encode a protein phosphatase 2A regulatory B" subunit. PP2A phosphatases are protein complexes usually composed of three subunits, a catalytic C subunit, a scaffolding A subunit and a regulatory B subunit [START_REF] Wlodarchak | PP2A as a master regulator of the cell cycle[END_REF]. Most species have multiple forms of each subunit and there are four distinct classes of the B subunit (B/B55/PR55, Bʹ/B56/PR61, Bʹʹ/PR72/PR130 and Bʹʹʹ/Striatin), which are unrelated at the sequence level. An analysis of the Ectocarpus sp. genome revealed that it encodes B, B' and B" subunits, but not B'"/Striatin (Figure 5B). The BAS protein is predicted to belong to the B" class, homologous to the PR130/PR72 human protein (Figure 5B). RNA-seq data [START_REF] Bourdareau | Contrôle génétique et épigénétique des transitions du cycle de vie chez l'algue brune Ectocarpus sp[END_REF][START_REF] Coelho | OUROBOROS is a master regulator of the gametophyte to sporophyte life cycle transition in the brown alga Ectocarpus[END_REF][START_REF] Godfroy | DISTAG/TBCCd1 Is Required for Basal Cell Fate Determination in Ectocarpus[END_REF][START_REF] Macaisne | The Ectocarpus IMMEDIATE UPRIGHT gene encodes a member of a novel family of cysteine-rich proteins that have an unusual distribution across the eukaryotes[END_REF] were analysed to investigate BAS gene expression during the Ectocarpus life cycle. BAS transcripts were detected throughout development, during both the gametophyte and sporophyte generations (Figure 6A). The BAS transcript was most abundant at the gamete stage (after the release from plurilocular gametangia) and had decreased in abundance about one week after germination, at the 2-5 cell stage (Figure 6A; Tables S7). This pattern of expression is consistent with a role of BAS in the early divisions of the initial cells of the parthenosporophyte generation provided that the transcript and/or protein persists in the initial cell during the first cell division. Interestingly, during the life cycle, the abundance of the BAS transcript was inversely related to the abundance of the DIS transcript, which was at a very low level in gametes but increased in abundance at later stages of development (Figure 6, Table S7, S8). The similar phenotypes of dis and bas mutants suggest that the products of the two genes may play roles in common cellular processes. We investigated the expression of DIS in a bas background, and, conversely, the expression of BAS in a dis background. We noticed that in a bas background, DIS expression in early stage (2-5 cell) was significantly higher (p-value=4.13E-02 and p-value=7.66E-02 respectively for bas-1 and bas-2 comparison with wild-type at the 2-5 cell stage, Figure 6B; Table S7), whereas no difference was observed in the levels of expression of BAS in absence of dis gene product. Taken together, these analyses suggest that BAS expression levels are not affected by DIS, but, conversely, DIS gene expression is disturbed by mutations at the BAS locus. This observation, however, is unlikely to explain the phenotypic similarity between bas and dis mutants.

BAS expression during

Analysis of the bas transcriptome

To further characterize the bas phenotype, an RNA-seq approach was employed to study gene expression in the bas-1 and bas-2 mutants compared with wild-type. We focused on the 2-5 cell stage during gamete germination because the bas phenotype was prominent during the early stages of development. Given the subtle differences in phenotype between bas-1 and bas-2 (in particular the meiotic defect in bas-2) we also focused on comparing bas-1 and bas-2 transcriptional patterns.

Overall, gene expression patterns in bas-1 and bas-2 were comparable, and both were different from the wild-type samples (Figure S4-S5; Tables S7-S9). However, bas-2 exhibited more differences compared with wild-type samples (Figure S4-S5): 40% of the transcriptome was differentially regulated in the comparison bas-2 versus wild-type, whereas 20% of the genes was differentially expressed (DE) between bas-1 and wild-type (Figure S6, Table S8, see Material and Methods for thresholds). Most genes were expressed under all conditions, only about 4% of the genes were not expressed in any of the samples.

Differentially expressed (DE) genes exhibited very similar patterns of up and down regulation in the two bas mutants compared to wild-type. Only 11 genes exhibited divergent expression patterns, i.e., up-regulated in bas-1 and down-regulated in bas-2, and six genes were upregulated in bas-2 and down-regulated in bas-1. The vast majority of the DE genes in bas-1 vs WT were also differentially expressed in bas-2 vs wild-type comparison (76.69% of the downregulated genes and 76.73% of the up-regulated genes) (Figure 7A, Table S9).

Down-regulated genes in bas-1 compared to wild-type had, globally, lower expression levels in bas-2; and up-regulated genes in bas-1 were slightly more expressed in bas-2 (Figure 7B). Conversely, down-regulated genes in bas-2 compared to WT had expression levels in bas-1 similar to wild-type samples; while up-regulated genes in bas-2 had intermediate expression level compare to wild-type and bas-2 (Figure 7B, Table S9). Comparison of the fold-change distribution of the DE genes from bas-1 vs wild-type with those of the DE genes from bas-2 vs wild-type showed that DE genes exhibited greater fold changes in bas-2 than in bas-1 (Figure 7C, D). Overall, these observations indicate similar changes in transcriptome of both bas mutants, with a stronger effect in bas-2 than in bas-1 compared to wild-type. Those results are coherent with the predicted effects of the two mutations on the BAS protein. The bas-1 mutation may result in the production of a protein partially active whereas the bas-2 mutation leads to a truncated protein with probably no activity.

GO term enrichment analyses of DE genes revealed that functions related to photosynthesis and metabolism were down regulated in both bas mutants whereas upregulated genes in mutants were associated with intracellular protein transport transcription and protein synthesis (Figure S7, Table S10). It is interesting to note that some of those functions can be linked with the Golgi apparatus, which appears to be affected by the bas mutation (see above). Using the HECTAR predictor [START_REF] Gschloessl | HECTAR: a method to predict subcellular targeting in heterokonts[END_REF], we examined the enrichment of DE genes in particular sub-cellular localizations. In coherence with GO term enrichment analyses, we observed an enrichment in "chloroplast" localization of the down-regulated genes in both bas mutants. We also observed a slight but strongly significant enrichment in proteins with a "signal peptide" suggesting an impact of the bas mutation on the production of secreted proteins (Table S11), which, again, may be consistent with defects observed in bas mutants at the level of the Golgi. Finally, we noted that 13 out of the 26 predicted BASIC LEUCINE-ZIPPER (bZIP) transcription factors in Ectocarpus are differentially expressed in bas-2 mutant compared to wild-type (Table S9).

We identified 26 and 41 genes that were exclusively expressed in the bas-1 and bas-2 respectively compared with the wild-type (i.e., genes that had TMP=0 in wild-type samples).

Seventeen genes were silenced in the bas-1 and 29 in bas-2 compared with wild-type (Table S12 andS13, see Material and Methods for thresholds). Most of those genes have unknown functions and, due to the low numbers of genes, no significant functional enrichment could be established. However, it is worth noting several transcription factors, which may be potential effector genes: such as Ec-14_003940 and Ec-16_000350, which are silenced in bas-1 and/or bas-2 and Ec-00_004890, which is activated in both mutants compared with the wild-type; and numerous genes associated with "Cellular regulation and signalling" or "Membrane function and transport". Looking at the putative localisation of those proteins, we observed that about 30% of the genes specifically silenced in bas-1 and/or bas-2 have "signal peptide" or "signal anchor" prediction, suggested a membrane or cell-wall localization, whereas only 16% of the total predicted proteins in the genome of Ectocarpus present this signature (Table S12). This two-fold enrichment is even higher than the "signal peptide" enrichment found in downregulated genes (Table S11).

Discussion

The BAS gene is involved in apical-basal axis formation in Ectocarpus

The two Ectocarpus mutant alleles identified in this study, bas-1 and bas-2, lack basal structures during both the gametophyte and the sporophyte generations of the life cycle. Analysis of the initial cells of the bas mutant showed that the morphological phenotype was associated with several cellular anomalies during germination and the first cell division, including disorganisation of the microtubule network, an increase in the number of microtubule bundles and Golgi apparatus and unipolar, rather than bi-polar, germination patterns. These observations highlight a key role for the BAS protein during the development of Ectocarpus. In particular, the BAS protein appears to operate during key cell divisions during development: the (first) initial cell division and, at later stages of development, during meiosis. However, no cellular defect was observed prior to initial cell divisions, suggesting that BAS operates only after release of the initial cells from the reproductive structures. The meiotic defect observed in the bas-2 mutant suggests that the BAS protein may have also a role during meiotic cell division. The absence of this meiotic defect in the bas-1 may be explained by the production of a protein with sufficient activity in bas-1 to ensure its role during meiosis but not during the first cell division. Higher penetrance of the bas-2 mutation is also indicated by the greater proportion of differentially expressed genes in bas-2 compared to bas-1. Indeed, about 40% of the genome was significantly mis-regulated in bas-2 mutant during early stages of development, demonstrating that mutations at the BAS locus can lead to broad, large-scale modifications to the transcriptome. It is interesting to note that half of the bZIP transcription factors of Ectocarpus are differentially expressed in bas-2 compared to wild-type. This observation suggests that BAS may part of a pathway involving additional regulatory proteins that drives the establishment of apical-basal axis during the early development of Ectocarpus.

BAS Encodes a PP2A protein with roles in cellular organization and development in animals and plants

Ectocarpus BAS is predicted to encode a PP2A phosphatase regulatory B" subunit. In animals, PP2A phosphatases are involved in diverse cellular processes and constitute a major component of cellular serine/threonine phosphatase activity, dephosphorylating several hundred cellular substrates [START_REF] Reynhout | Physiologic functions of PP2A: Lessons from genetically modified mice[END_REF][START_REF] Wlodarchak | PP2A as a master regulator of the cell cycle[END_REF]. PP2A has been implicated in the reorganization of several cellular structures, playing key roles in nuclear envelope breakdown during mitosis, and chromosome segregation via effects on assembly of the mitotic spindle and attachment of cytoplasmic microtubules to kinetochores. It is also involved in rearrangement of endoplasmic reticulum and the Golgi apparatus (reviewed in [START_REF] Wlodarchak | PP2A as a master regulator of the cell cycle[END_REF][START_REF] Wurzenberger | Phosphatases: providing safe passage through mitotic exit[END_REF]. In particular, the PP2A B'' subunit PR130 associates with CG-NAP that localizes to centrosomes and the Golgi apparatus [START_REF] Takahashi | Characterization of a novel giant scaffolding protein, CG-NAP, that anchors multiple signaling enzymes to centrosome and the golgi apparatus[END_REF], and restacking of newly formed Golgi cisternae requires dephosphorylation of Golgi stacking proteins by PP2A [START_REF] Tang | Molecular mechanism of mitotic Golgi disassembly and reassembly revealed by a defined reconstitution assay[END_REF]. More broadly, the animal PP2A Bʹʹ subunit is critical for cell-cell communication, cell adhesion, migration, proliferation and differentiation during animal development [START_REF] Creyghton | PR72, a novel regulator of Wnt signaling required for Naked cuticle function[END_REF][START_REF] Zwaenepoel | Protein phosphatase 2A PR130/B''alpha1 subunit binds to the SH2 domain-containing inositol polyphosphate 5-phosphatase 2 and prevents epidermal growth factor (EGF)-induced EGF receptor degradation sustaining EGF-mediated signaling[END_REF]. Altogether, these observations link animal PP2A to roles in cell division, subcellular features and developmental pattern formation. In the multicellular brown alga Ectocarpus, our results are consistent with a similar role for PP2A in subcellular organisation, cell division and cell fate determination. Our results therefore provide an example of protein functional conservation across eukaryotes that evolved multicellularity independently, suggesting that the role of PP2A in multicellular development has likely been preserved across very divergent lineages.

Consistent with a conserved role for PP2A in development across multicellular eukaryotes, the Arabidopsis B''-δ/ε subunit of PP2A (At5g28900/At5g28850) interacts with BASIC LEUCINE-ZIPPER (bZIP) transcription factors, and is implicated in leaf and root development as well as mechanical stress response [START_REF] Tsugama | Protein phosphatase 2A regulates the nuclear accumulation of the Arabidopsis bZIP protein VIP1 under hypoosmotic stress[END_REF][START_REF] Van Leene | Functional characterization of the Arabidopsis transcription factor bZIP29 reveals its role in leaf and root development[END_REF]. The PP2A regulatory B" subunit FASS/TON2, is essential for the reorganisation of cortical microtubular arrays into a dense preprophase band preceding cell division. FASS-containing PP2A complexes are targeted to microtubules through an association with TONNEAU1 (TON1) and TON1-recruiting motif protein (TRM) [START_REF] Spinner | The function of TONNEAU1 in moss reveals ancient mechanisms of division plane specification and cell elongation in land plants[END_REF]. PP2A interacts with and dephosphorylates KATANIN, a evolutionarily conserved microtubule-severing enzyme, to promote the formation of circumferential cortical microtubule arrays in Arabidopsis [START_REF] Ren | PP2A interacts with KATANIN to promote microtubule organization and conical cell morphogenesis[END_REF]. The Ectocarpus BAS protein is related to the Arabidopsis FASS/TON2 protein but is orthologous to the AtB"-δ/ε protein mentioned above (At5g28900/At5g28850). The microtubule phenotype we describe here, where PP2A B'' disruption in brown algae is associated with microtubule disorganisation, may further underline a conserved role for PP2A across plants and brown algae.

Golgi vesicle transport has been shown to play an important role in the establishment of the Fucus zygote polar axis prior to the first cell division (reviewed in [START_REF] Bogaert | Polarization of brown algal zygotes[END_REF][START_REF] Shaw | The role of targeted secretion in the establishment of cell polarity and the orientation of the division plane in Fucus zygotes[END_REF]). It has been proposed that the selective targeting of Golgi vesicles to the plasma membrane locally modifies the cell wall, participating in the establishment of the asymmetry of the cell wall required for rhizoid differentiation [START_REF] Bogaert | Brown algae as a model for plant organogenesis[END_REF][START_REF] Bogaert | Polarization of brown algal zygotes[END_REF][START_REF] Goodner | Fucus Embryogenesis: A Model to Study the Establishment of Polarity[END_REF]. In this model, the cell wall plays a key role in the establishment of the initial cell asymmetry, which is interesting with regard to the dramatic changes in expression of numerous genes with putative "Membrane and transport" functions in bas mutants.

In animals, the B'' class of PP2A B subunit is thought to be involved in Ca 2+ signalling through the presence of multiple EF-hand domains [START_REF] Xu | Targeting of protein phosphatases PP2A and PP2B to the C-terminus of the L-type calcium channel Ca v1.2[END_REF]. Note that two EF-hand domains are predicted in the BAS protein, and these EF-hands are absent or non-functional in bas mutants, suggesting that Ca 2+ binding may be disturbed. In the brown alga Fucus, Ca 2+ gradients have been shown to have a crucial function in zygote polarization and the establishment of the apical/basal axis during first cell division (reviewed in [START_REF] Bogaert | Polarization of brown algal zygotes[END_REF][START_REF] Brownlee | Polarity determination in Fucus: from zygote to multicellular embryo[END_REF]. In particular, cytosolic free Ca 2+ accumulates on the side of the growing (basal) rhizoid, directly linking intracellular Ca 2+ signaling and acquisition of basal cell identity. In this context, we can speculate that the BAS protein may be a potential sensor of a pre-germination Ca 2+ signal.

BAS and DIS may act in concert to mediate cell fate determination during the first cell division Similar morphological and cellular phenotypes were observed for bas and dis mutant strains suggesting that BAS and DIS may be involved in similar cellular processes. However, some differences between the mutants are also apparent, such as the less marked Golgi fragmentation and no effect on nuclear positioning in early stages of development in bas, and also a meiotic defect was only observed in bas-2.

DIS is predicted to encode a TBCCd1 protein. This protein shares similarity with TBCC, which is a component of the complex (TBCA to TBCE) that mediates dimerization of a and b tubulin subunits to form microtubules [START_REF] Nithianantham | Tubulin cofactors and Arl2 are cage-like chaperones that regulate the soluble αβtubulin pool for microtubule dynamics[END_REF][START_REF] Tian | Pathway leading to correctly folded beta-tubulin[END_REF]. However, TBCCd1 lacks a conserved arginine residue that is essential for TBCC activity and is unable to complement TBCC in yeast indicating that the two proteins may have different biochemical functions [START_REF] Goncalves | TBCCD1, a new centrosomal protein, is required for centrosome and Golgi apparatus positioning[END_REF]. TBCCd1 has been localized to both the centrosome and the Golgi in humans, Chlamydomonas, and trypanosomes and there is evidence that TBCCd1 plays important roles in positioning organelles within the cells of these diverse organisms [START_REF] André | The tubulin cofactor C family member TBCCD1 orchestrates cytoskeletal filament formation[END_REF][START_REF] Feldman | ASQ2 encodes a TBCC-like protein required for motherdaughter centriole linkage and mitotic spindle orientation[END_REF][START_REF] Goncalves | TBCCD1, a new centrosomal protein, is required for centrosome and Golgi apparatus positioning[END_REF]. However, the molecular mechanisms underlying these cellular phenotypes are unclear and they may not involve direct effects on microtubule assembly [START_REF] Goncalves | TBCCD1, a new centrosomal protein, is required for centrosome and Golgi apparatus positioning[END_REF].

Interestingly, analysis of human protein phosphatase interactions revealed that TBCCd1 is a partner of PP2A regulatory subunit B'' [START_REF] Huttlin | The BioPlex Network: A Systematic Exploration of the Human Interactome[END_REF][START_REF] Huttlin | Architecture of the human interactome defines protein communities and disease networks[END_REF][START_REF] Yadav | Systematic Analysis of Human Protein Phosphatase Interactions and Dynamics[END_REF]. If this interaction also occurs in brown algae, it would provide a mechanism whereby DIS and BAS could act within the same pathway involved in cell fate determination, with BAS regulating the DIS protein. Further analysis of the biochemical functions of BAS and DIS will be necessary to test this hypothesis.

To summarise, both TBCCd1 and PP2A have been linked to cytoskeleton and Golgi function and both proteins have been shown to play important roles in the regulation of cellular architecture in diverse eukaryotic systems. These observations are consistent with the pleiotropic cellular phenotypes of both the dis and bas mutants. We suggest that the observed morphological and cell fate (loss of basal cells) phenotypes of the bas and dis mutants are a consequence of cellular defects during the first cell division, perhaps through disruption of the distribution of hypothetical cell-fate-determining factors during this critical step of development (see model proposed by [START_REF] Godfroy | DISTAG/TBCCd1 Is Required for Basal Cell Fate Determination in Ectocarpus[END_REF]. Combining information about the Ectocarpus DIS and BAS proteins with observations of Ca 2+ waves in the Fucus embryo, we can speculate that BAS may be involved in sensing an intracellular Ca 2+ signal which participates to the distribution of a cellfate determining factor through reorganization of cytoskeleton and Golgi function involving DIS.

Methods

UV Mutagenesis and isolation of mutant strains

Strain cultivation, genetic crosses, raising of sporophytes from zygotes, and isolation of meiotic families were performed as described previously (Coelho et al., 2012a;Coelho et al., 2012b;[START_REF] Coelho | Ectocarpus: an evo-devo model for the brown algae[END_REF][START_REF] Godfroy | DISTAG/TBCCd1 Is Required for Basal Cell Fate Determination in Ectocarpus[END_REF]. Ectocarpus sp. (species 7, [START_REF] Montecinos | Species delimitation and phylogeographic analyses in the Ectocarpus subgroup siliculosi (Ectocarpales, Phaeophyceae)[END_REF] gametes are able to develop parthenogenically to produce haploid partheno-sporophytes, which are identical morphologically to the sporophytes that develop from diploid zygotes [START_REF] Bogaert | Polarization of brown algal zygotes[END_REF][START_REF] Bothwell | Cell cycles and endocycles in the model brown seaweed, Ectocarpus siliculosus[END_REF][START_REF] Peters | Life-cycle-generation-specific developmental processes are modified in the immediate upright mutant of the brown alga Ectocarpus siliculosus[END_REF]. This phenomenon was exploited to screen directly, in a haploid population, for mutants affected in early sporophyte development. UV mutagenesis of gametes was performed as described previously [START_REF] Coelho | OUROBOROS is a master regulator of the gametophyte to sporophyte life cycle transition in the brown alga Ectocarpus[END_REF][START_REF] Godfroy | Genome-wide comparison of ultraviolet and ethyl methanesulphonate mutagenesis methods for the brown alga Ectocarpus[END_REF][START_REF] Godfroy | DISTAG/TBCCd1 Is Required for Basal Cell Fate Determination in Ectocarpus[END_REF] and mutant partheno-sporophytes lacking basal structures were identified by visual screening under a light microscope. Table S1 describes the strains used in this study.

Genetic analysis of bas mutants Genetic crosses were performed as in (Coelho et al., 2012a). The bas-1 mutant (Ec800) was crossed with the outcrossing line Ec568 to generate a segregating population of 38 individuals. Each of the 38 individuals was derived from a different unilocular sporangium (each unilocular sporangium contains 50-100 meio-spores, derived from a single meiosis followed by at least five mitotic divisions). The meio-spores germinated to produce gametophytes, which were isolated and allowed to produce gametes which germinated parthenogenically. The resulting partheno-sporophytes were then observed under a light microscope to determine whether they exhibited the bas phenotype. The presence of the bas-1 mutation was determined by Sanger sequencing of PCR products (Forward: TGACGAATGATGCTAAACTGGA, Reverse: GACAACGGAGCAGACGAAC) for each of the 38 individuals. The bas-2 mutant (Ec801) was not usable for crosses because it did not form functional unilocular sporangia, which are required for gametophyte production.

Identification of candidate mutations

Genomic DNA from Ec800 and Ec801 strains was sequenced on an Illumina HiSeq4000 platform (1/12 th lane; 2x150nt paired-end; 8.50 and 7.95 Gbp of data, respectively; Fasteris, Switzerland). After quality cleaning using Trimmomatic [START_REF] Bolger | Trimmomatic: a flexible trimmer for Illumina sequence data[END_REF], the reads were mapped onto the Ectocarpus sp. reference genome [START_REF] Cormier | Re-annotation, improved large-scale assembly and establishment of a catalogue of noncoding loci for the genome of the model brown alga Ectocarpus[END_REF] using Bowtie2 [START_REF] Langmead | Fast gapped-read alignment with Bowtie 2[END_REF]. Coverage depth and breadth were, respectively, 34x and 96.83% for Ec800 and 32x and 96.81% for Ec801. Variants were called and counted using bcftools mpileup (http://samtools.github.io/). These variants were compared with a list of variants identified in genome sequence data for 14 other Ectocarpus mutant lines in order to remove false positive mutations due, for example, to errors in the reference genome sequence.

Variants unique to strains Ec800 and Ec801 were quality filtered based on coverage depth (±50% of the genome mean), mapping quality (>20), variant quality (>50), variant frequency (>0.9) and variant support in both sequencing directions. A custom python script allowed the identification of variants in coding regions (CDS) and the effect of each CDS mutation on the predicted protein was accessed manually (Table S5). A scheme of the approach is depicted in Figure S7.

Immunostaining Ectocarpus samples were processed as described (Coelho et al., 2012c) using a protocol adapted from [START_REF] Bisgrove | Alignment of centrosomal and growth axes is a late event during polarization of Pelvetia compressa zygotes[END_REF]. Briefly, Ectocarpus cells were settled on cover slips and at appropriate times after settlement were rapidly frozen in liquid nitrogen and fixed in 2.5% glutaraldehyde and 3.2% paraformaldehyde for 1 h, then washed in PBS and treated with approximately 1 day at 14°C in 12 h light/12 h dark regime to produce two-to five-cell stage partheno-sporophytes. Cells in capillaries were frozen at high-pressure (HPF Compact 03, Engineering Office M. Wohlwend GmbH), freeze-substituted (AFS2, Leica Microsystems) with 0.2% OsO4 and 0.1% uranyl acetate in acetone containing 5% H2O as substitution medium [START_REF] Read | Quantitative Electron Microscopy to Study HCMV Morphogenesis[END_REF] and embedded in Epon. Ultrathin sections were stained with uranyl acetate and lead citrate and analysed with a Tecnai Spirit (Thermo Fisher Scientific) operated at 120 kV.

In order to identify a region of the sample containing algae at high density for FIB-SEM data acquisition, a 3D X-ray tomogram of the resin block was acquired with a Bruker Skyscan 1272. The region of interest was exposed using a 90˚ diamond trimming knife (Cryotrim 90, Diatome) mounted on a Leica UC7 ultramicrotome. The sample was then attached on a stub using conductive silver epoxy resin (Ted Pella) and gold sputter coated (Quorum Q150RS).

FIB-SEM imaging was performed with a Zeiss Crossbeam 540 or a Crossbeam 550, using Atlas 3D (FIBICS, Carl Zeiss Microscopy) for sample preparation and acquisition. After the deposition of a protecting platinum coat on the surface above the region of interest, a 60 µm wide trench was opened to identify and image several cells in parallel. During the stack acquisition, FIB slicing was done with at 30 kV and 700 pA current. The datasets were acquired at 8 nm isotropic voxel size with the SEM at 1.5 kV and 700 pA current, using a back-scattered electron detector (ESB). After acquisition, the image stacks were acquired using the Fiji plugin "Linear stack alignment with SIFT" [START_REF] Lowe | Distinctive Image Features from Scale-Invariant Keypoints[END_REF]. We acquired images for a total of 5 cells for wild-type and 5 bas-2 cells, and chose a representative image to present in Figure 4. Images containing Golgi stacks were retrieved from aligned image stacks and cropped to reduce the image dimensions for further segmentation with the IMOD software package (https://bio3d.colorado.edu/imod/).

Phylogenetic trees

Multiple alignments were generated with Muscle in MEGA7 [START_REF] Kumar | MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets[END_REF]. Phylogenetic trees were then generated with RAxML (Stamatakis, 2014) using 1000 bootstrap replicates and the most appropriate model.

BAS and DIS gene expression estimation during the Ectocarpus life cycle

Expression levels of the BAS and DIS genes were investigated using TPM values obtained after kallisto pseudo-mapping and calculation of the lengthScaledTPM using the tximport package in R (see 'Comparative Transcriptome Analyses' section for details). Previously generated RNA-seq data for wild type and dis samples (Table S7) was used for comparisons.

Comparative transcriptome analyses RNA-seq analysis was performed to compare the abundances of gene transcripts in the mutants bas-1 and bas-2 and wild-type sporophytes. RNA-seq datasets were generated from triplicate samples of each genotype, and individuals were grown synchronously as described previously in standard culture conditions (Coelho et al., 2012b;[START_REF] Cossard | Selection drives convergent gene expression changes during transitions to cosexuality in haploid sexual systems[END_REF]. Germlings were filtered through a nylon mesh to recover only thalli at the 2-5 cell stage. Each replicate contained between 10 4 and 10 6 individual germlings. Tissue samples were rapidly frozen in liquid nitrogen and processed for RNA extraction. Total RNA was extracted from each sample using the Qiagen Mini kit as previously described [START_REF] Lipinska | Sexual dimorphism and the evolution of sex-biased gene expression in the brown alga Ectocarpus[END_REF]. For each replicate, cDNA was produced by oligo(dT) priming, fragmented, and prepared for stranded 2× 150-bp paired-end sequencing on an Illumina HiSeq 3000 platform.

Raw and cleaned read quality was evaluated using fastQC (v. 0.11.9) and mutliQC (v. 1.9). Raw reads were trimmed and filtered based on quality score, and adapter sequences were removed using Trimmomatic (v. 0.39). Transcript abundance was evaluate using kallisto (v. 0.46.2) via pseudo mapping on CDS features. Then matrix of counts and TPMs for all samples, all replicates were generated in R using tximport package.

A gene was considered to be expressed if the TPMmean was above the 5 th percentile (as in [START_REF] Cossard | Selection drives convergent gene expression changes during transitions to cosexuality in haploid sexual systems[END_REF][START_REF] Lipinska | Rapid turnover of life-cycle-related genes in the brown algae[END_REF]. About 4% of the genes in each sample had TPMmean values under this threshold and were considered not to be expressed in our samples (Table S9). Differential gene expression was analysed using DESeq2 (Love et al., 2014, 2). Genes were considered to be differentially expressed when the log2 fold change was below or equal -1 (down-regulated, at least twice as weakly expressed) or above or equal to 1 (up-regulated, at least twice as strongly expressed) and adjusted p-value below or equal to 0.01. Genes were considered to be exclusively (uniquely) expressed in bas mutants when they were significantly up-regulated compared to wild-type and the wild-type mean TPM was equal to 0. Conversely, genes were considered to be specifically silenced in bas mutants when there were significantly down-regulated compared to wild-type and their TPM means were equal to 0.

GO term and HECTAR localisation enrichment were carried out on R using the "clusterProfiler" package [START_REF] Yu | clusterProfiler: an R package for comparing biological themes among gene clusters[END_REF].

Accession Numbers

Accession numbers are provided in Table S7. S7.

Figure 1 .

 1 Figure 1. Schematic view of the life cycle of Ectocarpus sp.. Like most brown algae, Ectocarpus has a haploid-diploid life cycle with haploid, genetic sex determination[START_REF] Coelho | Ectocarpus: an evo-devo model for the brown algae[END_REF][START_REF] Coelho | Ectocarpus: an evo-devo model for the brown algae[END_REF]. Male and female gametophytes produce male and female gametes, respectively, which are released into the seawater from plurilocular gametangia (pg). Gamete fusion produces a zygote which will initiate the (diploid) sporophyte generation. However, gametes that do not meet a gamete of the opposite sex settle and lose their flagella, and they may still function as initial cells of the sporophyte generation because they can develop parthenogenically into a (haploid) partheno-sporophyte. Note that there is no visible (morphological) difference between haploid and diploid sporophytes. Meiosis occurs in the sporophyte (in the unilocular sporangia, U), producing several hundred haploid meio-spores. These meio-spores are released into the seawater and develop into male or female gametophytes. Partheno-sporophytes and diploid sporophytes may also be maintained by production of mitospores in plurilocular sporangia (ps): released mito-spores recycle the partheno-sporophyte generation (circular arrow in the scheme).

  up into the medium from the extensive, prostrate basal system. Reproductive structures are produced on the apical system (Figure2D, 2E).

Figure 2 .

 2 Figure 2. Phenotypes of bas mutants. A) Wild-type gametophyte germling, the arrowhead indicates the rhizoid cell (basal structure). B) Three-day old wild-type gametophyte, the arrowhead indicates the rhizoid. C) Wild-type sporophyte generation composed of round prostrate filaments firmly attached to the substrate. D) Wild-type plurilocular sporangium containing mitotic spores, produced after 20 days in culture. E) Wild-type unilocular sporangium (where meiosis takes place) produced after 25 days in culture. A secondary rhizoid is indicated by asterisks (**). (F-J) Development of the gametophyte generation of the bas-1 mutant. (K-N) Development of the sporophyte generation of bas-1 mutant. O) Occasionally, the mutant strains produced enlarged, abnormal cells (asterisk). P) Plurilocular sporangium on a bas-1 mutant sporophyte. Q) Unilocular sporangium on a bas-1 mutant sporophyte. R) Plurilocular gametangium on a fertile bas-1 gametophyte. S) Initial cell division of a bas-2 gametophyte. T) Aborted unilocular sporangium on a mature bas-2 sporophyte (about 3 weeks after initial cell germination). U) Proportions of 10-day old bas-1 and wild-type germlings that exhibited unipolar germination. Plots represent the mean and SE of five replicate cultures, the total number of germlings scored are indicated in brackets. The photographs are of representative bas-1 and wild-type (WT) germlings, exhibiting uni-(one arrow) or bi-polar (two arrows) germination, respectively. Arrows indicate the direction of germination. Note that, in the bas-1

Figure

  Figure 5. Identification of mutations in the BAS gene and identification of protein phosphate 2A subunits in Ectocarpus. A) Diagram showing the domain structure of the BAS gene, indicating the positions of the bas-1 and bas-2 mutations. The point mutation in exon 2 in bas-1 results a lysine (K) being replaced with a glutamate (E) residue, whereas the point mutation in bas-2 results in the introduction of a stop codon into the coding region of the gene (represented by an asterisk). Blue boxes indicate exons, with dark-blue representing untranslated regions and light-blue the coding region. B) Ectocarpus protein phosphatase 2A subunits. Unrooted maximum likelihood trees of PPA2A subunits (LG+G model). Only bootstrap (1000 repetitions) values of >50 are shown. Ectocarpus proteins are shown in blue. Asterisks and double asterisks indicate best species-to-species reciprocal Blastp matches with the corresponding Ectocarpus protein. The Ectocarpus genome does not encode an orthologue of PP2A subunit B"'/striatin. The domain structures of five PP2A subunit B" proteins are shown with the EF-hand domains in brown and disordered domains in green. AA, amino acid; Esp, Ectocarpus sp.; Tgo, Toxoplasma gondii; Ath, Arabidopsis thaliana; Hsa, Homo sapiens; Dme, Drosophila melanogaster; Sce, Sccharomyces cerevisiae; Nca, Naumovozyma castelli. Ectocarpus locusIDs are abbreviated as in the following example: Esp_14_3830, Ectocarpus sp. Ec-15_003830.

  Ectocarpus life cycle and in other developmental mutantsz 241

Figure 6 .

 6 Figure 6. Abundance of the BAS and DIS transcripts (measured as transcripts per million, TPM) during the life cycle of Ectocarpus and in developmental mutants. A) BAS transcript abundance during several developmental stages of wild-type Ectocarpus and in dis and bas mutant strains. Note the high abundance of the BAS transcript in gametes. GA: gametophyte, pSP: partheno-sporophyte. Significant differences in expression (Tukey test) are indicated as different letters above the plots, and detailed statistics are presented in TableS14. B) Abundance of the DIS transcript in wild-type samples compared with developmental mutants. pSP: partheno-sporophyte; GA:

Figure 7 .

 7 Figure 7. Differential gene expression analysis. A) Venn diagram of intersects of DE gene sets in bas mutants compare to wild-type. B) Boxplot representation of the distribution of gene expression levels (in log2 of TPM values +1) of the DE gene sets from comparisons of either bas-1 or bas-2 with wild-type. For each gene category, significant differences in expression level across strains, according to the Wilcoxon test, are indicated as different letters above the plots (details in Table S15). C-D) Histogram representation of the distribution of fold changes of the DE gene sets from comparisons of bas-1 or bas-2 with WT; vertical dashed bars indicate the medians of the distributions. DE genes in bas-1 compared to wild-type (C); DE genes in bas-2 compared to wild-type (D).

Figure S2 .Figure S3 .Figure

 S2S3 Figure S2. Pedigree of the Ectocarpus strains used in this study. SP, diploid, hybrid sporophyte; WT, wild type; m, male; f, female.

Figure S6 .CFigure S8 .

 S6S8 Figure S6. Vulcano plots of all genes in pairwise comparisons between wild-type (WT) and mutants (bas-1 and bas-2). The log2 FC value was calculated based on the mean expression level (TPM) for each gene. Each dot represents one gene. Blue represents upregulated genes and green downregulated genes in each comparison.

  Ectocarpus proteins are shown in blue. Asterisks and double asterisks indicate best species-to-species reciprocal Blastp matches with the corresponding Ectocarpus protein. The Ectocarpus genome does not encode an orthologue of PP2A subunit B"'/striatin. The domain structures of five PP2A subunit B" proteins are shown with the EF-hand domains in brown and disordered domains in green. AA, amino acid; Esp, Ectocarpus sp.; Tgo, Toxoplasma gondii; Ath, Arabidopsis thaliana; Hsa, Homo sapiens; Dme, Drosophila melanogaster; Sce, Sccharomyces cerevisiae; Nca, Naumovozyma castelli. Ectocarpus locusIDs are abbreviated as in the following example: Esp_14_3830, Ectocarpus sp. Ec-15_003830.

5. Identification of mutations in the BAS gene and identification of protein phosphate 2A subunits in

Ectocarpus. A) Diagram showing the domain structure of the BAS gene, indicating the positions of the bas-1 and bas-2 mutations. The point mutation in exon 2 in bas-1 results a lysine (K) being replaced with a glutamate (E) residue, whereas the point mutation in bas-2 results in the introduction of a stop codon into the coding region of the gene (represented by an asterisk). Blue boxes indicate exons, with dark-blue representing untranslated regions and light-blue the coding region. B) Ectocarpus protein phosphatase 2A subunits. Unrooted maximum likelihood trees of PPA2A subunits (LG+G model). Only bootstrap (1000 repetitions) values of >50 are shown.

Acknowledgements

This work was supported by the CNRS, Sorbonne Université, the Max Planck Society and the European Research Council (grant agreement 864038 to SMC). HY was supported by a grant from the China Scholarship Council (grant number 201608310119). We thank Nana Kinoshita-Terauchi (Shimoda Marine Research Center, University of Tsukuba) and Iris Kock (MPI Tübingen) for helpful discussion and images of the mutants and Viktoria Buhanets for help with segmentation of Fib-Sem images.

5% triton overnight. Samples were then rinsed in PBS and 100 mM NaBH4 was added for 4 h. Cell walls were degraded with cellulase (1% w/v) and hemicellulase (4% w/v) for 1 h, and the preparation was then rinsed with PBS and blocked overnight in 2.5% non-fat dry milk in PBS. Samples were treated with an anti-tubulin antibody (1/200th, DM1A; Sigma-Aldrich) at 20°C overnight and then treated with the secondary antibody (AlexaFluor 488-conjugated goat antimouse IgG; Sigma-Aldrich; 1:1000 in PBS) at 20°C overnight. The preparation was rinsed with PBS and blocked overnight in 2.5% non-fat dry milk in PBS and then treated with an anti-centrin antibody (1/1000th anticentrin 1 ab11257; Abcam) at 20°C overnight, followed by the secondary antibody (1/1000th AlexaFluor 555-conjugated goat anti-rabbit IgG; Sigma-Aldrich) for 8 h. Samples were stained with 4ʹ,6-diamidino-2-phenylindole (DAPI; 0.5 µg/mL in PBS) for 10 min at room temperature and mounted in ProLong Gold (Invitrogen).

Confocal Microscopy

Confocal microscopy was conducted using an inverted SP8 laser scanning confocal microscope (Leica Microsystems) equipped with a compact supply unit which integrates a LIAchroic scan head, several laser lines (405 and 488 nm), and standard photomultiplier tube detectors. We used the oil immersion lens HC PL APO 63×/1.40 OIL CS2. The scanning speed was set at 400 Hz unidirectional. The pinhole was adjusted to one Airy unit for all channels. The spatial sampling rate was optimized according to Niquist criteria, generating a 0.058 × 0.058 × 0.299-µm voxel size (xyz). The Z-stack height fitted the specimen thickness. A two-step sequential acquisition was designed to collect the signal from three or four channels. The first step recorded the antitubulin fluorescence signal (excitation, 488 nm; emission, 530 nm) and the transmitted light. The second step acquired the DAPI fluorescence signal (excitation, 405 nm; emission, 415-480 nm). Signal intensity was averaged three times. The Fiji software was used to optimize the raw images, including maximum intensity projection and de-noising (3*3 median filter). For any given data, both wild-type and mutant images were analysed simultaneously with similar settings.

Tracking of microtubule bundles was performed on maximum intensity projections of z-planes covering the whole thickness of the cells. We drew a line transversely, perpendicular to the growth axis of the cell and crossing the nucleus. Peaks corresponding to the microtubule bundles were then identified in plots of intensity profiles and counted, in order to estimate the number of microtubule bundles in each cell. Note that in the bas mutants, due to the disorganized nature of the microtubule network, average bundle numbers may be somewhat underestimated. This is because this method is well adapted for tracking microtubule bundles oriented with their long axis parallel to the image plane, but we may have missed bundles that were perpendicular to the plane of the transection.

Supplemental Figures