How does haploid ulva reproduce

The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. Macroalgal blooms typically consist of large accumulations of ephemeral macroalgal biomass. These blooms occur worldwide, often in shallow areas with relatively low water mixing that are affected by coastal eutrophication, and they have the potential to cause severe ecological and economic damage [ 1 — 3 ].

The largest documented bloom on record occurred four weeks before the Beijing Olympics, with a bloom of an estimated 20 million tons of Ulva prolifera in the Yellow Sea near Qingdao, China [ 4 — 6 ]. The costs of clean up for the bloom were estimated at The ecological effects of macroalgal blooms are often far-reaching and indirect; algal blooms negatively affect seagrass beds, sessile invertebrates and perennial algae [ 7 — 9 ].

Large blooms can create hypoxic environments that contribute to mass fish and invertebrate die-offs [ 10 , 11 ] and hydrogen sulfide from decaying algal mats can cause symptoms such as difficulty breathing and nausea in humans [ 12 ]. Large macroalgal blooms decrease light attenuation and shade seagrass beds and benthic perennial algae [ 13 ].

Blooms have increased worldwide over the years in frequency and intensity [ 14 — 16 ]. The green macroalgal genus Ulva forms large and dense sheets, a phenomenon known as green tides, and can proliferate by asexual e. Green tides include many genera of green algae, such as Chaetomorpha , and affect at least 37 countries worldwide [ 20 , 21 ].

Ulva is one of the most common macroalgal bloom-forming genera present in green tides and is the focus of this study. Like many marine algae, Ulva has a biphasic life cycle consisting of an alternation between two free-living forms, a haploid phase 1N, gametophyte and a diploid phase 2N, sporophyte; Fig 1.

These phases of Ulva are isomorphic, meaning that the gametophyte and sporophyte are morphologically similar and cannot be visually distinguished in the field. This deviation from a 1 to 1 ratio is because Ulva is dioecious; each spore produced by the diploid sporophyte can potentially result in a haploid gametophyte, but only the female gametophyte can produce a sporophyte, resulting in a relative overabundance of adult gametophytes.

For isomorphic algal species, however, a wide range of distributions of ploidy ratio in have been documented in the field [ 23 ]. Ulva cycles between two morphologically similar multicellular adult phases, a haploid gametophyte and a diploid sporophyte. Diploid sporophytes produce haploid zoospores that develop into gametophytes. Haploid gametophytes produce haploid gametes. There are few published studies on the in situ life cycle dynamics of Ulva ; Hiraoka and Yoshida [ 19 ] found a non-seasonal alternating dominance of the two phases for U.

This lack of a broader understanding of Ulva life cycle dynamics may be due to the difficulty of discerning between isomorphic phases; however, ploidy can be rapidly determined using flow cytometry [ 26 — 28 ].

Flow cytometry quantitatively analyzes the DNA content of nuclei in a suspended solution and can allow for a convenient, fast, and reliable method for determining ploidy. Although isomorphic sporophytes and gametophytes appear identical, they can occupy different ecological niches [ 29 — 31 ].

For example, one phase may be responsible for forming blooms, while the other may occur during non-bloom forming months, although data on these dynamics are relatively unknown [ 32 , 33 ]. In addition, the two phases may vary in growth rates, temperature optima, or susceptibility to herbivores [ 34 ]. Similarly, phases could vary in their response to environmental variables such as temperature and nutrients [ 29 , 35 ]. If there are ecological differences between Ulva gametophytes and sporophytes, the distribution of life history phases will be partially dependent upon the physical and biological factors of the system.

Some advantages for sporophytes include the ability to mask deleterious mutations [ 36 ] resulting in increased genetic diversity, the accumulation of mutations at twice the rate of gametophytes [ 37 ], and extra flexible alleles that contribute to faster adaptation by evolving to serve new functions [ 38 ]. Advantages for gametophytes include the immediate elimination of deleterious mutations, faster evolution due to strong selection on beneficial alleles [ 39 ], and lower nutrient requirements [ 40 ].

We investigated life cycle dynamics in the bloom-forming macroalgae Ulva compressa L. Agardh, which are common in summer macroalgal blooms in the estuarine system of Narragansett Bay, Rhode Island [ 41 , 42 ]. Macroalgal densities comprised mostly of Ulva peak in the summertime and vary significantly across sites, seasons, and years [ 43 — 45 ].

Our research focuses on four central questions regarding the life cycles and biology of U. Firstly, what is the relative abundance of sporophytes and gametophytes of both species? Secondly, how do these relative abundances correlate with physical and biological factors? Thirdly, do the phases have different growth rates, and lastly, do the phases have cells of different sizes? We interpret our data in the context of macroalgal bloom dynamics and the impacts of environmental variables in structuring bloom formation.

We collected Ulva spp. We chose these sites to represent a range of typical Ulva spp. At each site, on each sampling date, we haphazardly collected individuals by hand from the shallow subtidal zone, put them in a plastic bag, and brought them back to the lab.

We selected a minimum of 16 individuals and maximum of 40 individuals on each sampling date. Later, we identified U. A recent study by Mao et al. Since there are morphological similarities between U. Overall, we collected and analyzed total Ulva individuals: U. Both species were collected at all sites, with a minimum of 10 individuals of each species at each site.

Due to the nature of sampling and length of time necessary for preparing flow cytometry samples which limited our ability to collect larger sample sizes , we present and analyze our data here in terms of the overall relative abundance of each Ulva species during the peak bloom-forming season at each site.

However, we use collection date and month as covariates in building our logistic regression models for predicting the relative abundance of each phase see Statistical Analysis section. We also determined Ulva biomass data from monthly subtidal surveys of the same sites, following the protocol in Guidone [ 44 ]. Briefly, at each site, we collected all algae in each of 0.

Prior to thallus destruction for flow cytometry, we took a microscopic photograph at X of each individual that was analyzed for ploidy content. Using ImageJ www. We examined the upper cell layer, as U.

We used flow cytometry to determine the relative abundance of gametophytes and sporophytes in U. Based on the C-values haploid genome sizes of U. We used an enzyme solution developed specifically for efficient production of Ulva protoplasts [ 48 ], along with a modified version of the LB01 nuclear isolation buffer.

Instead of the standard 0. We were concerned with successful protoplast isolation and not with the exact number of protoplasts obtained, so we chose a qualitative method for isolating protoplasts [ 48 ]. We weighed all Ulva s amples to 0. Ulva samples were chopped with a razor blade in a large 85 mm x 25 mm plastic Petri dish for one minute, and then the tissue was transferred into a small 55 mm x 15 mm Petri dish that contained 5 mL of enzyme solution [ 48 ]. A total of 2mL of supernatant was then removed and replaced with 2mL of sterile filtered seawater.

Centrifugation with subsequent replacement of fluid was repeated twice, and after the last round of centrifugation, all supernatant was removed and replaced with 1mL of sterile filtered seawater. We observed successful protoplast isolation via microscopic examination at X. To liberate the nuclei, we added 1 mL of modified LB01 nuclear buffer kept on ice to each sample, vortexed and tapped the tube occasionally for eight minutes, and then added 0. This machine was optimized for marine applications and is equipped with three lasers nm, nm, and nm.

We used a green nm or a blue nm laser and quantified fluorescence at nm 20 nm bandwidth on a linear scale. Since sporophytes have twice the amount of genetic material as gametophytes, sporophytes have twice the amount of fluorescence as gametophytes Fig 2.

To measure the spread of the distribution of the data we used the coefficient of variation CV , which is the standard deviation expressed as a percentage of the population mean. The graphs on the left A, C represent an U. Fig 2A and 2B show the forward scatter by fluorescence, while Fig 2C and 2D represent the count of nuclei from 20, events. The sporophyte B, D has twice the fluorescence as the gametophyte A, C , with the gametophyte mean fluorescence near 19, and the sporophyte mean fluorescence near 38, We assessed growth rates of gametophytes and sporophytes of U.

We collected healthy Ulva individuals from the shallow subtidal zone in Greenwich Bay in the summer of In total, we used 90 U. We conducted growth experiments in June, July, and August to assess differences in growth over the peak bloom-forming months S4 Table. In the lab, we determined the species identity of each specimen via microscopic examination. We then spun individuals 20 times in a salad spinner prior to separating 1. We placed one 1. For each month, we had a sample size of at least five up to a maximum of 36 individuals of each phase of each species, except for U.

All Ulva were spun 20 times in a salad spinner prior to each weighing on a digital scale to ensure consistent mass, and all individuals were analyzed using flow cytometry for ploidy content see above. Cleavage continues until 32 to 64 daughter protoplasts are formed.

Each daughter protoplast metamorphoses into a biflagellate gamete. Just before the cleavage of the protoplast each cell develops a beak like outgrowth as its outer face and it expends to the thallus surface.

Later on a pore is formed at the tip of this beak, through which the gametes are liberated. The gametes are smaller than zoospores. They are priform inshape with a single chloroplast and an eye spot. The gametophytes liberate gametes at the beginning of each series offspring tide. After fusion of the gametes quadriflagellate zygote is formed.

It swims foa r short time and then comes to rest, withdraws its flagella and secretes a wal around it. Within a day or two the germination of zygote takes place. Ulva elminthoides Velley Withering S.

Ulva elongata Schousboe P. Ulva endiviifolia Martius S. Ulva enteromorpha f. Agardh Le Jolis P. Ulva enteromorpha Le Jolis U. Ulva enteromorpha var. Ulva erecta Lyngbye Fries S. Ulva erecta f. Crouan P. Gardner C. Ulva fascia O. Ulva fascia var. Ulva fasciata Delile S. Ulva fasciata f. Howe S. Ulva fasciata S. Gray S. Ulva fasciata var. Agardh Montagne U. Ulva fasciculata A. Ulva fasciola Roth Martius S. Ulva fastigiata Clemente S.

Ulva fetida D. Vaucher P. Ulva filiformis Hornemann S. Ulva filosa C. Thunberg P. Ulva fistulosa Hudson S. Ulva flabelliformis Roth P. Ulva flabelliformis Wulfen P. Ulva flavescens Hudson U. Ulva flexuosa f. Wynne C. Ulva flexuosa subsp. Curiel C. Wynne S. Ulva flexuosa var. Tsarenko S. Ulva flexuosa Wulfen C.

Ulva fluviatilis Sommerfelt S. Ulva foeniculacea Hudson C. Ulva foliacea Poiret P. Ulva fulvescens C. Ulva fungosa Desfontaines Poiret P. Ulva furcellata Turner S. Ulva furfuracea Mertens ex Hornemann S.

Ulva fusca Hudson S. Ulva gayralii Cauro P. Ulva gelatinosa Vaucher S. Ulva geminoidea V. Ulva geminoidea var. Ulva glandiformis S. Gmelin S. Ulva gracillima G. Ulva granulata Linnaeus S. Ulva grevillei Thuret Le Jolis S. Ulva halleri De Candolle Poiret P. Ulva hendayensis P. Parriaud Ballesteros U. Ulva hopkirkii M'Calla ex Harvey P. Crouan S. Ulva howensis A. Lucas Kraft C. Ulva hutchinensis Poiret S. Ulva iliohaha H. Sherwood C. Ulva imbricata Schousboe P. Ulva implicata Schousboe P.

Ulva incrassata Hudson S. Ulva incrassata O. Ulva incurvata Parriaud U. Ulva indica P. Anand S. Ulva indica Roth U. Ulva infundibuliformis Turra U. Ulva insignis Areschoug Papenfuss S. Ulva interrupta A. Ulva interrupta Poiret S. Ulva intestinalis f. Crouan C. Taskin S. Ulva intestinalis Linnaeus C.

Ulva intestinalis var. Tsarenko C. Ulva intricata Clemente U. Ulva intricata Thuillier U. Ulva intybacea Lamarck S. Ulva involvens P. Savi S. Ulva japonica Holmes Papenfuss S. Ulva javanica N.

Burman U. Ulva jugoslavica Bliding Ballesteros S. Ulva kraftiorum Huisman C. Ulva kuckuckiana Schmidt S. Ulva kylinii Bliding H. Ulva labyrinthiformis Linnaeus S. Ulva laciniata Lightfoot S. Ulva laciniata var. Ulva lactuca f. Agardh De Toni S. Agardh De Toni U. Linnaeus Kylin P. De Toni P. Ulva lactuca Linnaeus C - type. Ulva lactuca var. Agardh Schiffner U. Taylor S. De Candolle S.

Crouan Bornet C. Agardh Montagne P. Schmidt Lami C. Agardh Le Jolis S. Ulva lactucaefolia S. Ulva lactucifolia S. Gray P. Ulva lacunosa Duby S. Ulva laingii V. Ulva lanceolata Linnaeus S. Ulva latissima Linnaeus S. Ulva latissima Gunnerus S.

Ulva latissima var. Ulva leptophylla Arasaki U. Ulva ligulata Woodward S. Ulva limnetica K. Shimada C. Ulva linearis var. Ulva linearis P. Ulva lingulata A.

Ulva linkiana Greville Trevisan S. Ulva linza f. Ulva linza Linnaeus C. Ulva linza var. Ulva lippii J. Ulva littorea Suhr U. Ulva longissima Gunnerus S. Ulva lubrica Roth S. Ulva lumbricalis Linnaeus S. Ulva maculata Poiret U.

Ulva maeotica Proshkina-Lavrenko P. Ulva marginata J. Ulva maxima Gunnerus S. Furnari S. Ulva melanoidea Schousboe P. Ulva membranacea Stackh. Kingston S. Ulva meridionalis R. Ulva mertensii C. Martius S. Ulva mesenteriformis Roth P. Ulva miniata C. Agardh Lyngbye S. Ulva minima Vaucher P. Ulva moccana J. Ulva montana Lightfoot S. Ulva multifida J. Smith U. Ulva multifida Turner S.

On the other hand, this postulate is not applicable to organisms with UV systems in which mutations in both sex chromosomes, named UV chromosomes, are not sheltered, because they have no allelic counterparts in the dominant haploid phase, leading to the expectation of different evolutionary patterns for UV chromosomes 7.

However, there have been very few empirical studies of the structure and evolution of UV chromosomes. In the green plant lineage, the genomic sequences of MT loci and SDRs on UV chromosomes have been reported in four species: the unicellular green alga Chlamydomonas Chlorophyta , the colonial green alga Gonium Chlorophyta , the multicellular alga Volvox Chlorophyta , and the liverwort Marchantia Marchantiophyta 9 , 10 , The three green algal MT loci and SDRs have been compared along with the evolution of multicellularity and oogamy because these algae evolved from an ancestral unicellular green alga, similar to Chlamydomonas , into Volvox with multicellularity and oogamy since least million years ago Mya The sizes of Volvox male and female SDRs are over 1.

Many genes of the Volvox SDR are the same as those located inside and outside of the Chlamydomonas MT locus, suggesting that expansion of the MT locus involves the surrounding genes 9. Other well-studied green lineages include bryophyte species, specifically the liverwort Marchantia Marchantiophyta and the moss Ceratodon Bryophyta. Marchantia has accumulated repeats in sex chromosomes, and gametologs are exposed to purifying selection 11 , 14 , Although the genomic sequences of Ceratodon have not been reported, population genetics and molecular evolutionary approaches indicate that non-recombination of SDRs exposes gametologs The genomic sequences of the MT loci have been reported in several species outside the green lineages.

The brown alga Ectocarpus has a non-recombining SDR in which gametologs are exposed In both cases, degeneration signals, such as transposable element accumulation and relaxed codon bias, are found, but there are no clear evolutionary strata. The rules governing the generation of these differences are not yet clear.

Schematics of the life cycles of organisms are shown, along with the features of UV chromosomes. In these organisms, sex determination occurs in the diploid phase, and a sex chromosome of a particular sex degenerates.

In UV systems of multicellular organisms, the diploid saprophyte generates haploid zoospores or spores via meiosis, and mating type or sex is genetically determined by the UV chromosomes. Spores or zoospores with U and V chromosomes are female and male, respectively, and they develop haploid gametophytes. In UV systems of unicellular organisms, diploid zygospores after fusion of gametes with opposite mating types, some of which are dormant, undergo germination and meiosis to generate haploid vegetative cells that reproduce themselves and generate gametes under particular conditions, such as nutrient starvation.

Genomic sequences of UV chromosomes are revealed in two types of life cycle: dominating haploid phase e. Degeneration of sex chromosomes is thought to be observed only in the moss, with the other organisms harboring sex-determining regions SDRs in the UV chromosomes. The green seaweed Ulva partita has a life cycle with even domination of haploid and diploid phases, but it is not oogamous, in which gametes differentiate into eggs and sperms. The biflagellate gametes are anisogamous, apart from their size and ultrastructure.

Gametes of opposite mating types fuse, and a zygote develops into a sporophyte that is identical to a gametophyte in terms of morphology. The sporophyte somatic cells differentiate into zoospores that have four flagellae and are slightly larger than the gametes. Thus, the sexually reproductive form of this species is the mating type. The other type of MT locus is found in the unicellular green alga Chlamydomonas and the colonial green alga Gonium.

Their MT locus sizes are smaller than the SDRs in the UV chromosomes, and they have a low diversity of gametologs, which are genes shared between the MT loci of individual mating types.

Green seaweeds of the Ulvophyceae are multicellular and grow in coastal areas worldwide 21 , Ulva partita is a species of the Ulvophyceae and shows representative features of the life cycle of this order Fig. The asymmetry between the mating structure and the eye spot is observed even in the isogamous green alga Chlamydomonas reinhardtii Thus, U.

Ulva species are anisogamous but not oogamous. Compared with the other previously analyzed organisms with UV systems, U. Isomorphism between gametophytes and sporophytes is expected to restrict the functions of the MT locus genes because they must function equally in the haploid gametophyte, haploid gamete, diploid sporophyte, and haploid zoospore. Natural populations of Ulva species show no dominance of haploid or diploid phases and no sexual bias between seasons, suggesting that isomorphism and sexuality do not affect fitness in either phase This is distinct from other organisms.

For example, the two mosses develop extremely heteromorphic gametophytes and sporophytes or egg, sperm, and spores, and not all SDR genes are necessarily required for both phases, resulting in evolutionary relaxation of selective pressure on particular genes. Ulva genetically determines mating type after meiosis by harboring individual UV chromosomes in gametophytes, and it may acquire a transcriptional regulation system between mating types at the gamete stage or during gametogenesis.

Chlorophyta contains several classes; the major classes are Prasinophyceae, Trebouxiophyceae, Chlorophyceae, and Ulvophyceae In all Chlorophyta, the only known sex- or mating-determining gene is the Chlamydomonas MID Mi nus dominance , encoding a putative transcription factor containing an RWP-RK domain, including a leucine zipper-like motif 29 , A MID ortholog has been found in the Volvox SDR, and its genetic manipulation results in the transformation of sex, from female to male or from male to female.

However, the expression level of this gene is constant during spermatogenesis in males, suggesting that this gene does not play a role in sex determination but instead has a male-specific function in the differentiation of male vegetative cells into sperm MID is highly conserved in the Chlorophyceae lineage, but it is unclear whether other green algal lineages also possess this gene.

With regard to green algal evolution, it is of interest to examine the conservation of MID among the distinct taxonomic classes Chlorophyceae and Ulvophyceae. Here, we report identification of the MT locus in a species with a haploid mating type determination system without oogamy.

The primary issue that this study aims to resolve is how much the genomic structures and evolutionary history of the MT locus in U. The mating type determination system of U. The isomorphism between the gametophyte in the haploid stage and the sporophyte in the diploid stage may affect the evolution of the MT locus. We also investigated the orthologs among the U. The PacBio long reads 1. Although comparison of the scaffolds with unassembled PacBio long reads revealed mating type-specific MTS PacBio long reads, these reads were distributed over many scaffolds Supplementary Tables 1 and 2 ; Supplementary Fig.

To select the MTS PacBio long reads located within a particular narrow region, the ratio of the sum of the lengths of 5—15 successive MTS PacBio long reads on the same scaffold per genomic length to that of the distal positions of the successive reads was determined Supplementary Fig. For reads located close together in a narrow region, the ratio reached 1 see Supplementary Text for detailed analysis.

This analysis identified a scaffold containing a region that was highly divergent between the two mating types Supplementary Fig. In addition, the mapping results of the Illumina short reads from the two mating type genomes and RNA-sequencing RNA-seq reads derived from gametes and gametophytes were mapped, and the gene models predicted by the RNA-seq assemblies are shown in Supplementary Fig. These regions had lower gene density than the surrounding regions 8.

Genomic structures of the mating type MT locus in the green seaweed Ulva partita. Numbers are scaffold numbers. Transposable elements TEs of the same type are indicated by the same colors. The colored vertical bars indicate individual mating type-specific genes. Light gray vertical bars indicate mating gametologs.

The presence of the U. PCR products of same primer sets were loaded on different gels Supplementary Fig. Captured EtBr fluorescence images of gels were cropped and images with low intensity were enhanced.

As no genome of Ulva relatives has yet been analyzed, there are no training data for gene prediction based on genome sequencing data.

Thus, for precise prediction of genes based on expression, sets of RNA-seq assemblies from gametes and gametophytes of the individual mating types were assembled and mapped on the scaffolds in and around the MT locus. The sets of RNA-seq assemblies were gathered based on homology, and then defined as genes. In XY and ZW systems, particular transposable elements accumulate in the sex chromosomes No such accumulation of transposable elements has been detected in the MT loci of Chlamydomonas and Gonium , but it has been found in Volvox or Ectocarpus , Marchantia 9 , 11 , Transposable elements were predicted based on homology with known transposable elements and comparison of the genome with itself.

All examined genes were linked to the mating types of the individual isolates Fig. To analyze the evolutionary history of the gametologs in the MT locus, the homologous sequences of an MT locus gene encoding proliferation-associated protein 1, PAR1, and a gene encoding G-strand telomere-binding protein 1, GTBP1, in a region neighboring the MT locus, were isolated from species related to U.

Molecular phylogenies were then reconstructed Fig. In all species examined, two types of homologous sequence were identified from the two distinct mating types, and the phylogenetic tree showed that the genes could be classified into two clades Fig. In addition, these two clades were associated with the previously determined mating types Supplementary Table 7.

In contrast, the neighboring-region genes in each species were almost identical and were not classified into different clades in the molecular phylogeny Fig. These data suggest that the investigated gametolog existed in the MT locus when this locus was established and evolved independently within the MT loci of the individual mating types. Evolution of the gametologs at the mating type MT locus. Molecular phylogenies of a gametolog in and a gene outside the MT locus.

The mating type of each strain of the species was determined previously see Supplementary Table 7 and is indicated after the species name. The numbers above the scale bar indicate nucleotide substitutions per site. C dS and dN values for C. D dS and dN values for Volvox carteri. E dS and dN values for Ulva partita. Up, U. Cr, C. Vc, V. MS, model selection method.

MYN, modified YN method. The shadowed region shows the MT locus. Dashed lines show the border of the scaffold.

Next, to determine the type of selective pressure exerted on the gametologs after their divergence, the nucleotide substitution rates at synonymous and non-synonymous sites dS and dN, respectively were estimated. Mean distances between individual genes on the plot were also calculated as an index to compare the divergence of MT locus genes Supplementary Tables 10 — Both maximum-likelihood and approximate methods showed that the synonymous substitution rates for the gametologs in U. Mean dS values were much higher in U.

In addition, the dN means of U. Means of all distances between the two dots for each estimation method were calculated as an index of scattering Supplementary Tables 10 — The mean distances for U. All mean distances were lower in Chlamydomonas than in U. Mean distances in Volvox were similar to those of U. The molecular phylogeny and nucleotide substitution rates suggest that the U.

The synonymous and nonsynonymous substitution rates of the genes around the MT locus were estimated by the two methods. The data showed that almost all synonymous and non-synonymous substitution rates of the genes around the MT locus were near zero or zero; additionally, the synonymous substitution rates were higher than those of the MT locus, and the non-synonymous substitution rates were slightly higher than those of the MT locus.

It has been reported that relaxed codon usage bias occurs with reduced recombination in sex chromosomes 33 , Furthermore, the codon usage in CDSs obtained from all mRNA data and the codon usage for the MT locus genes were compared with those of other autosomal locus genes Supplementary Table The codon usage patterns of all of the autosomal genes and the MT locus genes did not appear to differ Supplementary Fig.

We investigated whether there were orthologs among the U. In addition, genes encoding proteins containing the RWP-RK domain in Chlorophyta were collected from the annotated genes from the genomes of five species: Chlamydomonas reinhardtii , Volvox carteri , Gonium pectorale , Coccomyxa subellipsoidea , and Micromonas pusilla.

Although these genes encode proteins containing a single RWP-RK domain, the protein lengths are very diverse.