Unicellular and multicellular developmental variations in algal zygotes produce sporophytes

Abstract

The emergence of sporophytes, that is, diploid multicellular bodies in plants, facilitated plant diversification and the evolution of complexity. Although sporophytes may have evolved in an ancestral alga exhibiting a haplontic life cycle with a unicellular diploid and multicellular haploid (gametophyte) phase, the mechanism by which this novelty originated remains largely unknown. Ulotrichalean marine green algae (Ulvophyceae) are one of the few extant groups with haplontic-like life cycles. In this study, we show that zygotes of the ulotrichalean alga Monostroma angicava, which usually develop into unicellular cysts, exhibit a developmental variation producing multicellular reproductive sporophytes. Multicellular development likely occurred stochastically in individual zygotes, but its ratio responded plastically to growth conditions. Sporophytes showed identical morphological development to gametophytes, which should reflect the expression of the same genetic programme directing multicellular development. Considering that sporophytes were evolutionarily derived in Ulotrichales, this implies that sporophytes emerged by co-opting the gametophyte developmental programme to the diploid phase. This study suggests a possible mechanism of sporophyte formation in haplontic life cycles, contributing to the understanding of the evolutionary transition from unicellular to multicellular diploid body plans in green plants.

1. Introduction

In many plants (green, red, and brown macroalgae as well as streptophytes), fertilized zygotes develop into diploid multicellular sporophytes. Sporophyte acquisition is an evolutionary innovation in plants that has triggered the morphological diversification and flourishing of terrestrial and coastal ecosystems [1,2]. Sporophytes have evolved independently in multiple plant lineages [2]. In green plants (Viridiplantae), sporophytes may have evolved in a haplontic life cycle, in which zygotes develop into a unicellular body, and meiotically produced spores develop into a haploid multicellular gametophyte [2–4]. Some extant algae (e.g. charophytes) exhibit this life cycle [5,6]. Sporophytes may have evolved in haplontic ancestors; therefore, many plants undergo an alternation of gametophyte and sporophyte generations (haplo-diplontic life cycles) [1,2,6]. However, despite the century-long interest [3], little is known about the early stages in the evolutionary transition from unicellular diploid bodies towards multicellular sporophytes.

Recent studies on Viridiplantae, especially streptophytes, have proposed two mechanistic hypotheses for the origin of sporophytes: the de novo acquisition of a genetic programme directing sporophyte multicellular development or the partial or large-scale co-option of the pre-existing gametophytic multicellular development programme to the diploid phase [2,4] (electronic supplementary material, figure S1). As the activation of a generation-specific developmental programme is controlled by a few regulators in land plants (embryophytes) and brown algae [7,8], large-scale co-option may easily occur between the ploidy phases, potentially resulting in sporophytes exhibiting identical morphology (isomorphic) to gametophytes [2,9]. The emergence of isomorphic sporophytes via wholesale co-option may have also contributed to the transition from obligately matrotrophic sporophytes on gametophytes to free-living ones in embryophytes [9]. However, whether and how co-option occurs in the haplontic life cycle is largely unknown.

Qualitatively novel phenotypes, such as sporophytes, may emerge from intraspecific/within-genotypic phenotypic variations [10–13], which generally evolve and are maintained as adaptations to changing environments [10,14]. A novel phenotype can be expressed in response to or regardless of surrounding environments [10,11]. This can provide opportunities that selection acts on resulting in further elaboration [10–13]. Thus, intraspecific variations can be an important stage in the evolution of novelty [10,13]. Considering this concept, haplontic algae and plant ancestors might be capable of sporophyte development as an alternative phenotype at the origin of sporophytes.

Marine green macroalgae (Ulvophyceae) have acquired multicellularity, sporophytes, and alternation of generations independently of embryophytes [4,15,16]. Unlike embryophytes, in which one generation depends on the other, both generations of ulvophyceans are generally free-living. Ulvophyceae exhibit diverse life cycles [6]. For example, Ulvales alternate between gametophytes and isomorphic sporophytes [6]. Whereas, members of the sister group Ulotrichales exhibit a haplontic-like life cycle [4,5]: gametophytes grow from winter to spring, and in summer, they develop microscopic uninucleate unicellular diploid cysts to cope with seasonal stresses [17,18]. Additionally, some ulotrichalean algae (e.g. genus Eugomontia) have evolved isomorphic sporophytes [19]. In this study, we report that in Monostroma angicava, an ulotrichalean alga where zygotes usually develop into unicellular cysts [18], unicellular/multicellular developmental variation of zygotes produces sporophytes, providing evidence for the co-option of the gametophyte programme.

2. Material and methods

(a) . Zygote collection

Mature M. angicava gametophytes were collected at Botofurinai (42°31'N, 140°98'E), Muroran, Hokkaido, Japan. They released gametes immediately after collection [20]. Gametes produced by a dioecious gametophyte without meiosis are genetically identical (isogenic) [21]; therefore, crossing a pair of male and female gametophytes yields isogenic zygotes. From 2013 to 2022, we obtained 32 different genotypes of isogenic zygotes (1, 2, 2, 9, 16 and 2 genotypes from 2013, 2014, 2015, 2016, 2017 and 2022, respectively).

(b) . Developmental fates of zygotes

Immediately after collection, the isogenic zygotes were cultured in six-well plastic plates (IWAKI, Tokyo, Japan) with Provasoli's enriched seawater (PES) medium [22] under long-day conditions (14 L : 10 D; 14°C; and approximately 15 µmol photons m⁻² s⁻¹) similar to the climate conditions in the field (late spring–early summer) [17]. They were cultured at a density of approximately 4.0 × 10² zygotes ml⁻¹, and the number of multicellular bodies per growing individuals was counted. Individual zygotes collected in 2013 were isolated in 96-well plastic plates (IWAKI). One genotype of zygotes collected in 2017 was also cultured under 5, 10, 18°C, or nutrient-poor (without PES) conditions. Differences in the ratios of multicellular to unicellular development were analysed by a binomial generalized linear model with a logit link and likelihood ratio tests using R 4.2.1 [23].

(c) . Characterization of multicellular bodies

Sporophytes are generally diploid and possess both male- and female-specific genomic regions [6]. We measured the relative fluorescence intensity of 4′-6-diamidino-2-phenylindole (DAPI)-stained nuclei of M. angicava multicellular bodies using gametophytic nuclei as a standard (1C) (see [18]). We examined multicellular bodies by direct PCR using male- and female-specific molecular markers [24]. Additionally, we examined the nuclei of multicellular bodies by two-colour fluorescent in situ hybridisation (FISH) using DIG-anti DIG and streptavidin–biotin FITC systems that targeted male- and female-specific genomic regions different from those of the sex markers ([24]).

We collected small, young M. angicava plants (approx. 1–10 mm) in late spring 2017 at Botofurinai and in late spring 2019 at Oshoro (43°12′N, 140°51′E). These samples were analysed using the sex markers.

(d) . Life cycle pathway of multicellular bodies

We measured the relative fluorescence intensity of the DAPI-stained nuclei of swimming spores released from multicellular bodies under the long-day conditions using gametes as a standard (1 C) [18]. These spores were cultured under the same long-day conditions. Cysts developed from these spores and zoospores produced from cysts were cultured under the same conditions as regular cysts and zoospores of M. angicava [18,25].

(e) . Phylogenetic analysis

We obtained 18S rDNA sequences of ulotrichalean species with both haploid and diploid phases from the NCBI database (electronic supplementary material, table S1). Oltmannsiellopsidales species with microscopic unicellular or colonial bodies were used as the outgroup. The dataset length was 1748 bp with 218 variable bases. These sequences were aligned using MAFFT [26], and a maximum-likelihood phylogeny was constructed with 1000 bootstrap replicates using IQTREE [27].

3. Results

(a) . Discovery of multicellular sporophytes in M. angicava

We discovered that M. angicava isogenic zygotes collected from the field developed into multicellular bodies, as well as regular unicellular cysts (figure 1a). Notably, we observed within-genotypic unicellular/multicellular developmental variation even among neighbouring zygotes grown in the same dishes under the same conditions. Isolation experiments confirmed that 4.2% (n = 72) of individual zygotes developed into multicellular bodies. By contrast to spherical unicellular cysts with multiple pyrenoids, multicellular bodies consisted of vegetative cells with a single pyrenoid and developed from a filamentous to a disc-like form before reaching a macroscopic saccate form (figure 1b–h). Their morphologies and developmental processes were indistinguishable from those of gametophytes developing from zoospores (figure 1j–m).

Figure 1.

Developmental variations in M. angicava zygotes produce multicellular sporophytes. (a) Unicellular/multicellular developmental variation of genetically identical zygotes. Arrow: a multicellular body. Arrowheads: unicellular cysts. (b–h) Differential development of zygotes. (b) Zygotes usually develop into spherical unicellular cysts (c,d). (e) Some zygotes develop into filamentous multicellular bodies (f), which then grow into disc-like (g) and saccate forms (h). (i) Sex markers reveal that multicellular sporophytes have both male- and female-specific genomic regions; ‘m’: male marker. ‘f’: female marker. Side bar: 500 bp. (j) Zoospores develop into filamentous multicellular bodies (k), which then grow into disc-like (l) and saccate forms (m). Insets in (h) and (m) show male (green, arrowheads) and female (red, arrow) FISH signals on nuclei of a diploid sporophyte and haploid gametophyte, respectively. Scale bars: 1 µm. 1N: haploid; 2N: diploid.

Compared with dioecious haploid gametophytes, the formed multicellular bodies were diploid (2.0 ± 0.1 C, mean ± s.e., n = 50), had larger somatic cells (electronic supplementary material, figure S2), and had both male- and female-specific genomic regions (figure 1i). Furthermore, FISH analysis showed that multicellular bodies contained both sex-specific regions in the nucleus, unlike gametophytes (figure 1h,m, insets). These independent and congruent results demonstrated that the multicellular bodies were diploid sporophytes.

(b) . Plasticity in sporophyte development

The ratio of sporophyte development responded plastically to growth conditions (electronic supplementary material, figure S3). The ratio was significantly increased under warmer conditions, especially at 14°C (electronic supplementary material, figure S3a) and under nutrient-rich (PES) conditions (d.f. = 1, deviance = 5.7, p = 0.017).

(c) . Occurrence and maintenance of sporophytes in the field

We found that 30 different genotypes of isogenic zygotes collected between 2013 and 2017 exhibited the developmental variation. The ratio of sporophytes varied among genotypes (mean ± s.d.: 1.7 ± 1.5%, range: 0.1–4.9%, n = 30 genotypes and n = 915.2 ± 410.7 growing individuals for each genotype). We also confirmed sporophyte development in two genotypes collected in 2022. We found that sporophytes actually grew among young gametophytes at low frequency in two M. angicava field populations (2.7%, n = 27 in Botofurinai; 0.8%, n = 250 in Oshoro) (electronic supplementary material, figure S4).

(d) . Life cycle variation via sporophytes

Multicellular sporophytes released diploid (mean ± s.e.: 1.9 ± 0.1 C, n = 57) biflagellate swimming spores with negative phototaxis (figure 2a). These newly discovered spores exhibited different characteristics from those of other M. angicava swimming cells (electronic supplementary material, figure S5). The spores developed into unicellular cysts (figure 2b) that were indistinguishable from regular M. angicava cysts. These cysts produced zoospores that developed into gametophytes of either sex (figure 2c–e), as with the regular cysts. Thus, M. angicava exhibited an intraspecific life cycle variation (figure 2f).

Figure 2.

Sporophyte reproduction and life cycle variation in M. angicava. Sporophytes produce biflagellate swimming spores (a) that develop into unicellular cysts (b). Cysts produce quadriflagellate zoospores (c) that develop into gametophytes (d,e). Arrowheads: eyespots. (f) Life cycle schematic of M. angicava exhibiting two pathways: through only the cyst (grey) and through the sporophyte (orange) via zygote developmental variation. 1N: haploid; 2N: diploid.

(e) . Phylogeny

The constructed ulotrichalean tree revealed that the diploid phase of many species is unicellular, and that multicellular sporophytes have evolved independently in Eugomontia and M. angicava (electronic supplementary material, figure S6).

4. Discussion

This study demonstrated the emergence of multicellular sporophytes as part of the life cycle in the extant haplontic-like green alga, M. angicava, via developmental variations in the zygote. This life cycle pathway corresponds to an alternation of generations. The occurrence of sporophytes in late spring demonstrates that this developmental variation occurs in the field. As very few observable M. angicava plants grow in summer [17], sporophytes may reproduce in smaller sizes and produce unicellular cysts from late spring to early summer, which is consistent with the basic M. angicava phenology [17]. In plants, the known life cycle variations are apospory, in which somatic cells of sporophytes de-differentiate and re-develop into gametophyte morphology without changes in ploidy, and its inverse phenomenon, apogamy [28]. In some ulvophyceans, unfertilized gametes parthenogenetically develop into both haploid partheno-sporophytes and partheno-gametophytes [29]. However, to our knowledge, no precedent exists for Viridiplantae in which the developmental fate of sexually fertilised zygotes changes to produce distinct morphology and cellularity (multicellular versus unicellular). Although older (1960s) literature has implied zygote unicellular/multicellular developmental variations in at least five ulotrichalean algae (electronic supplementary material, table S2), cytological and molecular evidence and further life cycle investigations are needed. We are the first to confirm this previously uncertain phenomenon. Similar phenomena may have been overlooked but may be potentially widespread among green algae.

Our results suggest that environmental differences enhance the multicellular development of zygotes. Within the temperature range of the M. angicava growth period (5–14°C), warmer and nutrient-rich conditions increased the ratio of multicellular development of zygotes. These conditions can facilitate rapid growth [17,25]. The decrease in this ratio observed under summer temperatures (18°C) might be an adaptation for avoiding seasonal (summer) stresses. This plasticity may offer adaptive benefits for this alga [14]. Additionally, genetically identical zygotes exhibited developmental variation regardless of environmental conditions, suggesting that individual zygotes developed stochastically into sporophytes. Such variations may be adaptive as a risk spreading strategy under spatiotemporally fluctuating environments [14]. The detailed adaptive significance of this variation is, however, beyond the scope of this study and remains to be investigated. Although the ratio of sporophyte development was not very high, our long-term (10-year) observations of various genotypes suggest that this variation has been maintained within M. angicava populations under fluctuating coastal environments. This implies that the mechanistic innovation, that is, the expression of sporophyte development, alone is insufficient for the evolution of sporophytes under the current environment. Given that phenotypic variations, especially environmentally induced ones, can facilitate novelty evolution [10–13], the variation reported here may be an important stage in the evolutionary transition from unicellular cysts to multicellular isomorphic sporophytes in ulvophyceans [6,16,19].

The co-option hypothesis has been tested by comparing the roles of homologous regulators between analogous organs in gametophytes and sporophytes of distant embryophyte species, both of which have heteromorphic sporophytes [30]. Although these attempts have provided insights into the evolutionary processes after sporophyte acquisition, it might be difficult to discuss co-options occurring on a large scale and the emergence of sporophytes. Intraspecific comparisons of M. angicava revealed the indistinguishable morphological development of sporophytes and gametophytes, suggesting the expression of the same developmental programme. The swimming spores produced by the sporophytes developed into unicellular cysts, indicating that sporophyte development did not result from the mutational loss of regulatory factors directing cyst development. Ulotrichalean evolutionary history indicates that multicellular sporophytes are derived. Together, these results suggest that in this alga, isomorphic sporophytes emerged via wholesale co-option of the haploid gametophytic multicellular programme in the diploid phase, which is consistent with the co-option hypothesis [2,4].

Our findings suggest that sporophyte formation by co-opting gametophyte programmes may occur via zygote developmental variations in green algae and ancestral plants under fluctuating environments, such as aquatic habitats. Similar mechanisms may have played important roles in streptophyte sporophyte evolution, although piecemeal rather than wholesale co-option may have occurred at its early stages [30]. Phenotypic variations in zygote development may also be present in fossil records of ancestral plants [1,9,12]. Further investigations on developmental variations that express novel multicellular forms can contribute to the understanding of the evolution of sporophytes and life cycles in Viridiplantae and other taxa.

Ethics

This work did not require ethical approval from a human subject or animal welfare committee.

Data accessibility

Data are available from Figshare: https://doi.org/10.6084/m9.figshare.22283950 [31].

Supplementary material is available online [32].

Declaration of AI use

We have not used AI-assisted technologies in creating this article.

Authors' contributions

Y.H.: conceptualization, data curation, formal analysis, funding acquisition, investigation, methodology, project administration, supervision, validation, visualization, writing—original draft, writing—review and editing; T.T.: investigation, supervision, validation, writing—review and editing.

All authors gave final approval for publication and agreed to be held accountable for the work performed therein.

Conflict of interest declaration

We declare we have no competing interests.

Funding

Funding was provided by the Japan Society for the Promotion of Science (grant nos 22K20664 and 23K14260 to Y.H.).