Alternating nuclear DNA content in chrysophytes provides evidence of their isomorphic haploid-diploid life cycle
Introduction
Across eukaryotic organisms there is a considerable diversity of life cycles. Individuals at particular life cycle stages may differ, for example, by their overall morphology, environmental requirements, or by the number of chromosome sets in cell nuclei (ploidy level). Many organisms alternate between two stages, a haploid phase with one set of chromosomes reduced by meiosis and a duplicated diploid phase following the fusion of gametes [1], [2]. Depending on whether the both stages are more-or-less equally represented in the life cycle or one of them largely predominates, various life cycles can be recognized. In a diploid life cycle, organisms switch between a short haploid phase (usually restricted to unicellular gametes) and the prevailing diploid phase, only which is capable of mitotic growth. Such a life cycle occurs among diatoms, raphidophytes or budding yeasts; however, it is best known from animals, including humans [3], [4], [5], [6]. A haploid life cycle is characterized by mitosis restricted to the haploid phase, which also lasts for most of the organism's lifespan, as the only diploid stage is a unicellular zygote. The haploid life cycle evolved in stoneworts (charophytes) and in some other green algae [7]. However, probably the most widespread among organisms is a haploid-diploid life cycle. Here, both the haploid and diploid stages are capable of mitotic growth. This life strategy dominates in land plants, red and brown algae, basidiomycete fungi, but also occurs in many groups of green algae and various groups of unicellular algae [4], [7], [8], [9], [10]. A peculiar form of this life cycle evolved in some green and red algae, where the haploid and diploid phases are morphologically indistinguishable [11], [12]. This isomorphic haploid-diploid life cycle can be found, for example, in sea lettuce (Ulva lactuca; [11]).
Knowledge of the life cycle and identification of particular life cycle stages are key not only to understand the basic biology of studied organisms, but also to correctly assess their genome size (1C vs. 2C value), and hence to design an optimal sequencing strategy, and properly interpret population genetic or genomic data [13], [14], [15]. In algae, understanding the life cycles is also essential to predict formation of blooms and toxins production [4]. However, the knowledge of algal life cycles remains still largely fragmented, particularly for most unicellular algae. This could be attributed to their microscopic size and a frequent lack of pronounced morphological features, which makes life cycle stage transitions harder to detect. Here, we attempt to overcome the problem by measuring nuclear DNA contents and looking for shifts in a ploidy level that should be associated with the life cycle transitions.
To broaden our knowledge of algal life cycles, we chose chrysophytes as a model group. The chrysophytes, also known as golden-brown algae, are single-celled or colonial flagellates, which occur primarily in freshwater phytoplankton and their blooms can cause an unpleasant fishy odour in drinking water reservoirs [16]. In some taxa (e.g. among the representatives of the genera Synura and Chrysosphaerella), the cells are covered by species-specific silica scales [17]. However, not much is known about the chrysophyte life cycle. Undifferentiated cells may serve as gametes [18]. Fusion of the gametes was observed in several cases, specifically the apical fusion in Kephyrion, Stenocalyx, Chrysolykos and Dinobryon or the posterior fusion in Synura and Mallomonas [18], [19]. The fusion of gametes is followed by cyst formation [17]. Some colonial species even produce separate male and female colonies [20]. According to Sandgren and Flanagin [21], the genus Synura is heterothallic and its sexuality might be induced at high cell densities. In recent years, the chrysophytes have drawn attention due to their remarkable DNA content diversity, ranging from 0.09 to 24.85 pg (0.09 to 24.31 Gbp), accompanied by numerous cases of major intraspecific variability [22], [23], [24]. This variation was either attributed to polyploidization (i.e., whole-genome doubling) or its source remained unresolved [23], [24].
The present study was stimulated by our repeated detection of intraspecific DNA content variation arising in cultures of some of our investigated taxa that opened the question whether this could be attributed to unprecedented rates of certain evolutionary processes (e.g. polyploidization, aneuploidization, proliferation of transposable elements [25]) or whether it constitutes an inherent part of organisms' life cycles. By employing flow cytometry on selected, DNA content variable taxa we are asking the following specific questions: 1) What are the patterns of DNA content variation among and within strains; and do these correspond to ploidy level shifts (i.e., two-fold DNA content differences)? 2) Of what character are the temporal changes in the DNA content of strains over time in cultivation? 3) Is intraspecific / intra-strain DNA content variation linked with differences in genomic base composition (i.e. GC content; no differences expected under the scenario of whole genome duplication)? 4) Are there any apparent phenotypic differences between intraspecific strains with different DNA contents?
Section snippets
Origin and cultivation of the investigated strains
For this study, we selected chrysophyte taxa where intraspecific DNA content variation was detected during our previous unpublished work. Altogether 61 chrysophyte strains were obtained from 49 various freshwater localities across the Northern hemisphere, comprising 59 isolates of the genus Synura, one isolate of Ochromonas tuberculata and one isolate of Chrysosphaerella brevispina. The sampling details are listed in Supplementary data Table S1. To establish new cultures, water samples were
Nuclear DNA content variation in chrysophytes
Altogether, this study was performed on 68 strains representing three chrysophyte genera, Synura, Chrysosphaerella, and Ochromonas (Supplementary data Table S1), where we previously detected intraspecific DNA content variation. Using the nuclear ITS rDNA (nu ITS rDNA) molecular barcode, we identified nine species of Synura: S. americana, S. glabra, S. heteropora, S. hibernica, S. lanceolata, S. macropora, S. petersenii, S. soroconopea, and S. sphagnicola (Fig. 1). Although the nu ITS rDNA
Intraspecific ploidy level variation in chrysophytes
With the exception of a single species (S. macropora, see below), DNA content variation within and among conspecific strains was not random but corresponded to different ploidy levels. Two to three ploidy levels were detected within the particular chrysophyte species investigated, here for simplicity referred to as haploid (1x), diploid (2x) and tetraploid (4x).
Aside from the (almost) exactly two-fold differences in nuclear DNA contents, the intraspecific ploidy level variation was also
Conclusions
In this study, we revealed that chrysophytes have a haploid-diploid life cycle. Chrysophyte taxa alternate between two ploidy states, both of which are capable of mitotic propagation and long-term survival in cultivation. The two life cycle stages are morphologically undistinguishable, apart from a small increase in cell size with the higher ploidy level. This is the first report of an isomorphic haploid-diploid life cycle among unicellular algae. Interestingly, our flow cytometric measurements
Funding
This work was supported by the Charles University [GAUK, project no. 1304317]. Additional support was provided by the Czech Academy of Sciences (long-term research development project no. RVO 67985939) and by the Charles University Research Centre program No. 204069.
CRediT authorship contribution statement
Dora Čertnerová: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Resources, Writing – original draft. Martin Čertner: Funding acquisition, Writing – review & editing. Pavel Škaloud: Resources, Writing – review & editing.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
We are indebted to Martin Pusztai, Helena Bestová and Iva Jadrná (Department of Botany, Charles University) for their help with sample collection. We also thank to Yvonne Němcová (Department of Botany, Charles University) and Jens Boenigk (Department of Biodiversity, University of Duisburg-Essen) for providing us with the cultures of Ochromonas tuberculata and S. sphagnicola (LO234KE), respectively.
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