Alternating nuclear DNA content in chrysophytes provides evidence of their isomorphic haploid-diploid life cycle

Highlights

• Chrysophytes have an isomorphic haplo-diploid life cycle.

• Alternation of life stages is accompanied by small change in cell size, however, overall phenotypes are indistinguishable.

• Life-stage transitions, accompanied with ploidy level shifts, are synchronized among cells.

Abstract

Across eukaryotic organisms there is a great diversity of life cycles. This particularly applies to unicellular eukaryotes (protists), where the life cycles are still largely unexplored, although this knowledge is key to understanding their biology.

To detect the often inconspicuous transitions among life cycle stages, we focused at shifts in ploidy levels within strains of unicellular chrysophyte alga. Representatives of three genera (Chrysosphaerella, Ochromonas, and Synura) were analysed for nuclear DNA contents using a propidium iodide flow cytometry. Selected strains exhibiting ploidy level variation were also surveyed for DNA base composition (GC content) and cell size. Additionally, we tracked ploidy level changes in seven strains under long-term cultivation.

An alternation of two ploidy levels was revealed in the life cycle of chrysophytes with both life cycle stages capable of mitotic growth and long-term survival in cultivation. With the exception of a small increase in cell size with higher ploidy, both life cycle stages shared the same phenotype and also had highly similar genomic GC content. Further, we detected three ploidy levels in two Synura species (S. glabra, S. heteropora), where the highest ploidy (putatively 4x) most likely resulted from a polyploidization event.

Consequently, chrysophytes have a haploid-diploid life cycle with isomorphic life cycle stages. As far as we know, this is the first report of such life cycle strategy in unicellular algae. Life cycle stages and life stage transitions seem to be synchronized among all cells coexisting within a culture, possibly due to chemical signals. Particular life stages may be more successful under certain environmental conditions, for our studied strains the diploid stage prevailed in cultivation.

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?