Genome sequence of Ostreococcus tauri virus OtV-2 enlightens the role of picoeukaryote niche separation in the ocean Running title: Genome sequence of a low light Ostreococcus tauri virus

Ostreococcus tauri, a unicellular marine green alga, is the smallest known free-living eukaryote and is ubiquitous in the surface oceans. The ecological success of this organism has been attributed to distinct lowand high-light adapted ecotypes existing in different niches at a range of depths in the ocean. Viruses have already been characterised that infect the high-light adapted strains. Ostreococcus tauri virus isolate OtV-2 is a large double stranded DNA algal virus that infects a low-light adapted strain of O. tauri and was assigned to the algal virus family Phycodnaviridae, genus Prasinovirus. Our working hypothesis for this study was that different viruses infecting high-light vs. low-light adapted O. tauri strains would provide clues to propagation strategies that would give them selective advantages within their particular light niche. Sequence analysis of the 184,409 base pair linear OtV-2 genome revealed a range of core functional genes exclusive to this low-light genotype and included a variety of unexpected genes, such as those encoding a RNA polymerase sigma factor, at least four DNA methyltransferases, a cytochrome b5 and a high affinity phosphate transporter. It is clear that OtV-2 has acquired a range of potentially functional genes from its host, other eukaryotes and even bacteria over evolutionary time. Such piecemeal accretion of genes is a trademark of large doublestranded DNA viruses that has allowed them to adapt their propagation strategies to keep up with host niche separation in the sunlit layers of the oceanic environment. Introduction Ostreococcus tauri is the smallest free-living eukaryote described to date, with a size of less than 1 μm (11). The cellular organisation of O. tauri is very simple with only a single chloroplast, a single mitochondrion, a single Golgi body and a very reduced cytoplasmic compartment (22). O. tauri also lacks flagella and there is no cell wall surrounding the cell membrane. The Ostreococcus genus includes distinct genotypes physiologically adapted to highor low-light environments, providing evidence of niche adaptation in eukaryotic picophytoplankton (39). Such adaptation has been well-characterised in recent studies on the diversity and ecophysiology of the cyanobacterium, Prochlorococcus. The global success of this abundant prokaryotic primary producer has been in part attributed to distinct lowand high-light adapted ecotypes existing in different niches and utilising different resources (38). Strains of O. tauri have been isolated in geographically different locations and depths and were shown to be genetically (based on 18S rRNA and internal transcribed spacer (ITS) sequencing) and physiologically (light-limited growth rates) different from one another (39). Growth rates of strains isolated from deep in the euphotic zone were reported to display severe photoinhibition at high light intensities (and are thus commonly referred to as lowlight-adapted strains), while strains isolated from surface waters have very slow growth rates at the lowest light intensities (and are thus commonly referred to as highlight-adapted strains). Genetic distances between isolates appear to result from a contrast in both light and nutrient conditions experienced between surface and deep isolates, which drive their genetic divergence (7, 39, 44). Another factor that has not been considered in determining niche separation in Ostreococcus spp. is the role that viruses play. There are two primary mechanisms that viruses use to shape the diversity and magnitude of microbial populations. The first is simply killing cells leading to host-specific lysis. Here, viruses exert an important influence on the biogeochemistry of the oceans as nutrients are shunted between the particulate and dissolved phases (20, 51). A second and arguably more important function that viruses play is their role in horizontal gene transfer (HGT). Viruses can simply be seen as vectors that facilitate gene shuttling, a role that has been poorly described in marine systems. However, genes transferred between hosts and viruses can give selective advantages in growth (for the host) or propagation (for the virus) in particular environmental niches. Information on virus propagation strategies and HGT events can be inferred and deduced, respectively, from genome sequence information. Ostreococcus spp. is an excellent model system since there are two host genomes, both of which are high-light adapted species (15, 32) and two virus genomes (14, 50) already sequenced. All grow or propagate in high-light adapted systems. Our working hypothesis for this study was that different viruses infecting high-light vs low-light adapted O. tauri strains would provide clues to propagation strategies that would give them selective advantages within their particular light niche. Here, we report the genomic sequence of a virus (OtV-2) that infects a low-light adapted strain of O. tauri and we compare inferred functionality of coding sequences with its high-light counterparts. Materials and methods