Viruses infecting a warm water picoeukaryote shed light on spatial co-occurrence dynamics of marine viruses and their hosts

Isolation and characterisation of viruses infecting the picoeukaryote Bathycoccus Clade BII

Bathycoccus BII isolates RCC716 and RCC715 used in our experiments were originally cultured from a nutrient-limited region in the Indian Ocean. Clade BII as a whole has been reported extensively in warm oligotrophic ocean gyres based on metagenome analyses [22,23,24]. Peak abundances occurr when well-developed deep chlorophyll maxima are present, or throughout the photic zone during mixing periods at Station ALOHA of the Hawaii Ocean Time-series [12]. We targeted BATS for viral isolation in springtime because Bathycoccus has been observed at relatively high abundance during this period using qPCR [74]. Here, three viruses were isolated against RCC716 [12] using seawater flown from BATS/Bermuda to the laboratory, obviating bringing this finicky strain into the field for use as a viral host. We then purified the viruses by serial dilutions and sequenced the partial PolB gene to determine whether they were evolutionarily different from other cultured viruses. BLASTn and preliminary phylogenetic analysis using GenBank nr sequences indicated they were distinct from described viruses with deposited sequences, with best BLASTn hits to Bathycoccus prasinos viruses (62–74% nucleotide identity). Transmission electron microscopy (TEM) revealed that all three viruses have similar morphology to other characterised prasinoviruses [75], with icosahedral capsids diameter ranging between 120 and 140 nm (Fig. 1A).

Genomic sequencing and multi-gene evolutionary analyses

Assembly of DNA sequences from the viral isolates after deep sequencing by Illumina rendered one complete dsDNA genome sequence (BII-V3), and two others may still be partial (Table 1). The BII-V2 genome, which was in one contig, was similar in size (~208 kb) to that of BII-V3 (~212 kb). The BII-V1 genome assembly was ~174 kb and comprised of four linear dsDNA scaffolds. The viral concentrate was deeply sequenced (>50x coverage) and minor fragmentation of the genome was partially related to repeats that were not resolved during assembly. The total number of putative open reading frames (ORFs) varied from 220 in BII-V1 to 235 in BII-V2 (Table 1). Gene synteny was globally well-conserved across the BII-Vs and the BpV1 and BpV2 viruses of B. prasinos (Fig. S1), with limited genomic rearrangements. Other genome characteristics such as the coding proportion (~90%) and G + C % (~36%) were similar to other described prasinoviruses infecting Mamiellophyceae [64, 75], for which the reported number of proteins range from 203 to 268 and G + C % from 37 to 45%. However, the full-length PolB gene from the genome assemblies differed for BII-V3 from the other two, in having a 329 amino acid intein (Supplementary information table S3). Likewise, inteins have been reported at the same PolB position in uncultivated prasinoviruses from the subtropical Pacific Ocean [76], where Bathycoccus BII is abundant [12].

To reconstruct a robust phylogeny for the new viruses, we employed 22 proteins previously identified as being shared across all available green algal virus genomes, including both prasinoviruses and chloroviruses [65]. We found all 22 in the predicted coding sequences of BII-V1; however, DNA helicase (SNF2) was not found in BII-V2 or -V3, FAD-dependent thymidylate synthase (thy1) and the topoisomerase IV were not found in BII-V2, nor was the prolyl 4-hydroxylase in the BII-V3 genome. Additional searches with tBLASTn did not recover these genes or fragments of them, suggesting they have been lost. Phylogenomic reconstruction grouped the three BII-Vs with the two BpVs [32], in a fully supported clade that branched adjacent to a large group of viruses that infect various species of Ostreococcus and Micromonas (Fig. 1B). The clade of Bathycoccus viruses was segregated in two subclades with BII-V2 and BII-V3 clustering together adjacent to BII-V1 and BpVs (Fig. 1B). While better resolution of the position of BII-V1 awaits greater taxonomic sampling, our results demonstrated that the three new viruses branch adjacent or basally to BpVs.

Variation in prasinovirus gene content and functions encoded

The three Bathycoccus Clade BII viruses had 72–77% of their proteins held in common, and ~30 unique proteins as well as a few proteins shared by just two of the three viruses (Fig. 1C). The 170 shared proteins had higher amino acid identities between BII-V2 and BII-V3 (73% aa identity) than to BII-V1 (69% and 68%, respectively). Generally, only 19–21% of Bathycoccus viral genes could be assigned a functional category, based on EggNOG classification. Similar functional category distributions were observed across both prasinoviruses and chloroviruses, including lipid metabolism, RNA processing and modification, and nucleotide metabolism and transport (Fig. 2A). Other functional categories were more variable, such as cell wall/membrane/envelope biogenesis genes prevalent in chloroviruses (potentially related to their enveloped nature), as well as genes involved in modification of the capsid with compounds such as with chitin and hyaluronan [77, 78] that are absent from prasinoviruses sequenced to date (Fig. 2A). Within prasinoviruses, most of the unique proteins in the Bathycoccus viruses lack defined functional categories. Among those with functional assignments, all five Bathycoccus viruses encoded a P2X receptor in the intracellular trafficking and secretion category, and both BII-V2 and -V3 encode two proteins putatively involved in degrading the aromatic compound 4-hydroxy-2-oxopentanoate to acetyl-CoA (secondary metabolite category), that otherwise are only encoded by one other prasinovirus, MpV1 [32]. Similar to the phylogenetic relationships, the functional category distributions of BII-V1 were closer to those of BpVs than to BII-Vs. The primary difference was in carbohydrate metabolism, where BII-V2 and -V3 each encodes ribulose-phosphate 3-epimerase (involved in the pentose phosphate pathway and carbon fixation; not found in any other available virus genomes, but encoded by B. prasinos) and TDP-glucose 4,6-dehydratase (involved in biosynthesis of rhamnose and encoded by most other chloroviruses and prasinoviruses [79]). Notably, the putative high-affinity phosphate transporter (PHO4, also termed HAPT) was only present in BII-V1 and BpV1, as well as OtV2 (isolated against the Ostreococcus Clade OII ecotype), and most sequenced viruses of O. lucimarinus (Supplementary information table S3). This gene is hypothesised to enhance phosphate uptake during infection under phosphorus‐limited host growth [25], as observed for the PstS phosphate transport system expressed by cyanophages [80], mitigating limitation of this key component of viral genomes. However, most isolated prasinovirus genomes come from waters that are not considered phosphate-limited, hence presence of this gene may connect to poising the host for responding to sudden availability of other nutrients, such as nitrogen, which is often limiting in the ecosystems from which these viruses were isolated. Studies of virus-cell responses under various limiting nutrients are required to understand the retention of this host-derived HGT.

Hierarchical clustering of orthologous proteins revealed patterns across prasinoviruses that generally corresponded with phylogenetic relationships. The BII- and Bp-viruses shared 130 orthologous proteins and hierarchical clustering (Fig. 2B) followed the clade structure of the phylogenomic reconstruction (Fig. 1B) with the exception of BII-V1 that grouped with BII-Vs, as well as OtV6, which grouped with Micromonas viruses. These orthologous proteins had on average 72% amino acid identity between BII-V2 and BII-V3, and 88% between the two B. prasinos viruses, but between 65 to 67% when comparing members of these two groups (Table 2). BII-V1 orthologs also had 67% and 66% amino acid identity to BII-V2 and BII-V3, respectively, while they had 83% and 80% identity to BpV1 and BpV2, respectively. Collectively, these results indicate that BII-V2 and -V3 diverged from BpVs prior to the divergence of BII-V1.

Of the 130 orthologous Bathycoccus virus proteins, 37% were assigned putative functions revealing core components of this viral group (Supplementary information table S3). These included genes involved in DNA replication and transcription, including PolB (type II), a DNA topoisomerase, a transcription factor S-II, mRNA capping enzymes, ribonucleases, a ribonucleotide reductase, and a dUTPase. Several others are necessary for viral particle synthesis, such as genes encoding structural elements for assembling the virion, including capsid proteins (5–6 copies per genome), as well as transcriptional regulators connected to the replication cycle. The BII viruses showed a number of differences among orthologous protein families. In addition to each having “unique” protein sets, there was a set of BII-V specific orthogroups, as well as some shared with BpVs, and/or other prasinoviruses (Fig. 1C and Supplementary information table S3). First, six predicted proteins showed orthologs across the three BII-Vs, but were not present in other prasinoviruses sequenced to date. Only one of these six was assigned putative function, belonging to the XRE family of transcriptional regulators. Additionally, all BII viruses harboured a tandem duplication of the FstH gene, while other sequenced prasinoviruses (including the two Clade BI viruses) have one copy (Supplementary information table S3). This ATP-dependent metalloprotease has been shown to be involved in photosystem II repair in cyanobacteria [81], and is present in genomes of photosynthetic eukaryotes, including all Mamiellophyceae [15, 16]. In Arabidopsis and Chlamydomonas it has been shown to be involved in protein quality control in the thylakoid membranes [82]. A gene of unknown function was also duplicated in the BII-virus genomes, that is a single copy in BpVs and absent from other sequenced prasinoviruses. Genes putatively encoding a glucose-1-phosphate adenylyltransferase, a glycosyltransferase and a thiamine pyrophosphate-requiring enzyme involved in amino acid biosynthesis were sporadically found in BII-viruses.

Considering the two Bathycoccus virus subclades (Fig. 1B), there is one predicted protein of unknown function exclusive to BpV1, BpV2 and BII-V1 and six predicted proteins shared only by BII-V2 and BII-V3. Among the latter, one belonged to the Ribulose-5-Phosphate-3-Epimerase (RPE) family, which catalyses the interconversion of D-ribulose 5-phosphate (Ru5P) into d-xylulose 5-phosphate, as part of the Calvin cycle (although no transit peptide was detected using TargetP) and in the oxidative pentose phosphate pathway. The ortholog analyses further showed that among prasinoviruses, 9, 17 and 18 genes were unique to BII-V1, BII-V2 and BII-V3, respectively (Fig. 2B). Apart from one nucleotidyltransferase and one glycosyltransferase (group 1) in BII-V1, none of these unique genes had known functions.

To study the evolutionary aspects of the shared prasinovirus proteins, we constructed and examined 130 phylogenies of orthogroups shared between Bathycoccus viruses. Nine showed a topology where all three BII-Vs grouped together with full support (100% bootstrap support), separate from the BpV orthologs, and in contrast to the multi-gene phylogeny where BII-V1 grouped with BpVs (Fig. 1B). The average amino acid similarities within each of these nine protein ortholog groups ranged from 85 to 88% between BII-Vs proteins, while they were 77 to 81% between BII-Vs and BpVs, different from overall amino acid similarity averages (Table 2). Interestingly, proteins from three of these nine ortholog groups, all lacking known functions, were adjacent to each other in the genome, or separated by only one gene. This synteny and co-location likely reflects the acquisition of these genes before co-infecting viruses diverged via viral recombination [83].

Infection dynamics of Bathycoccus viruses

General host specificity of BII-viruses was assessed using two B. prasinos isolates (CCMP1898 and RCC4222, Clade BI), the two available Clade BII isolates (RCC715 and RCC716), four Ostreococcus species and one Micromonas species (Table 3). None were able to infect the B. prasinos, Ostreococcus or Micromonas isolates tested, suggesting BII-V specificity for Bathycoccus Clade BII. Similar host specificity has been observed in O. lucimarinus viruses, none of which infect O. tauri [64], and other viruses of eukaryotic and prokaryotic algae [84, 85]. Some other prasinoviruses appear to have broader host ranges [85,86,87], or their host species are less divergent than the two known Bathycoccus clades. For example, generalist viruses isolated against Micromonas commoda can infect M. bravo [85]. Further investigations are necessary to determine the extent to which the six shared proteins in BII-Vs (absent from BpVs), are responsible for the differences in host and virus specificity of interactions, versus variations in the shared Bathycoccus virus proteins (65–83% similarity). Importantly, host specificity tests for the new viruses described herein were limited by weak sampling of Bathycoccus diversity (in culture; all that we could acquire were tested).

Although specific for the BII clade, the three BII-Vs exhibited variations in infectivity of the two cultured BII strains available, despite their isolation from the same sample and having identical ITS1 and ITS2 sequences. BII-V1 lysed and cleared RCC715 and RCC716 cultures after four days (Table 3). The same was true for BII-V2 and BII-V3, when incubated with RCC716. Different from results for BII-V1, we found that while BII-V2 and -V3 initially lysed RCC715 cultures, resistant populations became evident at day 7 of infectivity tests, and measureable lysis of RCC715 could not be achieved thereafter. These results underscored the need to further examine host-virus interactions for the three new viruses.

Infection dynamics over time course experiments further illuminated differences in BII-V impacts on hosts. In these experiments, growth rates of the uninfected (control) RCC715 and RCC716 cultures were 0.45 ± 0.04 day⁻¹ and 0.49 ± 0.06 per day, respectively, similar to rates during the pre-experiment acclimation period (T-test, p > 0.05). Host and virus dynamics were similar for RCC715 and RCC716 infected with BII-V1 (Fig. S2 and Fig. 3), with cell numbers starting to diverge from control abundances 10 h after inoculation (T-test, p < 0.05). Normalised chlorophyll-derived fluorescence of BII-V1 infected RCC715 and RCC716 was lower than in controls after two hours (Fig. S2C and Fig. S3A, T-test, p < 0.05), significantly in advance of host lysis and release of new viral particles which began 8-10 h after viral inoculation. Thus host physiology was markedly altered long before major cell lysis occurred.

Both BII-V2 and BII-V3 exhibited longer latent periods than BII-V1 in RCC716, such that host lysis and release of the new viral particles did not occur until 16–18 h after inoculation. Additionally, the estimated burst size of BII-V2 and BII-V3 (32 ± 3 and 140 ± 23 progeny virions per cell, respectively) was lower than that of BII-V1 (330 ± 267 progeny virions per cell, Supplementary information table S4). In all experiments, we strove to standardise the multiplicity of infection (MOI, ratio of infectious viruses to host cells), testing the MOI before and after each experiment. In these experiments, the measured MOI by MPN assays of BII-V3 (MOI = 0.01) deviated from initial characterisation, and as a result was lower than BII-V1 (MOI = 0.1) and BII-V2 (MOI = 0.1). This presumably made the contact rate of infectious BII-V3 and hosts lower, than for the other two viruses, however between BII-V1 and BII-V2 MOIs were consistent between experiments. Still, the viral-induced host mortalities by BII-V2 and BII-V3 at experiment end were 53 ± 7% and 35 ± 15%, lower than for BII-V1 (69 ± 13%; T-test, p < 0.01; Supplementary information table S5 and Fig. 3). Infectivity by prasinoviruses of O. lucimarinus also varies (reportedly from 14 to 32% infectivity during one less-controlled experiment) across closely related viruses that share higher average ortholog identity (90%) [30] than do the BII-Vs. Although it is tempting to compare with other phycodnaviruses, the host-virus models are often rapidly evolving systems and the methods are not consistent especially as many studies measure MOI once, at initial characterisation and not subsequently, and may, or may not, use a host strain grown in a defined state [88]. Our data show that a 10-fold variation occurs even between viruses that would typically be considered closely related. Thus, seemingly small genetic distances in common marker genes like PolB can reflect major differences in ecological impact of eukaryotic viruses.

Given that a resistant RCC715 population proliferated during BII-V2 and BII-V3 cross-infectivity tests, we expected infection dynamics of viruses inoculated into RCC715 cultures to differ from RCC716 experiments. Indeed, the MOI of BII-V2 (MOI = 0.004) and BII-V3 (MOI = 0.001) during RCC715 experiments were at least an order lower than in RCC716 infections. Although RCC715 lysis was observed under BII-V2 and BII-V3 infections, cell abundance did not always differ significantly from uninfected controls (Fig. S2A). Resistance responses have been reported for B. prasinos inoculated with BpV2, and M. pusilla against MpV1 in batch cultures [89], where lysis was observed after viral inoculation, but a fraction of cells always survived. Collectively, these results may reflect how arms-race dynamics [90] could lead to a range of infection phenotypes in closely related viruses like the BII-Vs.

Integrating evolutionary and experimental results

Our multi-gene and PolB phylogenies suggested BII-V1 was evolutionarily more closely related to B. prasinos viruses than to the BII-V2 and BII-V3 viruses (Fig. 1B). Such incongruencies between host and viral phylogenies have been observed in other prasinoviruses at a taxonomic level finer than the genus [87, 91], but are not well understood. Such incongruencies are often interpreted as reflecting host-switching events. Indeed, the Bathycoccus clades can overlap in the field [12], thus there does not appear to be an ecological barrier to host switching for Bathycoccus viruses, even though those isolated here show a high species- and strain-specificity. Moreover, our phylogenetic analyses of genes shared by Bathycoccus viruses resolved nine genes (including three that were co-localised in the genomes) that formed supported clades that separated BII-V proteins from the BpV proteins. These proteins could enable BII-V1 to infect Clade BII strains like BII-V2 and BII-V3, while it was evolutionary closer to BpVs. However, BII-V1 did not infect B. prasinos and multiple other scenarios are possible. Moreover, unlike the multi-gene phylogeny, the distribution of orthogroups (Fig. 2B) indicated a close relationship between BII-Vs in terms of gene content, wherein BII-V1 clusters with BII-V2 and -V3, and not with BpVs, contributing more complexity to understanding the mechanistic basis for the differences observed in virulence and potential resistance mechanisms.

There are several factors arising from prior studies that likely contribute to the differential patterns in infectivity and virulence of the BII-Vs on Clade BII members. First, variations in host resistance levels have been proposed to connect to the small outlier chromosomes (SOC), possessed by all Mamiellophyceae sequenced thus far, potentially linking to a virus-immune state based on a study of O. tauri and its viruses [92]. The SOC has fragmented genes and greater variation than other genomic regions across species, that could contribute to differing viral immune responses across strains. Although this region cannot be assembled from metagenomic data, and hence is not known for Clade BII members, mapping of metagenomic reads to the B. prasinos genome demonstrates microdiversity within B. prasinos populations [23]. Thus, the genomes of RCC716 and RCC715 might well possess SOC divergence that underpins observed differences in host-virus dynamics.

An important consideration with respect to the dynamics we observed is that field studies of specific genes point to a greater diversity within the Bathycoccus genus than currently recognised, including uncultured strains within the Clade BII that are more diverged than RCC716 and RCC715. In this case, while we isolated viruses against Bathycoccus RCC716 (i.e., BII-V1), among the BII-Vs there could either be different strategies, or they could be optimised for different but closely related hosts. In tropical Atlantic waters, a study using targeted metagenomics and subsequent PCR-based sequencing of the spliceosomal gene, PRP8, identified molecular diversity within Bathycoccus Clade BII, with two co-existing variants [22]. Additionally, among 13 Bathycoccus environmental clones annotated as Bathycoccus Clade BII rRNA/ITS sequences [12], four from the tropical Pacific and Atlantic (KY382370, KY382373, KY382374, KY368637) present two single nucleotide polymorphisms (see taxonomic revision section). Together with the analyses herein, an interpretation of these results is that BII-V1 dynamics arise from it being a lineage of BII viruses, with different host-optimisation than BII-V2 and -V3. In this scenario, addition of other BII-V1-like viruses would lead to a BII-V1-clade branching separately from both the BII-V2 and -V3-like viruses and B. prasinos viruses in both phylogenomic and orthology presence/absence analyses.

Distributions of host and virus based on marine metagenome surveys

The diversity and distributions of the specific marine eukaryotic hosts (prasinophytes and beyond) in connection to their viruses in the field have not yet been well characterised. Therefore, we next examined the distributions of the Bathycoccus viruses in nature, alongside the distributions of the two known Bathycoccus ecotypes, BI (B. prasinos) and BII (RCC716/715). Alongside a quantitative study demonstrating the two ecotypes occupy different marine water types, with some overlap [12], metagenome analyses show variations in the presence/absence of BI and BII in the Eastern North Pacific (ENP) [23] and Tara Oceans [24]. Further, sequences affiliated to previously available BpV sequences have been reported in cellular-fraction filtered seawater samples from the ENP and in Tara Oceans [22, 93]. We recruited reads from two Tara metagenome studies [4, 94] to Bathycoccus virus core genes (requiring 95% nucleotide identity) and compared their distributions to previously reported [24] relative abundances of Bathycoccus Clade BI and BII-affiliated reads in Tara data. One or both Bathycoccus Clades were detected in all 54 metagenomes analysed, while BI and BII viruses were detected in 31 out of 54 corresponding viromes (size fraction < 0.22 μm; Fig. 4A).

The distributions of the Bathycoccus Clades and their respective viruses showed strong co-occurrence patterns. Viruses infecting B. prasinos were concomitantly recovered with the Bathycoccus Clade BI host (Fig. 4B; Spearman’s correlation, p-value < 10⁻⁵, r² = 0.61). Similarly, viruses infecting the Bathycoccus Clade BII were concomitantly recovered with the Bathycoccus Clade BII host (Spearman’s correlation, p-value < 10⁻⁴, r² = 0.46). Out of the 31 samples where viruses and hosts were co-detected, the more abundant virus-type (BpVs or BII-Vs) corresponded with higher abundance of the known host clade in 29 of them. The other two stations were in frontal regions: Tara Station 70_SRF exhibited more BpV than BII-V (67% BpV of total Bathycoccus virus reads), while 96% of the Bathycoccus host reads were attributed to Clade BII (Fig. 4B); Tara Station 78-SRF, exhibited the opposite pattern with 83% BII-V comprising total Bathycoccus virus reads, while host reads predominantly mapped to Bathycoccus Clade BI (Fig. 4B). Stations 70, 72 and 76 are in the subtropical Atlantic basin and stations 68 and 78 sampled Agulhas rings from the South Atlantic Ocean (which behave as subpolar environments travelling across this subtropical region) [95]. Stations to the north (76, 72) have warmer waters and showed dominance of the BII host Clade, while the lower temperature adapted B. prasinos (BI) alone was detected in those to the south (82, 68). Mixing of Bathycoccus Clades has been reported in an eastern North Pacific transition zone [12], and supporting growth of multiple Ostreococcus Clades in Pacific transitional and frontal regions [96, 97]. Here, stations 70 and 78 appear to be similarly transitional, with the relative mapping abundances potentially reflecting differences of dispersion of free virions compared to host cells, or differences of viral decay rates or, from a technical perspective, statistical noise with the number of reads mapped in these two transitional samples that were lower compared to other viromes. To examine whether environmental factors would constrain Bathycoccus host and virus distribution, we performed a CCA using the relative abundance of Bathycoccus hosts and viruses alongside available environmental data (temperature, light, oxygen, salinity, phosphate and nitrite+nitrate). Temperature was identified as being significantly associated with the host and virus distribution (p-value < 0.01, Fig. S4), aligning with prior work indicating temperature, or a co-associated (but unmeasured) parameter, is one of the major drivers of the Bathycoccus ecotype distribution [12].

Ecologically the results further emphasise dynamics among different viruses and their epidemiology. Our analyses are based on mapping data to two previously available genomes for BI viruses and our three new BII viruses (all were isolated against cultured hosts), and therefore would not detect as yet uncharacterised/unsequenced viruses. Further, other viral genotypes could be present in metagenomic data, but not detected due to limitations connected to size-fractionation or depth of sequencing. Keeping these two caveats in mind, our results add a new dimension to environmental studies of eukaryotic algal viruses. Species-level specificity was previously observed in the field for viruses infecting Ostreococcus Clade OI, which flourishes in coastal and mesotrophic waters [97], but was not recovered in low latitude (warm) water samples [64] where Ostreococcus ecotype (Clade OII) usually thrives. Exploration of within-region dynamics showed that the haptophyte Emiliania huxleyi co-occurs with four to six different viral genotypes in the North Sea, but only two of these viruses dominated during and after blooms based on DGGE data from mesocosm studies [98]. In our study, the high intra-ecotype specificity observed, at least for the limited number of viruses isolated against members of the same Bathycoccus Clade, is notable. We found co-occurrences of different Bathycoccus viral types (two and sometimes three for BII viruses) infecting the same host Clade in 23 of the Tara Oceans samples analysed (Fig. 4C, Supplementary information table S6). Moreover, while BII-Vs often co-occurred in data from within individual sites (as did BpVs), generally only one viral type dominated (Fig. 4C). This is consistent with the Bank model [99], suggesting only a small fraction of a virus community is active and abundant at any given time while most populations are rare and dormant, forming a seed-bank that can ‘Kill-the-Winner’ when hosts reach critical abundance thresholds [100].

Prasinovirus diversity in nature

We examined prasinovirus distributions in nature without a cultivation step by searching Tara Oceans metagenome assemblies [46] and other environmental data for related PolB sequences. One hundred and seventy three sequences longer than 130 aa affiliated to prasinoviruses using BLASTp and preliminary tree analyses. Phylogenetic analysis revealed that 51% of these belonged to Bathycoccus virus lineages, 6% were attributed to Ostreococcus and Micromonas viruses (Fig. 4D), and the rest could not be assigned to known prasinoviruses. Multiple Bathycoccus virus PolB sequences belonged to supported clades that contained the newly isolated viruses. BII-V1 and BII-V2 formed clades with nine and 14 environmental sequences (>98% bootstrap support), respectively (Fig. S5). BII-V3 formed a clade with eight environmental sequences, two of which contained almost identical PolB inteins to that in BII-V3. These two viral PolB sequences were recovered from subtropical/tropical North Pacific samples (stations 132 and 138). Members of the broader environmental BII-V3-like clade (21 sequences), lacked the intein. Inteins in the spliceosomal protein PRP8 of wild Bathycoccus BII [22] lack amino acid similarity with the viral inteins. Multiple other BII-V-like sequences grouped in clades related to those of the isolated BII-Vs. Overall the bulk of prasinovirus sequences recovered from existing metagenomics data were from BII-V related lineages, not from BpVs.

We observed that one PolB from cold Arctic waters (contig_35, Fig. S5) grouped with BII-V sequences. A prior study recovered B. prasinos metagenomic reads from the same sample and emphasised the possible importance of viruses in controlling Bathycoccus populations in the Amundsen Gulf [51]. Our findings of a BII-V affiliated sequence could reflect the presence of yet another undescribed Bathycoccus host lineage, adapted to cold waters, or a more generalist BII-V that can infect B. prasinos. However, high dispersion rates of BII viruses through transport from the North Pacific could also account for this result. Co-occurrence analyses of hosts and their specific viruses, with attention to currents and water mass transport, are needed to resolve this question.

Analysis of PolB diversity also identified viral clades that differed with respect to environmental parameters. Temperature at the time of collection differed between BII-V1 (24.4 ± 1.9 °C) and both BII-V2 and BII-V3 (19.2 ± 4.3^oC and 19.12 ± 4.6^oC, respectively; Mann-Whitney, p < 0.01) clades. Likewise, the BpV-related viruses were from significantly lower temperature waters (13.2 ± 8.6^oC) than BII-V1, but not significantly different for BII-V2 and BII-V3. This mirrors CCA results from Bathycoccus host and virus distributions in metagenomics data and significant association with temperature (Fig. S4). Further, seven of the nine BII-V1 sequences came from the persistently warmest ocean in the world, the Indian Ocean, where the hosts we used for viral bait were also isolated. Additionally, two Indian Ocean studies noted diversity of potential Bathycoccus viruses, based on partial PolB gene sequences [33, 34]. Sequence comparisons with our data indicated these were BII-Vs (25 out of 28 Bathycoccus viral OTUs; data not shown) and only ~10% were BpVs, although the sequences were too short for inclusion in our phylogenetic reconstruction. We posit BII-V1 is a virus optimised for Bathycoccus Clade BII strains prevalent in warm waters (e.g., 28 °C) such as those at BATS in mid-summer. In contrast, BII-V2 and -V3 may specialise on another type of BII, or an as yet unidentified, but related Bathycoccus lineage at BATS in spring, when we collected our viral isolation sample. Here again, additional data collected with attention to temporal dynamics and ocean physics is needed to resolve drivers behind the observed patterns. The fact that viruses from a habitat rarely sampled in viral isolation efforts (i.e., open-ocean waters) revealed marked divergence, both from each other and from BpVs, emphasises the importance of such efforts for interpretation of sequence-based environmental surveys.

Taxonomic revision of the genus Bathycoccus

The differences observed herein with respect to Bathycoccus virus distributions and viral exclusivity mirror support for the hosts, Bathycoccus Clade BII and Bathycoccus Clade BI, as being different species. Further, the Clade BII Internal Transcribed Spacer (ITS) sequences delineate it from Clade BI ITS [12] at a level generally considered appropriate for species designation. As observed by Limardo and collaborators (2017), and herein using different imaging methodologies (Fig. 5A), there is no apparent morphological differences between representatives from both clades. We, therefore, name strain RCC716 Bathycoccus calidus based on molecular diagnoses and the protocols of the International Code of Nomenclature for Algae, Fungi and Plants. The species name refers to RCC716 being isolated from warm (28^oC) ocean waters, akin to distributions observed by qPCR [12], and metagenomics/transcriptomic read distributions [23, 24, 101]. This naming will avoid confusion arising from distributions of Clade BII and Clade BI being merged as “B. prasinos” patterns. BI is represented by the species B. prasinos [11] and was described prior to the availability of molecular data or isolation of RCC716.

Revision of the genus Bathycoccus

Bathycoccus Eikrem & Throndsen, 1990, emend. Bachy, Yung and Worden.

Genus characteristics as previously described [11]. Three nuclear pores clustered at a single basal location similar to those in O. tauri [102]. Type species Bathycoccus prasinos Eikrem & Throndsen.

Emendation of the species Bathycoccus prasinos Eikrem & Throndsen, 1990, emend. Bachy, Yung and Worden.

Description – characters of the genus. Images and sequences describe the type specimen (CCMP1898). The latter are available in GenBank under the accession number JX625115 (partial nuclear-encoded rRNA transcriptional unit).

Emended diagnosis – in the ITS2 of the nuclear-encoded rRNA transcriptional unit, universal helix 1 contains a 10 nt loop composed of 5′-CUUUUAUUUU-3′ from positions 2104-2113 of JX625115 (Fig. 5B). Differences from other Mamiellophyceae are also exhibited in the ITS1 [103].

Holotype – strain CCMP1898, also known as SCCAP K-0417, is the type specimen and is preserved in a metabolically inactive state at the NCMA (https://ncma.bigelow.org/). It was isolated by J. Throndsen from the surface waters at 40°45′00″N, 14°19′48″E in the Gulf of Naples, Italy (17 April 1986).

Habitat and ecology – temperate to high-latitude marine waters and coastal regions. Distributed in samples analysed herein ranging from 8 to 25 °C and salinities of 30–35 ppt. Niche-specific Tara samples 7.3 to 17.0 °C (exclusively Clade BI, no Clade BII). Not detected in oligotrophic marine regions.

Bathycoccus calidus Bachy, Yung and Worden, sp. nov.

Description − characters of the genus [11]. Sequences describe the holotype (RCC716) and are available in GenBank under the accession KY563784 (partial nuclear-encoded rRNA transcriptional unit).

Validating illustration – Fig. 5A and [12].

Molecular diagnosis − in the second internal transcribed spacer (ITS2) of the nuclear-encoded rRNA transcriptional unit, nucleotide 5 of the linker between the additional helix specific to Bathycoccaceae [103] and helix 4 is C, and base pairs 16/17 of helix 4 are U-A/C-G (Fig. 5B).

Holotype − cells of B. calidus strain RCC716 are preserved in a permanently metabolically inactive state for electron microscopy (in resin) in the University and Jepson Herbaria (UC/JEPS) at the University of California Berkeley (access number UC 2084460). DNA is also preserved (at −80 °C) in the Worden lab. RCC716 was collected on 6 November 2003 by Fabrice Not at 70 m depth in the Indian Ocean at a station (14°28’48”S, 113°27’00”E) with a surface temperature of 28 °C and purified from other algae by Florence Le Gall on 4 August 2003 using lab-based flow cytometry sorting. The living strain is maintained at the Roscoff Culture Collection (roscoff-culture-collection.org).

Habitat and ecology – present in warm oligotrophic ocean gyres, peak abundance typically in well-developed deep-chlorophyll maxima or throughout the photic zone during mixing periods. Distributed in samples analysed herein having salinities ranging from 33 to 36 ppt and temperatures from 10 to 29 °C (at the time of collection). Niche-specific Tara samples 18.6–27.7 °C (exclusively BII, no BI). Maximum reported abundance in multi-depth time-series at Station ALOHA occurred at 35 ppt, 23 °C [12].

Source: Ecology - nature.com