Time Series Data Provide Insights into the Evolution and Abundance of One of the Most Abundant Viruses in the Marine Virosphere: The Uncultured Pelagiphages vSAG 37-F6

Abstract

Viruses play a pivotal role in ecosystems by influencing biochemical cycles and impacting the structure and evolution of their host cells. The widespread pelagiphages infect Pelagibacter spp., the most abundant marine microbe on Earth, and thus play a significant role in carbon transformation through the viral shunt. Among these viruses, the uncultured lytic pelagiphage vSAG 37-F6, uncovered by single-virus genomics, is likely the most numerous virus in the ocean. While previous research has delved into the diversity and spatial distribution of vSAG 37-F6, there is still a gap in understanding its temporal dynamics, hindering our insight into its ecological impact. We explored the temporal dynamics of vSAG 37-F6, assessing periodic fluctuations in abundance and evolutionary patterns using long- and short-term data series. In the long-term series (7 years), metagenomics showed negative selection acting on all viral genes, with a highly conserved overall diversity over time composed of a pool of yearly emergent, highly similar novel strains that exhibited a seasonal abundance pattern with two peaks during winter and fall and a decrease in months with higher UV radiation. Most non-synonymous polymorphisms occurred in structural viral proteins located in regions with low conformational restrictions, suggesting that many of the viral genes of this population are highly purified over its evolution. At the fine-scale resolution (24 h time series), combining digital PCR and metagenomics, we identified two peaks of cellular infection for the targeted vSAG 37-F6 viral strain (up to approximately 10³ copies/ng of prokaryotic DNA), one before sunrise and the second shortly after midday. Considering the high number of co-occurring strains of this microdiverse virus, the abundance values at the species or genus level could be orders of magnitudes higher. These findings represent a significant advancement in understanding the dynamics of the potentially most abundant oceanic virus, providing valuable insights into ecologically relevant marine viruses.

Keywords: Pelagibacter; SAR11; metagenomics; pelagiphage; single-virus genomics; vSAG 37-F6; viruses.

Figure 1

Geographical locations of the sampling points along with a summary of procedures for conducting the time series experiments and research questions addressed (text to the right in figure panels). Two different geographic locations were studied: seawater samples collected from Cape Huertas (Alicante, Spain) were used for the 1-year and 24 h temporal samplings, while seawater from the Blanes Bay Microbial Observatory (Blanes, Spain) was used for the 7-year temporal sampling. Different procedures were conducted as follows: for the case 7-year and 24 h temporal samplings, DNA from microbial fractions were obtained for sequencing (i.e., microbial metagenomes) and digital PCR, respectively. For the 1-year temporal sampling, the viral fraction was purified and the DNA extracted and sequenced, obtaining viral metagenomes.

Figure 2

Temporal sampling across a 7-year time series. (A) Synonymous (grey) and non-synonymous (black) SNPs from the genomic consensus of each monthly sample and their respective positions within the 37-F6 genome. (B) Tridimensional structure of two proteins showing that the placement of the non-synonymous SNPs is primarily situated within the loops of the secondary structure. (C) Monthly measurement of viral abundance for 37-F6 over 7 years, categorized at the species level (black color), assessed using KPKG. CP: Capsid protein; SNPs: single-nucleotide polymorphisms.

Figure 3

Comparison of the ANI among different sample pairs. Relationship between the ANI of two samples and their spatial difference. The red trend line is horizontal, indicating the independence of both parameters. This observation was confirmed by a Pearson’s correlation test, which yielded a coefficient of −0.02. ANI: Average nucleotide identity.

Figure 4

Annual pattern of the 37-F6 virus. (A) Mapping of the reads of each month against the 37-F6 genome; all reads are depicted as matched with an identity of over 70 and a query coverage of over 50 (at the viral genus level). (B) Abundance of the virus measured by the reads recruited by the 37-F6 genome normalized by the viral genome length and the virome length (KPKG). (C) Absolute quantification of 37-F6 by digital PCR, showing the number of viral copies per mL of seawater and ng of viral fraction. CP: Capsid protein; KPKG: kilobase of reads per kilobase of the genome per gigabase of reads.

Figure 5

Diel cycling of the 37-F6 virus. Viral abundance represented and measured by PCR in number of copies of 37-F6 per mL of seawater (black) and copies of 37-F6 per ng of the prokaryotic fraction (red) compared with the total amount of viruses per mL of seawater (grey), measured by staining with SYBR Gold and counting using epifluorescence microscopy.