Microbial communities are critical to ecosystem dynamics and biogeochemical cycling in the open oceans. Viruses are essential elements of these communities, influencing the productivity, diversity, and evolution of cellular hosts. To further explore the natural history and ecology of open-ocean viruses, we surveyed the spatiotemporal dynamics of double-stranded DNA (dsDNA) viruses in both virioplankton and bacterioplankton size fractions in the North Pacific Subtropical Gyre, one of the largest biomes on the planet. Assembly and clustering of viral genomes revealed a peak in virioplankton diversity at the base of the euphotic zone, where virus populations and host species richness both reached their maxima. Simultaneous characterization of both extracellular and intracellular viruses suggested depth-specific reproductive strategies. In particular, analyses indicated elevated lytic interactions in the mixed layer, more temporally variable temperate phage interactions at the base of the euphotic zone, and increased lysogeny in the mesopelagic ocean. Furthermore, the depth variability of auxiliary metabolic genes suggested habitat-specific strategies for viral influence on light-energy, nitrogen, and phosphorus acquisition during host infection. Most virus populations were temporally persistent over several years in this environment at the 95% nucleic acid identity level. In total, our analyses revealed variable distributional patterns and diverse reproductive and metabolic strategies of virus populations in the open-ocean water column.

Viruses are the most abundant biological entities on Earth and play key roles in host ecology, evolution, and horizontal gene transfer. Despite recent progress in viral metagenomics, the inherent genetic complexity of virus populations still poses technical difficulties for recovering complete virus genomes from natural assemblages. To address these challenges, we developed an assembly-free, single-molecule nanopore sequencing approach, enabling direct recovery of complete virus genome sequences from environmental samples. Our method yielded thousands of full-length, high-quality draft virus genome sequences that were not recovered using standard short-read assembly approaches. Additionally, our analyses discriminated between populations whose genomes had identical direct terminal repeats versus those with circularly permuted repeats at their termini, thus providing new insight into native virus reproduction and genome packaging. Novel DNA sequences were discovered, whose repeat structures, gene contents, and concatemer lengths suggest they are phage-inducible chromosomal islands, which are packaged as concatemers in phage particles, with lengths that match the size ranges of co-occurring phage genomes. Our new virus sequencing strategy can provide previously unavailable information about the genome structures, population biology, and ecology of naturally occurring viruses and viral parasites.

Bacteriophages are numerically the most abundant DNA-containing entities in the oligotrophic ocean, yet how specific phage populations vary over time and space remains to be fully explored. Here, we conducted a metagenomic time-series survey of double-stranded DNA phages throughout the water column in the North Pacific Subtropical Gyre, encompassing 1.5 years from depths of 25 to 1,000 m. Viral gene sequences were identified in assembled metagenomic samples, yielding an estimated 172,385 different viral gene families. Viral marker gene distributions suggested that lysogeny was more prevalent at mesopelagic depths than in surface waters, consistent with prior prophage induction studies using mitomycin C. A total of 129 ALOHA viral genomes and genome fragments from 20 to 108 kbp were selected for further study, which represented the most abundant phages in the water column. Phage genotypes displayed discrete population structures. Most phages persisted throughout the time-series and displayed a strong depth structure that mirrored the stratified depth distributions of co-occurring bacterial taxa in the water column. Mesopelagic phages were distinct from surface water phages with respect to diversity, gene content, putative life histories, and temporal persistence, reflecting depth-dependent differences in host genomic architectures and phage reproductive strategies. The spatiotemporal distributions of the most abundant open-ocean bacteriophages that we report here provide new insight into viral temporal persistence, life history, and virus-host-environment interactions throughout the open-ocean water column.

Many plants attract insect pollinators with floral nectar (FN) and ant "bodyguards" with extrafloral nectar (EFN). If nectar production is costly or physiologically linked across glands, investment in one mutualism may trade off with investment in the other. We confirmed that changes in FN and EFN availability alter pollination and ant defense mutualisms in a field population of Turnera ulmifolia. Plants with additional FN tended to produce more seeds, while plants with reduced EFN production experienced less florivory. We then mimicked the consumptive effects of mutualists by removing FN or EFN daily for 50 days in a full factorial design using three Turnera species (T. joelii, T. subulata, and T. ulmifolia) in a glasshouse experiment. For T. ulmifolia and T. subulata, but not T. joelii, removing either nectar reduced production of the other, showing for the first time that EFN and FN production can trade off. In T. subulata, increased investment in FN decreased seed set, suggesting that nectar production can have direct fitness costs. Through the linked expression of EFN and FN, floral visitors may negatively affect biotic defense, and extrafloral nectary visitors may negatively affect pollination.

Time-series can provide critical insights into the structure and function of microbial communities. The analysis of temporal data warrants statistical considerations, distinct from comparative microbiome studies, to address ecological questions. This primer identifies unique challenges and approaches for analyzing microbiome time-series. In doing so, we focus on (1) identifying compositionally similar samples, (2) inferring putative interactions among populations, and (3) detecting periodic signals. We connect theory, code and data via a series of hands-on modules with a motivating biological question centered on marine microbial ecology. The topics of the modules include characterizing shifts in community structure and activity, identifying expression levels with a diel periodic signal, and identifying putative interactions within a complex community. Modules are presented as self-contained, open-access, interactive tutorials in R and Matlab. Throughout, we highlight statistical considerations for dealing with autocorrelated and compositional data, with an eye to improving the robustness of inferences from microbiome time-series. In doing so, we hope that this primer helps to broaden the use of time-series analytic methods within the microbial ecology research community.