Surveys of microbial communities across transitions coupled with contextual measures of the environment provide a useful approach to dissect the factors determining distributions of microorganisms across ecological niches. Here, monthly time-series samples of surface seawater along a transect spanning the nearshore coastal environment within Kāneʻohe Bay on the island of Oʻahu, Hawaiʻi, and the adjacent offshore environment were collected to investigate the diversity and abundance of SAR11 marine bacteria (order Pelagibacterales) over a 2-year time period. Using 16S ribosomal RNA gene amplicon sequencing, the spatiotemporal distributions of major SAR11 subclades and exact amplicon sequence variants (ASVs) were evaluated. Seven of eight SAR11 subclades detected in this study showed distinct subclade distributions across the coastal to offshore environments. The SAR11 community was dominated by seven (of 106 total) SAR11 ASVs that made up an average of 77% of total SAR11. These seven ASVs spanned five different SAR11 subclades (Ia, Ib, IIa, IV, and Va), and were recovered from all samples collected from either the coastal environment, the offshore, or both. SAR11 ASVs were more often restricted spatially to coastal or offshore environments (64 of 106 ASVs) than they were shared among coastal, transition, and offshore environments (39 of 106 ASVs). Overall, offshore SAR11 communities contained a higher diversity of SAR11 ASVs than their nearshore counterparts, with the highest diversity within the little-studied subclade IIa. This study reveals ecological differentiation of SAR11 marine bacteria across a short physiochemical gradient, further increasing our understanding of how SAR11 genetic diversity partitions into distinct ecological units.

High-throughput dilution culture has proved to be a successful approach to bring some difficult-to-isolate planktonic microorganisms into culture, including the highly abundant SAR11 lineage of marine bacteria. While the long-term preservation of bacterial isolates by freezing them in the presence of cryoprotectants, such as glycerol, has been shown to be an effective method of storing viable cells over long time periods (i.e., years), to our knowledge it had not previously been tested for its efficacy in preserving raw seawater for later use as an inoculum for high-throughput cultivation experiments.

In this study, we sought a means to increase current culture collections of SAR11 marine bacteria by testing the use of seawater cryopreserved with glycerol as an inoculum. In July 2017, raw seawater was collected outside of Kaneohe Bay, Hawai'i, in the tropical Pacific Ocean. A portion of this sample was diluted in seawater-based growth medium to create 576 x 2 mL dilution cultures containing 5 cells each and incubated for a high-throughput cultivation experiment, while another portion was cryopreserved in 10% glycerol. After ten months, a cryopreserved aliquot of seawater was thawed, diluted in seawater-based growth medium, and distributed to create a second high-throughput cultivation experiment of 480 x 2 mL dilution cultures containing 5 cells each and 94 cultures containing 105 cells each. The raw seawater cultivation experiment resulted in the successful isolation of 54 monocultures and 29 mixed-cultures, while cryopreserved seawater resulted in 59 monocultures and 29 mixed cultures. Combined, the cultures included 51 SAR11 isolates spanning 11 unique 16S rRNA gene amplicon sequence variants (ASVs) from raw seawater inoculum and 74 SAR11 isolates spanning 13 unique ASVs from cryopreserved seawater. A vast majority (115 of 125) of SAR11 isolates from the two HTC experiments were members of SAR11 subclade Ia, though isolates of subclades IIIa and Va were also recovered from cryopreserved seawater and subclade Ib was recovered from both. The four most abundant SAR11 subclade Ia ASVs found in the initial seawater sample used to create both culture experiments were isolated by both approaches.