Recent studies have uncovered evidence for a huge store of diversity in marine planktonic protists (protozoa and other eukaryotic microorganisms), matching or exceeding the incredible diversity observed for marine bacteria and archaea. Understanding relationships between this diversity and ocean ecosystem function is critical as humans navigate the effects of global change such as climate warming and ocean acidification. The two overall goals of this project are: 1) to discover and compare the diversity of marine protists across ocean gradients, and 2) to generate reference genomes of dominant, uncultured protist taxonomic groups, to serve as a resource for a broad scientific community.
This proof-of-concept project teams Bigelow Laboratory for Ocean Sciences' new Single Cell Genomics Center with the Tara Oceans Expedition to explore protistan diversity across important oceanographic gradients. Tara Oceans is an independently-funded, three-year, circum-global expedition to describe the diversity of marine plankton (oceans.taraexpeditions.org). The boat, Tara, is a modern 110-ft sailing schooner with wet and dry laboratories and a complete suite of oceanographic instruments. The expedition is collecting samples for high-throughput pipelines for genome sequencing as well as digital imaging protist morphology. Single protistan cells will be sorted from samples collected on Tara. The genomes of sorted single protist cells will be amplified, and these single amplified genomes (SAGs) will be phylogentically screened using DNA bar codes to identify the most abundant and diversed taxa for further genomic characterization. The French national sequencing center, Genoscope, has agree to sequence hundreds of SAGs (at no cost to this project) to form reference genomes for subsequent community-level molecular analyses (parallel samples will also be sequenced by other Tara Oceans partners using state-of the-art methods such as metagenomics, meta-transcriptomics, and pyrotagging).
The broader impacts of this project include the training of undergraduate and postdoctoral students, and involvement in various established education and outreach programs at Bigelow Laboratory and via the Tara Oceans platform. In addition the project will create a large library of marine protist SAGs to be archived and shared with other researchers interested in screening them for specific genes or performing whole genome sequencing in the future.
This was a proof-of-concept project to demonstrate that the diversity of micro-eukaryotes (protists) from surface marine waters could be sampled and measured using the single cell genomics approach that has been used successfully for marine bacteria. The diversity of microscopic life in the oceans is known to be large, but very poorly studied. Tara Oceans (www.oceans.taraexpeditions.org), an international expedition that went around the world to study the diversity of planktonic ecosystems in surface ocean waters, gave us the opportunity to test these innovative methods to understand the diversity of planktonic marine life. The goal of the Tara Oceans expedition was to better understand planktonic ecosystems as well as their interactions within the marine environment. For this project, we used the single cell approach to estimate diversity and retrieve whole genomes of uncultured micro-eukaryotic cells. The single cell genomics approach is divided into a series of 4 steps; 1) single cell isolation into a multi-well plate from a water sample using cell sorting technology (flow cytometry), 2) lysis of the sorted single cells using a freeze-thaw method and chemical disruption, 3) whole genome amplification using previously published techniques, and 4) whole genome sequencing, assembly and analysis. For this pilot project, we chose 8 Tara Oceans samples from the Mediterranean Sea and the Indian Ocean to test the single cell genomics approach. These samples represented a wide latitudinal range, from 42 °N to 21 °S, and a range of marine ecosystems including a coral atoll, the Adriatic Sea, and equatorial Indian Ocean waters. The two most important findings of this project were 1) that the single cell genomics approach was able to sample the diversity of the dominant micro-eukaryotes without major biases, and 2) we could distinguish cell types that were predominantly photosynthetic from those that primarily ingested other cells for energy. An added bonus of this approach is that we can retrieve whole genomes of individual, uncultured marine microbes to study their biology more generally, such as their exact identities, metabolic potentials, viral infections, recently ingested prey organisms, and symbiotic associations. The whole genome analysis is outside the scope of this project, but this work is underway in collaboration with others researchers and DNA sequencing facilities. Another important outcome of this study is the demonstration of the large taxonomic diversity that is contained in very small volumes of surface ocean water. Our samples were about 5 milliliters (1 teaspoon) each, and the amount of sample actually isolated and examined was even smaller. Yet these samples routinely contained eukaryotic organisms from groups across the whole tree of life (Fig. 1). This means that these diverse organisms are living and co-existing at very small scales in the oceans, a concentration of diversity not observed in larger scale ecosystems.
