MASTDIEV · Diversity patterns across lineages and evolutionary hierarchies in marine unicellular eukaryotes

Diversity patterns across lineages and evolutionary hierarchies in marine unicellular eukaryotes

In the oceans, microbes are prevalent and have a key role for the functioning of the global ecosystem. Marine microbes include members belonging to the three main domains of life: Bacteria, Eukarya and Archaea. Bacteria and to a more limited extent Archaea have been the focus of several studies. Eukarya still remain as the less known group of marine microbes. Among eukaryotes, Stramenopiles represent one of the major branches of the tree of life, encompassing several lineages that are microbial. In this project (MASTDIEV) we have investigated several aspects of one specific set of marine microeukaryotes, the Marine Stramenopiles (MAST). These groups (probably < 20) have been recently discovered and are in several cases phylogenetically unrelated. In most cases, MAST cells appear to be heterotrophic (bacterivorous) flagellates. Some of the MAST groups (e. g. MAST-4) can be abundant in marine microbial assemblages and therefore have an important impact in bacterial communities. We have studied several ecological and phylogenetic aspects of MAST species using Next Generation Sequencing techniques (454 and Illumina). In the following section, we summarise our findings (note that studies c-f are still ongoing): a) Study 1: Here we have investigated diversity patterns and activity of MAST species. We have used rDNA and rRNA from environmental samples (including several geographic locations) and 454 sequencing. We found different amounts of diversity and activity patterns within different MAST groups. In addition, 454 allowed us to approach to the saturation of diversity in different groups. More information about this study can be found in: Logares, R., Audic, S., Santini, S., Pernice, M. C., de Vargas, C., Massana, R. 2012. Diversity patterns and activity of uncultured marine heterotrophic flagellates unveiled with pyrosequencing. The ISME journal. In press b) Study 2: This study investigated diversity patterns and potential sexual compatibility within MAST-4, one of the most abundant MAST groups. Here we use a combination of Sanger and 454 sequencing. We found that MAST-4 contains a very limited phylogenetic diversity and includes a handful groups. In addition. We we have obtained evidence for sexual incompatibility between some MAST-4 lineages, indicating that they are different species. All results of this project can be found in: Rodriguez-Martinez R, Rocap G, Logares R, Romac S, Massana R. 2012. Low evolutionary diversification in a widespread and abundant uncultured protist (MAST-4). Molecular Biology and Evolution. In press. c) Study 3: Here we have revisited MAST phylogeny using a large amount of Sanger sequences from public databases plus 454 sequences. The analyses indicate that only a handful groups are missing from what is known so far. Thus, this study strengthen the phylogeny known for MAST groups and unveils a few new groups as well as improves the topology withing some groups. Large-scale phylogenies were carried out using the Supercomputer MareNostrum in Barcelona. This allowed us to carry out what are probably the largest phylogenetic analysis in a marine microeukaryote. d) Study 4: In this study we are investigating the genomic composition in different MAST-4 strains. MAST-4 single cells have been isolated using Flow Citometry. The genomes of these cells have been amplified using Multiple Displacement Amplification (MDA) generating Single Amplified Genomes (SAGs). These SAGs have been adapted for Illumina sequencing. Genomic comparisons between different strains of MAST-4 will allow us to determine the amount of genomic differentiation among evolutionary closely related strains. In particular, we will be able to determine whether local adaptation has occurred among strains from different locations. e) Study 5: In this study we are mapping sequences from metagenomic libraries to one large genomic fragment of MAST4. Using this approach, we will be able to find genomic variations in this flagellate species associated to different locations or environments. f) Study 6: In this study we are investigating the genetic diversity of MAST-4 at the population level using 454 sequencing of several marine environmental samples. Different to Study 2, here we aim at investigating population differentiation patterns using the highly variable Internal Transcribed Spacer 1 (intron). This permits us to look deeper into populations within putative species. Altogether, these different studies allows us to obtain a deeper understanding on the diversity and roles of MAST lineages in the oceans. Considering MAST species, as well as other flagellates, in our studies of microeukaryote communities is important for a more holistic view of the functioning of oceanic ecosystems. Although this knowledge is still building up, it will be important for policy makers in a few years, as understanding the functioning of the oceans is vital for understanding how the biosphere works as well as how humans may impact such functioning.

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