Low Evolutionary Diversification in a Widespread and Abundant Uncultured Protist (MAST-4)

Abstract

Recent culture-independent studies of marine planktonic protists have unveiled a large diversity at all phylogenetic scales and the existence of novel groups. MAST-4 represents one of these novel uncultured lineages, and it is composed of small (~2 μm) bacterivorous eukaryotes that are widely distributed in marine systems. MAST-4 accounts for a significant fraction of the marine heterotrophic flagellates at the global level, playing key roles in the marine ecological network. In this study, we investigated the diversity of MAST-4, aiming to assess its limits and structure. Using ribosomal DNA (rDNA) sequences obtained in this study (both pyrosequencing reads and clones with large rDNA operon coverage), complemented with GenBank sequences, we show that MAST-4 is composed of only five main clades, which are well supported by small subunit and large subunit phylogenies. The differences in the conserved regions of the internal transcribed spacers 1 and 2 (ITS1 and ITS2) secondary structures strongly suggest that these five clades are different biological species. Based on intraclade divergence, ITS secondary structures and comparisons of ITS1 and ITS2 trees, we did not find evidence of more than one species within clade A, whereas as many as three species might be present within other clades. Overall, the genetic divergence of MAST-4 was surprisingly low for an organism with a global population size estimated to be around 10(24), indicating a very low evolutionary diversification within the group.

Biogeography of the uncultured marine picoeukaryote MAST-4: temperature-driven distribution patterns

“…In a previous study, we showed that MAST-4 was composed of five main clades (Rodríguez-Martínez et al, 2012). To verify the robustness of these clades, we obtained 228 additional sequences of 500-650 bp encompassing the V9 region of the 18S rDNA and the ITS1 region from five different locations (Figure 1), including the locations from the previous study and one additional site.…”

“…It had 42 mismatches (except for one sequence with one mismatch and three sequences with two mismatches) to non-target sequences from the SILVA database (Pruesse et al, 2007). The reverse primer M4.58S-R (5 0 -GTTGCGAGAACCTA-GAC-3 0 ), located in the 5.8S rDNA, was designed to have a perfect match with the 22 MAST-4 sequences (Rodríguez-Martínez et al, 2012). This primer had at least two mismatches with all stramenopile sequences extracted from GenBank, except for some Labyrinthulida sequences (3 with no mismatches and 62 with one mismatch).…”

“…Sequences from clone libraries, together with sequences from the 'SSU-LSU' dataset (Rodríguez-Martínez et al, 2012), were aligned with MAFFT v6.853 (Katoh and Toh, 2008) with the E-INS-I algorithm, using a MAST-7 sequence as an outgroup. The alignment was inspected visually and modified using secondary structure models folded in mFOLD (Zuker, 2003) as explained before (Rodríguez-Martínez et al, 2012).…”

“…The alignment was inspected visually and modified using secondary structure models folded in mFOLD (Zuker, 2003) as explained before (Rodríguez-Martínez et al, 2012). A maximum likelihood (ML) phylogenetic tree was done with RAxML v7.0.4 MPI version (Stamatakis, 2006), using the General Time Reversible model of nucleotide substitution and a Gamma distributed rate of variation across sites.…”

“…In a previous study (Rodríguez-Martínez et al, 2012), we investigated the diversity of an important uncultured picoeukaryote, the MAST-4 (marine stramenopile group 4) lineage , which is widespread in surface marine waters (except polar systems) and represents approximately 9% of heterotrophic flagellates , Rodríguez-Martínez et al, 2009. We observed that despite its huge number of cells in the oceans, MAST-4 has a very low genetic divergence and is composed of only five main clades, each representing at least one biological species.…”