Shimodaira-Hasegawa (SH) and approximately unbiased (AU) tests for alternative monophyletic relationships of parabasalian classes.

<p>Abbreviations of the classes are shown in the footnote of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029938#pone-0029938-t001" target="_blank">Table 1</a> or the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029938#pone-0029938-g001" target="_blank">Figure 1</a>. Asterisks indicate that the tested monophyly was significantly different from the best ML topology at <i>P</i><0.05. Each of the monophyletic groupings of C+Tt and Tm+Tn appeared in the best ML topology.</p

Proposed evolutionary relationships of parabasalids.

<p>The tree shows the relationships of the six parabasalian classes. Flagellar multiplication in a single mastigont system has occurred independently in the boxed classes. The multiplications have occurred ancestrally in two classes (marked with filled circles) and probably twice within the other class (open circle). Triangles indicate the occurrence of cytoskeletal simplification in undulating membrane (UM) and costa. See the text for details.</p

Maximum likelihood phylogenetic analyses of parabasalids based on GAPDH (A), actin (B), and EF-1α (C) sequences.

<p>Unambiguously aligned protein sequences of 278 (A), 280 (B) and 274 (C) sites were used for phylogenetic inference. The species names of the parabasalids except for the genus <i>Trichonympha</i> are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029938#pone-0029938-t001" target="_blank">Table 1</a>. The GAPDH sequences of <i>Trichonympha acuta</i> and <i>Eucomonympha imla</i> and the EF-1α sequence of <i>Trichomonas tenax</i> (not shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029938#pone-0029938-t001" target="_blank">Table 1</a>) were also included in the analyses. Although the EF-1α sequences published for <i>Pentatrichomonas</i> and <i>Tritrichomonas </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029938#pone.0029938-Malik1" target="_blank">[57]</a> were not included in C because of their shorter sequence length, the analysis with a reducing number of sites (219 sites) demonstrated that they were related very closely to the sequences from the same taxa shown in C. The sequence accession number was indicated for each taxon. The sequences used for the concatenation are in bold. The trees were estimated in RAxML and the numbers near the nodes indicate the bootstrap values. Values below 50% are not shown. Vertical bars to the right of the trees represent the parabasalian classes according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029938#pone.0029938-Cepicka2" target="_blank">[26]</a>: C, Cristamonadea; S, Spirotrichonymphea; Tn, Trichonymphea; Tt, Tritrichomonadea; H, Hypotrichomonadea; and Tm, Trichomonadea. Scale bars correspond to 0.10 substitutions per site.</p

Phylogenetic relationship of parabasalids inferred from the concatenated dataset.

<p>The concatenated dataset comprising 278 amino acid sites of GAPDH, 280 amino acid sites of actin, 274 amino acid sites of EF-1α, and 1338 nucleotide sites of SSU rRNA gene sequences was analyzed in 28 parabasalian species. The tree was estimated in RAxML using separate models with the parameters and branch length optimized for each gene partitions individually. The supporting values (bootstrap in RAxML/Bayesian posterior probability) are indicated at the nodes. Values below 50% or 0.5 are indicated with hyphens. When the site-heterogeneous CAT model was used in each partition, the identical tree topology with similar bootstrap values was obtained (data not shown). Vertical bars to the right of the tree represent the parabasalian classes. The scale bar corresponds to 0.10 substitutions per site.</p

<i>In situ</i> survey from morphological observations.

<p>Temporal variation of biomasses (µg C L⁻¹) of (a) Phytoplankton and (b) micro- (ciliates and dinoflagellates) and nanoheterotrophic protists (represented mainly by heterotrophic nanoflagellates HF) at the coastal station R1 (50°48′ N, 1°34′ E) in the eastern English Channel (Strait of Dover) from March to April 2009. Results are shown here in terms of biomass – calculated based on abundance and cell size – (see Method section) and to better represent the stocks of the different compartments.</p

Rarefaction curves and richness estimator.

<p>Rarefaction curves representing the numbers of Operational Taxonomic Units (OTU) <i>versus</i> the number of reads plotted from tag pyrosequencing data. The OTUs were determined using the program Uclust <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039924#pone.0039924-Edgar1" target="_blank">[38]</a>, with a cutoff value set to 0.03 (OTUs were grouped when their level of sequence similarity ≥97%) for the analysis. The table indicates the number of reads, the number of OTU, the richness estimator (Schao1 and Margalef indices), and the heterogeneity of the diversity (Shannon and Simpson indices) for each sampling date.</p

Differential infra-specific diversity of Haptophyceae before and during their spring bloom.

<p>Tag pyrosequencing reads assigned to the group of Haptophyceae were independently extracted for both sampling dates. Reads sharing more than 97% sequence identity were grouped under the same OTUs using Uclust <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039924#pone.0039924-Edgar1" target="_blank">[38]</a>. All the reads having a size above 400bp and representative of an OTU were aligned together with reference sequences using Clustalw <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039924#pone.0039924-Thompson1" target="_blank">[84]</a>. The representative sequences and reference sequences were aligned using Muscle <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039924#pone.0039924-Edgar1" target="_blank">[38]</a>. The resulting alignments, manually curated using the Bioedit software (<a href="http://www.mbio.ncsu.edu/bioedit/bioedit.html" target="_blank">http://www.mbio.ncsu.edu/bioedit/bioedit.html</a>), were used to build phylogenetic trees. For tree construction, the Seaview 4.0 software (<a href="http://pbil.univ-lyon1.fr/software/seaview.html" target="_blank">http://pbil.univ-lyon1.fr/software/seaview.html</a>) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039924#pone.0039924-Gouy1" target="_blank">[39]</a> was used with Neighbor-joining (NJ) and K2P substitution method. Bootstrap values were estimated from 1000 replications. The unrooted phylogenetic tree displayed 21 OTUs inferred from 299 reads corresponding to March 31^st sample, and 56 OTUs inferred from 2,759 reads for April 21^st sample. On each branch are indicated the sampling dates with “1-” corresponding to March 31^st and “2-” to April 21^st, the reads I.D. as it was submitted into the SRA database (SRX031036) and between brackets the number of reads composing the OTU. The taxonomical subdivisions are indicated on the left side of the figure. For relevant clades, the sum of reads “n” composing a cluster was given for each sampling dates.</p

Proportion of taxonomic groups identified in the English Channel using pyrosequencing of 18S rRNA gene hypervariable regions.

<p>The reads obtained from pyrosequencing of 18S rRNA hypervariable region were subject to BlastN <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039924#pone.0039924-Altschul1" target="_blank">[34]</a> search against the Silva SSU rRNA database (<a href="http://www.arb-silva.de/" target="_blank">http://www.arb-silva.de/</a>) to assign a taxonomic group. The pie diagram displayed the proportion of reads, obtained at both sampling dates, belonging to a particular taxonomic group (threshold E = 10⁻⁵). The composition of the Alveolates, being the most dominant group, was displayed on an additional smaller pie diagram. “Not assigned” correspond to reads having a match in the Silva database, but without a precise taxonomic assignment. The percentage of reads assigned to a specific group was given when above 1%.</p