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

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

Abundance and α-diversity comparison of bacteriome and mycobiome between PMA-pretreated and untreated samples.

<p>Abundance and α-diversity comparison of bacteriome and mycobiome between PMA-pretreated and untreated samples.</p

Venn diagram representing number of shared and specific bacterial (A) and fungal (B) genera between PMA-treated and untreated samples.

<p>In each case, the first 5 most prevalent genera are listed.</p

Comparison of the enumeration of <i>P</i>. <i>aeruginosa</i> cells between cultures and qPCRs of PMA-treated and untreated sputum samples.

<p>Comparison of the enumeration of <i>P</i>. <i>aeruginosa</i> cells between cultures and qPCRs of PMA-treated and untreated sputum samples.</p

Effect of PMA on the composition of mycobiome of each sample.

<p><b>(A)</b>: Relative abundance of fungal genera of each sample (“yes” or “no” indicates the samples with or without PMA-pretreatment). <b>(B)</b>: PCA plot of the first two components of mycobiome of samples with and without PMA-pretreatment. Each marker represents treatment conditions (filled triangle symbols PMA-treated samples, filled square symbols untreated samples). Each color represents a given sputum sample (G040.III: dark blue; G088.II: chartreuse; G088.III: dark green; G172.I: yellow; G172.II: orange; G172.III: dark orange; G014.II: dark grey; G014.III: black; G176.II: firebrick; G176.III: dark red).</p

<i>p-values</i> and <i>r-values</i> of similarity analysis (one-way ANOSIM) of the bacterial and fungal OTU data.

<p><i>p-values</i> and <i>r-values</i> of similarity analysis (one-way ANOSIM) of the bacterial and fungal OTU data.</p

Abundance and α-diversity comparison of bacteriome and mycobiome between PMA-pretreated and untreated samples.

<p>Abundance and α-diversity comparison of bacteriome and mycobiome between PMA-pretreated and untreated samples.</p