Best tree of the Spirotrichea inferred by two-gene combined sequences (Atub-SSU).

<p>Bootstrap values for branches of the ML tree is given on nodes. The scale bar corresponds to 5 substitutions per 100 nucleotide positions. Dargyrome patterns and natural habitats are given after species name of euplotids by symbols. Clades I-IV for euplotids were designated according to Petroni <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040635#pone.0040635-Petroni1" target="_blank">[42]</a> and Yi et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040635#pone.0040635-Yi4" target="_blank">[38]</a>.</p

Assessing Whether Alpha-Tubulin Sequences Are Suitable for Phylogenetic Reconstruction of Ciliophora with Insights into Its Evolution in Euplotids

<div><p>The current understanding of ciliate phylogeny is mainly based on analyses of a single gene, the small subunit ribosomal RNA (SSU-rDNA). However, phylogenetic trees based on single gene sequence are not reliable estimators of species trees, and SSU-rDNA genealogies are not useful for resolution of some branches within Ciliophora. Since congruence between multiple loci is the best tool to determine evolutionary history, we assessed the usefulness of alpha-tubulin gene, a protein-coding gene that is frequently sequenced, for ciliate phylogeny. Here, we generate alpha-tubulin gene sequences of 12 genera and 30 species within the order Euplotida, one of the most frequently encountered ciliate clades with numerous apparently cosmopolitan species, as well as four genera within its putative sister order Discocephalida. Analyses of the resulting data reveal that: 1) the alpha-tubulin gene is suitable phylogenetic marker for euplotids at the family level, since both nucleotide and amino acid phylogenies recover all monophyletic euplotid families as defined by both morphological criteria and SSU-rDNA trees; however, alpha-tubulin gene is not a good marker for defining species, order and subclass; 2) for seven out of nine euplotid species for which paralogs are detected, gene duplication appears recent as paralogs are monophyletic; 3) the order Euplotida is non-monophyletic, and the family Uronychiidae with sequences from four genera, is non-monophyletic; and 4) there is more genetic diversity within the family Euplotidae than is evident from dargyrome (geometrical pattern of dorsal “silverline system” in ciliates) patterns, habit and SSU-rDNA phylogeny, which indicates the urgent need for taxonomic revision in this area.</p> </div

Support for Major Clades of Spirotrichean Species in Analyses Based on Five Datesets.

<p>NOTE.<b>-</b>nm = nonmonophyletic.</p

Euplotid Species for Which Alpha-Tubulin Genes Were Sequenced in the Present Work.

<p>Euplotid Species for Which Alpha-Tubulin Genes Were Sequenced in the Present Work.</p

Intraspecific Distances Between/Among α-Tubulin Clones and Between/Among Paralogs.

<p>NOTE.<b>-</b><i>N,</i> number of clones; <i>d</i>, number of nucleotide substitutions per site calculated using Tamura-Nei model; <i>dA</i>, number of amino acid substitutions per site calculated using Dayhoff model; R/S, number of replacement site substitutions/number of synonymous substitutions among clones.</p>#<p>Fixed between paralogs.</p>**<p><i>Euplotes sinicus</i> population I: Clone 1–4; <i>E. sinicus</i> population II: Clone 5.</p

Best tree of the Spirotrichea inferred by Maximum likelihood of Dataset Atub_n74.

<p>Species newly sequenced in the present study are shown in bold type. Bootstrap values for branches of the ML tree and posterior probability values for BI tree, respectively, are given on nodes. Fully supported (100%/1.00) branches are marked with solid circles. The scale bar corresponds to 10 substitutions per 100 nucleotide positions. Dargyrome patterns and natural habitats are given after species name of euplotids by symbols. Clades I-IV for euplotids were designated according to Petroni <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040635#pone.0040635-Petroni1" target="_blank">[42]</a> and Yi et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040635#pone.0040635-Yi4" target="_blank">[38]</a>.</p

Representative euplotid species from live material and after protargol impregnation.

<p>Representative euplotid species from live material and after protargol impregnation.</p

Genetic Differentiation of the Mitochondrial Cytochrome Oxidase <i>c</i> Subunit I Gene in Genus <i>Paramecium</i> (Protista, Ciliophora)

<div><p>Background</p><p>The mitochondrial cytochrome <i>c</i> oxidase subunit I (<i>COI</i>) gene is being used increasingly for evaluating inter- and intra-specific genetic diversity of ciliated protists. However, very few studies focus on assessing genetic divergence of the <i>COI</i> gene within individuals and how its presence might affect species identification and population structure analyses.</p><p>Methodology/Principal findings</p><p>We evaluated the genetic variation of the <i>COI</i> gene in five <i>Paramecium</i> species for a total of 147 clones derived from 21 individuals and 7 populations. We identified a total of 90 haplotypes with several individuals carrying more than one haplotype. Parsimony network and phylogenetic tree analyses revealed that intra-individual diversity had no effect in species identification and only a minor effect on population structure.</p><p>Conclusions</p><p>Our results suggest that the <i>COI</i> gene is a suitable marker for resolving inter- and intra-specific relationships of <i>Paramecium</i> spp.</p></div

Table S1 from Species delimitation for the molecular taxonomy and ecology of a widely distributed microbial eukaryotes genus <i>Euplotes</i> (Alveolata, Ciliophora)

Euplotes species analyzed in this study, and their reliability assessment based on rDNA sequences (Bold numbers were newly sequenced) and other integrated information

Phylogenetic tree of the barcoding region of 263 cytochrome <i>c</i> oxidase subunit I (<i>COI</i>) gene sequences of the genus <i>Paramecium</i> and genera <i>Lembadion</i> and, <i>Tetrahymena</i> inferred by Bayesian Inference (BI) analysis based on dataset <i>COI</i>-f.

<p>The branches are shaded according to subgenera <i>Chloroparamecium</i>, <i>Helianter</i>, <i>Cypriostomum</i>, <i>Paramecium</i>, proposed by Fokin <i>et al. </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077044#pone.0077044-Fokin1" target="_blank">[28]</a>. The scale bar corresponds to 30 substitutions per 100 nucleotide positions. For <i>P. bursaria</i>, Clade H includes populations sampled from Australia, Germany, and Poland; Clade I and J include populations sampled from Russia and Poland, Germany, Ukraine, and Canada; Clade K includes populations sampled from China (Pb1C1-4 & Pb2C &Pb3C2-3), Austria, Japan, and Italy; Clade L includes populations sampled from China (Pb3C1), Russia, and Japan (see details in Fig. S2 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077044#pone.0077044.s003" target="_blank">file S3</a>). For <i>P.caudatum</i>, Clade A includes populations sampled from China (PcC1-4 and AM072774), Australia, USA, and Brazil while members of Clade B were sampled from Germany, Italy, Russia, UK, Norway, Hungary, Slovenia, and Austria (see details in Fig. S3 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077044#pone.0077044.s003" target="_blank">file S3</a>). Inconsistent sequences (FJ905146, FJ905147, EU056259, EU056258, DQ837977, DQ837982, JF741258, JF304183) are marked in red <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077044#pone.0077044-Tarcz1" target="_blank">[14]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077044#pone.0077044-Barth3" target="_blank">[42]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077044#pone.0077044-StrderKypke2" target="_blank">[47]</a>.</p