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

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

Haplotype network of <i>Paramecium bursaria</i> generated on the basis of the maximum-likelihood tree.

<p>Black circles indicate intermediate or unsampled haplotypes, while lines between points represent nucleotide substitutions. Wherever there are more than four substitutions, they are indicated by numbers. Clades K, L, H, I, J are marked to match the corresponding clades in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077044#pone-0077044-g004" target="_blank">Figure 4</a>. Colored circles and squares indicate haplotypes whose size is proportional to the number of individuals showing that haplotype. Haplotype_7 is represented by 4 clones of Pb1C1; haplotype_14 is represented by 4 clones of Pb1C2 and 7 clones of Pb1C3; haplotype_22 is represented by 5 clones of Pb1C4 and 3 clones of Pb2C1; haplotype_25 is represented by 3 clones of Pb3C1.</p

Haplotype network of <i>Paramecium</i> sp. (A) based on the dataset <i>COI</i>_nw, <i>P. nephridiatum</i> (B) based on dataset <i>COI</i>_nn, <i>P. duboscqui</i> (C), based on dataset <i>COI</i>_nd and <i>P. caudatum</i> (D) based on dataset <i>COI</i>_nc generated on the basis of the maximum-likelihood tree.

<p>Each line between points represents a single mutational step. A haplotype is represented by a circle whose size is proportional to the number of individuals showing that haplotype. Haplotypes are colored to match the respective population in the map. A) Haplotype_1 is represented by 2 clones of PwC1; Haplotype_3 is represented by 4 clones of PwC1 and 2 clones of PwC2; B) Haplotype_4 is represented by 1 clone of PdC1, 1 clone of PdC2, 1 clone of PdC3 and 2 clones of PdC4; C) Haplotype_2 is represented by 4 clones of PnC1 and 1 clone of PnC3; D) Haplotype_6 is represented by 4 clones of PcC1 and 5 clones of PcC3; Haplotype_9 is represented by 6 clones of PcC2 and 5 clones of PcC4; Haplotype_13 is represented by 3 clones of PcC3.</p

Variable site details of <i>Paramecium bursaria</i> and <i>Paramecium caudatum</i>.

<p>Alignment of amplified <i>COI</i> sequences (primer binding regions excluded) based on datasets <i>COI</i>_nb and <i>COI</i>_nc. The nucleotides shaded with rectangles and circles are used to illustrate the levels of diversity found among different clones.</p

Spliceosomal introns in <i>Blastocystis</i>.

<p>(A) Sequences flanking the predicted exon–intron junctions in subtype (ST) 1 were aligned separately for each intron category and visualized with WebLogo3 (<a href="http://weblogo.threeplusone.com/" target="_blank">http://weblogo.threeplusone.com/</a>). The category and number (<i>N</i>) of each spliceosomal intron type are shown on the right. (B) Distribution of intron size in 3 sequenced <i>Blastocystis</i> ST genomes. Data for this figure can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2003769#pbio.2003769.s032" target="_blank">S11 Data</a>.</p

Peroxisomal machinery genes in selected organisms.

<p>Black circles indicate the gene is present. Grey circles indicate multiple isoforms.</p

Conserved polyadenylation motif sites (TGTTTGTT) in the genomes of <i>Blastocystis</i> ST1, ST4, and ST7.

<p>Conserved polyadenylation motif sites (TGTTTGTT) in the genomes of <i>Blastocystis</i> ST1, ST4, and ST7.</p

A comparison of unique genes between <i>Blastocystis</i> subtype (ST) pairs to pairs of protistan pathogens.

<p>The percentage of an organism’s protein-coding gene set, which is unique when compared to another organism’s protein-coding gene set and vice versa, is denoted by the width of the ribbon between the 2 as well as being indicated on the ribbon. For example, in a comparison between ST7 and ST1, 20% of the genes in ST7 are not represented in the ST1 set, while 10% of ST1's genes are not found in ST7. Comparisons are based on BLASTp results with an expect value (e-value) threshold of 1e-30 and >50% coverage of the query. <b>Abbreviations:</b> <i>C</i>., <i>Cryptosporidium</i>; <i>L</i>., <i>Leishmania</i>; <i>T</i>., <i>Theileria</i>. Plots were generated using Circos.</p

Randomized Axelerated Maximum Likelihood (RAxML) tree of kinases identified from <i>Blastocystis</i> subtypes (STs) 1, 4, and 7.

<p>Blue lines correspond to proteins from ST1, black from ST4, and red from ST7. Kinase families are indicated on the periphery.</p