Description of the genomic DNAs used in this study.

a<p>Letters A to H correspond to the <i>Symbiodinium</i> clades. Species names of outgroup samples are indicated: <i>Gymnodinium simplex</i>, <i>Pelagodinium beii</i>, and <i>Polarella glacialis</i>.</p>b<p>Alpha-numeric names correspond to <i>Symbiodinium ITS-2</i> rDNA molecular taxonomy sensu <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029816#pone.0029816-Pochon3" target="_blank">[71]</a>. Letters correspond to the <i>Symbiodinium</i> clades, and numbers correspond to a specific <i>ITS-2</i> sequence. All samples are genetically distinct, except for <i>Symbiodinium</i> A2, which was found in two distinct cultures and referred here to as A2_1 and A2_2. Sample D1.2 corresponds to the PSP1-05 sample originally isolated from the sponge <i>Haliclona koremella</i> (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029816#pone.0029816-Pochon3" target="_blank">[71]</a> for details).</p>c<p>Culture names of DNAs extracted from <i>Symbiodinium</i> cultures. N/A = Not Available.</p><p>*Indicates new sequences.</p

Estimation of divergence rates between markers for <i>Symbiodinium</i> types C1, C15, C90, and C91.

<p>The minimum, maximum and averaged uncorrected genetic distances among clade C <i>Symbiodinium</i> types are indicated for each marker investigated. Calculations for <i>calmodulin</i>, <i>rad24</i>, and <i>actin</i> were made on sequence alignments excluding (−) and including (+) introns.</p>a<p>No sequences were obtained for <i>Symbiodinium</i> C90 and C91 so only two types were compared.</p>b<p>No sequences were obtained for <i>Symbiodinium</i> C90, so only three types were compared.</p

Topological comparison of benchmark <i>nr28S</i> and four selected candidate genes.

<p>(<b>A</b>) 71 nuclear large subunit (<i>nr28S</i>) sequences (alignment size: 915 bp) (<b>B</b>) 26 cytochrome oxidase subunit 1 (<i>coI</i>) sequences (1057 bp), (<b>C</b>) 92 <i>calmodulin</i> sequences (154 bp), (<b>D</b>) 73 <i>rad24</i> sequences (580 bp), and (<b>E</b>) 71 <i>actin</i> sequences (925 bp). The <i>nr28S</i> topology is used here as the benchmark marker, with colors corresponding to clades A (red), B (pink), C (green), D (brown), E (orange), F (dark blue), G (yellow), and H (light blue). Phylogenies are rooted using the dinoflagellates <i>Gymnodinium simplex</i>, <i>Pelagodinium beii</i>, and <i>Polarella glacialis</i>. Detailed phylogenetic reconstructions, including node support values from the ML bootstrap analyses and Bayesian posterior probabilities, as well as the GenBank accession numbers, are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029816#pone.0029816.s001" target="_blank">Figures S1</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029816#pone.0029816.s002" target="_blank">S2</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029816#pone.0029816.s003" target="_blank">S3</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029816#pone.0029816.s004" target="_blank">S4</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029816#pone.0029816.s005" target="_blank">S5</a>.</p

Intron position mapping of three intron-containing genes.

<p>Positions and numbers of coding (exons [E]; shown in green) and non-coding regions (introns [I]; shown in red) in the genes <i>calmodulin</i> (<b>A</b>), <i>rad24</i> (<b>B</b>), and <i>actin</i> (<b>C</b>). The sizes of the non-coding regions indicated here depict the maximum intron size recorded in genomic samples in each <i>Symbiodinium</i> clade. DNA alignments ranged from 1,107 bp to 3,087 bp in length and letters A to H correspond to the eight <i>Symbiodinium</i> clades.</p

Flow diagram of the step-wise procedure for molecular marker identification.

<p>Flow diagram of the step-wise procedure for molecular marker identification.</p

Characteristics of the four genes (<i>coI</i>, <i>calmodulin</i>, <i>rad24</i>, and <i>actin</i>) selected for in-depth phylogenetic analyses.

<p>Characteristics of the four genes (<i>coI</i>, <i>calmodulin</i>, <i>rad24</i>, and <i>actin</i>) selected for in-depth phylogenetic analyses.</p

Seven out of eighty-four candidate genes selected for downstream analyses.

<p>Genes are sorted by decreasing level of transcript abundance in the two <i>Symbiodinium</i> EST libraries combined. Details of the EST libraries displaying hits in BLASTn are indicated for each gene by letters A, C, Ac, At, Ht, Kb, Km, and Lp, which correspond to EST libraries <i>Symbiodinium</i> A, <i>Symbiodinium</i> C, <i>Amphidinium carterae</i>, <i>Alexandrium tamarense</i>, <i>Heterocapsa triquetra</i>, <i>Karenia brevis</i>, <i>Karlodinium micrum</i>, and <i>Lingulodinium polyedrum</i>, respectively. Protein descriptions were obtained using BLASTx. The complete list of candidate genes (n = 84) identified after BLASTn comparisons of eight dinoflagellate EST libraries is presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029816#pone.0029816.s006" target="_blank">Table S1</a>.</p

<em>Cthulhu Macrofasciculumque</em> n. g., n. sp. and <em>Cthylla Microfasciculumque</em> n. g., n. sp., a Newly Identified Lineage of Parabasalian Termite Symbionts

<div><p>The parabasalian symbionts of lower termite hindgut communities are well-known for their large size and structural complexity. The most complex forms evolved multiple times independently from smaller and simpler flagellates, but we know little of the diversity of these small flagellates or their phylogenetic relationships to more complex lineages. To understand the true diversity of Parabasalia and how their unique cellular complexity arose, more data from smaller and simpler flagellates are needed. Here, we describe two new genera of small-to-intermediate size and complexity, represented by the type species <i>Cthulhu macrofasciculumque</i> and <i>Cthylla microfasciculumque</i> from <i>Prorhinotermes simplex</i> and <i>Reticulitermes virginicus</i>, respectively (both hosts confirmed by DNA barcoding). Both genera have a single anterior nucleus embeded in a robust protruding axostyle, and an anterior bundle flagella (and likely a single posterior flagellum) that emerge slightly subanteriorly and have a distinctive beat pattern. <i>Cthulhu</i> is relatively large and has a distinctive bundle of over 20 flagella whereas <i>Cthylla</i> is smaller, has only 5 anterior flagella and closely resembles several other parababsalian genera. Molecular phylogenies based on small subunit ribosomal RNA (SSU rRNA) show both genera are related to previously unidentified environmental sequences from other termites (possibly from members of the Tricercomitidae), which all branch as sisters to the Hexamastigitae. Altogether, <i>Cthulhu</i> likely represents another independent origin of relatively high cellular complexity within parabasalia, and points to the need for molecular characterization of other key taxa, such as <i>Tricercomitus</i>.</p> </div

Tertiary Endosymbiosis in Two Dinotoms Has Generated Little Change in the Mitochondrial Genomes of Their Dinoflagellate Hosts and Diatom Endosymbionts

<div><h3>Background</h3><p>Mitochondria or mitochondrion-derived organelles are found in all eukaryotes with the exception of secondary or tertiary plastid endosymbionts. In these highly reduced systems, the mitochondrion has been lost in all cases except the diatom endosymbionts found in a small group of dinoflagellates, called ‘dinotoms’, the only cells with two evolutionarily distinct mitochondria. To investigate the persistence of this redundancy and its consequences on the content and structure of the endosymbiont and host mitochondrial genomes, we report the sequences of these genomes from two dinotoms.</p> <h3>Methodology/Principal Findings</h3><p>The endosymbiont mitochondrial genomes of Durinskia baltica and Kryptoperidinium foliaceum exhibit nearly identical gene content with other diatoms, and highly conserved gene order (nearly identical to that of the raphid pennate diatom Fragilariopsis cylindrus). These two genomes are differentiated from other diatoms' by the fission of nad11 and by an insertion within nad2, in-frame and unspliced from the mRNA. Durinskia baltica is further distinguished from K. foliaceum by two gene fusions and its lack of introns. The host mitochondrial genome in D. baltica encodes cox1 and cob plus several fragments of LSU rRNA gene in a hugely expanded genome that includes numerous pseudogenes, and a trans-spliced cox3 gene, like in other dinoflagellates. Over 100 distinct contigs were identified through 454 sequencing, but intact full-length genes for <em>cox1</em>, <em>cob</em> and the 5′ exon of <em>cox3</em> were present as a single contig each, suggesting most of the genome is pseudogenes. The host mitochondrial genome of K. foliaceum was difficult to identify, but fragments of all the three protein-coding genes, corresponding transcripts, and transcripts of several LSU rRNA fragments were all recovered.</p> <h3>Conclusions/Significance</h3><p>Overall, the endosymbiont and host mitochondrial genomes in the two dinotoms have changed surprisingly little from those of free-living diatoms and dinoflagellates, irrespective of their long coexistence side by side in dinotoms.</p> </div

Number of inversions for the inter-conversions of the mitochondrial genomes of the two dinotoms and those of diatoms (predicted by GRIMM).

<p>Number of inversions for the inter-conversions of the mitochondrial genomes of the two dinotoms and those of diatoms (predicted by GRIMM).</p