Morphological features and origin of the newly obtained samples within this study.

<p>CZ – Czech Republic, SK – Slovakia, UK – England; OW – oocyst wall, MP – micropyle, OR – oocyst residuum.</p

Taxa and sequences included in the phylogenetic analyses.

*<p>: sequences included in the <i>Skeleton</i> matrix.</p><p>•: taxa used as outgroups for the phylogenetic analyses.</p><p>– : the sequence is not available.</p><p>Taxa for which new sequences were obtained in this study and Accession numbers of these sequences are printed in bold.</p

A <i>Skeleton</i> tree.

<p><i>Skeleton</i> tree (ML and BI) of the taxa for which all 3 genes (<i>18S rDNA</i>, <i>ORF 470</i> and <i>COI</i>) are available.</p

<i>Concatenated</i> ML tree.

<p> Letters A–D indicate clusters delimited according to the <i>Skeleton</i> tree (taxa present in the <i>Skeleton</i> tree are labeled with asterisks). Clades A and B are supported by both BI and ML analyses of the <i>Concatenated</i> and <i>Skeleton</i> matrices. The red node indicates a cluster with weak host specificity. Numbers 1–4 indicate lineages that are also supported by BI analyses of the following matrices: 1, <i>Concatenated</i>; 2, <i>ORF 470</i>; 3, <i>COI</i>; 4, <i>18S rDNA</i>. The newly added samples are printed in bold; coccidia from rodents are printed in blue. To decrease the size of the tree for the printed presentation, we removed several of the most basal outgroups.</p

<em>Candidatus</em> Sodalis melophagi sp. nov.: Phylogenetically Independent Comparative Model to the Tsetse Fly Symbiont <em>Sodalis glossinidius</em>

<div><p>Bacteria of the genus <em>Sodalis</em> live in symbiosis with various groups of insects. The best known member of this group, a secondary symbiont of tsetse flies <em>Sodalis glossinidius</em>, has become one of the most important models in investigating establishment and evolution of insect-bacteria symbiosis. It represents a bacterium in the early/intermediate state of the transition towards symbiosis, which allows for exploring such interesting topics as: usage of secretory systems for entering the host cell, tempo of the genome modification, and metabolic interaction with a coexisting primary symbiont. In this study, we describe a new <em>Sodalis</em> species which could provide a useful comparative model to the tsetse symbiont. It lives in association with <em>Melophagus ovinus</em>, an insect related to tsetse flies, and resembles <em>S. glossinidius</em> in several important traits. Similar to <em>S. glossinidius</em>, it cohabits the host with another symbiotic bacterium, the bacteriome-harbored primary symbiont of the genus <em>Arsenophonus</em>. As a typical secondary symbiont, <em>Candidatus</em> Sodalis melophagi infects various host tissues, including bacteriome. We provide basic morphological and molecular characteristics of the symbiont and show that these traits also correspond to the early/intermediate state of the evolution towards symbiosis. Particularly, we demonstrate the ability of the bacterium to live in insect cell culture as well as in cell-free medium. We also provide basic characteristics of type three secretion system and using three reference sequences (16 S rDNA, <em>groEL</em> and <em>spaPQR</em> region) we show that the bacterium branched within the genus <em>Sodalis</em>, but originated independently of the two previously described symbionts of hippoboscoids. We propose the name <em>Candidatus</em> Sodalis melophagi for this new bacterium.</p> </div

Morphology and ultrastructure of <i>Candidatus</i> Sodalis melophagi.

<p><b>A</b>: <i>In vitro</i> cell culture in Nomarski contrast. <b>B</b>, <b>C</b>: Cells of <i>Candidatus</i> Sodalis melophagi in bacteriome. Black arrows – cells of <i>Candidatus</i> Sodalis melophagi, white arrows – cells of the primary endosymbiont of the genus <i>Arsenophonus</i>.</p

Phylogenetic trees derived from <i>groEL</i> matrices by BI in MrBayes.

<p>New <i>Sodalis</i> lineages added in this study are printed in red. <b>A</b>: The tree inferred from aminoacid matrix. Posterior probabilities are indicated by the numbers at the nodes. <b>B</b>: The tree inferred from nucleotide matrix restricted taxonomically to the <i>Sodalis</i> branch. The numbers at the nodes show the posterior probabilities and bootstrap values from the identical topology obtained by ML in PhyML.</p

Phylogenetic tree derived from <i>spaPQR</i> region by BI in MrBayes.

<p>The numbers at the nodes show the posterior probabilites and bootstrap values from the identical topology obtained by ML in PhyML. New <i>Sodalis</i> lineages added in this study are printed in red.</p

16 S rDNA tree derived by BI analysis in MrBayes.

<p>Posterior probabilities are indicated by the numbers at the nodes. New <i>Sodalis</i> lineages added in this study are printed in red.</p

Composition of SSR-2 and SSR-3 copies of <i>Candidatus</i> Sodalis melophagi TTSS.

<p>Ψ – putative pseudogene (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040354#s4" target="_blank">Material and Methods</a>).</p