Molecular phylogeny of the endosymbiotic ciliates (Litostomatea: Trichostomatia) of vertebrate animals inferred from 18SrRNA gene sequences

Complete 18S rRNA sequences were elucidated from (1) six entodiniomorphid rumen ciliates, Diplodinium, Entodinium, Epidinium, Eudiplodinium, Ophryoscolex, and Polyplastron, (2) three vestibuliferid rumen ciliates, Balantidium, Dasytricha, Isotricha intestinalis, and I. prostoma, (3) two marsupial ciliates, Cycloposthium and Macropodinium, and (4) three free-living ciliates, Didinium, Dileptus, and Enchelyodon, likely the closest relatives to these endosymbionts.; Phylogenetic analysis of these 15 new sequences revealed that ophryoscolecids are a monophyletic group that is divided into three lineages corresponding to the subfamilial divisions of the Ophryoscolecidae, with Entodinium branching first. The rumen ciliates are the sister group to Cycloposthium consistent with their placement into the order Entodiniomorphida. Macropodinium does not group with the other entodiniomorphids, but basal to the vestibuliferid-entodiniomorphid clade. Together, the endosymbionts are the sister group to the free-living haptorians, together constituting the class Litostomatea.; The rate of nucleotide substitution for ciliates was calibrated to be 1% divergence per 72 to 80 million years (My). The origin of ciliates (i.e. crown eukaryotes) is calculated to be much older than previously speculated, dating back to the Paleoproterozoic, 1,980 to 2,200 million years ago. It was also determined that the rate of nucleotide substitution for rumen ciliates is almost a magnitude faster (1% per 8-11 My) than that for free-living ciliates. This faster clock might be explained by intense selection on survivability as they invaded the rumen, or by the relatively high ambient temperature (39{dollar}spcirc{dollar}C) of the rumen environment, as such high temperatures are known to decrease the efficiency of DNA repair mechanisms leading to higher mutation rates.; Intraspecific sequence variation among different hosts and geographical locations was examined using the ITS-1/5.8S/ITS-2 region. Analysis of this region from I. prostoma from Canadian, American, and Australian cattle and sheep, showed that there is no sequence variation within this region. This suggests that populations of I. prostoma on two continents are very recently diverged, consistent with human colonisation and migration of domestic animals in the 18{dollar}rm sp{lcub}th{rcub}{dollar}and 19{dollar}rm sp{lcub}th{rcub}{dollar} centuries. Finally, secondary structure of these sequences reveal that members of the class Litostomatea have "lost" helix E23-5, indicating a new molecular diagnostic feature for this class of ciliates.