Evolutionary conservation of the circadian gene timeout in Metazoa

<i>Timeless </i>(<i>Tim</i>) is considered to function as an essential circadian clock gene in <i>Drosophila</i>. Putative homologues of the <i>Drosophila timeless </i>gene have been identified in both mice and humans. While <i>Drosophila </i>contains two paralogs, <i>timeless </i>and <i>timeout</i>, acting in clock/light entrainment and chromosome integrity/photoreception, respectively, mammals contain only one <i>Tim </i>homolog. In this paper, we study the phylogeny of the <i>timeless</i>/<i>timeout </i>family in 48 species [including 1 protozoan (<i>Guillardia theta</i>), 1 nematode (<i>Caenorhabditis elegans</i>), 8 arthropods and 38 chordates], for which whole genome data are available by using MEGA (Molecular Evolutionary Genetics Analysis). Phylogenetic Analysis by Maximum Likelihood (PAML) was used to analyze the selective pressure acting on metazoan <i>timeless</i>/<i>timeout </i>genes. Our phylogeny clearly separates insect <i>timeless </i>and <i>timeout </i>lineages and shows that non-insect animal <i>Tim </i>genes are homologs of insect <i>timeout</i>. In this study, we explored the relatively rapidly evolving <i>timeless </i>lineage that was apparently lost from most deuterostomes, including chordates, and from <i>Caenorhabditis elegans</i>. In contrast, we found that the <i>timeout </i>protein, often confusingly called “<i>timeless</i>” in the vertebrate literature, is present throughout the available animal genomes. Selection results showed that <i>timeout </i>is under weaker negative selection than <i>timeless</i>. Finally, our phylogeny of <i>timeless</i>/<i>timeout </i>showed an evolutionary conservation of the circadian clock gene <i>timeout </i>in Metazoa. This conservation is in line with its multifunctionality, being essential for embryonic development and maintenance of chromosome integrity, among others

Principal coordinate analysis of the community membership (A) and structure (B) using Jaccard and Theta YC distances, respectively.

<p>Green squares and yellow circles represent captive and wild red panda bacterial communities, respectively. Distances between symbols on the ordination plot reflect relative dissimilarities in community memberships or structures.</p

Characterization of the Gut Microbiota in the Red Panda (<i>Ailurus fulgens</i>)

<div><p>The red panda is the only living species of the genus <i>Ailurus</i>. Like giant pandas, red pandas are also highly specialized to feed mainly on highly fibrous bamboo. Although several studies have focused on the gut microbiota in the giant panda, little is known about the gut microbiota of the red panda. In this study, we characterized the fecal microbiota from both wild (n = 16) and captive (n = 6) red pandas using a pyrosequecing based approach targeting the V1-V3 hypervariable regions of the 16S rRNA gene. Distinct bacterial communities were observed between the two groups based on both membership and structure. Wild red pandas maintained significantly higher community diversity, richness and evenness than captive red pandas, the communities of which were skewed and dominated by taxa associated with Firmicutes. Phylogenetic analysis of the top 50 OTUs revealed that 10 of them were related to known cellulose degraders. To the best of our knowledge, this is the first study of the gut microbiota of the red panda. Our data suggest that, similar to the giant panda, the gut microbiota in the red panda might also play important roles in the digestion of bamboo.</p></div

Comparison of community alpha diversities between the wild and captive red pandas.

<p>Diversity was measured by inverse Simpson (A) and Shannon index (B); Richness (C) and evenness (D) were measured by the number of observed OTUs and Shannon Evenness index, respectively. The top and bottom boundaries of each box indicate the 75^th and 25^th quartile valudes, respectively. The black lines within each box represent the median values. Different lowercase letters above the boxplots indicate significant differences in alpha diversities between wild and captive pandas (P<0.001, Mann Whitney test).</p

Neighbor-joining tree showing the phylogenetic relationship among the top 50 OTUs in this study (red) with known cellulose degraders (blue) and OTUs identified in the giant panda (green).

<p>Different phyla were shaded by different colors: green, Firmicutes; purple, Actinobacteria; orange, Bacteroidetes; blue, Proteobacteria. All bootstrap values > 50% were shown on the tree.</p

Relative abundance of OTUs at the phylum (A) and genus (B) level in the fecal microbiota from wild and captive red pandas.

<p>Relative abundance of OTUs at the phylum (A) and genus (B) level in the fecal microbiota from wild and captive red pandas.</p

OTUs differentially represented between wild and captive red pandas identified by linear discriminant analysis coupled with effect size (LEfSe).

<p>A. Histogram showing OTUs that are more abundant in wild (green color) or captive (red color) red pandas ranked by effect size. The distribution of the most differentially distributed OTUs: OTU001 (more abundant in captive red pandas) and OTU003 (more abundant in wild red pandas) were illustrated in B and C, respectively.</p

Phylogenetic relationship of <i>Enterocytozoon bieneusi</i> groups, the relationship between <i>E</i>. <i>bieneusi</i> genotypes identified in this study and other known genotypes deposited in the GenBank was inferred by a neighbor-joining analysis of ITS sequences based on genetic distance by the Kimura-2-parameter model.

<p>The numbers on the branches represent percent bootstrapping values from 1,000 replicates, with more than 50% shown in tree. Each sequence is identified by its accession number, genotype designation, and host origin. The group terminology for the clusters is based on the work of Zhao et al [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163605#pone.0163605.ref028" target="_blank">28</a>]. Genotypes with <i>black circles</i> and <i>open circles</i> are novel and known genotypes identified in this study, respectively.</p

Distribution of <i>E</i>. <i>bieneusi</i> genotypes in red-bellied tree squirrels from different countries.

<p>Distribution of <i>E</i>. <i>bieneusi</i> genotypes in red-bellied tree squirrels from different countries.</p

Gene locus, primer sequences, annealing temperatures and fragment length for the identification of <i>E</i>. <i>bieuensi</i> used in this study.

<p>Gene locus, primer sequences, annealing temperatures and fragment length for the identification of <i>E</i>. <i>bieuensi</i> used in this study.</p