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Bacterial Diversity In The Intestinal Tract Of Wolves And Comparative And Phylogenetic Analysis Of Canidae Mitochondrial Genomes

Posted on:2011-12-15Degree:DoctorType:Dissertation
Country:ChinaCandidate:L ChenFull Text:PDF
GTID:1100360308971076Subject:Conservation and Utilization of Wild Fauna and Flora
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Canidae, as the most widely distributed taxa in Carnivora, is inextricably linked to our life. Among the studies on canine animals, the researches of digestive physiology play a very important role, and the study on the digestive tract microbial communities is becoming a much hotter topic in recent years. Although the microbial communities in the different segments of the intestinal tract of domestic dogs (Canis lupus familiaris) have been good characterized, to our knowledge, no molecular study has assessed the composition of the microbial communities in the intestinal tracts of wild wolves(Canis lupus). Therefore, the first purpose of the present study is to characterize the microbial diversity in the distal gut of a group of healthy wolves by using the approach of 16S rRNA gene sequences analysis. Fecal samples were collected from 3 healthy adult wolves and we created clone libraries for the samples from each wolf separately. A total of 550 clones were randomly picked from each library aND307 non-chimeric near-full-length sequences were finally used for subsequent phylogenetic analysis. With 97% sequence similarity, a total of 65 OTUs were defined. Seventeen OTUs (31.9% of total clones) showed less than 98% sequence similarity to existing 16S rRNA gene sequences in the GenBank and RDP databases, and may represent uncharacterized bacterial species. Five major phylogenetic lineages were identified:Firmicutes (60%), Bacteroidetes (16.9%), Proteobacteria (9.2%), Fusobacteria (9.2%) and Actinobacteria (4.6%). The majority of clones fell within the order Clostridiales (53.8% of total OTUs). It was predominantly affiliated with five families: Lachnospiraceae was the most diverse bacterial family in this order, followed by Ruminococcaceae, Clostridiaceae, Peptococcaceae and Peptostreptococcaceae. We compared our results with human beings and other mammals and got a similar composition among the microbial communities in the intestinal tracts of them. The differences of the intestinal flora between wolves and domestic dogs were mainly in the component and diversity of bacteria in the phyla Actinobacteria and Fusobacteriales.Mitochondrial genome as an extra-chromosomal genome is extensively used for testing hypotheses of microevolution, studying population structure and phylogenetic relationships at various taxonomic levels. Since the domestic dog's mitochondrial genome was sequenced in 1997, there are four Canidae species whose mitochondrial genomes have been reported, they are Canis lupus, Canis familiaris, Canis latrans and Vulpes Vulpes. In 2008, we sequenced the mitochondrial genome of Tibetan wolves (Canis lupus laniger) captured from the Qinghai province of China. But the mitochondrial genomes of wolves in other regions of China have not been reported till now. So in the second part of this article, we sequenced the mitochondrial genomes of Mongolian wolf and other two species of Canidae in China, dhole and raccoon dog, by using the long and accurate polymerase chain reaction. We compared our results with the mitochondrial genomes of other canine animals for the purpose of detecting the common characteristic of the mitochondrial genomes of Canidae. Our results were as follows:The lengths of the mitochondrial genomes of Mongolian wolf, dhole and raccoon dog were 16709 bp,16672 bp aND16713 bp, respectively. The entire mitochondrial genome sequence contained 2 ribosomal RNA genes,13 protein-coding genes,22 transfer RNA genes aND1 non-coding region. A strong A-T bias was found in the mitochondrial genomes of the three canine animals. All the transfer RNA genes could be folded into the typical cloverleaf-shaped structure except tRNA-Ser (AGY), which lacked the dihydrouridine arm. The overall codon usages in the mitochondrial genome protein-coding genes were unification among canine animals. The frequencies of CTA (Leu), ATT (Ile), ATC (Ile), ATA (Ile) were higher than those of other codons. Some of the start or stop codons were different among wolves, dhole and raccoon dog. The comparison of the mitochondrial genome sequences of 4 genera (representing 8 canine species or subspecies) of Canidae exhibited a very high consistency among different canine animals. The differences of the Canidae animals were mainly found in the two peripheral domains (the ETAS domain and the CSB domain) of the control region of the mitochondrial genome. The difference in the length of the mitochondrial genomic sequences was mainly cause by the different number of the repeat units in the repeat sequence (RS-3). From the comparison of the substitution rate of the mitochondrial genomes we found that same gene in different animals had a similar substitution rate while different genes in the mitochondrial genome of the same animal had different rates. Different protein-coding genes also had different substitution rates and the ND6 gene expressed the most prominent difference to other protein-coding genes. Among all the mitochondrial genes, tRNA genes had the lowest substitution rate and implicated a slower evolution rate.The discussion about the relationships and the evolution mechanisms of the Canidae animals is another hot topic which has been considerable studied based on a lot of morphological data and various molecular data. But because of the limits of the sequence method and the spare of complete mitochondrial genome sequence in the database, there is no report about the phylogenetic relationship of Canidae based on the complete mitochondrial genome sequence. Thus, as the third composition of this article, we analyzed the phylogenetic relationship of different subspecies of wolves by using the concatenated data set of 12S rRNA +16S rRNA+12 heavy-strand encoded protein-coding genes and reconstructed the phylogenetic relationship of the suborder Caniformia by using 23 representative species, and pay a special attention on the evolutionary relationships of Canidae animals. Our result indicates that, within the subspecies datas of Canis lupus, Canis lupus laniger was polarized first, then was Canis lupus arabs. Canis lupus chanco and Canis lupus lupus were sister species, which had a similar phylogenetic position. Within the suborder Caniformia, there was a basal caniform divergence between Cynoidea and Arctoidea. The four genera in Canidae were divided into three clades with an equal phylogenetic position. From the estimates of divergence time of canine animals we found that the genus Nyctereutes splited first. The result of the maximum parsimony, maximum likelihood and Bayesian inferences all indicated that the genus Canis is a unilateral unit besides the Canis latrans. All the topology of the three inference methods indicated that Canis lupus familiaris should be defined as a subspecies of Canis lupus. The estimated divergence time of dog and wolf was 1.56~1.92 million years before present.
Keywords/Search Tags:Canis lupus, Cuon alpinus, Nyctereutes procyonoides, Canidae, 16S rRNA, Intestinal microbiota, Phylogenetics
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