Study On The Mitochondrial Genome And Its Phylogenetic Development

Mitochondria are very important organelles providing energy for various life activities in animal cells. Because of the characteristics of the genome in mitochondria, such as easy to obtain, fast evolutionary rate and maternal inheritance, it has been more and more used as an important molecular marker in the analysis of population genetic structure, phylogenetic research and adaptive evolution study of energy metabolism. In addition, mitochondrial genome is conducive to further studies on animal taxonomy and conservation. Canidae is a family of widespread dog-like carnivores including 36 extant species all over the world. Within the Canidae, four distinct monophyletic groupings are apparent, the fox-like canids, the wolf-like canids, the South American foxes, the maned wolf(Chysocyon brachyurus) and bush dog(Speothos venaticus). Chysocyon brachyurus, as the unique canid in South America, is significant to explore the phylogenetic relationship between the unique canids in South America and others. And there used to be controversial in taxonomic status of Vulpes zerda. In this study, the whole mitochondrial genome of Chysocyon brachyurus and Vulpes zerda were sequenced and compared with other Canidae species. In order to have an insight into the classification and phylogenetic relationships of canids, phylogenetic analysis based on the whole mitochondrial genome and partial genes were executed among 8 Canidae species. The results are as follows.1.The complete mitochondrial genome sequence of Chysocyon brachyurus and Vulpes zerda are 16, 760 bp and16, 734 bp, respectively. They are identical to the general features of the Canidae mitochondrial genome, and consist of 13 protein-coding genes, 22 tRNA genes, 2 rRNA gene and the control region, which locations and arrangements are highly conservatived in complete mitochondrial genome. The A+T bias is detected in the base composition of the mitochondrial genome, which is also evident in tRNA genes, r RNA genes and protein-coding genes. The base usage of the mitochondrial genome is A>T>C>G in common except Vulpes zerda(A>C>T>G). Most of the protein-coding genes take ATG as initiation codon, expect for ND2, ND3 and ND5 genes start with ATA. Most of these protein-coding genes terminate with TAA and the incomplete stop codons(TA- and T--) while Cytb gene terminate with AGA. The tRNA genes of Chysocyon brachyurus and Vulpes zerda are in accordance with other canids in the total length, locations and the secondary structure. The tRNA genes all form typical cloverleaf-shaped secondary structure, while tRNASer(AGY) has a DHU arm deletion. In addition, 7 unmatched base pairs are detected at the same locations in all canids tRNA genes. The length of the control region of Chysocyon brachyurus and Vulpes zerda are 1298 bp and 1271 bp respectively. And the variable tandem repeats in control region are reckoned as the main reason of the length variability. Eight putative conserved sequence blocks(CSB-B ~ CSB-F and CSB-1 ~ CSB-3) previously found in mammals are detected, and the variable tandem repeats(5’-ACACGTA(G)CGT-3’ in Chysocyon brachyurus, 5’- ACACACGT-3’and 5’-ACACGT-3’ in Vulpes zerda) are located between CSB-1and CSB-2.2. Phylogenetic relationships in Canidae were reconstructed with neighbor joining, maximum parsimony, maximum likelihood and Bayesian Inference methods respectively. The result showed that the canids divide into two clades, Chysocyon, Cuon and Canis belonged to the same one, and Chysocyon brachyurus splitted off firstly from this clade. Vulpes and Nyctereutes belonged to the other one, Vulpes zerda separated firstly from Vulpes common ancestor. The phylogenetic analysis based on Cytb gene, COXI gene and COXII gene confirmed that four distinct monophyletic groupings were apparent, and the unique canids in South America was a sister group.3. The BEAST analysis conducted on the mitochondrial genome showed that the divergence time between the two clades was dated back to 8.37 Ma. Chysocyon and the common ancestor of Cuon + Canis diverged at 4.77 Ma, and Vulpes zerda diverged at 3.85 Ma. It’s in accordance with the genetic relationship inferred from the phylogenetic tree and other former researches. Besides, the divergence time of Nyctereutes +Vulpes was 7.20 Ma, which help to infer that the radiation of canids took place earlier than the fossil records.4. Vulpes zerda has been classified under the genus Canis or its own genus Fennecus. In this study, the mitochondrial genome of Vulpes zerda was compared with the other one in the Genbank database. It’s indicated that the genetic distances are much greater than the intraspecific distances, and they hold different phylogenetic position and divergence time. The phylogenetic analysis of the data in this study is in accordance with former researches, and it is possible to infer that the data in the Genbank database is from Vulpes rueppelli. It’s validated that Vulpes zerda is classified under the genus Vulpes.