Plateau Partridge, Tibetan Snow Cock, Tibetan Eared Chicken Whole Mitochondrial Genome Sequence Determination And Phylogenetic Analysis

Phasianidae, the largest families of Galliformes, plays an important role with respects of the economy and scientific research. With extensive application of techniques and theory of molecular biology, the great progress of phylogenetic relationships among Phasianidae has been made. while some of the results challenges the traditional classifications. However, due to a rapid radiation evolutionary events in the history, the taxonomic status of some genera remains controversial.The complete mitochondrial genome of three Phasianidae species (Perdix hodgsoniae,Tetraogallus tibetanus and Crossoptilon harmani) were sequenced by primer-walking method based on the L-PCR products amplified with 6 pairs of primers, and compared with respects of gene order, base composition bias and codon usage. Combining the other 23 species with complete mitochondrian genome deposited in GenBank, the phylogenetic relationship of 26 Phasianidae species were reconstructed using Maximum likelihood, Bayesian inference and Pasimony methods with Numida meleagris as an outgoup. The main conclusion drawn as following:1. The complete mitochondrial genome size of Perdix hodgsoniae,Tetraogallus tibetanus and Crossoptilon harmani is 16698bp,16686bp and 16682bp respectively, containing 13 protein coding genes,2 rRNA,22 tRNA and a non-coding control region.2. The gene order of three mitochondrial genomes is similar to that of the most other birds, and the gene order between Cyt b and 12S rRNA is Cytb/tRNAThr/tRNAPro/ND6/tRNAGlu/D-loop/tRNAPhe /12S rRNA.3. All protein-coding genes initiate with ATG, except for COI, which begins with GTG, while most of the protein-coding genes had the stop codon TAA and TAG, whereas COI and ND6 of Crossoptilon harmani terminate with the stop codon AGG, besides two genes appear to end in an incomplete stop codons T (ND4,COIII).4. The lengths of the 12S rRNA and 16S rRNA are 964 bp,974 bp,964 bp, and 1609 bp,1605 bp, 1609 bp, respectively.The 12S rRNA and 16S rRNA genes located between the tRNAPhe and tRNALeu(UUR), and separated by the tRNAVal gene. Perdix hodgsoniae, Tetraogallus tibetanus and Crossoptilon harmani mtDNA encode 22 tRNA genes, and each tRNA can be folded into a clover-leaf secondary structure with the exception of tRNASer(AGY). Lots of G-U atypical pairings were identified in the stem regions, which is useful to stability of tRAN secondary structure. The control region of the Perdix hodgsoniae, Tetraogallus tibetanus and Crossoptilon harmani mitochondrial genome is 1150bp,1155bp and 1146bp in length respectively, and is located between the tRNAphe and tRNAGlu genes.The AT content of control regions is 58.8%,59.7%and 58.3%.5. The phylogenetic relationships of 26 Phasianidae species were determined using different databases from the complete mitochondrial genomes, using maximum parsimony, maximum likelihood and Bayesian analysis. The analysis of mitochondrial genomes suggest that the genera within Phasianidae have the following relationship:((Coturnix+Tetraogallus)+((((((Lophura+ Crossoptilon)+Phasianus)+Syrmaticus)+Perdix)+(Lophophorus+Tragopan))+((Gallus+ Bambusicola)+Francolinus))), but Arborophila is far away from other Phasianidae. The placement of Polyplectron and Pavo within the Phasianidae remains undetermined.6. Phasianidae are generally thought to be comprised of Pheasants and Partridges in traditional classification system, however, our analyses did not support that. The analyses of nucleic acid and amino acid sequences of the mitochondrial genomes strongly support the monophyly of Lophur, Coturnix, Syrmaticus and Gallus. Lophura, Crossoptilon and Phasianus show a close relationship, while Lophophorus and Tragopan, Gallus Bambusicola and Francolinus, Coturnix and Tetraogallus are closely related in the reconstructed tree.7. In addition, we also discussed the phylogenetic performance and utility of mitonchondrial genes, including the constant test of molecular evolution rate, the resolution of different levels and the sensitivity of different methods in constructing phylogenetic trees. The results of KH test and SH test show that likelihood score of MP, ML and BI trees was no significant difference (P>0.05), therefore, we considered that the topology of these trees could be well explained the dataset. ND5 and ATP6 are the most appropriate on the analyses of nucleotide and amino acid sequences in reflecting the phylogenetic relationships of Phasianidae species. At the level of genus, both nucleotide and amino acid sequences of ND4 and ND5 are good, by contrast, Cytb and ND4L are the worest.Comprehensive consideration of the species and genus, ND4 and ND5 is the most effective genes based on both nucleotide and amino acid sequences.8. We computed the molecular dating among Gallus, Bambusicola and Francolinus,and the results shows that the divergence time of Gallus/Bambusicola is (33.9579±4.5277)* 106aBP under 1+2 Ts nucleotide alternative model in ND5 gene,the divergence time of Gallus/Bambusicola is (36.2350±7.8771)*106aBP under 1+2Ts nucleotide alternative model in Cytb gene,the divergence time of Francolinus/Bambusicola+Gallus is (40.9843±8.6145)*106aBP under 1+2+3Ts nucleotide alternative model in ND5 gene.