The Complete Mitochondrial Genomes Of Three Pycnonotidae Species And Evolutionary Analyses

As a branch of vertebrates, aves act as an important role in the ecosystem. There are 10,560 extant species of birds of the world which belong to 2278 genus, 240 families and 40 orders.In order to analyze the characteristics of Pycnonotidae mitogenome, we sequenced the complete mitochondrial genomes of three species(Pycnonotus sinensis, P. melanicterus and Spizixos semitorques) in the family Pycnonotidae. In order to compare the evolutionary rate across different families of Passeriformes, we sequenced 34 species of nucleotide gene musk. Based on mitochondrial gene nad2 and nuclear gene musk, combined the data in the Gen Bank, we performed evolutionary rate analysis across 22 families of Passeriformes. In order to compare the phylogenetic relationships among passeriforms based on different dataset, we used the 13 mitochondrial PCGs sequences from 84 species and nad2 + musk gene sequences from 321 species, respectively.In order to estimate the divergence time of Passeriformes and Pycnonotidae, we sequenced the musk, CHD1-16 and NIPBL-16 genes from 20 avian species. Combined with cox1、atp6、12S r RNA、nad2 and cob genes, we constructed phylogenetic trees using ML and BI methods, respectively. Furthermore, we estimated the divergence time of Passeriformes and Pycnonotidae using BEAST software.Based on the date we sequenced, the main results are summarized as follows:1. The sequencing and analysis of the complete mitochondrial genomes of three species of Passeriformes In this study, we sequenced the complete mitochondrial genomes of three species in the family of Pycnonotidae, the length of three complete mitogenomes is 16,922 bp( P. sinensis), 17,011 bp( P. melanicterus) and 17,045 bp( S. semitorques), respectively. Their structures of genome, base compositions, condon usage and t RNA structures are similar to other reported Passeriformes species. Notably, a noval CAA stop condon at the end of cox2 gene( P. sinensis and P. melanicterus), and atp6 gene( P. melanicterus) was detected.2. Comparative analysis of phylogenetic relationships of major lineages of Passeriformes Based on maximum likelihood(ML) method, we construct phylogenetic tree of Passeriformes using 13 mitochondrial PCGs sequences from 84 species, and compared with the phylogenetic tree of Passeriformes based on 321 nad2 and musk gene sequences. Both in the phylogenetic tree based on 13 PCGs and nad2 + musk, Suboscines is at the basal of the phylogenetic tree with high bootstrap support, and Corvoidea is at the basal of the oscines clade. However, the monophyly of Muscicapoidea is not well supported, and the phylogenetic position of Muscicapoidea, Paridae and Certhioidea is different.3. Evolutionary rate analysis across major families of Passeriformes We construct rate heterogeneity analysis based on mitochondrial gene nad2 and nuclear gene musk across 22 families of Passeriformes, the highest evolutionary rate was observed for Vireonidae, and the lowest for Timaliidae.4. Estimated the divergence time of the Passeriformes and Pycnonotidae We estimated the divergence time of the Passeriformes and Pycnonotidae baesd on nuclear genes( CHD1-16, NIPBL-16 and musk) intron sequences and mitochondrial genes(cox1、atp6、12S r RNA、nad2 and cob genes) used BEAST software. Fossil calibration points are used in the study, the divergence time of Galliformes and Anseriformes(101.7, SD = 8.0), the divergence time of Galloanserae and Neoaves(122.2,SD = 8.6) and the divergence time of Coturnix coturnix and Gallus gallus(35,SD = 1.83), respectively. In the study, the divergence time of Passeriformes is 93.64 Mya( 73.79-112.91 Mya), and the divergence time of Pycnonotidae is 40.90 Mya( 10.48- 53.17 Mya). Neoaves divergenced in the Late Cretaceous, and the divergence between Neognathae and Palaognathae is in the late Cretaceous, our results consistent with the previous study. Most of the modern bird started to diverge after the CretaceousPaleocene. Our results suggest that a large number of lineages survived from the Cretaceous, and birds gene transformation in different ecosystems was take place in the Late Cretaceous, our result is not consistent with the theory of “Big bang for tertiary birds” by Feduccia.5. Characteristics of gene evolution Average rate heterogeneity and selection pressure analysis of 13 mitochondrial PCGs were analyzed. In the study, the lowest selection pressure and the highestevolutionary rate were observed for atp8 and nad6 gene, the highest selection pressure and the lowest evolutionary rate were observed for cox1 and cox3 gene. The rate of nad2 and cob gene is in the average level of 13 PCGs, the rate heterogeneity in nad2 and cob gene is quite stable, and cox1、atp6、nad2 and cob genes were selected from the 13 mitochondrial PCGs as the representative of mitochondrial genes and work as a molecular mark in the phylogenetic analysis. The rate heterogeneity of nuclear gene musk, CHD1 Z and NIPBLZ is above 0.2, and the rate heterogeneity of CHD1Z-16 and NIPBLZ-16 is much faster than CHD1W-16 and NIPBLW-16, but CHD1-16 gene sequence in the chromosome Z and W is more differentiated than NIPBL-16 gene. The phylogenetic analyses based on CHD1-16 indicate that CHD1Z-CHD1 W diverged in the neognathae ancestor, but the phylogenetic analyses based on NIPBL-16 indicate that NIPBLZ- NIPBLW differentiated until the diverge of Galloanserae and Neoaves, and the diverge of NIPBLZ-NIPBLW is not well supported. 6. Develop a new molecular marker for avian sex identification Our results indicate that the homologous of NIPBL-16 intron sequences on the sex chromosome Z and W result in different length across birds, which is similar to the sex identification gene CHD1, the results indicate that NIPBL-16 intron sequence can be used as a new sex identification molecular marker in Neoaves. In conclusion, we sequenced and analyzed the complete mitochondrial genomes of 3 Passeriformes species, a noval CAA stop condon at the end of cox2 and atp6 gene was detected. Major lineages of Passeriformes analysis indicate that Suboscines is at the basal of the Passeriformes clade, and Corvoidea is at the basal of the Oscines clade. The highest evolutionary rate was observed for Vireonidae, and the lowest for Timaliidae by the evolutionary rate analysis across major families of Passeriformes. In the study, the divergence time of Passeriformes is 93.64 Mya( 73.79-112.91 Mya), and Pycnonotidae is 40.90 Mya( 10.48- 53.17 Mya). Besides, we also found that the evolutionary rate of nad2 and cob gene is in the average level of 13 PCGs, and NIPBL-16 intron sequences can used as a new sex identification molecular marker in Neoaves.