Chen Siang Ng, Cheng-Kuo Lai, Huei-Mien Ke, Hsin-Han Lee, Chih-Feng Chen, Pin-Chi Tang, Hsu-Chen Cheng, Meiyeh J. Lu, Wen-Hsiung Li, Isheng Jason Tsai, Genome Assembly and Evolutionary Analysis of the Mandarin Duck Aix galericulata Reveal Strong Genome Conservation among Ducks, Genome Biology and Evolution, Volume 14, Issue 6, June 2022, evac083,
Aix galericulata and Tadorna ferruginea are two Anatidae species representing different taxonomic groups of Anseriformes. We used a PCR-based method to determine the complete mtDNAs of both species, and estimated phylogenetic trees based on the complete mtDNA alignment of these and 14 other Anseriforme species, to clarify Anseriform phylogenetics. Phylogenetic trees were also estimated using a multiple sequence alignment of three mitochondrial genes (Cyt b, ND2, and COI) from 68 typical species in GenBank, to further clarify the phylogenetic relationships of several groups among the Anseriformes. The new mtDNAs are circular molecules, 16,651 bp (Aix galericulata) and 16,639 bp (Tadorna ferruginea) in length, containing the 37 typical genes, with an identical gene order and arrangement as those of other Anseriformes. Comparing the protein-coding genes among the mtDNAs of 16 Anseriforme species, ATG is generally the start codon, TAA is the most frequent stop codon, one of three, TAA, TAG, and T-, commonly observed. All tRNAs could be folded into canonical cloverleaf secondary structures except for tRNASer (AGY) and tRNALeu (CUN), which are missing the \"DHU\" arm.Phylogenetic relationships demonstrate that Aix galericula and Tadorna ferruginea are in the same group, the Tadorninae lineage, based on our analyses of complete mtDNAs and combined gene data. Molecular phylogenetic analysis suggests the 68 species of Anseriform birds be divided into three families: Anhimidae, Anatidae, and Anseranatidae. The results suggest Anatidae birds be divided into five subfamilies: Anatinae, Tadorninae, Anserinae, Oxyurinae, and Dendrocygninae. Oxyurinae and Dendrocygninae should not belong to Anserinae, but rather represent independent subfamilies. The Anatinae includes species from the tribes Mergini, Somaterini, Anatini, and Aythyini. The Anserinae includes species from the tribes Anserini and Cygnini.
Citation: Liu G, Zhou L, Li B, Zhang L (2014) The Complete Mitochondrial Genome of Aix galericulata and Tadorna ferruginea: Bearings on Their Phylogenetic Position in the Anseriformes. PLoS ONE 9(11): e109701.
Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. The complete mtDNA sequences of Aix galericulata and Tadorna ferruginea have been deposited in the GenBankof NCBI database under accession numbers KF437906 and KF684946.
Mitochondrial DNA is a powerful, increasingly popular, and widely used molecular marker for the estimation of the animal phylogenetic relationships. It has become a major tool of comparative genomics and plays an important role in phylogenetic studies, comparative and evolutionary genomics, and molecular evolutionary analyses, owing to its maternal inheritance, lack of recombination, and accelerated nucleotide substitution rates compared with those of the nuclear DNA , . Here, we attempt to resolve the controversial Aves species using mtDNA analyses. Our newly completed mitochondrial genomes should provide new insights into the phylogenetic position of important species, and yield insight into the higher-level systematics of Anseriform birds. Early molecular work disentangling the phylogeny of the Anseriformes was mostly based on one or a few mitochondrial loci, almost always including Cyt b, ND2, and/or control region (CR) sequences , . However, complete mtDNA sequences have become increasingly important for comprehensive evolutionary and phylogenetic studies , , . Several analyses have demonstrated that complete mtDNA provides higher levels of phylogenetic support than those based on individual or partial mitogenomes , , , , , . Complete mtDNA sequences are not only more informative than shorter sequences of individual genes, but also provide reliable information toward the inference of phylogenetic relationships among controversial animals , , , . Consequently, complete mtDNA genomes are becoming a preferred marker for resolving controversial species relationships, and are increasingly important for comprehensive evolutionary studies , , , , . However, very few Anseriform birds are currently represented with complete mtDNAs, consequently, a number of Anseriform species and their phylogenetic relationships remain unresolved. We sequenced the complete mtDNA of two important Anseriform birds, from the genera Aix and Tadorna, in this study. We also analyzed the nucleotide composition, codon usage, and compositional biases of the mitogenomes. Our phylogenomic analysis should shed increased light on the phylogenetic status of Aix galericulata and Tadorna ferruginea, and on the phylogenetic relationships of other important groups of Anseriformes.
The complete mtDNAs of Aix galericulata and Tadorna ferruginea are 16,651 and 16,639 bp in length, respectively. Both contain the typical set of 37 genes, including 13 protein-coding genes (PCGs) (ATP6, ATP8, COI-III, ND1-6, ND4L, and Cyt b), two rRNAs (12S rRNA and 16S rRNA), 22 tRNAs and a putative CR (D-loop) (Table 1). The heavy (H-) DNA strand carries most of the genes, 12 PCGs, two rRNAs, and 14 tRNAs. ND6 and eight tRNAs are located on the light (L-) strand (Table 1).
Through the 13 protein-coding genes, ATG is the start codon in nine of the 13 PCGs in Aix galericulata, but ND4 starts with ATT, while COI, COII, and ND5 begin with the nonstandard start codon GTG. In Tadorna ferruginea ten PCGs start with ATG, and ATT is the start codon only in ND4, while COI and ND5 begin with GTG. The standard stop termination codon TAA occurs in most of the same genes in the two birds' mtDNAs, except ND2 stops with TAA in Aix galericulata and TAG in Tadorna ferruginea. Furthermore, AGG terminates the ND1 and COI genes, TAG terminates the ND3 and ND6 gene, and the incomplete termination codon T- occurs in the COIII and ND4 genes in both birds.
In Aix galericulata and Tadorna ferruginea 12S rRNA (997 bp and 982 bp, respectively) and 16S rRNA (1,604 bp and 1,610 bp, respectively) genes are located between the tRNAPhe and tRNALeu genes, separated by the tRNAVal gene. The two complete mtDNAs contain 22 tRNAs genes, and except for tRNASer (AGY) and tRNALeu (CUN), which lack dihydrouridine (DHU) arms, all other tRNAs could be folded into the typical cloverleaf structure. The longest tRNAs are tRNAAsn (78 bp) and tRNAGlu (87 bp) in Aix galericulata and Tadorna ferruginea, respectively, and the shortest is tRNACys (65 bp in both).
Non-coding regions in the mtDNAs include the CRs and a few intergenic spacers. The CRs are located between the tRNAGlu and tRNAPhe genes, which are 1,071 bp and 1,077 bp, respectively, in Aix galericulata and Tadorna ferruginea. Additionally, 11 gene junction regions spacer by a total of 31 bp, with the longest one being 10 bp between ND6 and tRNAGlu in Aix galericulata (Table 1). There are a total of 55 bp spacer region at 12 gene junctions in Tadorna ferruginea (Table 1).
Our chosen 16 Anseriforme species represent two major branches of the Anseriformes phylogeny with highly similar topologies and only slight differences in bootstrap support and posterior probability values (Figure 1). The first branch is Anatidae and the second is Anseranatidae. Anatidae contains Anatinae, Tadorninae, Anserinae, and Dendrocygninae; and Anseranatidae only contains Anseranas semipalmata. Anatinae and Tadorninae are sister groups, grouped together nestled within the clades Anserinae and Dendrocygninae. Mergini, Anatini, and Aythyini form Anatinae; Anserinae contains Anserini and Aythyini. Aix galericulata and Tadorna ferruginea are in the Tadorninae group.
We compared the total length of the 13 PCGs in Aix galericulata and Tadorna ferruginea with other Anseriform birds. Lengths among them are quite similar and very conservative; the longest one is Aix galericulata (11,403 bp) and the shortest is Anser fabalis (11,328 bp). The 13 PCGs have a total length of 11,331 bp in Aix galericulata and 11,385 bp in Tadorna ferruginea, which is 68.05% and 68.42% of each entire mtDNA genome, respectively. In both species, the longest gene is ND5, located between the tRNALeu (CUN) and Cyt b genes, and the shortest is ATP8, which is between the tRNALys and ATP6 genes. Most PCGs used ATG as start codons, only a few start with GTG, GTC, or ATA. Stop codons are also similar across species, with TAA, TAG, and T- occurring most frequently. In Tadorna ferruginea and Tadorna ferruginea, the start condons are ATG, GTG and ATT, and TAA, AGG, TAG and T- as stop termination codons occur in most of the same genes in the two birds' mtDNAs. Among the 13 PCGs, specific examples include the following: the COI initiation codon is GTG and the termination codon is AGG in all 16 species; Cyt b starts with ATG and ends with TAA; COII starts with ATG and ends with TAG, except in Branta canadensis, where it starts with GTC; ND6 starts with ATG and ends with TAG, except in Anser fabalis it ends with TAA; ND1 starts with ATG and ends with AGG, except in Anser albifrons where the stop codon is TAA; and ND2 starts with ATG and ends with TAG, except in Anseranas semipalmata, Cygnus atratus, and Tadorna ferruginea, where the stop codon is TAA (Table 3).
The CR is the only major non-coding segment of mtDNA, and has higher variability, evolving three to five times more rapidly, than other vertebrate mtDNAs . Its primary function is thought to be the regulation of replication and transcription . In Aves the CR is located between the tRNAGlu and tRNAPhe genes. Sequence variation in the CR results in length variability