New Insight into Parrots' Mitogenomes Indicates That Their Ancestor Contained a Duplicated Region

Abstract

Mitochondrial genomes of vertebrates are generally thought to evolve under strong selection for size reduction and gene order conservation. Therefore, a growing number of mitogenomes with duplicated regions changes our view on the genome evolution. Among Aves, order Psittaciformes (parrots) is especially noteworthy because of its large morphological, ecological, and taxonomical diversity, which offers an opportunity to study genome evolution in various aspects. Former analyses showed that tandem duplications comprising the control region with adjacent genes are restricted to several lineages in which the duplication occurred independently. However, using an appropriate polymerase chain reaction strategy, we demonstrate that early diverged parrot groups contain mitogenomes with the duplicated region. These findings together with mapping duplication data from other mitogenomes onto parrot phylogeny indicate that the duplication was an ancestral state for Psittaciformes. The state was inherited by main parrot groups and was lost several times in some lineages. The duplicated regions were subjected to concerted evolution with a frequency higher than the rate of speciation. The duplicated control regions may provide a selective advantage due to a more efficient initiation of replication or transcription and a larger number of replicating genomes per organelle, which may lead to a more effective energy production by mitochondria. The mitogenomic duplications were associated with phenotypic features and parrots with the duplicated region can live longer, show larger body mass as well as predispositions to a more active flight. The results have wider implications on the presence of duplications and their evolution in mitogenomes of other avian groups.

Fig. 1.

Comparison of mitochondrial gene orders. The region between gene ND5 and gene 12S rRNA is shown for: typical vertebrate gene order (A), typical avian gene order (B), hypothetical gene order ancestral for Psittaciformes (C) assuming tandem duplication of the cytb to CR segment, and gene orders with duplicated CRs found for parrot species (D). ND5, NADH dehydrogenase subunit 5 gene; cytb, cytochrome b gene; T, tRNA gene for threonine; P, tRNA gene for proline; ND6, NADH dehydrogenase subunit 6; E, tRNA gene for glutamic acid; F, tRNA gene for phenylalanine; 12S, 12S rRNA gene. Pseudogenes are marked by ψ and colored correspondingly to their functional gene copy. The solid blue line above the ND6 pseudogene in GO-1 gene order indicates a fragment identical with ND6 gene; the dashed line above this pseudogene and the tRNA-Glu gene indicates substantial degeneration of their sequences; the solid red line above this pseudogene with the letter “i” indicates an insertion within the pseudogene sequence.

Fig. 2.

Location of primer pairs and relative differences in the length of diagnostic fragments used to determine CR state as single or duplicated. Segments 15, 16, and ND6 correspond to the strategy proposed by Schirtzinger et al. (2012) for mitogenomes comprising: typical avian gene order (A), duplicated gene order identified for Amazona species (B), tandem duplication of the tRNA-Thr to CR segment (C and D). A strategy proposed in this study for identification of all variants of gene orders with duplicated CRs using the diagnostic Fragment 5 (E). Sequences of diagnostic primers D-F and D-R are listed in table 1. ND5, NADH dehydrogenase subunit 5 gene; cytb, cytochrome b gene; T, tRNA gene for threonine; P, tRNA gene for proline; ND6, NADH dehydrogenase subunit 6; E, tRNA gene for glutamic acid; F, tRNA gene for phenylalanine; 12S, 12S rRNA gene. Pseudogenes are marked by ψ and colored correspondingly to their functional gene copy.

Fig. 3.

Phylogenetic trees obtained in PhyloBayes based on all available parrot sequences of CRs (A) and those from Amazona and Pionus (B). The blue and red colors correspond to the first and the second copy of CR, respectively. The values at nodes, in the order shown, indicate posterior probabilities found in PhyloBayes (PB) and MrBayes (MB) as well as SH-aLRT (SH) and nonparametric bootstrap (BP) percentages calculated in IQ-TREE. The posterior probabilities <0.5 and the percentages <50% were omitted or indicated by a dash “-.”

Fig. 4.

Box plots of AT-skew (ATs) and GC-skew (GCs) calculated for the entire genome as well as all sites in PCGs and 4-fold degenerate (4FD) sites in mitogenomes with duplicated (D) or single (S) regions. The thick line indicates median, the boxes show quartile range, and the whiskers denote the range without outliers. P-values of the null hypothesis assuming no differences in DNA asymmetry between the mitogenomes are shown.

Fig. 5.

Box plots of morphological features for parrots with mitogenomes containing duplicated (D) or single (S) regions. M, body mass; W, wingspan; T, tail length; L, total body length. The thick line indicates median, the boxes show quartile range, and the whiskers denote the range without outliers. P-values of the null hypothesis assuming no differences in a given feature between the parrots are shown.

Fig. 6.

Box plots of the maximum life span in years for parrots with mitogenomes containing duplicated (D) or single (S) regions. The thick line indicates median, the boxes show quartile range, and the whiskers denote the range without outliers. P-value of the null hypothesis assuming no differences in the maximum life span between the parrots is shown.

Fig. 7.

Maximum likelihood reconstruction of ancestral states and mapping of mitogenomic duplications onto the ND2 phylogenetic tree of parrots with verified presence or absence of the duplication. The area of colors at nodes corresponds to the probability of the given state, single or duplicated region. The probability value for a more probable state was given.

Fig. 8.

Maximum parsimony reconstruction of ancestral states and mapping of mitogenomic duplications onto the ND2 phylogenetic tree of parrots with verified presence or absence of the duplication. Two-colored branches correspond to the equal probability of two states, single or duplicated region.

Fig. 9.

Maximum likelihood (A) and maximum parsimony (B) reconstruction of ancestral states and mapping of mitogenomic duplications onto the parrot phylogenetic tree based on 37 mitochondrial genes. Other explanation as in figures 7 and 8.