Bird mitochondrial gene order: insight from 3 warbler mitochondrial genomes

Two main gene orders exist in birds: the ancestral gene order and the remnant control region (CR) 2 gene order. These gene orders differ by the presence of 1 or 2 copies of the CR, respectively. Among songbirds, Oscines were thought to follow the ancestral gene order, with the exception of the lyrebird and Phylloscopus warblers. Here, we determined the complete mitochondrial genome sequence of 3 non-Phylloscopus warblers species and found that the blackcap (Sylvia atricapilla) and the reed warbler (Acrocephalus scirpaceus) have 2 almost identical copies of the CR, whereas the eastern orphean warbler (Sylvia crassirostris) follows the remnant CR 2 gene order. Our results contradict previous studies suggesting that Acrocephalus and most sylvioid warblers exhibit the ancestral gene order. We were able to trace this contradiction to a misidentification of gene order from polymerase chain reaction length determination. We thus suggest that passerine gene order evolution needs to be revised.     

Passerine diversity in the late Oligocene of Germany: earliest evidence for the sympatric coexistence of Suboscines and Oscines

A passerine avifauna from the late Oligocene ( c. 26–25 mya) of Germany was characterized by a high diversity of conspicuously small birds ranging in size from the smallest known Oscines to moderately small forms. The avifauna comprised both Oscines and Suboscines. Other passerine fragments showed such an unexpected mosaic of characters that it was impossible to assign them with certainty to any subordinate clade within the Passeriformes. The isolated remains of oscine passerines are the earliest evidence of this taxon in the Northern Hemisphere. Coexistence of oscine and suboscine passerines during the late Oligocene is also documented for the first time in the Northern Hemisphere. These finds reduce a major gap in the passerine fossil record and allow new insights into the composition and natural history of ancient avifaunas.     

Mhc diversity in two passerine birds: no evidence for a minimal essential Mhc

Humans express an array of Mhc genes, while the chicken has an Mhc that is relatively small and compact with fewer expressed genes. Here we ask whether the "minimal essential Mhc" of the chicken is representative for birds. We investigated the RFLP genotypes in 55 great reed warblers Acrocephalus arundinaceus and 10 willow warblers Phylloscopus trochilus to obtain an overview of the number of class II B genes. There were 13-17 bands per individual in the great reed warblers and 25-30 in the willow warblers, and every individual had a unique RFLP genotype. The high number of RFLP bands indicates that both species have a large number of class II B genes although some may be pseudogenes. Seven different class II B sequences were detected in a great reed warbler cDNA library. There was considerable sequence divergence between the cDNA sequences in exon 2 (peptide-binding region, PBR), whereas they were very similar in exon 3. The cDNA sequences were easily alignable to a classical chicken class II B sequence, and balancing selection was acting in the PBR. One of the cDNA sequences had two deletions and is likely nonfunctional. Finally, the polymorphic class I and class II B RFLP fragments seemed to be linked in the five studied great reed warbler families. These and previous results suggest that birds of the order Passeriformes in general have more Mhc class I and II B genes than birds of the order Galliformes. This difference could be caused by their phylogenetic past, and/or by variance in the selection pressure for maintaining a high number of Mhc genes.     

Complete mitochondrial genome of Odontobutis haifengensis (Perciformes,Odontobutiae): A unique rearrangement of tRNAs and additional non-coding regions identified in the genus Odontobutis

Herein, the complete mitochondrial genome of Odontobutis haifengensis was sequenced for the first time. The O. haifengensis mitogenome was 17,016 bp in length and included 13 protein-coding genes, 22 transfer RNAs (tRNAs), 2 ribosomal RNAs (rRNAs), and a control region (CR). The genome organization, base composition, codon usage, and gene rearrangement was similar to other Odontobutis species. Furthermore, a tRNA gene rearrangement within the SLH cluster was found to be identical to other Odontobutis species. Moreover, the gene order and the positions of additional intergenic non-coding regions suggests that the observed unique gene rearrangement resulted from a tandem duplication and random loss of large-scale gene regions. Additionally, phylogenetic analysis showed that Odontobutis species form a monophyletic clade due to the conserved mitochondrial gene rearrangement. This study provides useful information that aids in a better understanding of mitogenomic diversity and evolutionary patterns of Odontobutidae species.     

Characterization of the red knot (Calidris canutus) mitochondrial control region.

We sequenced the complete mitochondrial control regions of 11 red knots (Calidris canutus). The control region is 1168 bp in length and is flanked by tRNA glutamate (glu) and the gene ND6 at its 5' end and tRNA phenylalanine (phe) and the gene 12S on its 3' end. The sequence possesses conserved sequence blocks F, E, D, C, CSB-1, and the bird similarity box (BSB), as expected for a mitochondrial copy. Flanking tRNA regions show correct secondary structure, and a relative rate test indicated no significant difference between substitution rates in the sequence we obtained versus the known mitochondrial sequence of turnstones (Charadriiformes: Scolopacidae). These characteristics indicate that the sequence is mitochondrial in origin. To confirm this, we sequenced the control region of a single individual using both purified mitochondrial DNA and genomic DNA. The sequences were identical using both methods. The sequence and methods presented in this paper may now serve as a reference for future studies using knot and other avian control regions. Furthermore, the discovery of five variable sites in 11 knots towards the 3' end of the control region, and the variability of this region in contrast to the more conserved central domain in the alignment between knots and other Charadriiformes, highlights the importance of this area as a source of variation for future studies in knots and other birds.     

Variations in mitochondrial tRNA gene organization of reptiles as phylogenetic markers

Amplification and sequencing of mitochondrial DNA regions corresponding to three major clusters of transfer RNA genes from a variety of species representing major groups of birds and reptiles revealed some new variations in tRNA gene organization. First, a gene rearrangement from tRNA(His)-tRNA(Ser)(AGY)-tRNA(Leu)(CUN) to tRNA(Ser)(AGY)- tRNA(His)tRNA(Leu)(CUN) occurs in all three crocodilians examined (alligator, caiman, and crocodile). In addition an exceptionally long spacer region between the genes for NADH dehydrogenase subunit 4 and tRNA(Ser)(AGY) is found in caiman. Second, in congruence with a recent finding by Seutin et al., a characteristic stem-and-loop structure for the putative light-strand replication origin located between tRNA(Asn) and tRNA(Cys) genes is absent for all the birds and crocodilians. This stem-and-loop structure is absent in an additional species, the Texas blind snake, whereas the stem-and-loop structure is present in other snakes, lizards, turtles, mammals, and a frog. The disappearance of the stem-and-loop structure in the blind snake most likely occurred independently of that on the lineage leading to birds and crocodilians. Finally, the blind snake has a novel type of tRNA gene arrangement in which the tRNA(Gln) gene moved from one tRNA cluster to another. Sequence substitution rates for the tRNA genes appeared to be somewhat higher in crocodialians than in birds and mammals. As regards the controversial phylogenetic relationship among the Aves, Crocodilia, and Mammalia, a sister group relationship of birds and crocodilians relative to mammals, as suggested from the common loss of the stem-and- loop structure, was supported with statistical significance by molecular phylogenetic analyses using the tRNA gene sequence data.      

Phylogeny of major lineages of suboscines (Passeriformes) analysed by nuclear DNA sequence data

Phylogenetic relationships among major groups of passeriform birds were studied by analyses of nucleotide sequence data from two nuclear genes, c- myc and RAG-1. The results corroborated both the monophyly of the order Passeriformes, and the major dichotomy into oscine and suboscine passerines previously suggested based on syringeal morphology and DNA-DNA hybridizations. The representatives of the Old World suboscines (families Eurylaimidae, Philepittidae and Pittidae) formed a monophyletic clade. The New World suboscines clustered into two clades. The first contained Conopophaga (Conopophagidae), Furnarius (Furnariidae), Lepidocolaptes (Dendrocolaptidae), Thamnophilus (Formicariidae), and Rhinocrypta (Rhinocryptidae). Previously, the monophyly of this group has been inferred from their possession of a unique, "tracheophone" syrinx, and from DNA-DNA hybridisation data. The second clade of New World suboscines includes Gubernetes and Muscivora (Tyrannidae), Phytotoma (Phytotomidae), Tityra (Cotingidae) and Pipra (Pipridae). This group of families have been considered monophyletic based on morphology (although ambiguously) and DNA-DNA hybridisation. The sister group relationship of Tityra and Phytotoma supports the previously supposed cotingid affinity of Phytotoma . Nuclear DNA data also unambiguously group the lyrebirds Menura with the oscines.
The presented results from the analysis of nuclear DNA agree well with morphology and DNA-DNA hybridisation data. The precise age of the divergences studied herein are unknown but based on interpretations of the fossil record of passerine birds many of them might date back to the early Tertiary. The agreement between data from the nuclear DNA and other sources, along with the fact that neither of the studied genes showed sign of saturation, indicate the great potential of these two nuclear genes to resolve very old divergences in birds.     

Genetic evidence supports song learning in the three-wattled bellbird Procnias tricarunculata (Cotingidae)

Vocal learning is thought to have evolved in three clades of birds (parrots, hummingbirds, and oscine passerines), and three clades of mammals (whales, bats, and primates). Behavioural data indicate that, unlike other suboscine passerines, the three-wattled bellbird Procnias tricarunculata (Cotingidae) is capable of vocal learning. Procnias tricarunculata shows conspicuous vocal ontogeny, striking geographical variation in song, and rapid temporal change in song within a population. Deprivation studies of vocal development in P. tricarunculata are impractical. Here, we report evidence from mitochondrial DNA sequences and nuclear microsatellite loci that genetic variation within and among the four allopatric breeding populations of P. tricarunculata is not congruent with variation in vocal behaviour. Sequences of the mitochondrial DNA control region document extensive haplotype sharing among localities and song types, and no phylogenetic resolution of geographical populations or behavioural groups. The vocally differentiated, allopatric breeding populations of P. tricarunculata are only weakly genetically differentiated populations, and are not distinct taxa. Mitochondrial DNA and microsatellite variation show small (2.9% and 13.5%, respectively) but significant correlation with geographical distance, but no significant residual variation by song type. Estimates of the strength of selection that would be needed to maintain the observed geographical pattern in vocal differentiation if songs were genetically based are unreasonably high, further discrediting the hypothesis of a genetic origin of vocal variation. These data support a fourth, phylogenetically independent origin of avian vocal learning in Procnias. Geographical variations in P. tricarunculata vocal behaviour are likely culturally evolved dialects.     

The mitochondrial NADH dehydrogenase subunit 6 (ND6) gene in Murres: relevance to phylogenetic and population studies among birds.

The nucleotide sequences of the mitochondrial ND6 and tRNA(Glu) genes and part of the displacement loop region in two closely related seabird species are presented. A chicken type gene organization in which the tRNA(Glu), ND6, and displacement loop are localized next to each other was found in these species and suggests that this is a conserved feature of avian mitochondrial DNA. The nucleotide and amino acid divergences of ND6 at different taxonomic levels are assessed, and its relevance to phylogenetic studies in birds is discussed.     

Resistance of flight feathers to mechanical fatigue covaries with moult strategy in two warbler species

Flight feather moult in small passerines is realized in several ways. Some species moult once after breeding or once on their wintering grounds; others even moult twice. The adaptive significance of this diversity is still largely unknown. We compared the resistance to mechanical fatigue of flight feathers from the chiffchaff Phylloscopus collybita, a migratory species moulting once on its breeding grounds, with feathers from the willow warbler Phylloscopus trochilus, a migratory species moulting in both its breeding and wintering grounds. We found that flight feathers of willow warblers, which have a shaft with a comparatively large diameter, become fatigued much faster than feathers of chiffchaffs under an artificial cyclic bending regime. We propose that willow warblers may strengthen their flight feathers by increasing the diameter of the shaft, which may lead to a more rapid accumulation of damage in willow warblers than in chiffchaffs.     

Conflicting patterns of mitochondrial and nuclear DNA diversity in Phylloscopus warblers

Molecular variation is often used to infer the demographic history of species, but sometimes the complexity of species history can make such inference difficult. The willow warbler, Phylloscopus trochilus, shows substantially less geographical variation than the chiffchaff, Phylloscopus collybita, both in morphology and in mitochondrial DNA (mtDNA) divergence. We therefore predicted that the willow warbler should harbour less nuclear DNA diversity than the chiffchaff. We analysed sequence data obtained from multiple samples of willow warblers and chiffchaffs for the mtDNA cytochrome b gene and four nuclear genes. We confirmed that the mtDNA diversity among willow warblers is low (pi = 0.0021). Sequence data from three nuclear genes (CHD-Z, AFLP-WW1 and MC1R) not linked to the mitochondria demonstrated unexpectedly high nucleotide diversity (pi values of 0.0172, 0.0141 and 0.0038) in the willow warbler, on average higher than the nucleotide diversity for the chiffchaff (pi values of 0.0025, 0.0017 and 0.0139). In willow warblers, Tajima's D analyses showed that the mtDNA diversity, but not the nuclear DNA diversity, has been reduced relative to the neutral expectation of molecular evolution, suggesting the action of a selective sweep affecting the maternally inherited genes. The large nuclear diversity seen within willow warblers is not compatible with processes of neutral evolution occurring in a population with a constant population size, unless the long-term effective population size has been very large (N(e) > 10(6)). We suggest that the contrasting patterns of genetic diversity in the willow warbler may reflect a more complex evolutionary history, possibly including historical demographic fluctuations or historical male-biased introgression of nuclear genes from a differentiated population of Phylloscopus warblers.     

Evidence of a neo-sex chromosome in birds

Neo-sex chromosomes often originate from sex chromosome–autosome fusions and constitute an important basis for the study of gene degeneration and expression in a sex chromosomal context. Neo-sex chromosomes are known from many animal and plant lineages, but have not been reported in birds, a group in which genome organization seems particularly stable. Following indications of sex linkage and unexpected sex-biased gene expression in warblers (Sylvioidea; Passeriformes), we have conducted an extensive marker analysis targeting 31 orthologues of loci on zebra finch chromosome 4a in five species, representative of independent branches of Passerida. We identified a region of sex linkage covering approximately the first half (10 Mb) of chromosome 4a, and associated to both Z and W chromosomes, in three Sylvioidea passerine species. Linkage analysis in an extended pedigree of one species additionally confirmed the association between this part of chromosome 4a and the Z chromosome. Markers located between 10 and 21 Mb of chromosome 4a showed no signs of sex linkage, suggesting that only half of the chromosome was involved in this transition. No sex linkage was observed in non-Sylvioidea passerines, indicating that the neo-sex chromosome arose at the base of the Sylvioidea branch of the avian phylogeny, at 47.4–37.6 millions years ago (MYA), substantially later than the ancestral sex chromosomes (150 MYA). We hypothesize that the gene content of chromosome 4a might be relevant in its transition to a sex chromosome, based on the presence of genes (for example, the androgen receptor) that could offer a selective advantage when associated to Z-linked sex determination loci.     

Improved COI barcoding primers for Southeast Asian perching birds (Aves: Passeriformes)

The All Birds Barcoding Initiative aims to assemble a DNA barcode database for all bird species, but the 648-bp 'barcoding' region of cytochrome c oxidase subunit I (COI) can be difficult to amplify in Southeast Asian perching birds (Aves: Passeriformes). Using COI sequences from complete mitochondrial genomes, we designed a primer pair that more reliably amplifies and sequences the COI barcoding region of Southeast Asian passerine birds. The 655-bp region amplified with these primers overlaps the COI region amplified with other barcoding primer pairs, enabling direct comparison of sequences with previously published DNA barcodes.     

Duplication and Concerted Evolution of the Mitochondrial Control Region in the Parrot Genus Amazona

We report a duplication and rearrangement of the mitochondrialgenome involving the control region of parrots in the genusAmazona. This rearrangement results in a gene order of cytochromeb/tRNA^Thr/pND6/pGlu/CR1/tRNA^Pro/NADH dehydrogenase 6/tRNA^Glu/CR2/tRNA^Phe/12srRNA, where CR1 and CR2 refer to duplicate control regions,and pND6 and pGlu indicate presumed pseudogenes. In contrastto previous reports of duplications involving the control regionsof birds, neither copy of the parrot control region shows anyindications of degeneration. Rather, both copies contain manyof the conserved sequence features typically found in aviancontrol regions, including the goose hairpin, TASs, the F, C,and D boxes, conserved sequence box 1 (CSB1), and an apparenthomolog to the mammalian CSB3. We conducted a phylogenetic analysisof homologous portions of the duplicate control regions from21 individuals representing four species of Amazona (A. ochrocephala,A. autumnalis, A. farinosa, and A. amazonica) and Pionus chalcopterus.This analysis revealed that an individual's two control regioncopies (i.e., the paralogous copies) were typically more closelyrelated to one another than to corresponding segments of otherindividuals (i.e., the orthologous copies). The average sequencedivergence of the paralogous control region copies within anindividual was 1.4%, versus a mean value of 4.1% between controlregion orthologs representing nearest phylogenetic neighbors.No differences were found between the paralogous copies in eitherthe rate or the pattern in which the two copies accumulatedbase pair changes. This pattern suggests concerted evolutionof the two control regions, perhaps through occasional geneconversion events. We estimated that gene conversion eventsoccurred on average every 34,670 ± 18,400 years basedon pairwise distances between the paralogous control regionsequences of each individual. Our results add to the growingbody of work indicating that under some circumstances duplicatedmitochondrial control regions are retained through evolutionarytime rather than degenerating and being lost, presumably dueto selection for a small mitochondrial genome.     

The use of AFLP to find an informative SNP: genetic differences across a migratory divide in willow warblers

We used the amplified fragment length polymorphism (AFLP) method to obtain genetic markers distinguishing two subspecies of willow warblers Phylloscopus trochilus that have different migratory behaviours but are not differentiated in mitochondrial DNA or at several microsatellite loci. With the inverse-polymerase chain reaction (PCR) approach we converted a dominant AFLP-marker to a codominant single nucleotide polymorphism (SNP). Across Scandinavia we typed 621 birds at the SNP locus AFLP-WW1 and we found a sigmoid change in allele frequencies centred around 62 degrees latitude. North of the latitudinal cline was a west-east cline. Both clines are narrower than one would expect from dispersal distances in willow warblers, which suggests that these are maintained by selection. The latitudinal cline at the locus AFLP-WW1 is paralleled by changes in several other traits, all of which might be maintained by a single selective force. The most plausible selection factor that we have identified is selection against hybrids because of inferior migratory behaviour. The selective force maintaining the east-west cline is less obvious. We discuss alternatives to the selection scenario, involving colonization history and asymmetric gene flow.     

Description of a new species of Phylloscopus warbler from Vietnam and Laos

A new species of Phylloscopus warbler, which we name Phylloscopus calciatilis Limestone Leaf Warbler, is described from central and northern Vietnam and central and northern Laos; it probably also breeds in southernmost China. In morphology, the new species is very similar to Sulphur-breasted Warbler Phylloscopus ricketti , but it is smaller with a proportionately larger bill and rounder wing. Its song and calls are diagnostic. Based on mitochondrial and nuclear DNA, the new species is most closely related to P. ricketti and Yellow-vented Warbler Phylloscopus cantator , and it is inferred to be sister to the latter. The mitochondrial divergences between these three species are at the low end of the variation found in other species of Phylloscopus and Seicercus warblers, but greater than in other taxa generally treated as subspecies. Possible introgressive hybridization between the new species and P. ricketti is discussed, but more data are needed to establish whether it does occur and, if it does, to what extent. The new species appears to have a restricted breeding range in limestone karst environments, where it is locally common and therefore not under any immediate threat. In view of the recognition of the new species, all previous records of P. ricketti sensu lato need to be re-evaluated.     

The complete mitochondrial genomes of two schizothoracine fishes (Teleostei,Cypriniformes): A novel minisatellite in fish mitochondrial genomes

Minisatellites, a class of variable number tandem repeats (VNTRs), are abundant throughout the control region in animal mitochondrial DNA (mtDNA) but rare in other regions of animal mtDNA. Here, we reported a novel minisatellite in fish mitochondrial genomes. We first determined the complete mitochondrial genomes of two schizothoracine fishes (Herzensteinia microcephalus and Schizopygopsis pylzovi) and found a type of minisatellites in a novel region between the tRNA‐Thr and tRNA‐Pro genes in their mtDNA. To explore the origin and evolution of the minisatellites in different schizothoracine and closely related fishes, we analyzed the available 80 fish mitogenomes which represent five closely related tribes of cyprinine fishes. The results from the phylogenetic analyses show that the schizothoracine fishes sensu stricto is not a monophyletic group and is divided into two clades (Schizothoracini and Schizopygopsini); and the minisatellite is only present in Schizopygopsini distributed in the region between the two tRNA genes (tRNA‐Thr and tRNA‐Pro) of the mtDNA. This is the first record of a minisatellite in a non‐control region of fish mitogenome.