Evolution of hominoid mitochondrial DNA with special reference to the silent substitution rate over the genome

Summary Focusing on the synonymous substitution rate, we carried out detailed sequence analyses of hominoid mitochondrial (mt) DNAs of ca. 5-kb length. Owing to the outnumbered transitions and strong biases in the base compositions, synonymous substitutions in mtDNA reach rapidly a rather low saturation level. The extent of the compositional biases differs from gene to gene. Such changes in base compositions, even if small, can bring about considerable variation in observed synonymous differences and may result in the region-dependent estimate of the synonymous substitution rate. We demonstrate that such a region dependency is due to a failure to take proper account of heterogeneous compositional biases from gene to gene but that the actual synonymous substitution rate is rather uniform. The synonymous substitution rate thus estimated is 2.37 ± 0.11 × 10^–8 per site per year and comparable to the overall rate for the noncoding region. On the other hand, the rate of nonsynonymous substitutions differs considerably from gene to gene, as expected under the neutral theory of molecular evolution. The lowest rate is 0.8 × 10^–9 per site per year forCOI and the highest rate is 4.5 × 10^–9 forATPase 8, the degree of functional constraints (measured by the ratio of the nonsynonymous to the synonymous substitution rate) being 0.03 and 0.19, respectively. Transfer RNA (tRNA) genes also show variability in the base contents and thus in the nucleotide differences. The average rate for 11 tRNAs contained in the 5-kb region is 3.9 × 10^–9 per site per year. The nucleotide substitutions in the genome suggest that the transition rate is about 17 times faster than the transversion rate.     

Nucleotide Substitution Rate of Mammalian Mitochondrial Genomes

We present here for the first time a comprehensive study based on the analysis of closely related organisms to provide an accurate determination of the nucleotide substitution rate in mammalian mitochondrial genomes. This study examines the evolutionary pattern of the different functional mtDNA regions as accurately as possible on the grounds of available data, revealing some important ``genomic laws.' The main conclusions can be summarized as follows. (1) High intragenomic variability in the evolutionary dynamic of mtDNA was found. The substitution rate is strongly dependent on the region considered, and slow- and fast-evolving regions can be identified. Nonsynonymous sites, the D-loop central domain, and tRNA and rRNA genes evolve much more slowly than synonymous sites and the two peripheral D-loop region domains. The synonymous rate is fairly uniform over the genome, whereas the rate of nonsynonymous sites depends on functional constraints and therefore differs considerably between genes. (2) The commonly accepted statement that mtDNA evolves more rapidly than nuclear DNA is valid only for some regions, thus it should be referred to specific mitochondrial components. In particular, nonsynonymous sites show comparable rates in mitochondrial and nuclear genes; synonymous sites and small rRNA evolve about 20 times more rapidly and tRNAs about 100 times more rapidly in mitochondria than in their nuclear counterpart. (3) A species-specific evolution is particularly evident in the D-loop region. As the divergence times of the organism pairs under consideration are known with sufficient accuracy, absolute nucleotide substitution rates are also provided. Received: 11 May 1998 / Accepted: 2 September 1998     

Micro-scale signature of purifying selection in Marburg virus genomes

In the seven protein-coding genes in the Marburg virus (MARV) genome, the synonymous nucleotide diversity substantially exceeded the nonsynonymous nucleotide diversity, indicating strong purifying selection. Likewise, there was evidence of purifying selection on 5'UTR and 3'UTR, where nucleotide diversity (pi) was significantly less than piS in the coding regions. Nonsynonymous polymorphic sites showed significantly reduced mean gene diversity in comparison to other polymorphic sites, indicating that purifying selection at certain slightly deleterious nonsynonymous polymorphisms is ongoing. Moreover, nonsynonymous polymorphic sites showed significantly reduced gene diversity in comparison to adjacent synonymous sites, even though the vast majority of such adjacent synonymous sites were in the same codon or an adjacent codon. Thus purifying selection, in conjunction with recombination and/or backward mutation, can act to break up linkage relationships at a micro-scale in the MARV genome. The ability of purifying selection to break up linkage between synonymous and nonsynonymous polymorphisms on such a fine scale has not been reported in any other genome.     

Complete mtDNA genome of Otus sunia (Aves,Strigidae) and the relaxation of selective constrains on Strigiformes mtDNA following evolution

Most of owls are nocturnal raptor and usually use their soft and fluffy feathers to flight silently to catch prey while other diurnal raptors prefer fierce attack and swift flight. For energy cost of these different hunting strategies can be greatly different, we speculate that mitochondrial gene of owls may undergo a different evolution pattern following raptors evolution. To test our hypothesis, we sequenced the mtDNA genome of Otus sunia and calculated the ratio of nonsynonymous to synonymous nucleotide substitutions (ω, Ka/Ks, dN/dS) of raptors. The mtDNA genome of O. sunia was 17,609 bp in length, containing 13 PCGs, 2 ribosomal RNAs, 22 transfer RNAs and a control region. Secondly structure of tRNAs and rRNAs were predicted and conserved sequence blocks (CSBs) on control region were identified. The Bayesian inference tree and maximum likelihood tree based on 13 PCGs and 2 rRNAs suggested the owls were related to other raptors. Finally, calculation of ω-values of each owls and other raptors mtDNA PCGs indicated that owls accumulated more nonsynonymous nucleotide substitutions relative to synonymous substitutions compared to other raptors. For mtDNA PCGs associated with energy metabolism, this finding may reveal the degeneration of flight abilities of owls.     

Phylogeny and evolution of chloroplast tRNAs in Adoxaceae

Chloroplasts are semiautonomous organelles found in photosynthetic plants. The major functions of chloroplasts include photosynthesis and carbon fixation, which are mainly regulated by its circular genomes. In the highly conserved chloroplast genome, the chloroplast transfer RNA genes (cp tRNA) play important roles in protein translation within chloroplasts. However, the evolution of cp tRNAs remains unclear. Thus, in the present study, we investigated the evolutionary characteristics of chloroplast tRNAs in five Adoxaceae species using 185 tRNA gene sequences. In total, 37 tRNAs encoding 28 anticodons are found in the chloroplast genome in Adoxaceae species. Some consensus sequences are found within the Ψ‐stem and anticodon loop of the tRNAs. Some putative novel structures were also identified, including a new stem located in the variable region of tRNA^Tyr in a similar manner to the anticodon stem. Furthermore, phylogenetic and evolutionary analyses indicated that synonymous tRNAs may have evolved from multiple ancestors and frequent tRNA duplications during the evolutionary process may have been primarily caused by positive selection and adaptive evolution. The transition and transversion rates are uneven among different tRNA isotypes. For all tRNAs, the transition rate is greater with a transition/transversion bias of 3.13. Phylogenetic analysis of cp tRNA suggested that the type I introns in different taxa (including eukaryote organisms and cyanobacteria) share the conserved sequences “U‐U‐x2‐C” and “U‐x‐G‐x2‐T,” thereby indicating the diverse cyanobacterial origins of organelles. This detailed study of cp tRNAs in Adoxaceae may facilitate further investigations of the evolution, phylogeny, structure, and related functions of chloroplast tRNAs.     

A mitochondria-specific mutational signature of aging: increased rate of A > G substitutions on the heavy strand

The mutational spectrum of the mitochondrial DNA (mtDNA) does not resemble any of the known mutational signatures of the nuclear genome and variation in mtDNA mutational spectra between different organisms is still incomprehensible. Since mitochondria are responsible for aerobic respiration, it is expected that mtDNA mutational spectrum is affected by oxidative damage. Assuming that oxidative damage increases with age, we analyse mtDNA mutagenesis of different species in regards to their generation length. Analysing, (i) dozens of thousands of somatic mtDNA mutations in samples of different ages (ii) 70053 polymorphic synonymous mtDNA substitutions reconstructed in 424 mammalian species with different generation lengths and (iii) synonymous nucleotide content of 650 complete mitochondrial genomes of mammalian species we observed that the frequency of A_H > G_H substitutions (H: heavy strand notation) is twice bigger in species with high versus low generation length making their mtDNA more A_H poor and G_H rich. Considering that A_H > G_H substitutions are also sensitive to the time spent single-stranded (TSSS) during asynchronous mtDNA replication we demonstrated that A_H > G_H substitution rate is a function of both species-specific generation length and position-specific TSSS. We propose that A_H > G_H is a mitochondria-specific signature of oxidative damage associated with both aging and TSSS.     

Deleterious mutations at the mitochondrial ND3 gene in South American marsh rats (Holochilus).

Statistical analyses of DNA sequences have revealed patterns of nonneutral evolution in mitochondrial DNA of mice, humans, and Drosophila. Here we report patterns of mitochondrial sequence evolution in South American marsh rats (genus Holochilus). We sequenced the complete mitochondrial ND3 gene in 82 Holochilus brasiliensis and 21 H. vulpinus to test the neutral prediction that the ratio of nonsynonymous to synonymous nucleotide changes is the same within and between species. Within H. brasiliensis we observed a greater number of amino acid polymorphisms than expected based on interspecific comparisons. This contingency table analysis suggests that many amino acid polymorphisms are mildly deleterious. Several tests of the frequency distribution also revealed departures from a neutral, equilibrium model, and these departures were observed for both nonsynonymous and synonymous sites. In general, an excess of rare sites was observed, consistent with either a recent selective sweep or with populations not at mutation-drift equilibrium.     

Analysis of polymorphisms in the major expressed class I locus (B-FIV) of the chicken

 We analyzed the polymorphic nature of eleven alleles expressed by the major class I locus (B-FIV) in chickens. Similar to mammalian class I loci, the nucleotide substitutions with high variability occur in exons 2 and 3 encoding the α1 and α2 domains. However, the nonsynonymous to synonymous ratio of nucleotide substitutions in exon 3 encoding the α helix and β sheets is reversed compared with HLA. The region of exon 3 encoding the α2 helix demonstrates a much lower nonsynonymous to synonymous ratio, suggesting evolutionary selection of a more conserved α2 helix in B-FIV compared with HLA. Amino acid residues with high Wu-Kabat variability are typically located in positions predicted to impact antigen presentation. B-FIV amino acid residues predicted to interact with the CDR1α region of the T-cell receptor (Tcr) demonstrate less variability than in mouse and human class I alleles. The combination of a reduced nonsynonymous to synonymous ratio in exon 3 encoding the α2 helix and the limited variability in CDR1α contact residues is discussed with regard to concerted evolution between a minimal major histocompatibility complex and compaction of Tcr variable gene segments in the chicken. Received: 18 Juli 1997 /  Revised: 21 November 1997     

Lineage-specific selection in human mtDNA: lack of polymorphisms in a segment of MTND5 gene in haplogroup J

Human mitochondrial DNA (mtDNA) is a nonrecombining genome that codes for 13 subunits of the mitochondrial oxidative phosphorylation system, 2 rRNAs, and 22 tRNAs. Mutations have accumulated sequentially in mtDNA lineages that diverged tens of thousands of years ago. The genes in mtDNA are subject to different functional constraints and are therefore expected to evolve at different rates, but the rank order of these rates should be the same in all lineages of a phylogeny. Previous studies have indicated, however, that specific regions of mtDNA may have experienced different histories of selection in different lineages, possibly because of lineage-specific interactions or environmental factors such as climate. We report here on a survey for lineage-specific patterns of nucleotide polymorphism in human mtDNA. We calculated molecular polymorphism indices and neutrality tests for classes of functional sites and genes in 837 human mtDNA sequences, compared the results between continent-specific mtDNA lineages, and used two sliding window methods to identify differences in the patterns of polymorphism between haplogroups. A general correlation between nucleotide position and the level of nucleotide polymorphism was identified in the coding region of the mitochondrial genome. Nucleotide diversity in the protein-coding sequence of mtDNA was generally not much higher than that found for many genes in nuclear DNA. A comparison of nonsynonymous/synonymous rate ratios in the 13 protein-coding genes suggested differences in the relative levels of selection between haplogroups, including the European haplogroup clusters. Interestingly, a segment of the MTND5 gene was found to be almost void of segregating sites and nonsynonymous mutations in haplogroup J, which has been associated with susceptibility to certain complex diseases. Our results suggest that there are haplogroup-specific differences in the intensity of selection against particular regions of the mitochondrial genome, indicating that some mutations may be non-neutral within specific phylogenetic lineages but neutral within others.     

Sequence Heteroplasmy of D-Loop and rRNA Coding Regions in Mitochondrial DNA from Holstein Cows of Independent Maternal Lineages

A mitochondrial DNA (mtDNA) fragment containing the D-loop, phenylalanine tRNA, valine tRNA, and 12S and 16 rRNA genes was cloned and sequenced from 36 cows of 18 maternal lineages to identify the polymorphic sites within those regions and to detect the existence of heteroplasmic mtDNA in cows. Seventeen variable sites were observed within the D-loop and rRNA coding regions of bovine mtDNA within a 2.5-kb span. The hypervariable sites in the D-loop and rRNA coding regions were identified at nucleotide positions 169, 216, and 1594. Heteroplasmic mtDNA (variable mtDNA within a tissue) existed extensively in cows and was detected within the above regions in 11 of 36 cows sequenced. The insertion, deletion, and nucleotide transversion polymorphisms were found only in homopolymer regions. Heteroplasmy was observed frequently and seemingly is persistent in cattle. Though heteroplasmy was demonstrated, most lineages and mtDNA sites showed no heteroplasmy.     

Disrupted tRNA Gene Diversity and Possible Evolutionary Scenarios

The following unusual tRNAs have recently been discovered in the genomes of Archaea and primitive Eukaryota: multiple-intron-containing tRNAs, which have more than one intron; split tRNAs, which are produced from two pieces of RNA transcribed from separate genes; tri-split tRNAs, which are produced from three separate genes; and permuted tRNA, in which the 5′ and 3′ halves are encoded with permuted orientations within a single gene. All these disrupted tRNA genes can form mature contiguous tRNA, which is aminoacylated after processing by cis or trans splicing. The discovery of such tRNA disruptions has raised the question of when and why these complex tRNA processing pathways emerged during the evolution of life. Many previous reports have noted that tRNA genes contain a single intron in the anticodon loop region, a feature common throughout all three domains of life, suggesting an ancient trait of the last universal common ancestor. In this context, these unique tRNA disruptions recently found only in Archaea and primitive Eukaryota provide new insight into the origin and evolution of tRNA genes, encouraging further research in this field. In this paper, we summarize the phylogeny, structure, and processing machinery of all known types of disrupted tRNAs and discuss possible evolutionary scenarios for these tRNA genes.     

Identifying the ligated amino acid of archaeal tRNAs based on positions outside the anticodon

Proper recognition of tRNAs by their aminoacyl-tRNA synthetase is essential for translation accuracy. Following evidence that the enzymes can recognize the correct tRNA even when anticodon information is masked, we search for additional nucleotide positions within the tRNA molecule that potentially contain information for amino acid identification. Analyzing 3936 sequences of tRNA genes from 86 archaeal species, we show that the tRNAs’ cognate amino acids can be identified by the information embedded in the tRNAs’ nucleotide positions without relying on the anticodon information. We present a small set of six to 10 informative positions along the tRNA, which allow for amino acid identification accuracy of 90.6% to 97.4%, respectively. We inspected tRNAs for each of the 20 amino acid types for such informative positions and found that tRNA genes for some amino acids are distinguishable from others by as few as one or two positions. The informative nucleotide positions are in agreement with nucleotide positions that were experimentally shown to affect the loaded amino acid identity. Interestingly, the knowledge gained from the tRNA genes of one archaeal phylum does not extrapolate well to another phylum. Furthermore, each species has a unique ensemble of nucleotides in the informative tRNA positions, and the similarity between the sets of positions of two distinct species reflects their evolutionary distance. Hence, we term this set of informative positions a “tRNA cipher.” It is tempting to suggest that the diverging code identified here might also serve the aminoacyl tRNA synthetase in the task of tRNA recognition.     

Comparison of codon usage and tRNAs in mitochondrial genomes of Candida species

To gain insight into the nature of the mitochondrial genomes (mtDNA) of different Candida species, the synonymous codon usage bias of mitochondrial protein coding genes and the tRNAs in C. albicans, C. parapsilosis, C. stellata, C. glabrata and the closely related yeast Saccharomyces cerevisiae were analyzed. Common features of the mtDNA in Candida species are a strong A+T pressure on protein coding genes, and insufficient mitochondrial tRNA species are encoded to perform protein synthesis. The wobble site of the anticodon is always U for the NNR (NNA and NNG) codon families, which are dominated by A-ending codons, and always G for the NNY (NNC and NNU) codon families, which is dominated by U-ending codons, and always U for the NNN (NNA, NNU, NNC and NNG) codon families, which are dominated by A-ending codons and U-ending codons. Patterns of synonymous codon usage of Candida species can be classified into three groups: (1) optimal codon-anticodon usage, Glu, Lys, Leu (translated by anti-codon UAA), Gln, Arg (translated by anti-codon UCU) and Trp are containing NNR codons. NNA, whose corresponding tRNA is encoded in the mtDNA, is used preferentially. (2) Non-optimal codon-anticodon usage, Cys, Asp, Phe, His, Asn, Ser (translated by anti-codon GCU) and Tyr are containing NNY codons. The NNU codon, whose corresponding tRNA is not encoded in the mtDNA, is used preferentially. (3) Combined codon-anticodon usage, Ala, Gly, Leu (translated by anti-codon UAG), Pro, Ser (translated by anti-codon UGA), Thr and Val are containing NNN codons. NNA (tRNA encoded in the mtDNA) and NNU (tRNA not encoded in the mtDNA) are used preferentially. In conclusion, we propose that in Candida species, codons containing A or U at third position are used preferentially, regardless of whether corresponding tRNAs are encoded in the mtDNA. These results might be useful in understanding the common features of the mtDNA in Candida species and patterns of synonymous codon usage.     

Mitochondrial DNA polymorphisms as risk factors for Parkinson’s disease and Parkinson’s disease dementia

The activity of complex I of the mitochondrial respiratory chain has been found to be decreased in patients with Parkinsons disease (PD), but no mutations have been identified in genes encoding complex I subunits. Recent studies have suggested that polymorphisms in mitochondrial DNA (mtDNA)-encoded complex I genes (MTND) modify susceptibility to PD. We hypothesize that the risk of PD is conveyed by the total number of nonsynonymous substitutions in the MTND genes in various mtDNA lineages rather than by single mutations. To test this possibility, we determined the number of nonsynonymous substitutions of the seven MTND genes from 183 Finns. The differences in the total number of nonsynonymous substitutions and the nonsynonymous to synonymous substitution rate ratio (K_a/K_s) of MTND genes between the European mtDNA haplogroup clusters (HV, JT, KU, IWX) were analysed by using a statistical approach. Patients with PD (n=238) underwent clinical examination together with mtDNA haplogroup analysis and the clinical features between patient groups defined by the number of nonsynonymous substitutions were compared. Our analysis revealed that the haplogroup clusters HV and KU had a lower average number of amino acid replacements and a lower K_a/K_s ratio in the MTND genes than clusters JT and IWX. Supercluster JTIWX with the highest number of amino acid replacements was more frequent among PD patients and even more frequent among patients with PD who developed dementia. Our results suggest that a relative excess of nonsynonymous mutations in MTND genes in supercluster JTWIX is associated with an increased risk of PD and the disease progression to dementia.     

High‐altitude adaptation in vertebrates as revealed by mitochondrial genome analyses

The high‐altitude environment may drive vertebrate evolution in a certain way, and vertebrates living in different altitude environments might have different energy requirements. We hypothesized that the high‐altitude environment might impose different influences on vertebrate mitochondrial genomes (mtDNA). We used selection pressure analyses and PIC (phylogenetic independent contrasts) analysis to detect the evolutionary rate of vertebrate mtDNA protein‐coding genes (PCGs) from different altitudes. The results showed that the ratio of nonsynonymous/synonymous substitutions (dN/dS) in the mtDNA PCGs was significantly higher in high‐altitude vertebrates than in low‐altitude vertebrates. The seven rapidly evolving genes were shared by the high‐altitude vertebrates, and only one positive selection gene (ND5 gene) was detected in the high‐altitude vertebrates. Our results suggest the mtDNA evolutionary rate in high‐altitude vertebrates was higher than in low‐altitude vertebrates as their evolution requires more energy in a high‐altitude environment. Our study demonstrates the high‐altitude environment (low atmospheric O₂ levels) drives vertebrate evolution in mtDNA PCGs.     

The complete nucleotide sequence of the mitochondrial genome of <Emphasis Type="Italic">Phthonandria atrilineata</Emphasis> (Lepidoptera: Geometridae)

Using long-polymerase chain reaction (Long-PCR) method, we determined the complete nucleotide sequence of the mitochondrial genome (mitogenome) of Phthonandria atrilineata. The complete mtDNA from P. atrilineata was 15,499 base pairs in length and contained 13 protein-coding genes (PCGs), 2 rRNA genes, 22 tRNA genes, and a control region. The P. atrilineata genes were in the same order and orientation as the completely sequenced mitogenomes of other lepidopteran species. The nucleotide composition of P. atrilineata mitogenome was biased toward A + T nucleotides (81.02%), and the 13 PCGs show different A + T contents that range from 73.25% (cox1) to 92.12% (atp8). Phthonandria had the canonical set of 22 tRNA genes, that fold in the typical cloverleaf structure described for metazoan mt tRNAs, with the unique exception of trnS(AGN). The phylogenetic relationships were reconstructed with the concatenated sequences of the 13 PCGs of the mitochondrial genome, which confirmed that P. atrilineata is most closely related to the superfamily Bombycoidea.     

Departure from neutrality at the mitochondrial NADH dehydrogenase subunit 2 gene in humans,but not in chimpanzees.

To test whether patterns of mitochondrial DNA (mtDNA) variation are consistent with a neutral model of molecular evolution, nucleotide sequences were determined for the 1041 bp of the NADH dehydrogenase subunit 2 (ND2) gene in 20 geographically diverse humans and 20 common chimpanzees. Contingency tests of neutrality were performed using four mutational categories for the ND2 molecule: synonymous and nonsynonymous mutations in the transmembrane regions, and synonymous and nonsynonymous mutations in the surface regions. The following three topological mutational categories were also used: intraspecific tips, intraspecific interiors, and interspecific fixed differences. The analyses reveal a significantly greater number of nonsynonymous polymorphisms within human transmembrane regions than expected based on interspecific comparisons, and they are inconsistent with a neutral equilibrium model. This pattern of excess nonsynonymous polymorphism is not seen within chimpanzees. Statistical tests of neutrality, such as TAJIMA''s D test, and the D and F tests proposed by FU and LI, indicate an excess of low frequency polymorphisms in the human data, but not in the chimpanzee data. This is consistent with recent directional selection, a population bottleneck or background selection of slightly deleterious mutations in human mtDNA samples. The analyses further support the idea that mitochondrial genome evolution is governed by selective forces that have the potential to affect its use as a "neutral" marker in evolutionary and population genetic studies.     

Codon Usage Bias and tRNA Abundance in Drosophila

Codon usage bias of 1,117 Drosophila melanogaster genes, as well as fewer D. pseudoobscura and D. virilis genes, was examined from the perspective of relative abundance of isoaccepting tRNAs and their changes during development. We found that each amino acid contributes about equally and highly significantly to overall codon usage bias, with the exception of Asp which had very low contribution to overall bias. Asp was also the only amino acid that did not show a clear preference for one of its synonymous codons. Synonymous codon usage in Drosophila was consistent with ``optimal' codons deduced from the isoaccepting tRNA availability. Interestingly, amino acids whose major isoaccepting tRNAs change during development did not show as strong bias as those with developmentally unchanged tRNA pools. Asp is the only amino acid for which the major isoaccepting tRNAs change between larval and adult stages. We conclude that synonymous codon usage in Drosophila is well explained by tRNA availability and is probably influenced by developmental changes in relative abundance. Received: 5 December 1996 / Accepted: 14 June 1997     

“Balancing” balancing selection? Assortative mating at the major histocompatibility complex despite molecular signatures of balancing selection

In vertebrate animals, genes of the major histocompatibility complex (MHC) determine the set of pathogens to which an individual's adaptive immune system can respond. MHC genes are extraordinarily polymorphic, often showing elevated nonsynonymous relative to synonymous sequence variation and sharing presumably ancient polymorphisms between lineages. These patterns likely reflect pathogen‐mediated balancing selection, for example, rare‐allele or heterozygote advantage. Such selection is often reinforced by disassortative mating at MHC. We characterized exon 2 of MHC class II, corresponding to the hypervariable peptide‐binding region, in song sparrows (Melospiza melodia). We compared nonsynonymous to synonymous sequence variation in order to identify positively selected sites; assessed evidence for trans‐species polymorphisms indicating ancient balancing selection; and compared MHC similarity of socially mated pairs to expectations under random mating. Six codons showed elevated ratios of nonsynonymous to synonymous variation, consistent with balancing selection, and we characterized several alleles similar to those occurring in at least four other avian families. Despite this evidence for historical balancing selection, mated pairs were significantly more similar at MHC than were randomly generated pairings. Nonrandom mating at MHC thus appears to partially counteract, not reinforce, pathogen‐mediated balancing selection in this system. We suggest that in systems where individual fitness does not increase monotonically with MHC diversity, assortative mating may help to avoid excessive offspring heterozygosity that could otherwise arise from long‐standing balancing selection.