Convergent evolution of major histocompatibility complex molecules in humans and New World monkeys

  In both Old World and New World monkeys Mhc-DRB sequences have been found which resemble human DRB1*03 and DRB3 genes in their second exon. The resemblance is shared sequence motifs and clustering of the genes or the encoded proteins in phylogenetic trees. This similarity could be due to common ancestry, convergence at the molecular level, or chance. To test which of these three explanations applies, we sequenced segments of New World monkey and macaque genes which encompass the entire second exon and large parts of both flanking introns. The test strongly supports the monophyly of New World monkey DRB intron sequences. The phylogenies of introns 1 and 2 from DRB1*03-like and DRB3-like genes are congruent, but both are incongruent with the exon 2-based phylogeny. The matching of intron 1- and intron 2-based phylogenies with each other suggests that reciprocal recombination has not played a major role in exon 2 evolution. Statistical comparisons of exon 2 from different DRB1*03 and DRB3 lineages indicate that it was neither gene conversion (descent), nor chance, but molecular convergence that has shaped their characteristic motifs. The demonstration of convergence in anthropoid Mhc-DRB genes has implications for the classification, age, and mechanism of generation of DRB allelic lineages. Received: 30 August 1999 / Revised: 19 October 1999     

Mhc-DRB genes of platyrrhine primates

The two infraorders of anthropoid primates, Platyrrhini (New World monkeys) and Catarrhini (Old World monkeys and the hominoids) are estimated to have diverged from a common ancestor 37 million years ago. The major histocompatibility complex class II DRB gene and haplotype polymorphism of the Catarrhini has been characterized in several recent studies. The present study was undertaken to obtain information on the DRB polymorphism of the Platyrrhini. Fifty-five complete exon 2 DRB sequences were obtained from six species of Platyrrhini representing both the Callitrichidae and the Cebidae families. Combined with the results of a parallel contig mapping study, our data indicate that at least three loci (DRB1*03, DRB3, and DRB5) are shared by the Catarrhini and the Platyrrhini. However, the three loci are occupied by functional genes in the former infraorder and mostly by pseudogenes in the latter. Instead of the pseudogenes, the Platyrrhini have evolved a new set of apparently functional genes — DRB11 and DRB*W12 through DRB*W19, which have thus far not been found in the Catarrhini. The DRB*W13, *W14, *W15, *W17, *W18, and *W19 genes seem to be restricted to the Cebidae family, whereas the DRB*W16 locus has so far been documented in the Callitrichidae family only. The DRB alleles of the cotton-top tamarin, and perhaps also those of the common marmoset (both members of the family Callitrichidae), are characterized by low nucleotide diversity, possibly indicating that they diverged from a common ancestral gene relatively recently. Correspondence to: J. Klein.     

Analysis of the sequence variations in the Mhc DRB1-like gene of the endangered Humboldt penguin (Spheniscus humboldti)

The Major Histocompatibility Complex (Mhc) genomic region of many vertebrates is known to contain at least one highly polymorphic class II gene that is homologous in sequence to one or other of the human Mhc DRB1 class II genes. The diversity of the avian Mhc class II gene sequences have been extensively studied in chickens, quails, and some songbirds, but have been largely ignored in the oceanic birds, including the flightless penguins. We have previously reported that several penguin species have a high degree of polymorphism on exon 2 of the Mhc class II DRB1-like gene. In this study, we present for the first time the complete nucleotide sequences of exon 2, intron 2, and exon 3 of the DRB1-like gene of 20 Humboldt penguins, a species that is presently vulnerable to the dangers of extinction. The Humboldt DRB1-like nucleotide and amino acid sequences reveal at least eight unique alleles. Phylogenetic analysis of all the available avian DRB-like sequences showed that, of five penguin species and nine other bird species, the sequences of the Humboldt penguins grouped most closely to the Little penguin and the mallard, respectively. The present analysis confirms that the sequence variations of the Mhc class II gene, DRB1, are useful for discriminating among individuals within the same penguin population as well those within different penguin population groups and species.The nucleotide sequence and amino acid sequence data reported in this paper have been submitted to the DDBJ database and have been assigned the accession numbers AB088371–AB088374, AB089199, AB154393–AB154399, and AB162144.     

Evolution of Mhc–DRB Introns: Implications for the Origin of Primates

Introns are generally believed to evolve too rapidly and too erratically to be of much use in phylogenetic reconstructions. Few phylogenetically informative intron sequences are available, however, to ascertain the validity of this supposition. In the present study the supposition was tested on the example of the mammalian class II major histocompatibility complex (Mhc) genes of the DRB family. Since the Mhc genes evolve under balancing selection and are believed to recombine or rearrange frequently, the evolution of their introns could be expected to be particularly rapid and subject to scrambling. Sequences of intron 4 and 5 DRB genes were obtained from polymerase chain reaction-amplified fragments of genomic DNA from representatives of six eutherian orders—Primates, Scandentia, Chiroptera, Dermoptera, Lagomorpha, and Insectivora. Although short stretches of the introns have indeed proved to be unalignable, the bulk of the intron sequences from all six orders, spanning >85 million years (my) of evolution, could be aligned and used in a study of the tempo and mode of intron evolution. The analysis has revealed the Mhc introns to evolve at a rate similar to that of other genes and of synonymous sites of non-Mhc genes. No evidence of homogenization or large-scale scrambling of the intron sequences could be found. The Mhc introns apparently evolve largely by point mutations and insertions/deletions. The phylogenetic signals contained in the intron sequences could be used to identify Scandentia as the sister group of Primates, to support the existence of the Archonta superorder, and to confirm the monophyly of the Chiroptera. Received: 26 October 1998 / Accepted: 21 December 1998     

Identification of new Mamu-DRB alleles using DGGE and direct sequencing

 Rhesus macaques represent important animal models for biomedical research. The ability to identify macaque major histocompatibility complex (Mhc) alleles is crucial for fully understanding these models of autoimmune and infectious disease. Here we describe a rapid and unambiguous way to distinguish DRB alleles in the rhesus macaque using the polymerase chain reaction, denaturing gradient gel electrophoresis (DGGE), and direct sequencing. The highly variable second exon of Mamu-DRB alleles was amplified using generic DRB primers and alleles were separated by DGGE. DNA was then reamplified from plugs removed from the gel and alleles were determined using fluorescent-based sequencing. Validity of this typing procedure was confirmed by identification of all DRB alleles for three macaques previously characterized by cloning and sequencing techniques. Importantly, our analysis revealed DRB alleles not previously identified in the three reference animals. Using this technique, we identified 40 alleles in fifteen unrelated macaques. On the basis of phylogenetic tree analyses, 14 new DRB alleles were assigned to 10 different Mhc-DRB lineages. Interestingly, two of the new DRB6 lineages had previously been identified in prosimians and pigtailed macaques. Whereas traditional DRB typing methods provide limited information, our new technique provides a simple and relatively rapid way of identifying DRB alleles for tissue typing, determining individual identification and studies of disease association and susceptibility. This new technique should also contribute to ongoing studies of Mhc function and evolution in many different species of nonhuman primates. Received: 29 May 1996 / Revised: 8 August 1996     

Comparative genetics of a highly divergent DRB microsatellite in different macaque species

The DRB region of the major histocompatibility complex (MHC) of cynomolgus and rhesus macaques is highly plastic, and extensive copy number variation together with allelic polymorphism makes it a challenging enterprise to design a typing protocol. All intact DRB genes in cynomolgus monkeys (Mafa) appear to possess a compound microsatellite, DRB-STR, in intron 2, which displays extensive length polymorphism. Therefore, this STR was studied in a large panel of animals, comprising pedigreed families as well. Sequencing analysis resulted in the detection of 60 Mafa-DRB exon 2 sequences that were unambiguously linked to the corresponding microsatellite. Its length is often allele specific and follows Mendelian segregation. In cynomolgus and rhesus macaques, the nucleotide composition of the DRB-STR is in concordance with the phylogeny of exon 2 sequences. As in humans and rhesus monkeys, this protocol detects specific combinations of different DRB-STR lengths that are unique for each haplotype. In the present panel, 22 Mafa-DRB region configurations could be defined, which exceeds the number detected in a comparable cohort of Indian rhesus macaques. The results suggest that, in cynomolgus monkeys, even more frequently than in rhesus macaques, new haplotypes are generated by recombination-like events. Although both macaque species are known to share several identical DRB exon 2 sequences, the lengths of the corresponding microsatellites often differ. Thus, this method allows not only fast and accurate DRB haplotyping but may also permit discrimination between highly related macaque species.     

Characterization and evolution of major histocompatibility complex class II genes in the aye-aye, Daubentonia madagascariensis

Major histocompatibility complex genes (Mhc-DQB and Mhc-DRB) were sequenced in seven aye-ayes (Daubentonia madagascariecsis), which is an endemic and endangered species in Madagascar. An aye-aye from a north-eastern population showed genetic relatedness to individuals of a north-western population and had a somewhat different repertoire from another north-eastern individual. These observations suggest that the extent of genetic variation in Mhc genes is not excessively small in the aye-aye in spite of recent rapid destruction of their habitat by human activities. In light of Mhc gene evolution, trans-species and allelic polymorphisms can be estimated to have been retained for more than 50 Ma (million years) based on the time scale of lemur evolution.     

Mhc Allelic Diversity and Modern Human Origins

Thirty complete coding sequences of human major histocompatibility complex (Mhc) class II DRB alleles, spanning 237 codons, were analyzed for phylogenetic information using distance, parsimony, and likelihood approaches. Allelic genealogies derived from different parts of the coding sequence (exon 2, the 5′ and 3′ ends of exon 2, respectively, and exons 3–6) were compared. Contrary to prior assertions, a rigorous analysis of allelic genealogies in this gene family cannot be used to justify the claim that the lineage leading to modern humans contained on average at least 100,000 individuals. Phylogenetic inferences based upon the exon 2 region of the DRB loci are complicated by selection and recombination, so this part of the gene does not provide a complete and accurate view of allelic relationships. Attempts to reconstruct human history from genetic data must use realistic models which consider the complicating factors of nonequilibrium populations, recombination, and different patterns of selection. Received: 19 February 1997 / Accepted: 12 June 1997     

Mhc class II B gene evolution in East African cichlid fishes

 A distinctive feature of essential major histocompatibility complex (Mhc) loci is their polymorphism characterized by large genetic distances between alleles and long persistence times of allelic lineages. Since the lineages often span several successive speciations, we investigated the behavior of the Mhc alleles during or close to the speciation phase. We sequenced exon 2 of the class II B locus 4 from 232 East African cichlid fishes representing 32 related species. The divergence times of the (sub)species ranged from 6000 to 8.4 million years. Two types of evolutionary analysis were used to elucidate the pattern of exon 2 sequence divergence. First, phylogenetic methods were applied to reconstruct the most likely evolutionary pathways leading from the last common ancestor of the set to the extant sequences, and to assess the probable mechanisms involved in allelic diversification. Second, pairwise comparisons of sequences were carried out to detect differences seemingly incompatible with origin by nonparallel point mutations. The analysis revealed point mutations to be the most important mechanism behind allelic divergences, with recombination playing only an auxiliary part. Comparison of sequences from related species revealed evidence of random allelic (lineage) losses apparently associated with speciation. Sharing of identical alleles could be demonstrated between species that diverged 2 million years ago. The phylogeny of the exon was incongruent with that of the flanking introns, indicating either a high degree of convergent evolution at the peptide-binding region-encoding sites, or intron homogenization. Received: 6 December 1999 / Revised: 15 February 2000     

Evolutionary relationship ofHLA-DRB genes inferred from intron sequences

The major histocompatibility complex (Mhc) consists of class I and class II genes. In the humanMhc (HLA) class II genes, nineDRB loci have been identified. To elucidate the origin of these duplicated loci and allelic divergences at the most polymorphicDRBI locus, introns 4 and 5 as well as the 3′ untranslated region (altogether approximately 1,000 base pairs) of sevenHLA-DRB loci, threeHLA-DRBI alleles, and nine nonhuman primateDRB genes were examined. It is shown that there were two major diversification events inHLA-DRB genes, each involving gene duplications and allelic divergences. Approximately 50 million years (my) ago,DRBI ^*04 and an ancestor of theDRB1 ^*03 cluster (DRBI ^*03, DRBI^*15, andDRB3) diverged from each other andDRB5, DRB7, DRB8, and an ancestor of theDRB2 cluster (DRB2, DRB4, andDRB6) arose by gene duplication. Later, about 25 my ago,DRBI ^*15 diverged fromDRBI*03, andDRB3 was duplicated fromDRBI ^*03. Then, some 20 my ago, the lineage leading to theDRB2 cluster produced two new loci,DRB4 andDRB6. TheDRBI ^*03 andDRBI ^*04 allelic lineages are extraordinarily old and have persisted longer than some duplicated genes. The orthologous relationships ofDRB genes between human and Old World monkeys are apparent, but those between Catarrhini and New World monkeys are equivocal because of a rather rapid expansion and contraction of primateDRB genes by duplication and deletion. Correspondence to: Y. Satta     

<Emphasis Type="Italic">Mhc</Emphasis> class II diversity and balancing selection in greater prairie-chickens

The major histocompatibility complex (Mhc) of domestic chickens has been characterized as small and relatively simple compared with that of mammals. However, there is growing evidence that the Mhc of many bird lineages may be more complex, even within the Order Galliformes. In this study, we measured genetic variation and balancing selection at Mhc loci in another galliform, the greater prairie-chicken. We cloned and sequenced a 239 bp fragment of Mhc Class II β-chain (BLB) exon 2 in 14 individuals. There was a total of 10 unique sequences and a minimum of four BLB loci. The d _N/d _S ratio at peptide-binding codons was significantly greater than one, suggesting balancing selection is acting on the BLB. We also recovered two YLB sequences, which clustered tightly with YLB sequences from three other species: domestic chicken, black grouse and common quail. The relatively large number of loci revealed in our study suggests that even closely related galliforms differ in the level of Mhc variation and structure.     

Trans-specific Mhc polymorphism and the origin of species in primates

The major histocompatibility complex (Mhc) is a cluster of loci controlling the specific immune response in vertebrates. Mhc alleles often differ by a large number of nucleotide substitutions, some of which began to accumulate before the emergence of extant species. We have applied the theory of allelic genealogy to the primate Mhc genes with the aim of estimating the size of the founding populations. The calculations indicate that the long-term effective population size of the studied species was between 10⁴ and 10⁵ individuals and that it most likely never dropped below 10³ individuals.     

Extensive sharing of MHC class II alleles between rhesus and cynomolgus macaques

In contrast to rhesus monkeys, substantial knowledge on cynomolgus monkey major histocompatibility complex (MHC) class II haplotypes is lacking. Therefore, 17 animals, including one pedigreed family, were thoroughly characterized for polymorphic Mhc class II region genes as well as their mitochondrial DNA (mtDNA) sequences. Different cynomolgus macaque populations appear to exhibit unique mtDNA profiles reflecting their geographic origin. Within the present panel, 10 Mafa-DPB1, 14 Mafa-DQA1, 12 Mafa-DQB1, and 35 Mafa-DRB exon 2 sequences were identified. All of these alleles cluster into lineages that were previously described for rhesus macaques. Moreover, about half of the Mafa-DPB1, Mafa-DQA1, and Mafa-DQB1 alleles and one third of the Mafa-DRB exon 2 sequences are identical to rhesus macaque orthologues. Such a high level of Mhc class II allele sharing has not been reported for primate species. Pedigree analysis allowed the characterization of nine distinct Mafa class II haplotypes, and seven additional ones could be deduced. Two of these haplotypes harbor a duplication of the Mafa-DQB1 locus. Despite extensive allele sharing, rhesus and cynomolgus monkeys do not appear to possess identical Mhc class II haplotypes, thus illustrating that new haplotypes were generated after speciation by recombination-like processes.     

Molecular cloning of major histocompatibility complex class II B gene cDNA from the Bengalese finch Lonchura striata

The only avian major histocompatibility complex (Mhc) genes thus far identified are from species of the relatively small order of Galliformes, while by far the largest order of Passeriformes (songbirds), containing some 60% of extant bird species, has not been studied at all in this regard. The Galliformes emerged more than 55 million years (my) ago, the Passeriformes some 25 my ago. Because of the potential for the use of Mhc genes as markers in the study of songbird populations, an attempt was made to clone class II B genes of a passeriform species, the Bangalese finch Lonchura striata acuticauda. Using a set of primers designed on the basis of known sequences, a probe corresponding to part of exon II was obtained by the polymerase chain reaction. The probe was then used to screen a Bengalese finch cDNA library and to isolate and sequence two nearly full-length clones. The sequences reveal the presence of one presumably functional class II B locus in this bird species.     

Sequence analysis of a polymorphic Mhc class II gene in Pacific salmon

Polymorphism of the nucleotide sequences encoding 149 amino acids of linked major histocompatibility complex (Mhc) class II 131 and 132 peptides, and of the intervening intron (548–773 base pairs), was examined within and among seven Pacific salmon (Oncorhynchus) species. Levels of nucleotide diversity were higher for theB1 sequence than forB2 or the intron in comparisons both within and between species. For the codons of the peptide binding region of the BI sequence, the level of nonsynonymous nucleotide substitution (d_N) exceeded the level of synonymous substitution (d_S) by a factor of ten for within-species comparisons, and by a factor of four for between-species comparisons. The excess of d_N indicates that balancing selection maintains diversity at this salmonidMhc class II locus, as is common forMhc loci in other vertebrates. Levels of nucleotide diversity for both the exon and intron sequences were greater among than within species, and there were numerous species-specific nucleotides present in both the coding and noncoding regions. Thus, neighbor-joining analysis of both the intron and exon regions provided phylogenies in which the sequences clustered strongly by species. There was little evidence of shared ancestral (trans-species) polymorphism in the exon phylogeny, and the intron phylogeny depicted standard relationships among the Pacific salmon species. The lack of shared allelicB1 lineages in these closely related species may result from severe bottlenecks that occurred during speciation or during the ice ages that glaciated the rim of the north Pacific Ocean approximately every 100 000 years in the Pleistocene.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers U34692-U34720     

Evolution of MHC-<Emphasis Type="Italic">DRB</Emphasis> class II polymorphism in the genus <Emphasis Type="Italic">Apodemus</Emphasis> and a comparison of <Emphasis Type="Italic">DRB</Emphasis> sequences within the family Muridae (Mammalia: Rodentia)

Allelic diversity at major histocompatibility complex (MHC) genes is thought to be maintained by balancing selection over long periods of time, even across multiple speciation events. Trans-species sharing of MHC alleles among genera has been supported by many studies on mammals and fish, but in rodents, the results are ambiguous. We investigated natural levels of MHC-DRB variability and evolutionary processes in the wood mouse (Apodemus sylvaticus) and the yellow-necked mouse (Apodemus flavicollis), which are common, sympatric murid rodents in European forests. Using single-strand conformation polymorphism analysis and DNA sequencing, 38 DRB exon 2 alleles were detected among 162 A. sylvaticus from nine different locations in Germany and Switzerland, and 15 DRB exon 2 alleles were detected among 60 A. flavicollis from three different locations in northern Germany. There was evidence for balancing selection in both species. Phylogenetic analysis, including additional murid taxa, showed that the DRB exon 2 sequences did not separate according to species, consistent with trans-species evolution of the MHC in these taxa.     

Evolutionary Significance of the Entepicondylar Foramen of the Humerus in New World Monkeys (Platyrrhini)

Although consistently absent in extant catarrhine monkeys, the presence and absence of the entepicondylar foramen of the humerus (EEF) vary greatly among living and fossil platyrrhines. Aiming to test the mode of evolution of this character in platyrrhine phylogeny, we performed stochastic character mapping of the presence and absence of the EEF based on museum material and literature research. We also tested for phylogenetic signal of the EEF and its correlation with semi-brachation, vertical clinging, and manipulative foraging in New World monkeys. We found more losses than gains of the EEF in the Haplorhini and Platyrrhini phylogenies. The EEF showed a strong phylogenetic signal congruent with a Brownian model of evolution. All models that assumed a dependence between foraging and locomotion behaviors showed a better fit to the data, but the differences from an independent model were not statistically significant. Thus, we assume that the observed pattern could have originated from genetic drift, except in Leontopithecus where the EEF appears to have been lost due to the narrowing of the distal humerus. Although not functionally relevant, the presence and absence of EEF is taxonomically useful for discriminating between the Aotus and Callicebus species groups, and diagnoses large platyrrhine clades such as marmosets and Atelidae. Thus, it should be considered when inferring systematic relationships among living and extinct platyrrhines.     

Polymorphism and transcription of Mhc class I genes in a passerine bird, the great reed warbler

 The class I genes of the major histocompatibility complex (Mhc) are here investigated for the first time in a passerine bird. The great reed warbler is a rare species in Sweden with a few semi-isolated populations. Yet, we found extensive Mhc class I variation in the study population. The variable exon 3, corresponding to the α₂ domain, was amplified from genomic DNA with degenerated primers. Seven different genomic class I sequences were detected in a single individual. One of the sequences had a deletion leading to a shift in the reading frame, indicating that it was not a functional gene. A randomly selected clone was used as a probe for restriction fragment length polymorphism (RFLP) studies in combination with the restriction enzyme Pvu II. The RFLP pattern was complex with 21–25 RFLP fragments per individual and extensive variation. Forty-nine RFLP genotypes were detected in 55 tested individuals. To study the number of transcribed genes, we isolated 14 Mhc class I clones from a cDNA library from a single individual. We found eight different sequences of four different lengths (1.3–2.2 kilobases), suggesting there are at least four transcribed loci. The number of nonsynonymous substitutions (d _N ) in the peptide binding region of exon 3 were higher than the number of synonymous substitutions (d _S ), indicating balancing selection in this region. The number of transcribed genes and the numerous RFLP fragments found so far suggest that the great reed warbler does not have a "minimal essential Mhc" as has been suggested for the chicken. Received: 13 May 1998 / Revised: 18 August 1998