Rapid Evolution of the Ribonuclease A Superfamily: Adaptive Expansion of Independent Gene Clusters in Rats and Mice

The two eosinophil ribonucleases, eosinophil-derived neurotoxin (EDN/RNase 2) and eosinophil cationic protein (ECP/RNase 3), are among the most rapidly evolving coding sequences known among primates. The eight mouse genes identified as orthologs of EDN and ECP form a highly divergent, species-limited cluster. We present here the rat ribonuclease cluster, a group of eight distinct ribonuclease A superfamily genes that are more closely related to one another than they are to their murine counterparts. The existence of independent gene clusters suggests that numerous duplications and diversification events have occurred at these loci recently, sometime after the divergence of these two rodent species (∼10–15 million years ago). Nonsynonymous substitutions per site (d _N) calculated for the 64 mouse/rat gene pairs indicate that these ribonucleases are incorporating nonsilent mutations at accelerated rates, and comparisons of nonsynonymous to synonymous substitution (d _N / d _S) suggest that diversity in the mouse ribonuclease cluster is promoted by positive (Darwinian) selection. Although the pressures promoting similar but clearly independent styles of rapid diversification among these primate and rodent genes remain uncertain, our recent findings regarding the function of human EDN suggest a role for these ribonucleases in antiviral host defense. Received: 8 April 1999 / Accepted: 22 June 1999     

Adaptive Diversification of Vomeronasal Receptor 1 Genes in Rodents

The vomeronasal receptor 1 (V1R) are believed to be pheromone receptors in rodents. Here we used computational methods to identify 95 and 62 new putative V1R genes from the draft rat and mouse genome sequence, respectively. The rat V1R repertoire consists of 11 subfamilies, 10 of which are shared with the mouse, while rat appears to lack the H and I subfamilies found in mouse and possesses one unique subfamily (M). The estimations of the relative divergence times suggest that many subfamilies originated after the split of rodents and primates. The analysis also reveals that these clusters underwent an expansion very close to the split of mouse and rat. In addition, maximum likelihood analysis showed that the nonsynonymous and synonymous rate ratio for most of these clusters was much higher than one, suggesting the role of positive selection in the diversification of these duplicated V1R genes. Because V1R are thought to mediate the process of signal transduction in response to pheromone detection, we speculate that the V1R genes have evolved under positive Darwinian selection to maintain the ability to discriminate between large and complex pheromonal mixtures.Reviewing Editor: Dr. Rasmus Nielsen     

The ABCs of MHC

The major histocompatibility complex (MHC) contains the most diverse genes known in vertebrates. These genes encode cell‐surface molecules that play a central role in controlling immunological activity and, as a consequence, in tissue rejection, autoimmunity, and immune responses to infectious diseases. In vertebrates, there are many different MHC genes, most with many alleles. This is true for all primates studied thus far. Multiple loci and alleles allow for an increased peptide‐binding repertoire; their variety has a profound impact on an organism's ability to battle constantly evolving pathogens. The argument that infectious disease is a driving force for MHC variability is supported by observations that most of the allelic variation centers on the amino acid residues that directly interact with foreign peptides. However, while MHC diversity could be maintained through heterozygote advantage, frequency‐dependent selection, or both, the direct evidence that natural selection enhances diversity is limited. Indeed, it is not wholly clear whether selection operates only with respect to disease resistance or if behavioral and biological mechanisms also contribute to the extreme variation that has been observed for many species. Furthermore, reproductive behavior and biology may also help to maintain genetic variability at MHC loci.     

Trans-species polymorphism and evidence of selection on class II MHC loci in tuco-tucos (Rodentia: Ctenomyidae)

Balancing selection acting over the evolutionary history of a lineage can result in the retention of alleles among species for longer than expected under neutral evolution. The associated pattern of trans-species polymorphism, in which similar or even identical alleles are shared among species, is often used to infer that balancing selection has occurred. The genes of the major histocompatibility complex (MHC) are thought to be subject to balancing selection that maintains alleles associated with response to specific pathogens. To explore the role of balancing selection in shaping MHC diversity in ctenomyid rodents, we examined allelic variability at the class II DRB and DQA loci in 18 species in the genus Ctenomys. Previous studies of four of these species had revealed significant within-population evidence of positive selection on MHC loci. The current study expands upon these analyses to (1) evaluate among-species evidence of positive selection and (2) explore the potential for balancing selection on MHC genes. Interspecific nucleotide sequence variation revealed significant evidence of positive selection on the DRB and DQA loci. At the same time, comparisons of phylogenetic trees for these MHC loci with a putative species tree based on mitochondrial sequence data revealed multiple examples of trans-specific polymorphism, including sharing of identical DRB and DQA alleles among distantly related species of Ctenomys. These findings suggest that MHC genes in these animals have historically been subject to balancing selection and yield new insights into the complex suite of forces shaping MHC diversity in free-living vertebrates.     

High-resolution copy-number variation map reflects human olfactory receptor diversity and evolution

Olfactory receptors (ORs), which are involved in odorant recognition, form the largest mammalian protein superfamily. The genomic content of OR genes is considerably reduced in humans, as reflected by the relatively small repertoire size and the high fraction ( approximately 55%) of human pseudogenes. Since several recent low-resolution surveys suggested that OR genomic loci are frequently affected by copy-number variants (CNVs), we hypothesized that CNVs may play an important role in the evolution of the human olfactory repertoire. We used high-resolution oligonucleotide tiling microarrays to detect CNVs across 851 OR gene and pseudogene loci. Examining genomic DNA from 25 individuals with ancestry from three populations, we identified 93 OR gene loci and 151 pseudogene loci affected by CNVs, generating a mosaic of OR dosages across persons. Our data suggest that approximately 50% of the CNVs involve more than one OR, with the largest CNV spanning 11 loci. In contrast to earlier reports, we observe that CNVs are more frequent among OR pseudogenes than among intact genes, presumably due to both selective constraints and CNV formation biases. Furthermore, our results show an enrichment of CNVs among ORs with a close human paralog or lacking a one-to-one ortholog in chimpanzee. Interestingly, among the latter we observed an enrichment in CNV losses over gains, a finding potentially related to the known diminution of the human OR repertoire. Quantitative PCR experiments performed for 122 sampled ORs agreed well with the microarray results and uncovered 23 additional CNVs. Importantly, these experiments allowed us to uncover nine common deletion alleles that affect 15 OR genes and five pseudogenes. Comparison to the chimpanzee reference genome revealed that all of the deletion alleles are human derived, therefore indicating a profound effect of human-specific deletions on the individual OR gene content. Furthermore, these deletion alleles may be used in future genetic association studies of olfactory inter-individual differences.     

Predicting Species Numbers Using Species–Area and Endemics–Area Relations

Current ecological theory predicts an allometric relation between the number of species with restricted range size (endemics) and area (the endemics–area relation EAR), a pattern similar to the common species–area relation (SAR). Using SARs and EARs we can estimate species loss after habitat loss. A comparison of the predictive power of both approaches (using a patch occupancy model and data from European butterflies) revealed that the EAR approach is less reliable than the SAR. Contrary to current theory it appeared that EARs are relations in their own right that describe spatial distributions of endemic species. They do not simply follow from the underlying SAR. The implications of these results for the applicability of SARs and EARs in biodiversity forecasting are discussed.     

The effect of immune selection on the structure of the meningococcal opa protein repertoire

The opa genes of the Gram negative bacterium Neisseria meningitidis encode Opacity-associated outer membrane proteins whose role is to promote adhesion to the human host tissue during colonisation and invasion. Each meningococcus contains 3-4 opa loci, each of which may be occupied by one of a large number of alleles. We analysed the Opa repertoire structure in a large, well-characterised collection of asymptomatically carried meningococci. Our data show an association between Opa repertoire and meningococcal lineages similar to that observed previously for meningococci isolated from cases of invasive disease. Furthermore, these Opa repertoires exhibit discrete, non-overlapping structure at a population level, and yet low within-repertoire diversity. These data are consistent with the predictions of a mathematical model of strong immune selection upon a system where identical alleles may occupy different loci.     

Population differences in the human functional olfactory repertoire

Olfactory receptors (OR) constitute the molecular basis for the sense of smell. They are encoded by a large multigene family that in humans includes approximately 400 functional genes and approximately 600 putative pseudogenes, distributed on all but two chromosomes. To examine the ethnogeographic variability in the functional chemosensory repertoire, we resequenced 32 OR loci reported to contain a single coding region disruption in seven Caucasians and seven Pygmies. Thirteen of the 32 OR loci were found to have an interrupted coding region in all 28 alleles sampled, seven had an intact form in all the individuals examined, and 12 were polymorphic, segregating both the intact and the null variants. Among the latter loci, the frequency of the null allele was higher in Caucasians than in Pygmies, suggesting that African populations may have a larger repertoire of functional OR genes. Interestingly, when analyzing the entire OR coding regions, we find an excess of high-frequency derived alleles at many loci in the Caucasian sample but less so in the Pygmy sample. Our observations are unlikely to be accounted for by simple demographic models but may be explained by positive selection acting on OR loci in Caucasians.     

Allelic Genealogy under Overdominant and Frequency-Dependent Selection and Polymorphism of Major Histocompatibility Complex Loci

To explain the long-term persistence of polymorphic alleles (trans-specific polymorphism) at the major histocompatibility complex (MHC) loci in rodents and primates, a computer simulation study was conducted about the coalescence time of different alleles sampled under various forms of selection. At the same time, average heterozygosity, the number of alleles in a sample, and the rate of codon substitution were examined to explain the mechanism of maintenance of polymorphism at the MHC loci. The results obtained are as follows. (1) The coalescence time for neutral alleles is too short to explain the trans-specific polymorphism at the MHC loci. (2) Under overdominant selection, the coalescence time can be tens of millions of years, depending on the parameter values used. The average heterozygosity and the number of alleles observed are also high enough to explain MHC polymorphism. (3) The pathogen adaptation model proposed by Snell is incapable of explaining MHC polymorphism, since the coalescence time for this model is too short and the expected heterozygosity and the expected number of alleles are too small. (4) From the mathematical point of view, the minority advantage model of frequency-dependent selection is capable of explaining a high degree of polymorphism and trans-specific polymorphism. (5) The molecular mimicry hypothesis also gives a sufficiently long coalescence time when the mutation rate is low in the host but very high in the parasite. However, the expected heterozygosity and the expected number of alleles tend to be too small. (6) Consideration of the molecular mechanism of the function of MHC molecules and other biological observations suggest that the most important factor for the maintenance of MHC polymorphism is overdominant selection. However, some experiments are necessary to distinguish between the overdominance and frequency-dependent selection hypotheses.     

A Novel System of Polymorphic and Diverse NK Cell Receptors in Primates

There are two main classes of natural killer (NK) cell receptors in mammals, the killer cell immunoglobulin-like receptors (KIR) and the structurally unrelated killer cell lectin-like receptors (KLR). While KIR represent the most diverse group of NK receptors in all primates studied to date, including humans, apes, and Old and New World monkeys, KLR represent the functional equivalent in rodents. Here, we report a first digression from this rule in lemurs, where the KLR (CD94/NKG2) rather than KIR constitute the most diverse group of NK cell receptors. We demonstrate that natural selection contributed to such diversification in lemurs and particularly targeted KLR residues interacting with the peptide presented by MHC class I ligands. We further show that lemurs lack a strict ortholog or functional equivalent of MHC-E, the ligands of non-polymorphic KLR in “higher” primates. Our data support the existence of a hitherto unknown system of polymorphic and diverse NK cell receptors in primates and of combinatorial diversity as a novel mechanism to increase NK cell receptor repertoire.     

Class I MHC expression in the yellow baboon

MHC class I molecules play a crucial role in the immune response to pathogens and vaccines and in self/non-self recognition. Therefore, characterization of MHC class I gene expression of Papio subspecies is a prerequisite for studies of immunology and transplantation in the baboon (papio hamadryas). To elucidate MHC class I expression and variation within Papio subspecies and to further investigate the evolution of A and B loci in Old World primates, we have characterized the expressed class I repertoire of the yellow baboon (Papio hamadryas cynocephalus) by cDNA library screening. A total of nine distinct MHC class I cDNAs were isolated from a spleen cDNA library. The four A alleles and four B alleles obtained represent four distinct loci indicating that a duplication of the A and B loci has taken place in the lineage leading to these Old World primates. No HLA--C homologue/orthologue was found. In addition a single, nonclassical homologue of HLA--E was characterized. Examination of nucleotide and extrapolated protein sequences indicates that alleles at the two B loci are much more diversified than the alleles at the A loci. One of the A loci in particular appears to display very limited polymorphism in both Papio hamadryas cynocephalus and Papio hamadryas anubis subspecies. The failure to detect a homologue of HLA--C in the baboon provides additional evidence for the more recent origin of this locus in the pongidae and hominidae: Further comparative analysis with MHC sequences among the primate species reveals specific patterns of divergence and conservation within class I molecules of the yellow baboon.     

The strength of selection on ultraconserved elements in the human genome

Ultraconserved elements are stretches of consecutive nucleotides that are perfectly conserved in multiple mammalian genomes. Although these sequences are identical in the reference human, mouse, and rat genomes, we identified numerous polymorphisms within these regions in the human population. To determine whether polymorphisms in ultraconserved elements affect fitness, we genotyped unrelated human DNA samples at loci within these sequences. For all single-nucleotide polymorphisms tested in ultraconserved regions, individuals homozygous for derived alleles (alleles that differ from the rodent reference genomes) were present, viable, and healthy. The distribution of allele frequencies in these samples argues against strong, ongoing selection as the force maintaining the conservation of these sequences. We then used two methods to determine the minimum level of selection required to generate these sequences. Despite the lack of fixed differences in these sequences between humans and rodents, the average level of selection on ultraconserved elements is less than that on essential genes. The strength of selection associated with ultraconserved elements suggests that mutations in these regions may have subtle phenotypic consequences that are not easily detected in the laboratory.     

Inability of bm14 mice to respond to Theiler's murine encephalomyelitis virus is caused by defective antigen presentation, not repertoire selection

Natural selection drives diversification of MHC class I proteins, but the mechanism by which selection for polymorphism occurs is not known. New variant class I alleles differ from parental alleles both in the nature of the CD8 T cell repertoire formed and the ability to present pathogen-derived peptides. In the current study, we examined whether T cell repertoire differences, Ag presentation differences, or both account for differential viral resistance between mice bearing variant and parental alleles. We demonstrate that nonresponsive mice have inadequate presentation of viral Ag, but have T cell repertoires capable of mounting Ag-specific responses. Although previous work suggests a correlation between the ability to present an Ag and the ability to generate a repertoire responsive to that Ag, we show that the two functions of MHC class I are independent.     

Evolutionary Dynamics of the Interferon-Induced Transmembrane Gene Family in Vertebrates

Vertebrate interferon-induced transmembrane (IFITM) genes have been demonstrated to have extensive and diverse functions, playing important roles in the evolution of vertebrates. Despite observance of their functionality, the evolutionary dynamics of this gene family are complex and currently unknown. Here, we performed detailed evolutionary analyses to unravel the evolutionary history of the vertebrate IFITM family. A total of 174 IFITM orthologous genes and 112 pseudogenes were identified from 27 vertebrate genome sequences. The vertebrate IFITM family can be divided into immunity-related IFITM (IR-IFITM), IFITM5 and IFITM10 sub-families in phylogeny, implying origins from three different progenitors. In general, vertebrate IFITM genes are located in two loci, one containing the IFITM10 gene, and the other locus containing IFITM5 and various numbers of IR-IFITM genes. Conservation of evolutionary synteny was observed in these IFITM genes. Significant functional divergence was detected among the three IFITM sub-families. No gene duplication or positive selection was found in IFITM5 sub-family, implying the functional conservation of IFITM5 in vertebrate evolution, which is involved in bone formation. No IFITM5 locus was identified in the marmoset genome, suggesting a potential association with the tiny size of this monkey. The IFITM10 sub-family was divided into two groups: aquatic and terrestrial types. Functional divergence was detected between the two groups, and five IFITM10-like genes from frog were dispersed into the two groups. Both gene duplication and positive selection were observed in aquatic vertebrate IFITM10-like genes, indicating that IFITM10 might be associated with the adaptation to aquatic environments. A large number of lineage- and species-specific gene duplications were observed in IR-IFITM sub-family and positive selection was detected in IR-IFITM of primates and rodents. Because primates have experienced a long history of viral infection, such rapid expansion and positive selection suggests that the evolution of primate IR-IFITM genes is associated with broad-spectrum antiviral activity.     

Arrival and diversification of caviomorph rodents and platyrrhine primates in South America

Platyrrhine primates and caviomorph rodents are clades of mammals that colonized South America during its period of isolation from the other continents, between 100 and 3 million years ago (Mya). Until now, no molecular study investigated the timing of the South American colonization by these two lineages with the same molecular data set. Using sequences from three nuclear genes (ADRA2B, vWF, and IRBP, both separate and combined) from 60 species, and eight fossil calibration constraints, we estimated the times of origin and diversification of platyrrhines and caviomorphs via a Bayesian relaxed molecular clock approach. To account for the possible effect of an accelerated rate of evolution of the IRBP gene along the branch leading to the anthropoids, we performed the datings with and without IRBP (3768 sites and 2469 sites, respectively). The time window for the colonization of South America by primates and by rodents is demarcated by the dates of origin (upper bound) and radiation (lower bound) of platyrrhines and caviomorphs. According to this approach, platyrrhine primates colonized South America between 37.0 +/- 3.0 Mya (or 38.9 +/- 4.0 Mya without IRBP) and 16.8 +/- 2.3 (or 20.1 +/- 3.3) Mya, and caviomorph rodents between 45.4 +/- 4.1 (or 43.7 +/- 4.8) Mya and 36.7 +/- 3.7 (or 35.8 +/- 4.3) Mya. Considering both the fossil record and these molecular datings, the favored scenarios are a trans-Atlantic migration of primates from Africa at the end of the Eocene or beginning of the Oligocene, and a colonization of South America by rodents during the Middle or Late Eocene. Based on our nuclear DNA data, we cannot rule out the possibility of a concomitant arrival of primates and rodents in South America. The caviomorphs radiated soon after their arrival, before the Oligocene glaciations, and these early caviomorph lineages persisted until the present. By contrast, few platyrrhine fossils are known in the Oligocene, and the present-day taxa are the result of a quite recent, Early Miocene diversification.     

Test for Selection on Polymorphic Isozyme Genes Using the Population Cage Method

Published experimental data of several species of Drosophila using the population cage method were reexamined. The authors of these works have claimed that selection operates on polymorphic isozyme genes. Since selection coefficients (s) estimated by us for the above data, assuming overdominance, or for some cases, additivity between alleles, were too large (s > 0.1), we conclude that these experimental results do not reflect the effects of the single loci in question; rather, we suggest that careful experimentation and judgment are required for testing with the population cage method whether or not selection actually operates on single genes.     

Significance of Population Size on the Fixation of Nonsynonymous Mutations in Genes Under Varying Levels of Selection Pressure

Previous studies observed a higher ratio of divergences at nonsynonymous and synonymous sites (ω = d_N/d_S) in species with a small population size compared to that estimated for those with a large population size. Here we examined the theoretical relationship between ω, effective population size (N_e), and selection coefficient (s). Our analysis revealed that when purifying selection is high, ω of species with small N_e is much higher than that of species with large N_e. However the difference between the two ω reduces with the decline in selection pressure (s → 0). We examined this relationship using primate and rodent genes and found that the ω estimated for highly constrained genes of primates was up to 2.9 times higher than that obtained for their orthologous rodent genes. Conversely, for genes under weak purifying selection the ω of primates was only 17% higher than that of rodents. When tissue specificity was used as a proxy for selection pressure we found that the ω of broadly expressed genes of primates was up to 2.1-fold higher than that of their rodent counterparts and this difference was only 27% for tissue specific genes. Since most of the nonsynonymous mutations in constrained or broadly expressed genes are deleterious, fixation of these mutations is influenced by N_e. This results in a higher ω of these genes in primates compared to those from rodents. Conversely, the majority of nonsynonymous mutations in less-constrained or tissue-specific genes are neutral or nearly neutral and therefore fixation of them is largely independent of N_e, which leads to the similarity of ω in primates and rodents.     

Insight into gene evolution within Cervidae and Bovidae through genetic variation in MHC-DQA in the black muntjac (Muntiacus crinifrons)

The critical role that the major histocompatibility complex plays in the immune recognition of parasites and pathogens makes its evolutionary dynamics exceptionally relevant to ecology, population biology, and conservation studies. The black muntjac is a rare deer endemic to a small mountainous region in eastern China. We found that this species has two DQA loci through cDNA expression and sequence variation analysis. The level of variation at both DQA loci was found to be extremely low (three alleles for DQA1 and four alleles for DQA2), possibly because of past bottlenecks and the species' relatively solitary behavior pattern. The ratio of d(N)/d(S) in the putative peptide binding region of the DQA2 locus (13.36, P = 0.012) was significantly larger than one but not that of DQA1 (0.94, P = 0.95), suggesting strong positive selection at the DQA2 but not at the DQA1 locus. This difference might reflect different sets of evolutionary selection pressures acting on the two loci. The phylogenetic tree showed that DQA1 alleles from two species of Cervidae and two of Bovidae grouped together, as did the DQA2 alleles. However, different genes from the four species were located in separate branches. These results lead us to suggest that these DQA alleles are derived from primordial DQA genes from a common ancestor and are maintained in Cervidae and Bovidae since their divergence around 25.5-27.8 million years ago.