Episodic evolution of growth hormone in primates and emergence of the species specificity of human growth hormone receptor

Growth hormone (GH) evolution is very conservative among mammals, except for primates and ruminant artiodactyls. In fact, most known mammalian GH sequences differ from the inferred ancestral mammalian sequence by only a few amino acids. In contrast, the human GH sequence differs from the inferred ancestral sequence by 59 amino acids. However, it is not known when this rapid evolution of GH occurred during primate evolution or whether it was due to positive selection. Also, human growth hormone receptor (GHR) displays species specificity; i.e., it can interact only with human (or rhesus monkey) GH, not with nonprimate GHS: The species specificity of human GHR is largely due to the Leu-->Arg change at position 43, and it has been hypothesized that this change must have been preceded by the His-->Asp change at position 171 of GH. Is this hypothesis true? And when did these changes occur? To address the above issues, we sequenced GH and GHR genes in prosimians and simians. Our data supported the above hypothesis and revealed that the species specificity of human GHR actually emerged in the common ancestor of Old World primates, but the transitional phase still persists in New World monkeys. Our data showed that the rapid evolution of primate GH occurred during a relatively short period (in the common ancestor of higher primates) and that the rate of change was especially high at functionally important sites, suggesting positive selection. However, the nonsynonymous rate/synonymous rate ratio at these sites was <1, so relaxation of purifying selection might have played a role in the rapid evolution of the GH gene in simians, possibly as a result of multiple gene duplications. Similar to GH, GHR displayed an accelerated rate of evolution in primates. Our data revealed proportionally more amino acid replacements at the functionally important sites in both GH and GHR in simians but, surprisingly, showed few coincidental replacements of amino acids forming the same intermolecular contacts between the two proteins.     

The gene of retroviral origin Syncytin 1 is specific to hominoids and is inactive in Old World monkeys

Syncytin 1 is one of the best known examples of recent acquisition of a new gene from an endogenous retrovirus (HERV) in the human genome and has been implicated in placental physiology. Within primates, Syncytin 1 is conserved in all hominoids but has not been characterized in Old World monkeys (OWMs). In this study, we investigated the status of Syncytin 1 in 14 hominoid and OWM species. We show that although the HERV-W provirus responsible for the origin of this gene was present in the genome of the most recent common ancestor of hominoids and OWMs, Syncytin 1 is inactive in OWMs. In addition, we were able to determine that the evolution of Syncytin 1 in hominoids involved an accumulation of amino acid changes and showed signatures of both positive and purifying selection. Our results indicate that Syncytin 1 is indeed a hominoid-specific gene and illustrate the complex and dynamic process associated with the origin of new genes.     

Rapid expansion of killer cell immunoglobulin-like receptor genes in primates and their coevolution with MHC Class I genes

The gene family of killer cell immunoglobulin-like receptors (KIRs) in primates provides the first line of defense against virus infection and tumor transformation. Interacting with MHC class I molecules, KIRs can regulate the cytotoxic activity of natural killer (NK) cells and distinguish the tumor and virus infected cells from normal body cells. Phylogenetic analysis and comparison of domain structures identified three major groups of KIR genes (group I, II, and III genes). These groups of KIR genes, generated by a series of gene duplications, have acquired different MHC-binding specificity. Inference of ancestral KIR sequences suggested that the functional divergence of group I genes from group II genes occurred by positive selection at the MHC-binding sites after duplication. Our evolutionary study has shown that group I genes diverged from group II genes about 17 million years ago (Mya) apparently after separation of hominoids from Old World (OW) monkeys. Around the same time, gene duplication generating the class I MHC-C locus appears to have occurred. These findings suggest that KIR and MHC class I genes have coevolved as an interacting system. The KIR gene family has experienced a rapid expansion in primate species. The rate of expansion of this gene family seems to be one of the highest among all hominoid gene families. The KIR gene family is also subject to birth-and-death evolution.     

The natural history of class I primate alcohol dehydrogenases includes gene duplication, gene loss, and gene conversion

Background

Gene duplication is a source of molecular innovation throughout evolution. However, even with massive amounts of genome sequence data, correlating gene duplication with speciation and other events in natural history can be difficult. This is especially true in its most interesting cases, where rapid and multiple duplications are likely to reflect adaptation to rapidly changing environments and life styles. This may be so for Class I of alcohol dehydrogenases (ADH1s), where multiple duplications occurred in primate lineages in Old and New World monkeys (OWMs and NWMs) and hominoids.

Methodology/Principal Findings

To build a preferred model for the natural history of ADH1s, we determined the sequences of nine new ADH1 genes, finding for the first time multiple paralogs in various prosimians (lemurs, strepsirhines). Database mining then identified novel ADH1 paralogs in both macaque (an OWM) and marmoset (a NWM). These were used with the previously identified human paralogs to resolve controversies relating to dates of duplication and gene conversion in the ADH1 family. Central to these controversies are differences in the topologies of trees generated from exonic (coding) sequences and intronic sequences.

Conclusions/Significance

We provide evidence that gene conversions are the primary source of difference, using molecular clock dating of duplications and analyses of microinsertions and deletions (micro-indels). The tree topology inferred from intron sequences appear to more correctly represent the natural history of ADH1s, with the ADH1 paralogs in platyrrhines (NWMs) and catarrhines (OWMs and hominoids) having arisen by duplications shortly predating the divergence of OWMs and NWMs. We also conclude that paralogs in lemurs arose independently. Finally, we identify errors in database interpretation as the source of controversies concerning gene conversion. These analyses provide a model for the natural history of ADH1s that posits four ADH1 paralogs in the ancestor of Catarrhine and Platyrrhine primates, followed by the loss of an ADH1 paralog in the human lineage.     

Novel member of the CD209 (DC-SIGN) gene family in primates

Two CD209 family genes identified in humans, CD209 (DC-SIGN) and CD209L (DC-SIGNR/L-SIGN), encode C-type lectins that serve as adhesion receptors for ICAM-2 and ICAM-3 and participate in the transmission of human and simian immunodeficiency viruses (HIV and SIV, respectively) to target cells in vitro. Here we characterize the CD209 gene family in nonhuman primates and show that recent evolutionary alterations have occurred in this family across primate species. All of the primate species tested, specifically, Old World monkeys (OWM) and apes, have orthologues of human CD209. In contrast, CD209L is missing in OWM but present in apes. A third family member, that we have named CD209L2, was cloned from rhesus monkey cDNA and subsequently identified in OWM and apes but not in humans. Rhesus CD209L2 mRNA was prominently expressed in the liver and axillary lymph nodes, although preliminary data suggest that levels of expression may vary among individuals. Despite a high level of sequence similarity to both human and rhesus CD209, rhesus CD209L2 was substantially less effective at binding ICAM-3 and poorly transmitted HIV type 1 and SIV to target cells relative to CD209. Our data suggest that the CD209 gene family has undergone recent evolutionary processes involving duplications and deletions, the latter of which may be tolerated because of potentially redundant functional activities of the molecules encoded by these genes.     

Conservative evolution in duplicated genes of the primate Class I ADH cluster

Humans have seven alcohol dehydrogenase genes (ADH) falling into five classes. Three out of the seven genes (ADH1A, ADH1B and ADH1C) belonging to Class I are expressed primarily in liver and code the main enzymes catalyzing ethanol oxidization. The three genes are tandemly arrayed within the ADH cluster on chromosome 4 and have very high nucleotide similarity to each other (exons: >90%; introns: >70%), suggesting the genes have been generated by duplication event(s). One explanation for maintaining similarity of such clustered genes is homogenization via gene conversion(s). Alternatively, recency of the duplications or some other functional constraints might explain the high similarities among the genes. To test for gene conversion, we sequenced introns 2, 3, and 8 of all three Class I genes (total>15.0 kb) for five non-human primates--four great apes and one Old World Monkey (OWM)--and compared them with those of humans. The phylogenetic analysis shows each intron sequence clusters strongly within each gene, giving no evidence for gene conversion(s). Several lines of evidence indicate that the first split was between ADH1C and the gene that gave rise to ADH1A and ADH1B. We also analyzed cDNA sequences of the three genes that have been previously reported in mouse and Catarrhines (OWMs, chimpanzee, and humans) and found that the synonymous and non-synonymous substitution (dN/dS) ratios in all pairs are less than 1 representing purifying selection. This suggests that purifying selection is more important than gene conversion(s) in maintaining the overall sequence similarity among the Class I genes. We speculate that the highly conserved sequences on the three duplicated genes in primates have been achieved essentially by maintaining stability of the hetero-dimer formation that might have been related to dietary adaptation in primate evolution.     

Natural selection in the <Emphasis Type="Italic">TLR-</Emphasis>related genes in the course of primate evolution

The innate immune system constitutes the front line of host defense against pathogens. Toll-like receptors (TLRs) recognize molecules derived from pathogens and play crucial roles in the innate immune system. Here, we provide evidence that the TLR-related genes have come under natural selection pressure in the course of primate evolution. We compared the nucleotide sequences of 16 TLR-related genes, including TLRs (TLR1–10), MYD88, TILAP, TICAM1, TICAM2, MD2, and CD14, among seven primate species. Analysis of the non-synonymous/synonymous substitution ratio revealed the presence of both strictly conserved and rapidly evolving regions in the TLR-related genes. The genomic segments encoding the intracellular Toll/interleukin 1 receptor domains, which exhibited lower rates of non-synonymous substitution, have undergone purifying selection. In contrast, TLR4, which carried a high proportion of non-synonymous substitutions in the part of extracellular domain spanning 200 amino acids, was found to have been the suggestive target of positive Darwinian selection in primate evolution. However, sequence analyses from 25 primate species, including eight hominoids, six Old World monkeys, eight New World monkeys, and three prosimians, showed no evidence that the pressure of positive Darwinian selection has shaped the pattern of sequence variations in TLR4 among New World monkeys and prosimians. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Color vision of ancestral organisms of higher primates

The color vision of mammals is controlled by photosensitive proteins called opsins. Most mammals have dichromatic color vision, but hominoids and Old World (OW) monkeys enjoy trichromatic vision, having the blue-, green-, and red-sensitive opsin genes. Most New World (NW) monkeys are either dichromatic or trichromatic, depending on the sex and genotype. Trichromacy in higher primates is believed to have evolved to facilitate the detection of yellow and red fruits against dappled foliage, but the process of evolutionary change from dichromacy to trichromacy is not well understood. Using the parsimony and the newly developed Bayesian methods, we inferred the amino acid sequences of opsins of ancestral organisms of higher primates. The results suggest that the ancestors of OW and NW monkeys lacked the green gene and that the green gene later evolved from the red gene. The fact that the red/green opsin gene has survived the long nocturnal stage of mammalian evolution and that it is under strong purifying selection in organisms that live in dark environments suggests that this gene has another important function in addition to color vision, probably the control of circadian rhythms.      

Evolutionary relationships among the primate Mhc-DQA1 and DQA2 alleles

The variation of the Mhc-DQA1 and DQA2 loci of ten different primate species (hominoids and Old World monkeys) was studied in order to obtain an insight in the processes that generate polymorphism of major histocompatibility complex (Mhc) class II genes and to establish the evolutionary relationships of their alleles. To that end nucleotide sequences of 36 Mhc class II DQA1 and seven DQA2 second exons were determined and phylogenetic trees that illustrate their evolutionary relationships were constructed. We demonstrate the existence of four primate Mhc-DQA1 allele lineages, two of which probably existed before the separation of the ancestors of the hominoids and Old World monkeys (approximately 22–28 million years ago). Mhc-DQA2 sequences were found only in the hominoid species and showed little diversity. We found no evidence for a major contribution of recombinational events to the generation of allelic diversity of the primate Mhc-DQA1 locus. Instead, our data suggest that the primate Mhc-DQA1 and DQA2 loci are relatively stable entities that mutated primarily as a result of point mutations.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers M76186-M76229.     

Molecular evolution of proglucagon

The vertebrate proglucagon gene encodes glucagon, and the two glucagon-like peptides GLP-1 and GLP-2. To better understand the origin and diversification of the distinct hormonal roles of the three glucagon-like sequences encoded by the proglucagon gene, we have examined the evolution of this gene. The structure of proglucagon has been largely maintained within vertebrates. Duplication of the proglucagon gene or duplications of sequences within the proglucagon gene are rare. All proglucagon gene duplications are likely to be the result of genome duplication events. Examination of the rates of amino acid sequence evolution of each hormone reveals that they have not evolved in a uniform manner. Each hormone has evolved in an episodic fashion, suggesting that the selective constraints acting upon the sequence vary between, and within, vertebrate classes. Changes in selection on a sequence often reflect changes in the function of the sequence, such as the change in function of GLP-1 from a glucagon-like hormone in fish to an incretin in mammals. We found that the GLP-2 sequence underwent rapid sequence evolution in the early mammal lineage, therefore we have concluded that mammalian GLP-2 has acquired a new biological function that is not found in other vertebrates. Comparisons of the hormone sequences show that many amino acid residues that are functionally important in mammalian hormones are not conserved through vertebrate evolution. This observation suggests that the sequences involved in hormone action change through evolution.     

Molecular Evolution of Prolactin in Primates

Pituitary prolactin, like growth hormone (GH) and several other protein hormones, shows an episodic pattern of molecular evolution in which sustained bursts of rapid change contrast with long periods of slow evolution. A period of rapid change occurred in the evolution of prolactin in primates, leading to marked sequence differences between human prolactin and that of nonprimate mammals. We have defined this burst more precisely by sequencing the coding regions of prolactin genes for a prosimian, the slow loris (Nycticebus pygmaeus), and a New World monkey, the marmoset (Callithrix jacchus). Slow loris prolactin is very similar in sequence to pig prolactin, so the episode of rapid change occurred during primate evolution, after the separation of lines leading to prosimians and higher primates. Marmoset prolactin is similar in sequence to human prolactin, so the accelerated evolution occurred before divergence of New World monkeys and Old World monkeys/apes. The burst of change was confined largely to coding sequence (nonsynonymous sites) for mature prolactin and is not marked in other components of the gene sequence. This and the observations that (1) there was no apparent loss of function during the episode of rapid evolution, (2) the rate of evolution slowed toward the basal rate after this burst, and (3) the distribution of substitutions in the prolactin molecule is very uneven support the idea that this episode of rapid change was due to positive adaptive selection. In the slow loris and marmoset there is no evidence for duplication of the prolactin gene, and evidence from another New World monkey (Cebus albifrons) and from the chimpanzee and human genome sequences, suggests that this is the general position in primates, contrasting with the situation for GH genes. The chimpanzee prolactin sequence differs from that of human at two residues and comparison of human and chimpanzee prolactin gene sequences suggests that noncoding regions associated with regulating expression may be evolving differently from other noncoding regions.     

Species-specific size expansion and molecular evolution of the oleosins in angiosperms

Oleosins are hydrophobic plant proteins thought to be important for the formation of oil bodies, which supply energy for seed germination and subsequent seedling growth. To better understand the evolutionary history and diversity of the oleosin gene family in plants, especially angiosperms, we systematically investigated the molecular evolution of this family using eight representative angiosperm species. A total of 73 oleosin members were identified, with six members in each of four monocot species and a greater but variable number in the four eudicots. A phylogenetic analysis revealed that the angiosperm oleosin genes belonged to three monophyletic lineages. Species-specific gene duplications, caused mainly by segmental duplication, led to the great expansion of oleosin genes and occurred frequently in eudicots after the monocot–eudicot divergence. Functional divergence analyses indicate that significant amino acid site-specific selective constraints acted on the different clades of oleosins. Adaptive evolution analyses demonstrate that oleosin genes were subject to strong purifying selection after their species-specific duplications and that rapid evolution occurred with a high degree of evolutionary dynamics in the pollen-specific oleosin genes. In conclusion, this study serves as a foundation for genome-wide analyses of the oleosins. These findings provide insight into the function and evolution of this gene family in angiosperms and pave the way for studies in other plants.     

The evolutionary origin of human subtelomeric homologies--or where the ends begin

The subtelomeric regions of human chromosomes are comprised of sequence homologies shared between distinct subsets of chromosomes. In the course of developing a set of unique human telomere clones, we identified many clones containing such shared homologies, characterized by the presence of cross-hybridization signals on one or more telomeres in a fluorescence in situ hybridization (FISH) assay. We studied the evolutionary origin of seven subtelomeric clones by performing comparative FISH analysis on a primate panel that included great apes and Old World monkeys. All clones tested showed a single hybridization site in Old World monkeys that corresponded to one of the orthologous human sites, thus indicating the ancestral origin. The timing of the duplication events varied among the subtelomeric regions, from approximately 5 to approximately 25 million years ago. To examine the origin of and mechanism for one of these subtelomeric duplications, we compared the sequence derived from human 2q13--an ancestral fusion site of two great ape telomeric regions--with its paralogous subtelomeric sequences at 9p and 22q. These paralogous regions share large continuous homologies and contain three genes: RABL2B, forkhead box D4, and COBW-like. Our results provide further evidence for subtelomeric-mediated genomic duplication and demonstrate that these segmental duplications are most likely the result of ancestral unbalanced translocations that have been fixed in the genome during recent primate evolution.     

Genetic evidence for the coexistence of pheromone perception and full trichromatic vision in howler monkeys

Vertebrate pheromones are water-soluble chemicals perceived mainly by the vomeronasal organ (VNO) for intraspecific communications. Humans, apes, and Old World (OW) monkeys lack functional genes responsible for the pheromone signal transduction and are generally insensitive to vomeronasal pheromones. It has been hypothesized that the evolutionary deterioration of pheromone sensitivity occurred because pheromone communication became redundant after the emergence of full trichromatic color vision via the duplication of the X-chromosome-linked red/green opsin gene in the common ancestor of hominoids and OW monkeys. Interestingly, full trichromacy also evolved in the New World (NW) howler monkeys via an independent duplication of the same gene. Here we sequenced from three species of howler monkeys an essential component of the VNO pheromone transduction pathway, the gene encoding the ion channel TRP2. In contrast to those of hominoids and OW monkeys, the howler TRP2 sequences have none of the characteristics of pseudogenes. This and other observations indicate that howler monkeys have maintained both their systems of pheromone communication and full trichromatic vision, suggesting that the presence of full trichromacy alone does not lead to the loss of pheromone communication. We suggest that the ecological differences between OW and NW primates, particularly in habitat selection, may have also affected the evolution of pheromone perception.     

Human and ape molecular clocks and constraints on paleontological hypotheses

Although the relationships of the living hominoid primates (humans and apes) are well known, the relationships of the fossil species, times of divergence of both living and fossil species, and the biogeographic history of hominoids are not well established. Divergence times of living species, estimated from molecular clocks, have the potential to constrain hypotheses of the relationships of fossil species. In this study, new DNA sequences from nine protein-coding nuclear genes in great apes are added to existing datasets to increase the precision of molecular time estimates bearing on the evolutionary history of apes and humans. The divergence of Old World monkeys and hominoids at the Oligocene-Miocene boundary (approximately 23 million years ago) provides the best primate calibration point and yields a time and 95% confidence interval of 5.4 +/- 1.1 million years ago (36 nuclear genes) for the human-chimpanzee divergence. Older splitting events are estimated as 6.4 +/- 1.5 million years ago (gorilla, 31 genes), 11.3 +/- 1.3 million years ago (orangutan, 33 genes), and 14.9 +/- 2.0 million years ago (gibbon, 27 genes). Based on these molecular constraints, we find that several proposed phylogenies of fossil hominoid taxa are unlikely to be correct.     

Evolutionary rate variation among vertebrate beta globin genes: implications for dating gene family duplication events

A comprehensive dataset of 62 beta globin gene sequences from various vertebrates was compiled to test the molecular clock and to estimate dates of gene duplications. We found that evolution of the beta globin family of genes is not clock-like, a result that is at odds with the common use of this family as an example of a constant rate of evolution over time. Divergence dates were estimated either with or without assuming the molecular clock, and both analyses produced similar date estimates, which are also in general agreement with estimates reported previously. In addition we report date estimates for seven previously unexamined duplication events within the beta globin family. Despite multiple sources of rate variation, the average rate across the beta globin phylogeny yielded reasonable estimates of divergence dates in most cases. Exceptions were cases of gene conversion, where it appears to have led to underestimates of divergence dates. Our results suggest (i) the major duplications giving rise to the paralogous beta globin genes are associated with significant evolutionary rate variation among gene lineages; and (ii) genes arising from more recent gene duplications (e.g., tandem duplications within lineages) do not appear to differ greatly in rate. We believe this pattern reflects a complex interplay of evolutionary forces where natural selection for diversifying paralogous functions and lineage-specific effects contribute to rate variation on a long-term basis, while gene conversion tends to increase sequence similarity. Gene conversion effects appear to be stronger on recent gene duplicates, as their sequences are highly similar. Lastly, phylogenetic analyses do not support a previous report that avian globins are members of a relic lineage of omega globins.     

Evolution of the proximal promoter region of the mammalian growth hormone gene.

The evolutionary relationship between the proximal growth hormone (GH) gene promoter sequences of 12 mammalian species was explored by comparison of their trinucleotide composition and by multiple sequence alignment. Both approaches yielded results that were consistent with the known fossil record-based phylogeny of the analysed sequences, suggesting that the two methods of tree reconstruction might be equally efficient and reliable. The pattern of evolution inferred for the mammalian GH gene promoters was found to vary both temporally and spatially. Thus, two distinct regions devoid of any evolutionary changes exist in primates, but only one of these 'gaps' is also observed in rodents, and neither is seen in ruminants. Furthermore, different evolutionary rates must have prevailed during different periods of evolutionary time and in different lineages, with a dramatic increase in evolutionary rate apparent in primates. Since a similar pattern of discontinuity has been previously noted for the evolution of the GH-coding regions, it may reflect the action of positive selection operating upon the GH gene as a single cohesive unit. Strong evidence for the action of gene conversion between primate GH gene promoters is provided by the fact that the human GH1 and GH2 sequences, which are thought to have diverged before the divergence of Old World monkeys from great apes, are more similar to one another than either is to the rhesus monkey GH2 promoter. Finally, it was noted that a number of nucleotide positions in the GH1 gene promoter that are polymorphic in humans appear to be highly conserved in mammals. This apparent conundrum, which could represent a caveat for the interpretation of phylogenetic footprinting studies, is potentially explicable in terms either of reduced genetic diversity in highly inbred animal species or insufficient population data from non-human species.     

Papio cynocephalus endogenous retrovirus among old world monkeys: evidence for coevolution and ancient cross-species transmissions

To study the evolutionary history of Papio cynocephalus endogenous retrovirus (PcEV), we analyzed the distribution and genetic characteristics of PcEV among 17 different species of primates. The viral pol-env and long terminal repeat and untranslated region (LTR-UTR) sequences could be recovered from all Old World species of the papionin tribe, which includes baboons, macaques, geladas, and mangabeys, but not from the New World monkeys and hominoids we tested. The Old World genera Cercopithecus and Miopithecus hosted either a PcEV variant with an incomplete genome or a virus with substantial mismatches in the LTR-UTR. A complete PcEV was found in the genome of Colobus guereza-but not in Colobus badius-with a copy number of 44 to 61 per diploid genome, comparable to that seen in papionins, and with a sequence most closely related to a virus of the papionin tribe. Analysis of evolutionary distances among PcEV sequences for synonymous and nonsynonymous sites indicated that purifying selection was operational during PcEV evolution. Phylogenetic analysis suggested that possibly two subtypes of PcEV entered the germ line of a common ancestor of the papionins and subsequently coevolved with their hosts. One strain of PcEV was apparently transmitted from a papionin ancestor to an ancestor of the central African lowland C. guereza.