Molecular evolution of <Emphasis Type="Italic">PKD2</Emphasis> gene family in mammals

PKD2 gene encodes a critical cation channel protein that plays important roles in various developmental processes and is usually evolutionarily conserved. In the present study, we analyzed the evolutionary patterns of PKD2 and its homologous genes (PKD2L1, PKD2L2) from nine mammalian species. In this study, we demonstrated the orthologs of PKD2 gene family evolved under a dominant purifying selection force. Our results in combination with the reported evidences from functional researches suggested the entire PKD2 gene family are conserved and perform essential biological roles during mammalian evolution. In rodents, PKD2 gene family members appeared to have evolved more rapidly than other mammalian lineages, probably resulting from relaxation of purifying selection. However, positive selection imposed on synonymous sites also potentially contributed to this case. For the paralogs, our results implied that PKD2L2 genes evolved under a weaker purifying selection constraint than PKD2 and PKD2L1 genes. Interestingly, some loop regions of transmembrane domain of PKD2L2 exhibited higher P _N/P _S ratios than expected, suggesting these regions are more functional divergent in organisms and worthy of special attention. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Chun Ye, Huan Sun have contributed equally to this work.     

Sexual selection halts the relaxation of protamine 2 among rodents

Sexual selection has been proposed as the driving force promoting the rapid evolutionary changes observed in some reproductive genes including protamines. We test this hypothesis in a group of rodents which show marked differences in the intensity of sexual selection. Levels of sperm competition were not associated with the evolutionary rates of protamine 1 but, contrary to expectations, were negatively related to the evolutionary rate of cleaved- and mature-protamine 2. Since both domains were found to be under relaxation, our findings reveal an unforeseen role of sexual selection: to halt the degree of degeneration that proteins within families may experience due to functional redundancy. The degree of relaxation of protamine 2 in this group of rodents is such that in some species it has become dysfunctional and it is not expressed in mature spermatozoa. In contrast, protamine 1 is functionally conserved but shows directed positive selection on specific sites which are functionally relevant such as DNA-anchoring domains and phosphorylation sites. We conclude that in rodents protamine 2 is under relaxation and that sexual selection removes deleterious mutations among species with high levels of sperm competition to maintain the protein functional and the spermatozoa competitive.     

Adaptive Evolution of the First Extra Exon in the Murid Rodent Prolactin Gene Family

The prolactin gene family in rodents consists of multiple members that coordinate the processes of reproduction and pregnancy. Some members of this family acquired one or two additional exons between exon 2 and exon 3 of the prototypical 5-exon, 4-intron structure, but the evolutionary importance of this insertion is unclear. Here, we focus on those members and survey this question by molecular evolutionary methods. Phylogenetic analysis shows that those members cluster into two distinct groups. Further analysis shows that the two groups of genes originated before the divergence of mouse and rat but after that of rodents from other mammals. We compared the d _N/d _S values for each branch of the gene tree but found no evidence to support positive selection for any branch. We found strong evidence, however, that one site (11E) of the 13 sites of the first extra exon underwent positive selection by the site-specific models of the maximum-likelihood method. Combining our molecular evolutionary analysis with other known functional evidence, we believe that the insertion of the extra exon implies some functional adaptation.     

Extraordinary sequence divergence at Tsga8, an X-linked gene involved in mouse spermiogenesis

The X chromosome plays an important role in both adaptive evolution and speciation. We used a molecular evolutionary screen of X-linked genes potentially involved in reproductive isolation in mice to identify putative targets of recurrent positive selection. We then sequenced five very rapidly evolving genes within and between several closely related species of mice in the genus Mus. All five genes were involved in male reproduction and four of the genes showed evidence of recurrent positive selection. The most remarkable evolutionary patterns were found at Testis-specific gene a8 (Tsga8), a spermatogenesis-specific gene expressed during postmeiotic chromatin condensation and nuclear transformation. Tsga8 was characterized by extremely high levels of insertion-deletion variation of an alanine-rich repetitive motif in natural populations of Mus domesticus and M. musculus, differing in length from the reference mouse genome by up to 89 amino acids (27% of the total protein length). This population-level variation was coupled with striking divergence in protein sequence and length between closely related mouse species. Although no clear orthologs had previously been described for Tsga8 in other mammalian species, we have identified a highly divergent hypothetical gene on the rat X chromosome that shares clear orthology with the 5' and 3' ends of Tsga8. Further inspection of this ortholog verified that it is expressed in rat testis and shares remarkable similarity with mouse Tsga8 across several general features of the protein sequence despite no conservation of nucleotide sequence across over 60% of the rat-coding domain. Overall, Tsga8 appears to be one of the most rapidly evolving genes to have been described in rodents. We discuss the potential evolutionary causes and functional implications of this extraordinary divergence and the possible contribution of Tsga8 and the other four genes we examined to reproductive isolation in mice.     

Natural selection and mammalian BRCA1 sequences: elucidating functionally important sites relevant to breast cancer susceptibility in humans

Comparison of orthologous gene sequences is emerging as a powerful approach to elucidating functionally important positions in human disease genes. Using a diverse array of 132 mammalian BRCA1 (exon 11) sequences, we evaluated the functional significance of specific sites in the context of selection information (purifying, neutral, or diversifying) as well as the ability to extract such information from alignments that index varying degrees of mammalian diversity. Small data sets of either closely related taxa (Primates) or divergent placental taxa were unable to distinguish sites conserved due to purifying selection from sites conserved due to chance (false-positive rate = 65%-99%). Increasing the number of placental taxa to 57 greatly reduced the potential false-positive rate (0%-1.5%). Using the larger data set, we ranked the oncogenic risk of human missense mutations using a novel method that incorporates site-specific selection level and severity of the amino acid change evaluated against the amino acids present in other mammalian taxa. In addition to sites undergoing positive selection in Marsupialia, Laurasiatheria, Euarchontoglires, and Primates, we identified sites most likely to be undergoing divergent selection pressure in different lineages and six pairs of potentially interacting sites. Our results demonstrate the necessity of including large numbers of sequences to elucidate functionally important sites of a protein when using a comparative evolutionary approach.     

哺乳动物抗凝血酶基因的分子特征、微共线性与适应性进化

抗凝血酶(AT)是哺乳动物体内重要的天然抗凝血因子之一,它隶属于丝氨酸蛋白酶抑制物家族,主要参与并调节复杂的血凝过程。基于比较基因组学手段,共挖掘出17个哺乳动物抗凝血酶基因(AT),并剖析了它们的基因结构、微共线性、保守基序、功能结构域、以及系统进化关系。基因结构与微共线分析表明,哺乳动物AT基因具有5-12个外显子,大多数是7个外显子;不同物种AT基因所处区段之间具有较好的共线性。哺乳动物AT蛋白特征分析显示,SERPIN功能结构域与保守基序1、2、3、4、5、8和9存在相互重叠的部分。AT基因进化树揭示基因进化和通常认为的物种进化几乎一致。同时,利用PAML中Codeml的位点特异模型在AT基因中发掘了1个正选择位点328D。     

The voltage-gated potassium channel subfamily KQT member 4 (KCNQ4) displays parallel evolution in echolocating bats

Bats are the only mammals that use highly developed laryngeal echolocation, a sensory mechanism based on the ability to emit laryngeal sounds and interpret the returning echoes to identify objects. Although this capability allows bats to orientate and hunt in complete darkness, endowing them with great survival advantages, the genetic bases underlying the evolution of bat echolocation are still largely unknown. Echolocation requires high-frequency hearing that in mammals is largely dependent on somatic electromotility of outer hair cells. Then, understanding the molecular evolution of outer hair cell genes might help to unravel the evolutionary history of echolocation. In this work, we analyzed the molecular evolution of two key outer hair cell genes: the voltage-gated potassium channel gene KCNQ4 and CHRNA10, the gene encoding the α10 nicotinic acetylcholine receptor subunit. We reconstructed the phylogeny of bats based on KCNQ4 and CHRNA10 protein and nucleotide sequences. A phylogenetic tree built using KCNQ4 amino acid sequences showed that two paraphyletic clades of laryngeal echolocating bats grouped together, with eight shared substitutions among particular lineages. In addition, our analyses indicated that two of these parallel substitutions, M388I and P406S, were probably fixed under positive selection and could have had a strong functional impact on KCNQ4. Moreover, our results indicated that KCNQ4 evolved under positive selection in the ancestral lineage leading to mammals, suggesting that this gene might have been important for the evolution of mammalian hearing. On the other hand, we found that CHRNA10, a gene that evolved adaptively in the mammalian lineage, was under strong purifying selection in bats. Thus, the CHRNA10 amino acid tree did not show echolocating bat monophyly and reproduced the bat species tree. These results suggest that only a subset of hearing genes could underlie the evolution of echolocation. The present work continues to delineate the genetic bases of echolocation and ultrasonic hearing in bats.     

Sex chromosomes and sex determination in weird mammals

Weird mammals are of two types. Highly divergent mammals, such as the marsupials and monotremes, have informed us of the evolutionary history of the Y chromosome and sex-determining gene, and the recently specialized rodents can help us predict its future. The Y chromosome has had a short but eventful history, and is already heading briskly for oblivion. It originated as a homologous partner of the X when it acquired a sex-determining gene (not necessarily SRY). Most of the genes on the Y, even those with a male-specific function, evolved from genes now on the X. At the mercy of a high rate of variability and the forces of drift and selection, the Y has lost genes at a rate of 3-6 genes/million years, sparing those that acquired critical male-specific functions. Even these genes have disappeared from one mammalian lineage or another as their functions were usurped by genes elsewhere in the genome. The mammalian testis-determining gene, SRY, is a typical Y-borne gene. It arose by truncation of a gene (SOX3) on the X that is expressed in brain development, and it may work by interacting with (inhibiting?) related genes, including SOX9. Variant sex-determining systems in rodents show that the action of SRY can change, as it evidently has in the mouse, and SRY can be inactivated, as in akodont rodents, or even completely superseded, as in mole voles.     

Adaptive evolution of fertilization proteins within a genus: variation in ZP2 and ZP3 in deer mice (Peromyscus)

Rapid evolution of reproductive proteins has been documented in a wide variety of taxa. In internally fertilized species, knowledge about the evolutionary dynamics of these proteins between closely related taxa is primarily limited to accessory gland proteins in the semen of Drosophila. Investigation of additional taxa and functional classes of proteins is necessary in order to determine if there is a general pattern of adaptive evolution of reproductive proteins between recently diverged species. We performed an evolutionary analysis of 2 egg coat proteins, ZP2 and ZP3, in 15 species of deer mice (genus Peromyscus). Both of these proteins are involved in egg-sperm binding, a critical step in maintaining species-specific fertilization. Here, we show that Zp2 and Zp3 gene trees are not consistent with trees based on nonreproductive genes, Mc1r and Lcat, where species formed monophyletic clades. In fact, for both of the reproductive genes, intraspecific amino acid variation was extensive and alleles were sometimes shared across species. We document positive selection acting on ZP2 and ZP3 and identify specific amino acid sites that are likely targets of selection using both maximum likelihood approaches and patterns of parallel amino acid change. In ZP3, positively selected sites are clustered in and around the region implicated in sperm binding in Mus, suggesting changes may impact egg-sperm binding and fertilization potential. Finally, we identify lineages with significantly elevated rates of amino acid substitution using a Bayesian mapping approach. These findings demonstrate that the pattern of adaptive reproductive protein evolution found at higher taxonomic levels can be documented between closely related mammalian species, where reproductive isolation has evolved recently.     

Rapid evolution of a primate sperm protein: relaxation of functional constraint or positive Darwinian selection?

Protamines are arginine-rich proteins that replace histones and bind sperm DNA during spermatogenesis in vertebrates. Previous studies have shown that protamine exons evolve faster than does the protamine intron. It has been suggested that this is a result of a relaxation of functional constraint. However, a more likely explanation is that the evolutionary rate of exons has been accelerated by positive Darwinian selection, because introns are generally believed to evolve in a neutral fashion. Therefore, we examined the possibility that positive selection has been acting on the protamine genes of three groups of placental mammals: primates (hominoids and Old World monkeys), rodents (mice, rats, and guinea pigs), and pecoran ruminants (deer and bovids). We found that the nucleotide substitution rate at nonsynonymous sites is significantly higher than the rate at synonymous and intron sites for protamine P1 of hominoids and Old World monkeys. This result suggests that positive selection has been operating on protamine P1 of these species. In contrast, no clear-cut evidence of positive selection was found for protamine P1 of ruminants and rodents or protamine P2 of primates. The agent of positive selection on primate protamine P1 remains unknown, though sperm competition is a possibility. Further investigations on the function and intraspecific polymorphism of this protein are needed in order to identify the selection agent.     

Maximum‐likelihood approaches reveal signatures of positive selection in BMP15 and GDF9 genes modulating ovarian function in mammalian female fertility

Bone morphogenetic proteins (BMPs) and the growth factors (GDFs) play an important role in ovarian folliculogenesis and essential regulator of processes of numerous granulosa cells. BMP15 gene variations linked to various ovarian phenotypic consequences subject to the species, from infertility to improved prolificacy in sheep, primary ovarian insufficiency in women or associated with minor subfertility in mouse. To study the evolving role of BMP15 and GDF9, a phylogenetic analysis was performed. To find out the candidate gene associated with prolificacy in mammals, the nucleotide sequence of BMP15 and GDF9 genes was recognized under positive selection in various mammalian species. Maximum‐likelihood approaches used on BMP15 and GDF9 genes exhibited a robust divergence and a prompted evolution as compared to other TGFβ family members. Furthermore, among 32 mammalian species, we identified positive selection signals in the hominidae clade resulting to 132D, 147E, 163Y, 191W, and 236P codon sites of BMP15 and 162F, 188K, 206R, 240A, 244L, 246H, 248S, 251D, 253L, 254F and other codon sites of GDF9. The positively selected amino acid sites such as Alanine, Lucien, Arginine, and lysine are important for signaling. In conclusion, this study evidences that GDF9 and BMP15 genes have rapid evolution than other TGFß family members and was subjected to positive selection in the mammalian clade. Selected sites under the positive selection are of remarkable significance for the particular functioning of the protein and consequently for female fertility.     

Discordant evolution of the adjacent antiretroviral genes TRIM22 and TRIM5 in mammals

TRIM5alpha provides a cytoplasmic block to retroviral infection, and orthologs encoded by some primates are active against HIV. Here, we present an evolutionary comparison of the TRIM5 gene to its closest human paralogs: TRIM22, TRIM34, and TRIM6. We show that TRIM5 and TRIM22 have a dynamic history of gene expansion and loss during the evolution of mammals. The cow genome contains an expanded cluster of TRIM5 genes and no TRIM22 gene, while the dog genome encodes TRIM22 but has lost TRIM5. In contrast, TRIM6 and TRIM34 have been strictly preserved as single gene orthologs in human, dog, and cow. A more focused analysis of primates reveals that, while TRIM6 and TRIM34 have evolved under purifying selection, TRIM22 has evolved under positive selection as was previously observed for TRIM5. Based on TRIM22 sequences obtained from 27 primate genomes, we find that the positive selection of TRIM22 has occurred episodically for approximately 23 million years, perhaps reflecting the changing pathogenic landscape. However, we find that the evolutionary episodes of positive selection that have acted on TRIM5 and TRIM22 are mutually exclusive, with generally only one of these genes being positively selected in any given primate lineage. We interpret this to mean that the positive selection of one gene has constrained the adaptive flexibility of its neighbor, probably due to genetic linkage. Finally, we find a striking congruence in the positions of amino acid residues found to be under positive selection in both TRIM5alpha and TRIM22, which in both proteins fall predominantly in the beta2-beta3 surface loop of the B30.2 domain. Astonishingly, this same loop is under positive selection in the multiple cow TRIM5 genes as well, indicating that this small structural loop may be a viral recognition motif spanning a hundred million years of mammalian evolution.     

Positive selection of three chitinase genes of the family 18 of glycoside hydrolases in mammals

The digestive enzyme chitinase degrades chitin, and is found in a wide range of organisms, from prokaryotes to eukaryotes. Although mammals cannot synthesize or assimilate chitin, several proteins of the glycoside hydrolase (GH) chitinase family GH18, including some with enzymatic activity, have recently been identified from mammalian genomes. Consequently, there is growing interest in molecular evolution of this family of proteins. Here we report on the use of maximum likelihood methods to test for evidence of positive selection in three genes of the chitinase family GH18, all of which are found in mammals. These focal genes are CHIA, CHIT1 and CHI3L1, which encode the chitinase proteins acidic mammalian chitinase, chitotriosidase and cartilage protein 39, respectively. The results of our analyses indicate that each of these genes has undergone independent selective pressure in their evolution. Additionally, we have found evidence of a signature of positive natural selection, with most sites identified as being subject to adaptive evolution located in the catalytic domain. Our results suggest that positive selection on these genes stems from their function in digestion and/or immunity.     

Evolutionary History of Cathepsin L (L-like) Family Genes in Vertebrates

Cathepsin L family, an important cysteine protease found in lysosomes, is categorized into cathepsins B, F, H, K, L, S, and W in vertebrates. This categorization is based on their sequence alignment and traditional functional classification, but the evolutionary relationship of family members is unclear. This study determined the evolutionary relationship of cathepsin L family genes in vertebrates through phylogenetic construction. Results showed that cathepsins F, H, S and K, and L and V were chronologically diverged. Tandem-repeat duplication was found to occur in the evolutionary history of cathepsin L family. Cathepsin L in zebrafish, cathepsins S and K in xenopus, and cathepsin L in mice and rats underwent evident tandem-repeat events. Positive selection was detected in cathepsin L-like members in mice and rats, and amino acid sites under positive selection pressure were calculated. Most of these sites appeared at the connection of secondary structures, suggesting that the sites may slightly change spatial structure. Severe positive selection was also observed in cathepsin V (L2) of primates, indicating that this enzyme had some special functions. Our work provided a brief evolutionary history of cathepsin L family and differentiated cathepsins S and K from cathepsin L based on vertebrate appearance. Positive selection was the specific cause of differentiation of cathepsin L family genes, confirming that gene function variation after expansion events was related to interactions with the environment and adaptability.     

Positive Evolutionary Selection On the RIG-I-Like Receptor Genes in Mammals

The mammalian RIG-I-like receptors, RIG-I, MDA5 and LGP2, are a family of DExD/H box RNA helicases responsible for the cytoplasmic detection of viral RNA. These receptors detect a variety of RNA viruses, or DNA viruses that express unusual RNA species, many of which are responsible for a great number of severe and lethal diseases. Host innate sentinel proteins involved in pathogen recognition must rapidly evolve in a dynamic arms race with pathogens, and thus are subjected to long-term positive selection pressures to avoid potential infections. Using six codon-based Maximum Likelihood methods, we were able to identify specific codons under positive selection in each of these three genes. The highest number of positively selected codons was detected in MDA5, but a great percentage of these codons were located outside of the currently defined protein domains for MDA5, which likely reflects the imposition of both functional and structural constraints. Additionally, our results support LGP2 as being the least prone to evolutionary change, since the lowest number of codons under selection was observed for this gene. On the other hand, the preponderance of positively selected codons for RIG-I were detected in known protein functional domains, suggesting that pressure has been imposed by the vast number of viruses that are recognized by this RNA helicase. Furthermore, the RIG-I repressor domain, the region responsible for recognizing and binding to its RNA substrates, exhibited the strongest evidence of selective pressures. Branch-site analyses were performed and several species branches on the three receptor gene trees showed evidence of episodic positive selection. In conclusion, by looking for evidence of positive evolutionary selection on mammalian RIG-I-like receptor genes, we propose that a multitude of viruses have crafted the receptors biological function in host defense, specifically for the RIG-I gene, contributing to the innate species-specific resistance/susceptibility to diverse viral pathogens.     

Evidence for positive selection on the leptin gene in Cetacea and Pinnipedia

The leptin gene has received intensive attention and scientific investigation for its importance in energy homeostasis and reproductive regulation in mammals. Furthermore, study of the leptin gene is of crucial importance for public health, particularly for its role in obesity, as well as for other numerous physiological roles that it plays in mammals. In the present work, we report the identification of novel leptin genes in 4 species of Cetacea, and a comparison with 55 publicly available leptin sequences from mammalian genome assemblies and previous studies. Our study provides evidence for positive selection in the suborder Odontoceti (toothed whales) of the Cetacea and the family Phocidae (earless seals) of the Pinnipedia. We also detected positive selection in several leptin gene residues in these two lineages. To test whether leptin and its receptor evolved in a coordinated manner, we analyzed 24 leptin receptor gene (LPR) sequences from available mammalian genome assemblies and other published data. Unlike the case of leptin, our analyses did not find evidence of positive selection for LPR across the Cetacea and Pinnipedia lineages. In line with this, positively selected sites identified in the leptin genes of these two lineages were located outside of leptin receptor binding sites, which at least partially explains why co-evolution of leptin and its receptor was not observed in the present study. Our study provides interesting insights into current understanding of the evolution of mammalian leptin genes in response to selective pressures from life in an aquatic environment, and leads to a hypothesis that new tissue specificity or novel physiologic functions of leptin genes may have arisen in both odontocetes and phocids. Additional data from other species encompassing varying life histories and functional tests of the adaptive role of the amino acid changes identified in this study will help determine the factors that promote the adaptive evolution of the leptin genes in marine mammals.     

An Evolutionary Analysis of Antigen Processing and Presentation across Different Timescales Reveals Pervasive Selection

The antigenic repertoire presented by MHC molecules is generated by the antigen processing and presentation (APP) pathway. We analyzed the evolutionary history of 45 genes involved in APP at the inter- and intra-species level. Results showed that 11 genes evolved adaptively in mammals. Several positively selected sites involve positions of fundamental importance to the protein function (e.g. the TAP1 peptide-binding domains, the sugar binding interface of langerin, and the CD1D trafficking signal region). In CYBB, all selected sites cluster in two loops protruding into the endosomal lumen; analysis of missense mutations responsible for chronic granulomatous disease (CGD) showed the action of different selective forces on the very same gene region, as most CGD substitutions involve aminoacid positions that are conserved in all mammals. As for ERAP2, different computational methods indicated that positive selection has driven the recurrent appearance of protein-destabilizing variants during mammalian evolution. Application of a population-genetics phylogenetics approach showed that purifying selection represented a major force acting on some APP components (e.g. immunoproteasome subunits and chaperones) and allowed identification of positive selection events in the human lineage.We also investigated the evolutionary history of APP genes in human populations by developing a new approach that uses several different tests to identify the selection target, and that integrates low-coverage whole-genome sequencing data with Sanger sequencing. This analysis revealed that 9 APP genes underwent local adaptation in human populations. Most positive selection targets are located within noncoding regions with regulatory function in myeloid cells or act as expression quantitative trait loci. Conversely, balancing selection targeted nonsynonymous variants in TAP1 and CD207 (langerin). Finally, we suggest that selected variants in PSMB10 and CD207 contribute to human phenotypes. Thus, we used evolutionary information to generate experimentally-testable hypotheses and to provide a list of sites to prioritize in follow-up analyses.     

Accelerated evolution of small serum proteins (SSPs)-The PSP94 family proteins in a Japanese viper

Five small serum proteins (SSPs) with molecular masses of 6.5-10 kDa were detected in Habu (Trimeresurus flavoviridis) serum; this included two novel proteins SSP-4 and SSP-5. The amino acid sequences of these proteins and of SSP-1, SSP-2, and SSP-3, which were reported previously, were determined on the basis of the nucleotide sequences of their cDNAs. Although these proteins exhibited only limited sequence identity to mammalian prostatic secretory protein of 94 amino acids (PSP94), the topological pattern of disulfide bonds in SSPs was identical to that of the mammalian proteins. SSP-3 and SSP-4 lacked approximately 30 residues at the C-terminal. Each of the full-length cDNAs encoded a mature protein of 62-90 residues and a highly conserved signal peptide. The evolutionary distances between SSPs estimated on the basis of the amino acid changes were significantly greater than those of the synonymous nucleotide substitutions; these finding, together with results from analyses of nonsynonymous to synonymous rates of change (dN/dS) suggest that snake SSPs have endured substantial accelerated adaptive protein evolution. Such accelerated positive selection in SSPs parallels other findings of similar molecular evolution in snake venom proteins and suggests that diversifying selection on both systems may be linked, and that snake SSP genes may have evolved by gene duplication and rapid diversification to facilitate the acquisition of various functions to block venom activity within venomous snakes.