Strong Selective Constraints Research Articles

Complete mitochondrial genomes of eight seahorses and pipefishes (Syngnathiformes: Syngnathidae): insight into the adaptive radiation of syngnathid fishes

BackgroundThe evolution of male pregnancy is the most distinctive characteristic of syngnathids, and their specialized life history traits make syngnathid species excellent model species for many issues in biological evolution. However, the origin of syngnathids and the evolutionary divergence time of different syngnathid species remain poorly resolved. Comprehensive phylogenetic studies of the Syngnathidae will provide critical evidence to elucidate their origin, evolution, and dispersal patterns.ResultsWe sequenced the mitochondrial genomes of eight syngnathid species in this study, and the estimated divergence times suggested that syngnathids diverged from other teleosts approximately 48.8 Mya during the Eocene period. Selection analysis showed that many mitochondrial genes of syngnathids exhibited significantly lower Ka/Ks values than those of other teleosts. The two most frequently used codons in syngnathid fishes were different from those in other teleosts, and a greater proportion of the mitochondrial simple sequence repeats (SSRs) were distributed in non-coding sequences in syngnathids compared with other teleosts.ConclusionsOur study indicated that syngnathid fishes experienced an adaptive radiation process during the early explosion of species. Syngnathid mitochondrial OXPHOS genes appear to exhibit depressed Ka/Ks ratios compared with those of other teleosts, and this may suggest that their mitogenomes have experienced strong selective constraints to eliminate deleterious mutations.

The second life of Plasmodium in the mosquito host: gene regulation on the move.

Malaria parasites face dynamically changing environments and strong selective constraints within human and mosquito hosts. To survive such hostile and shifting conditions, Plasmodium switches transcriptional programs during development and has evolved mechanisms to adjust its phenotype through heterogeneous patterns of gene expression. In vitro studies on culture-adapted isolates have served to set the link between chromatin structure and functional gene expression. Yet, experimental evidence is limited to certain stages of the parasite in the vertebrate, i.e. blood, while the precise mechanisms underlying the dynamic regulatory landscapes during development and in the adaptation to within-host conditions remain poorly understood. In this review, we discuss available data on transcriptional and epigenetic regulation in Plasmodium mosquito stages in the context of sporogonic development and phenotypic variation, including both bet-hedging and environmentally triggered direct transcriptional responses. With this, we advocate the mosquito offers an in vivo biological model to investigate the regulatory networks, transcription factors and chromatin-modifying enzymes and their modes of interaction with regulatory sequences, which might be responsible for the plasticity of the Plasmodium genome that dictates stage- and cell type-specific blueprints of gene expression.

Pseudogenes Provide Evolutionary Evidence for the Competitive Endogenous RNA Hypothesis.

The competitive endogenous RNA (ceRNA) hypothesis is an attractively simple model to explain the biological role of many putatively functionless noncoding RNAs. Under this model, there exist transcripts in the cell whose role is to titrate out microRNAs such that the expression level of another target sequence is altered. That it is logistically possible for expression of one microRNA recognition element (MRE)-containing transcript to affect another is seen in the multiple examples of pathogenic effects of inappropriate expression of MRE-containing RNAs. However, the role, if any, of ceRNAs in normal biological processes and at physiological levels is disputed. By comparison of parent genes and pseudogenes we show, both for a specific example and genome-wide, that the pseudo-3′ untranslated regions (3′UTRs) of expressed pseudogenes are frequently retained and are under selective constraint in mammalian genomes. We found that the pseudo-3′UTR of BRAFP1, a previously described oncogenic ceRNA, has reduced substitutions relative to its pseudo-coding sequence, and we show sequence constraint on MREs shared between the parent gene, BRAF, and the pseudogene. Investigation of RNA-seq data reveals expression of BRAFP1 in normal somatic tissues in human and in other primates, consistent with biological ceRNA functionality of this pseudogene in nonpathogenic cellular contexts. Furthermore, we find that on a genome-wide scale pseudo-3′UTRs of mammalian pseudogenes (n = 1,629) are under stronger selective constraint than their pseudo-coding sequence counterparts, and are more often retained and expressed. Our results suggest that many human pseudogenes, often considered nonfunctional, may have an evolutionarily constrained role, consistent with the ceRNA hypothesis.

The Grayling Genome Reveals Selection on Gene Expression Regulation after Whole-Genome Duplication.

Whole-genome duplication (WGD) has been a major evolutionary driver of increased genomic complexity in vertebrates. One such event occurred in the salmonid family ∼80 Ma (Ss4R) giving rise to a plethora of structural and regulatory duplicate-driven divergence, making salmonids an exemplary system to investigate the evolutionary consequences of WGD. Here, we present a draft genome assembly of European grayling (Thymallus thymallus) and use this in a comparative framework to study evolution of gene regulation following WGD. Among the Ss4R duplicates identified in European grayling and Atlantic salmon (Salmo salar), one-third reflect nonneutral tissue expression evolution, with strong purifying selection, maintained over ∼50 Myr. Of these, the majority reflect conserved tissue regulation under strong selective constraints related to brain and neural-related functions, as well as higher-order protein–protein interactions. A small subset of the duplicates have evolved tissue regulatory expression divergence in a common ancestor, which have been subsequently conserved in both lineages, suggestive of adaptive divergence following WGD. These candidates for adaptive tissue expression divergence have elevated rates of protein coding- and promoter-sequence evolution and are enriched for immune- and lipid metabolism ontology terms. Lastly, lineage-specific duplicate divergence points toward underlying differences in adaptive pressures on expression regulation in the nonanadromous grayling versus the anadromous Atlantic salmon. Our findings enhance our understanding of the role of WGD in genome evolution and highlight cases of regulatory divergence of Ss4R duplicates, possibly related to a niche shift in early salmonid evolution.

Selective constraints in cold-region wild boars may defuse the effects of small effective population size on molecular evolution of mitogenomes.

Spatial range expansion during population colonization is characterized by demographic events that may have significant effects on the efficiency of natural selection. Population genetics suggests that genetic drift brought by small effective population size (N e) may undermine the efficiency of selection, leading to a faster accumulation of nonsynonymous mutations. However, it is still unknown whether this effect might be balanced or even reversed by strong selective constraints. Here, we used wild boars and local domestic pigs from tropical (Vietnam) and subarctic region (Siberia) as animal model to evaluate the effects of functional constraints and genetic drift on shaping molecular evolution. The likelihood‐ratio test revealed that Siberian clade evolved significantly different from Vietnamese clades. Different datasets consistently showed that Siberian wild boars had lower Ka/Ks ratios than Vietnamese samples. The potential role of positive selection for branches with higher Ka/Ks was evaluated using branch‐site model comparison. No signal of positive selection was found for the higher Ka/Ks in Vietnamese clades, suggesting the interclade difference was mainly due to the reduction in Ka/Ks for Siberian samples. This conclusion was further confirmed by the result from a larger sample size, among which wild boars from northern Asia (subarctic and nearby region) had lower Ka/Ks than those from southern Asia (temperate and tropical region). The lower Ka/Ks might be due to either stronger functional constraints, which prevent nonsynonymous mutations from accumulating in subarctic wild boars, or larger N e in Siberian wild boars, which can boost the efficacy of purifying selection to remove functional mutations. The latter possibility was further ruled out by the Bayesian skyline plot analysis, which revealed that historical N e of Siberian wild boars was smaller than that of Vietnamese wild boars. Altogether, these results suggest stronger functional constraints acting on mitogenomes of subarctic wild boars, which may provide new insights into their local adaptation of cold resistance.

Low evolutionary rate of infectious pancreatic necrosis virus (IPNV) in Italy is associated with reduced virulence in trout.

Infectious pancreatic necrosis virus (IPNV) is a naked double-stranded RNA virus with a bi-segmented genome that is classified within the family Birnaviridae, genus Aquabirnavirus. IPNV was first detected in Italian trout farms in the late 1970s and ultimately became endemic. To characterize the evolution of IPNV circulating in Italy, particularly whether there is a link between evolutionary rate and virulence, we obtained and analyzed the VP1 (polymerase) and the pVP2 (major capsid protein precursor) sequences from 75 IPNV strains sampled between 1978 and 2017. These data revealed that the Italian IPNV exhibit relatively little genetic variation over the sampling period, falling into four genetic clusters within a single genogroup (group 2 for VP1 and genogroup V for pVP2) and contained one example of inter-segment reassortment. The mean evolutionary rates for VP1 and pVP2 were estimated to be 1.70 and 1.45 × 10−4 nucleotide substitutions per site, per year, respectively, and hence significantly lower than those seen in other Birnaviruses. Similarly, the relatively low ratios of non-synonymous (dN) to synonymous (dS) nucleotide substitutions per site in both genes indicated that IPNV was subject to strong selective constraints, again in contrast to other RNA viruses infecting salmonids that co-circulate in the same area during the same time period. Notably, all the Italian IPNV harbored a proline at position 217 (P217) and a threonine at position 221 (T221) in pVP2, both of which are associated with a low virulence phenotype. We therefore suggest the lower virulence of IPNV may have resulted in reduced rates of virus replication and hence lower rates of evolutionary change. The data generated here will be of importance in understanding the factors that shape the evolution of Aquabirnaviruses in nature.

Patterns of Population Variation in Two Paleopolyploid Eudicot Lineages Suggest That Dosage-Based Selection on Homeologs Is Long-Lived.

Genes that are inherently subject to strong selective constraints tend to be overretained in duplicate after polyploidy. They also continue to experience similar, but somewhat relaxed, constraints after that polyploidy event. We sought to assess for how long the influence of polyploidy is felt on these genes’ selective pressures. We analyzed two nested polyploidy events in Brassicaceae: the At-α genome duplication that is the most recent polyploidy in the model plant Arabidopsis thaliana and a more recent hexaploidy shared by the genus Brassica and its relatives. By comparing the strength and direction of the natural selection acting at the population and at the species level, we find evidence for continued intensified purifying selection acting on retained duplicates from both polyploidies even down to the present. The constraint observed in preferentially retained genes is not a result of the polyploidy event: the orthologs of such genes experience even stronger constraint in nonpolyploid outgroup genomes. In both the Arabidopsis and Brassica lineages, we further find evidence for segregating mildly deleterious variants, confirming that the population-level data uncover patterns not visible with between-species comparisons. Using the A. thaliana metabolic network, we also explored whether network position was correlated with the measured selective constraint. At both the population and species level, nodes/genes tended to show similar constraints to their neighbors. Our results paint a picture of the long-lived effects of polyploidy on plant genomes, suggesting that even yesterday’s polyploids still have distinct evolutionary trajectories.

The Limits to Parapatric Speciation II: Strengthening a Preexisting Genetic Barrier to Gene Flow in Parapatry.

By encompassing the whole continuum between allopatric and sympatric scenarios, parapatric speciation includes many potential scenarios for the evolution of new species. Here, we investigate how a genetic barrier to gene flow, that relies on a single postzygotic genetic incompatibility, may further evolve under ongoing migration. We consider a continent island model with three loci involved in pairwise Dobzhansky–Muller incompatibilities (DMIs). Using an analytic approach, we derive the conditions for invasion of a new mutation and its consequences for the strength and stability of the initial genetic barrier. Our results show that the accumulation of genetic incompatibilities in the presence of gene flow is under strong selective constraints. In particular, preexisting incompatibilities do not always facilitate the invasion of further barrier genes. If new mutations do invade, they will often weaken or destroy the barrier rather than strengthening it. We conclude that migration is highly effective at disrupting the so-called “snowball effect”, the accelerated accumulation of DMIs that has been described for allopatric populations en route to reproductive isolation.

Connecting theory and data to understand recombination rate evolution.

Meiotic recombination is necessary for successful gametogenesis in most sexually reproducing organisms and is a fundamental genomic parameter, influencing the efficacy of selection and the fate of new mutations. The molecular and evolutionary functions of recombination should impose strong selective constraints on the range of recombination rates. Yet, variation in recombination rate is observed on a variety of genomic and evolutionary scales. In the past decade, empirical studies have described variation in recombination rate within genomes, between individuals, between sexes, between populations and between species. At the same time, theoretical work has provided an increasingly detailed picture of the evolutionary advantages to recombination. Perhaps surprisingly, the causes of natural variation in recombination rate remain poorly understood. We argue that empirical and theoretical approaches to understand the evolution of recombination have proceeded largely independently of each other. Most models that address the evolution of recombination rate were created to explain the evolutionary advantage of recombination rather than quantitative differences in rate among individuals. Conversely, most empirical studies aim to describe variation in recombination rate, rather than to test evolutionary hypotheses. In this Perspective, we argue that efforts to integrate the rich bodies of empirical and theoretical work on recombination rate are crucial to moving this field forward. We provide new directions for the development of theory and the production of data that will jointly close this gap.This article is part of the themed issue 'Evolutionary causes and consequences of recombination rate variation in sexual organisms'.

Plastome Evolution in the Sole Hemiparasitic Genus Laurel Dodder (Cassytha) and Insights into the Plastid Phylogenomics of Lauraceae.

To date, little is known about the evolution of plastid genomes (plastomes) in Lauraceae. As one of the top five largest families in tropical forests, the Lauraceae contain many species that are important ecologically and economically. Lauraceous species also provide wonderful materials to study the evolutionary trajectory in response to parasitism because they contain both nonparasitic and parasitic species. This study compared the plastomes of nine Lauraceous species, including the sole hemiparasitic and herbaceous genus Cassytha (laurel dodder; here represented by Cassytha filiformis). We found differential contractions of the canonical inverted repeat (IR), resulting in two IR types present in Lauraceae. These two IR types reinforce Cryptocaryeae and Neocinnamomum—Perseeae–Laureae as two separate clades. Our data reveal several traits unique to Cas. filiformis, including loss of IRs, loss or pseudogenization of 11 ndh and rpl23 genes, richness of repeats, and accelerated rates of nucleotide substitutions in protein-coding genes. Although Cas. filiformis is low in chlorophyll content, our analysis based on dN/dS ratios suggests that both its plastid house-keeping and photosynthetic genes are under strong selective constraints. Hence, we propose that short generation time and herbaceous lifestyle rather than reduced photosynthetic ability drive the accelerated rates of nucleotide substitutions in Cas. filiformis.

Differential evolutionary patterns and expression levels between sex-specific and somatic tissue-specific genes in peanut

The patterns of evolution and expression of tissue-specific genes are poorly understood beyond sex-specific genes. Accordingly, we identified 3,191 tissue-specific genes and 38,745 common genes using 22 RNA-seq datasets from cultivated peanut. The expression levels of tissue-specific genes were significantly lower than those of common genes. Further, the expression levels of sex-specific genes were significantly higher than those of somatic tissue-specific genes. Among sex-specific genes, the expression levels of gynoecium-specific genes were significantly higher than those of androecium-specific genes. Function-specific genes were lacking among tissue-specific genes, and tissue-specific gene annotations overlapped among different tissues. Duplicate gene pairs were classified as homogeneous pairs expressed within the same tissue or heterogeneous pairs expressed in different tissues. Heterogeneous gene pairs evolved more rapidly than homogeneous gene pairs. In addition, somatic tissue-specific genes evolved faster than sex-specific genes. Molecular signatures of selection indicated that somatic tissue-specific genes have mainly experienced relaxed selection, while sex-specific genes have been under stronger selective constraint. Somatic tissue-specific genes had higher codon usage bias than sex-specific genes. These contrasting patterns between somatic tissue-specific and sex-specific genes provide new insights into the basic biology and evolution of peanut, an important crop.

Comparative Analyses of Selection Operating on Nontranslated Intergenic Regions of Diverse Bacterial Species.

Nontranslated intergenic regions (IGRs) compose 10-15% of bacterial genomes, and contain many regulatory elements with key functions. Despite this, there are few systematic studies on the strength and direction of selection operating on IGRs in bacteria using whole-genome sequence data sets. Here we exploit representative whole-genome data sets from six diverse bacterial species: Staphylococcus aureus, Streptococcus pneumoniae, Mycobacterium tuberculosis, Salmonella enterica, Klebsiella pneumoniae, and Escherichia coli We compare patterns of selection operating on IGRs using two independent methods: the proportion of singleton mutations and the dI/dS ratio, where dI is the number of intergenic SNPs per intergenic site. We find that the strength of purifying selection operating over all intergenic sites is consistently intermediate between that operating on synonymous and nonsynonymous sites. Ribosome binding sites and noncoding RNAs tend to be under stronger selective constraint than promoters and Rho-independent terminators. Strikingly, a clear signal of purifying selection remains even when all these major categories of regulatory elements are excluded, and this constraint is highest immediately upstream of genes. While a paucity of variation means that the data for M. tuberculosis are more equivocal than for the other species, we find strong evidence for positive selection within promoters of this species. This points to a key adaptive role for regulatory changes in this important pathogen. Our study underlines the feasibility and utility of gauging the selective forces operating on bacterial IGRs from whole-genome sequence data, and suggests that our current understanding of the functionality of these sequences is far from complete.

The Relationship between Selection, Network Connectivity, and Regulatory Variation within a Population of Capsella grandiflora.

Interactions between genes can have important consequences for how selection shapes sequence variation at these genes. Specifically, genes that have pleiotropic effects by affecting the expression level of many other genes may be under stronger selective constraint. We used coexpression networks to measure connectivity between genes and investigated the relationship between gene connectivity and selection in a natural population of the plant Capsella grandiflora. We observed that network connectivity was negatively correlated with genetic divergence due to stronger negative selection on highly-connected genes even when controlling for variation in gene expression level. However, the presence of local regulatory variation for a gene’s expression level was also associated with reduced negative selection and lower gene connectivity. While it is difficult to disentangle the causal relationships between these factors, our results show that both connectivity and local regulatory variation are important factors for explaining variation in selection between genes.

2b or Not 2b: Experimental Evolution of Functional Exogenous Sequences in a Plant RNA Virus.

Horizontal gene transfer (HGT) is pervasive in viruses and thought to be a key mechanism in their evolution. On the other hand, strong selective constraints against increasing genome size are an impediment for HGT, rapidly purging horizontally transferred sequences and thereby potentially hindering evolutionary innovation. Here, we explore experimentally the evolutionary fate of viruses with simulated HGT events, using the plant RNA virus Tobacco etch virus (TEV), by separately introducing two functional, exogenous sequences to its genome. One of the events simulates the acquisition of a new function though HGT of a conserved AlkB domain, responsible for the repair of alkylation or methylation damage in many organisms. The other event simulates the acquisition of a sequence that duplicates an existing function, through HGT of the 2b RNA silencing suppressor from Cucumber mosaic virus. We then evolved these two viruses, tracked the maintenance of the horizontally transferred sequences over time, and for the final virus populations, sequenced their genome and measured viral fitness. We found that the AlkB domain was rapidly purged from the TEV genome, restoring fitness to wild-type levels. Conversely, the 2b gene was stably maintained and did not have a major impact on viral fitness. Moreover, we found that 2b is functional in TEV, as it provides a replicative advantage when the RNA silencing suppression domain of HC-Pro is mutated. These observations suggest a potentially interesting role for HGT of short functional sequences in ameliorating evolutionary constraints on viruses, through the duplication of functions.

Network Analysis of Genome-Wide Selective Constraint Reveals a Gene Network Active in Early Fetal Brain Intolerant of Mutation.

Using robust, integrated analysis of multiple genomic datasets, we show that genes depleted for non-synonymous de novo mutations form a subnetwork of 72 members under strong selective constraint. We further show this subnetwork is preferentially expressed in the early development of the human hippocampus and is enriched for genes mutated in neurological Mendelian disorders. We thus conclude that carefully orchestrated developmental processes are under strong constraint in early brain development, and perturbations caused by mutation have adverse outcomes subject to strong purifying selection. Our findings demonstrate that selective forces can act on groups of genes involved in the same process, supporting the notion that purifying selection can act coordinately on multiple genes. Our approach provides a statistically robust, interpretable way to identify the tissues and developmental times where groups of disease genes are active.

Genetic Links between Recombination and Speciation

Genetic Links between Recombination and Speciation

MC1R diversity in Northern Island Melanesia has not been constrained by strong purifying selection and cannot explain pigmentation phenotype variation in the region.

BackgroundVariation in human skin pigmentation evolved in response to the selective pressure of ultra-violet radiation (UVR). Selection to maintain darker skin in high UVR environments is expected to constrain pigmentation phenotype and variation in pigmentation loci. Consistent with this hypothesis, the gene MC1R exhibits reduced diversity in African populations from high UVR regions compared to low-UVR non-African populations. However, MC1R diversity in non-African populations that have evolved under high-UVR conditions is not well characterized. MethodsIn order to test the hypothesis that MC1R variation has been constrained in Melanesians the coding region of the MC1R gene was sequenced in 188 individuals from Northern Island Melanesia. The role of purifying selection was assessed using a modified McDonald Kreitman’s test. Pairwise FST was calculated between Melanesian populations and populations from the 1000 Genomes Project. The SNP rs2228479 was genotyped in a larger sample (n = 635) of Melanesians and tested for associations with skin and hair pigmentation.ResultsWe observe three nonsynonymous and two synonymous mutations. A modified McDonald Kreitman’s test failed to detect a significant signal of purifying selection. Pairwise FST values calculated between the four islands sampled here indicate little regional substructure in MC1R. When compared to African, European, East and South Asian populations, Melanesians do not exhibit reduced population divergence (measured as FST) or a high proportion of haplotype sharing with Africans, as one might expect if ancestral haplotypes were conserved across high UVR populations in and out of Africa. The only common nonsynonymous polymorphism observed, rs2228479, is not significantly associated with skin or hair pigmentation in a larger sample of Melanesians.ConclusionsThe pattern of sequence diversity here does not support a model of strong selective constraint on MC1R in Northern Island Melanesia This absence of strong constraint, as well as the recent population history of the region, may explain the observed frequencies of the derived rs2228479 allele. These results emphasize the complex genetic architecture of pigmentation phenotypes, which are controlled by multiple, possibly interacting loci. They also highlight the role that population history can play in influencing phenotypic diversity in the absence of strong natural selection.Electronic supplementary materialThe online version of this article (doi:10.1186/s12863-015-0277-x) contains supplementary material, which is available to authorized users.

Evolution and divergence of SBP-box genes in land plants.

BackgroundSquamosa promoter binding protein (SBP)-box family genes encode plant-specific transcription factors that control many important biological functions, including phase transition, inflorescence branching, fruit ripening, and copper homeostasis. Nevertheless, the evolutionary patterns of SBP-box genes and evolutionary forces driving them are still not well understood.Methods104 SBP-box gene candidates of five representative land plants were obtained from Phytozome database (v10.3). Phylogenetic combined with gene structure analyses were used to identify SBP-box gene lineages in land plants. Gene copy number and the sequence and structure features were then compared among these different SBP-box lineages. Selection analysis, relative rate tests and expression divergence were finally used to interpret the evolutionary relationships and divergence of SBP-box genes in land plants.ResultsWe investigated 104 SBP-box genes from moss, Arabidopsis, poplar, rice, and maize. These genes are divided into group I and II, and the latter is further divided into two subgroups (subgroup II-1 and II-2) based on phylogenetic analysis. Interestingly, subgroup II-1 genes have similar sequence and structural features to group I genes, whereas subgroup II-2 genes exhibit intrinsic differences on these features, including high copy numbers and the presence of miR156/miR529 regulation. Further analyses indicate that subgroup II-1 genes are constrained by stronger purifying selection and evolve at a lower substitution rate than II-2 genes, just as group I genes do when compared to II genes. Among subgroup II-2 genes, miR156 targets evolve more rapidly than miR529 targets and experience comparatively relaxed purifying selection. These results suggest that group I and subgroup II-1 genes under strong selective constraint are conserved. By contrast, subgroup II-2 genes evolve under relaxed purifying selection and have diversified through gene copy duplications and changes in miR156/529 regulation, which might contribute to morphological diversifications of land plants.ConclusionsOur results indicate that different evolutionary rates and selection strengths lead to differing evolutionary patterns in SBP-box genes in land plants, providing a guide for future functional diversity analyses of these genes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1998-y) contains supplementary material, which is available to authorized users.

Inference of Evolutionary Forces Acting on Human Biological Pathways.

Because natural selection is likely to act on multiple genes underlying a given phenotypic trait, we study here the potential effect of ongoing and past selection on the genetic diversity of human biological pathways. We first show that genes included in gene sets are generally under stronger selective constraints than other genes and that their evolutionary response is correlated. We then introduce a new procedure to detect selection at the pathway level based on a decomposition of the classical McDonald–Kreitman test extended to multiple genes. This new test, called 2DNS, detects outlier gene sets and takes into account past demographic effects and evolutionary constraints specific to gene sets. Selective forces acting on gene sets can be easily identified by a mere visual inspection of the position of the gene sets relative to their two-dimensional null distribution. We thus find several outlier gene sets that show signals of positive, balancing, or purifying selection but also others showing an ancient relaxation of selective constraints. The principle of the 2DNS test can also be applied to other genomic contrasts. For instance, the comparison of patterns of polymorphisms private to African and non-African populations reveals that most pathways show a higher proportion of nonsynonymous mutations in non-Africans than in Africans, potentially due to different demographic histories and selective pressures.

Distinctive patterns of evolution of the δ-globin gene (HBD) in primates.

In most vertebrates, hemoglobin (Hb) is a heterotetramer composed of two dissimilar globin chains, which change during development according to the patterns of expression of α- and β-globin family members. In placental mammals, the β-globin cluster includes three early-expressed genes, ε(HBE)-γ(HBG)-ψβ(HBBP1), and the late expressed genes, δ (HBD) and β (HBB). While HBB encodes the major adult β-globin chain, HBD is weakly expressed or totally silent. Paradoxically, in human populations HBD shows high levels of conservation typical of genes under strong evolutionary constraints, possibly due to a regulatory role in the fetal-to-adult switch unique of Anthropoid primates. In this study, we have performed a comprehensive phylogenetic and comparative analysis of the two adult β-like globin genes in a set of diverse mammalian taxa, focusing on the evolution and functional divergence of HBD in primates. Our analysis revealed that anthropoids are an exception to a general pattern of concerted evolution in placental mammals, showing a high level of sequence conservation at HBD, less frequent and shorter gene conversion events. Moreover, this lineage is unique in the retention of a functional GATA-1 motif, known to be involved in the control of the developmental expression of the β-like globin genes. We further show that not only the mode but also the rate of evolution of the δ-globin gene in higher primates are strictly associated with the fetal/adult β-cluster developmental switch. To gain further insight into the possible functional constraints that have been shaping the evolutionary history of HBD in primates, we calculated dN/dS (ω) ratios under alternative models of gene evolution. Although our results indicate that HBD might have experienced different selective pressures throughout primate evolution, as shown by different ω values between apes and Old World Monkeys + New World Monkeys (0.06 versus 0.43, respectively), these estimates corroborated a constrained evolution for HBD in Anthropoid lineages, which is unlikely to be related to protein function. Collectively, these findings suggest that sequence change at the δ-globin gene has been under strong selective constraints over 65 Myr of primate evolution, likely due to a regulatory role in ontogenic switches of gene expression.