Strong Functional Constraints Research Articles

Rare ecomorphological convergence on a complex adaptive landscape: Body size and diet mediate evolution of jaw shape in squirrels (Sciuridae).

Convergence is widely regarded as compelling evidence for adaptation, often being portrayed as evidence that phenotypic outcomes are predictable from ecology, overriding contingencies of history. However, repeated outcomes may be very rare unless adaptive landscapes are simple, structured by strong ecological and functional constraints. One such constraint may be a limitation on body size because performance often scales with size, allowing species to adapt to challenging functions by modifying only size. When size is constrained, species might adapt by changing shape; convergent shapes may therefore be common when size is limiting and functions are challenging. We examine the roles of size and diet as determinants of jaw shape in Sciuridae. As expected, size and diet have significant interdependent effects on jaw shape and ecomorphological convergence is rare, typically involving demanding diets and limiting sizes. More surprising is morphological without ecological convergence, which is equally common between and within dietary classes. Those cases, like rare ecomorphological convergence, may be consequences of evolving on an adaptive landscape shaped by many-to-many relationships between ecology and function, many-to-one relationships between form and performance, and one-to-many relationships between functionally versatile morphologies and ecology. On complex adaptive landscapes, ecological selection can yield different outcomes.

Measles Virus

Measles virus (MeV) is exceptionally contagious and still a major cause of death in child.However, recently significant progress towards the elimination of measles has been made through increased vaccination coverage of measles-containing vaccines. The hemagglutinin (H) protein of MeV interacts with a cellular receptor, and this interaction is the first step of infection. MeV uses two different receptors, signaling lymphocyte activation molecule (SLAM) and nectin-4 expressed on immune cells and epithelial cells, respectively. The interactions of MeV with these receptors nicely explain the immune suppressive and high contagious properties of MeV. Binding of the H protein to a receptor triggers conformational changes in the fusion (F) protein, inducing fusion between viral and host plasma membranes for entry. The stalk region of the H protein plays a key role in the F protein-triggering. Recent studies of the H protein epitopes have revealed that the receptor binding site of the H protein constitutes a major neutralizing epitope. The interaction with two proteinaceous receptors probably imposes strong functional constraints on this epitope for amino acid changes. This would be a reason why measles vaccines, which are derived from MV strains isolated more than 60 years ago, are still highly effective against all MV strains currently circulating.

Molecular Evolution and Characterization of Hemagglutinin (H) in Peste des Petits Ruminants Virus.

Peste des Petits Ruminants (PPR) is an acute, highly contagious, and febrile viral disease that affects both domestic and wild small ruminants. The disease has become a major obstacle to the development of sustainable Agriculture. Hemagglutinin (H), the envelope glycoprotein of Peste des Petits Ruminants Virus (PPRV), plays a crucial role in regulating viral adsorption and entry, thus determining pathogenicity, and release of newly produced viral particles. In order to accurately understand the epidemic of the disease and the interactions between the virus and host, we launch the work. Here, we examined H gene from all four lineages of the PPRV to investigate evolutionary and epidemiologic dynamics of PPRV by the Bayesian method. In addition, we predicted positive selection sites due to selective pressures. Finally, we studied the interaction between H protein and SLAM receptor based on homology model of the complex. Phylogenetic analysis suggested that H gene can also be used to investigate evolutionary and epidemiologic dynamics of PPRV. Positive selection analysis identified four positive selection sites in H gene, in which only one common site (aa246) was detected by two methods, suggesting strong operation structural and/or functional constraint of changes on the H protein. This target site may be of interest for future mutagenesis studies. The results of homology modeling showed PPRVHv-shSLAM binding interface and MVH-maSLAM binding interface were consistent, wherein the groove in the B4 blade and B5 of the head domain of PPRVHv bound to the AGFCC′ β-sheets of the membrane-distal ectodomain of shSLAM. The binding regions could provide insight on the nature of the protein for epitope vaccine design, novel drug discovery, and rational drug design against PPRV.

Molecular Evolution and Functional Divergence of Trace Amine-Associated Receptors.

Trace amine-associated receptors (TAARs) are a member of the G-protein-coupled receptor superfamily and are known to be expressed in olfactory sensory neurons. A limited number of molecular evolutionary studies have been done for TAARs so far. To elucidate how lineage-specific evolution contributed to their functional divergence, we examined 30 metazoan genomes. In total, 493 TAAR gene candidates (including 84 pseudogenes) were identified from 26 vertebrate genomes. TAARs were not identified from non-vertebrate genomes. An ancestral-type TAAR-like gene appeared to have emerged in lamprey. We found four therian-specific TAAR subfamilies (one eutherian-specific and three metatherian-specific) in addition to previously known nine subfamilies. Many species-specific TAAR gene duplications and losses contributed to a large variation of TAAR gene numbers among mammals, ranging from 0 in dolphin to 26 in flying fox. TAARs are classified into two groups based on binding preferences for primary or tertiary amines as well as their sequence similarities. Primary amine-detecting TAARs (TAAR1-4) have emerged earlier, generally have single-copy orthologs (very few duplication or loss), and have evolved under strong functional constraints. In contrast, tertiary amine-detecting TAARs (TAAR5-9) have emerged more recently and the majority of them experienced higher rates of gene duplications. Protein members that belong to the tertiary amine-detecting TAAR group also showed the patterns of positive selection especially in the area surrounding the ligand-binding pocket, which could have affected ligand-binding activities and specificities. Expansions of the tertiary amine-detecting TAAR gene family may have played important roles in terrestrial adaptations of therian mammals. Molecular evolution of the TAAR gene family appears to be governed by a complex, species-specific, interplay between environmental and evolutionary factors.

Constrained body shape among highly genetically divergent allopatric lineages of the supralittoral isopod Ligia occidentalis (Oniscidea).

Multiple highly divergent lineages have been identified within Ligia occidentalis sensu lato, a rocky supralittoral isopod distributed along a ~3000km latitudinal gradient that encompasses several proposed marine biogeographic provinces and ecoregions in the eastern Pacific. Highly divergent lineages have nonoverlapping geographic distributions, with distributional limits that generally correspond with sharp environmental changes. Crossbreeding experiments suggest postmating reproductive barriers exist among some of them, and surveys of mitochondrial and nuclear gene markers do not show evidence of hybridization. Populations are highly isolated, some of which appear to be very small; thus, the effects of drift are expected to reduce the efficiency of selection. Large genetic divergences among lineages, marked environmental differences in their ranges, reproductive isolation, and/or high isolation of populations may have resulted in morphological differences in L. occidentalis, not detected yet by traditional taxonomy. We used landmark-based geometric morphometric analyses to test for differences in body shape among highly divergent lineages of L. occidentalis, and among populations within these lineages. We analyzed a total of 492 individuals from 53 coastal localities from the southern California Bight to Central Mexico, including the Gulf of California. We conducted discriminant function analyses (DFAs) on body shape morphometrics to assess morphological variation among genetically differentiated lineages and their populations. We also tested for associations between phylogeny and morphological variation, and whether genetic divergence is correlated to multivariate morphological divergence. We detected significant differences in body shape among highly divergent lineages, and among populations within these lineages. Nonetheless, neither lineages nor populations can be discriminated on the basis of body shape, because correct classification rates of cross-validated DFAs were low. Genetic distance and phylogeny had weak to no effect on body shape variation. The supralittoral environment appears to exert strong stabilizing selection and/or strong functional constraints on body shape in L. occidentalis, thereby leading to morphological stasis in this isopod.

Phylogenetic significance of chaetal arrangement and chaetogenesis in Maldanidae (Annelida)

Chaetae are important structures to facilitate locomotion in annelids. Being at the interface between the organisms and its environment, chaetae are supposed to underlie strong functional constraints to optimize the relation between structure and function. As such chaetae are potentially susceptible for convergent evolution. On the other hand, chaetae gained enormous taxonomic importance due to their conservative structure in species and supraspecific taxa which reasonably can only be explained by strong evolutionary constrains that conserve their structure. In this paper, we study the chaetation and chaetogenesis in two species of Maldanidae, Clymenura clypeata Saint-Joseph 1894 and Johnstonia clymenoides Quatrefages 1866 to unravel conservative traits in their structure and development. In a literature survey across maldanids, we address questions on the ontogenetic variation, on homology and on the phylogenetic significance especially of the bearded hooked neurochaetae. We provide evidence that functionally constraint ontogenetic variation overlies historically (phylogenetically) constraint expression of structural information and can show that within maldanids a variety of different chaetal types must be homologous due to their ontogenetic continuity. Furthermore, we use chaetation and chaetal characters to discuss the subgroup relationships within Maldanomorpha in the light of recent cladistics analyses based on morphological and molecular data. This study shows that functional considerations need to use phylogenies as backbone.

Floral miniaturisation and autogamy in boreal-arctic plants are epitomised by Iceland's most frequent orchid, Platanthera hyperborea.

Background and Aims. This paper concludes our series of publications comparing island and mainland speciation in European butterfly-orchids, by studying the morphology, phylogenetics and reproductive biology of the controversial circum-arctic species Platanthera (Limnorchis) hyperborea—the most frequent of seven Icelandic orchids. We draw particular attention to its phylogenetic placement, remarkable reproductive biology and morphological convergence on other Platanthera lineages through floral miniaturisation.Methods. Five populations of P. hyperborea in southwest Iceland were measured for 33 morphological characters and subjected to detailed multivariate and univariate analyses, supported by light and scanning electron microscopy of selected flowers. Representative samples from six populations were sequenced for nrITS and placed in a taxonomically broader phylogenetic matrix derived from previous studies.Key Results . Section Limnorchis consists of three distinct ITS-delimited clades based on P. stricta, P. sparsifolia–limosa–aquilonis and P. dilatata–hyperborea. Within the latter group, supposed species boundaries overlap; instead, the data indicate a crude stepwise series of ribotypic transitions extending eastward from North America to Iceland. Morphometric data failed to identify any taxonomically meaningful partitions among Icelandic P. hyperborea populations, despite the presence of a distinct and apparently plesiomorphic ribotype at the most glacially influenced habitat sampled. Microscopic study of the flowers revealed several distinguishing features (some not previously reported), including resupinate lateral sepals, toothed bract margins, club-shaped papillae shared by both the interior of the labellar spur and the stigmatic surface, and an exceptionally adhesive stigma that is reliably covered in disaggregated pollen masses prior to anthesis; auricles are absent.Conclusions. Ribotypes suggest that Icelandic P. hyperborea represents the terminus of a migration route that may have begun in East Asia before passing through North America and presumably Greenland. The incohesive pollinia, rapidly desiccating anther locules, weakly developed rostellum, exceptionally adhesive stigma and the close juxtaposition of compact male and female reproductive organs together conspire to cause routine autogamy and frequent cleistogamy, despite the continued production of substantial nectar reservoirs in the spur and consequent ongoing attraction to the flowers of insects, including mosquitoes. When considered in combination with independently derived lineages of Platanthera on the Azorean and Hawaiian archipelagos also bearing small green flowers, our observations show allometric and paedomorphic reductions in flower size as the primary evolutionary driver, but also indicate strong developmental and functional constraints.

Sequence analysis of the Ras-MAPK pathway genes SOS1, EGFR & GRB2 in silver foxes (Vulpes vulpes): candidate genes for hereditary hyperplastic gingivitis.

Hereditary hyperplastic gingivitis (HHG) is an autosomal recessive disease that presents with progressive gingival proliferation in farmed silver foxes. Hereditary gingival fibromatosis (HGF) is an analogous condition in humans that is genetically heterogeneous with several known autosomal dominant loci. For one locus the causative mutation is in the Son of sevenless homologue 1 (SOS1) gene. For the remaining loci, the molecular mechanisms are unknown but Ras pathway involvement is suspected. Here we compare sequences for the SOS1 gene, and two adjacent genes in the Ras pathway, growth receptor bound protein 2 (GRB2) and epidermal growth factor receptor (EGFR), between HHG-affected and unaffected foxes. We conclude that the known HGF causative mutation does not cause HHG in foxes, nor do the coding regions or intron-exon boundaries of these three genes contain any candidate mutations for fox gum disease. Patterns of molecular evolution among foxes and other mammals reflect high conservation and strong functional constraints for SOS1 and GRB2 but reveal a lineage-specific pattern of variability in EGFR consistent with mutational rate differences, relaxed functional constraints, and possibly positive selection.

Vampire bats exhibit evolutionary reduction of bitter taste receptor genes common to other bats.

The bitter taste serves as an important natural defence against the ingestion of poisonous foods and is thus believed to be indispensable in animals. However, vampire bats are obligate blood feeders that show a reduced behavioural response towards bitter-tasting compounds. To test whether bitter taste receptor genes (T2Rs) have been relaxed from selective constraint in vampire bats, we sampled all three vampire bat species and 11 non-vampire bats, and sequenced nine one-to-one orthologous T2Rs that are assumed to be functionally conserved in all bats. We generated 85 T2R sequences and found that vampire bats have a significantly greater percentage of pseudogenes than other bats. These results strongly suggest a relaxation of selective constraint and a reduction of bitter taste function in vampire bats. We also found that vampire bats retain many intact T2Rs, and that the taste signalling pathway gene Calhm1 remains complete and intact with strong functional constraint. These results suggest the presence of some bitter taste function in vampire bats, although it is not likely to play a major role in food selection. Together, our study suggests that the evolutionary reduction of bitter taste function in animals is more pervasive than previously believed, and highlights the importance of extra-oral functions of taste receptor genes.

Origins and functional diversification of salinity‐responsive Na+, K+ATPase α1 paralogs in salmonids

The Salmoniform whole-genome duplication is hypothesized to have facilitated the evolution of anadromy, but little is known about the contribution of paralogs from this event to the physiological performance traits required for anadromy, such as salinity tolerance. Here, we determined when two candidate, salinity-responsive paralogs of the Na(+) , K(+) ATPase α subunit (α1a and α1b) evolved and studied their evolutionary trajectories and tissue-specific expression patterns. We found that these paralogs arose during a small-scale duplication event prior to the Salmoniform, but after the teleost, whole-genome duplication. The 'freshwater paralog' (α1a) is primarily expressed in the gills of Salmoniformes and an unduplicated freshwater sister species (Esox lucius) and experienced positive selection in the freshwater ancestor of Salmoniformes and Esociformes. Contrary to our predictions, the 'saltwater paralog' (α1b), which is more widely expressed than α1a, did not experience positive selection during the evolution of anadromy in the Coregoninae and Salmonine. To determine whether parallel mutations in Na(+) , K(+) ATPase α1 may contribute to salinity tolerance in other fishes, we studied independently evolved salinity-responsive Na(+) , K(+) ATPase α1 paralogs in Anabas testudineus and Oreochromis mossambicus. We found that a quarter of the mutations occurring between salmonid α1a and α1b in functionally important sites also evolved in parallel in at least one of these species. Together, these data argue that paralogs contributing to salinity tolerance evolved prior to the Salmoniform whole-genome duplication and that strong selection and/or functional constraints have led to parallel evolution in salinity-responsive Na(+) , K(+) ATPase α1 paralogs in fishes.

Genetically Diverse Variants of Sugarcane bacilliform virus Infecting Sugarcane in India and Evidence of a Novel Recombinant Badnavirus Variant

AbstractSugarcane bacilliform viruses (SCBV; genus Badnavirus) cause leaf fleck disease in sugarcane worldwide. SCBV was detected in 28 sugarcane cultivars originating from eight states of India. Eight representative SCBV isolates from five different states showed sequence variability up to 27% in the reverse transcriptase and RNase H (RT/RNase H) genetic region. Five isolates [SCBV‐AP (Co693077): HQ324248, SCBV‐Assam (CoBIN94063): HQ324246, SCBV‐Bihar (Bo141): HQ324245, SCBV‐UP (Co1424): KC013276 and SCBV‐UP (CoSe6460): KC013277] showed sequence identity of 86–88% with Sugarcane bacilliform IM virus (SCBIMV), but the other three isolates [SCBV‐Kerala (Co7219): HQ324249, SCBV‐TN (Co86032): GQ385038 and SCBV‐UP (CoSe92423): HQ324247] showed maximum sequence identity of 75–79% with Sugarcane bacilliform MO virus (SCBMOV). In phylogenetic analysis, the SCBV isolates segregated into two new subclades and were distinct from the known SCBV genotypes. The rates of non‐synonymous and synonymous (dN/dS) substitution indicated the signs of purifying selection with strong functional constraints for RT/RNase H region in SCBV population. A variant (SCBV‐UP, CoSe92423) was identified to be a recombinant isolate having two other Indian SCBV isolates as parents. Although RT/RNase H region is a recombination cold spot, a strong recombination might have played a key role in the evolution of this new variant of SCBV. Our study provides an insight into the diverse genetic structure of SCBV population and presence of a novel recombinant SCBV species/variant infecting sugarcane cultivars in India. Our results will be helpful in devising a robust detection procedure for quarantine and field testing of sugarcane germplasm.

Genome-wide Comparative Analysis of the GRAS Gene Family in Populus, Arabidopsis and Rice

GRAS genes belong to a gene family of transcription regulators that function in the regulation of plant growth and development. Our knowledge about the expansion and diversification of this gene family in flowering plants is presently limited to the herbaceous species Arabidopsis and rice. Numerous aspects, including the phylogenetic history, expansion, functional divergence and adaptive evolution await further study, especially in woody tree species. Based on the latest genome assemblies, we found 106, 34 and 60 putative GRAS genes in Populus, Arabidopsis and rice, respectively. Phylogenetic analysis revealed that GRAS proteins could be divided into at least 13 subfamilies. Tandem and segmental duplications are the most common expansion mechanisms of this gene family, and their frequent joint action may explain the rapid expansion in Populus. Site-specific shifts in evolutionary rates might be the main force driving subfamily-specific functional diversification. Adaptive evolution analysis revealed that GRAS genes have evolved mainly under purifying selection after duplication, suggesting that strong functional constraints have a bearing on the evolution of GRAS genes. Both expressed sequence tags (EST) and microarray data revealed that GRAS genes in Populus have broad expression patterns across a variety of organs/tissues. Expression divergence analyses between paralogous pairs of GRAS genes suggested that the retention of GRAS genes after duplication could be mainly attributed to substantial functional novelty such as neo-functionalization or sub-functionalization. Our study highlights the expansion and diversification of the GRAS gene family in Populus and provides the first comprehensive analysis of this gene family in the Populus genome.

Slow Co-Evolution of the MAGO and Y14 Protein Families Is Required for the Maintenance of Their Obligate Heterodimerization Mode

The exon junction complex (EJC) plays important roles in RNA metabolisms and the development of eukaryotic organisms. MAGO (short form of MAGO NASHI) and Y14 (also Tsunagi or RBM8) are the EJC core components. Their biological roles have been well investigated in various species, but the evolutionary patterns of the two gene families and their protein-protein interactions are poorly known. Genome-wide survey suggested that the MAGO and Y14 two gene families originated in eukaryotic organisms with the maintenance of a low copy. We found that the two protein families evolved slowly; however, the MAGO family under stringent purifying selection evolved more slowly than the Y14 family that was under relative relaxed purifying selection. MAGO and Y14 were obliged to form heterodimer in a eukaryotic organism, and this obligate mode was plesiomorphic. Lack of binding of MAGO to Y14 as functional barrier was observed only among distantly species, suggesting that a slow co-evolution of the two protein families. Inter-protein co-evolutionary signal was further quantified in analyses of the Tol-MirroTree and co-evolution analysis using protein sequences. About 20% of the 41 significantly correlated mutation groups (involving 97 residues) predicted between the two families was clade-specific. Moreover, around half of the predicted co-evolved groups and nearly all clade-specific residues fell into the minimal interaction domains of the two protein families. The mutagenesis effects of the clade-specific residues strengthened that the co-evolution is required for obligate MAGO-Y14 heterodimerization mode. In turn, the obliged heterodimerization in an organism serves as a strong functional constraint for the co-evolution of the MAGO and Y14 families. Such a co-evolution allows maintaining the interaction between the proteins through large evolutionary time scales. Our work shed a light on functional evolution of the EJC genes in eukaryotes, and facilitates to understand the co-evolutionary processes among protein families.

Using Pseudogene Database to Identify Lineage-Specific Genes and Pseudogenes in Humans and Chimpanzees

It has been revealed that gene content changes, or gene gains or losses, have played an important role in the evolution of modern humans. As one of the major players accounting for gene content changes, gene pseudogenization is abundant in mammalian genomes, and approximately 20000 pseudogenes have been identified in ape genomes. Therefore, it is an interesting question how to exploit rich information embedded in pseudogenes. Here, I present a bioinformatic pipeline that utilizes a pseudogene database to identify both lineage-specific genes and pseudogenes in humans and chimpanzees. I found 6 human-specific gene gains (HSGs), 1 chimpanzee-specific gene gain, and 4 chimpanzee-specific pseudogenes, most not discovered in previous studies. Further analysis showed that HSGs have been evolving under strong purifying selection and are broadly expressed, indicating strong functional constraint. This study demonstrates the usage of pseudogene information in comparative genomics and suggests that new genes during primate evolution may acquire essential functions in a short time. The pipeline developed here could also be applied to other species.

Molecular Evolution Constraints in the Floral Organ Specification Gene Regulatory Network Module across 18 Angiosperm Genomes

The gene regulatory network of floral organ cell fate specification of Arabidopsis thaliana is a robust developmental regulatory module. Although such finding was proposed to explain the overall conservation of floral organ types and organization among angiosperms, it has not been confirmed that the network components are conserved at the molecular level among flowering plants. Using the genomic data that have accumulated, we address the conservation of the genes involved in this network and the forces that have shaped its evolution during the divergence of angiosperms. We recovered the network gene homologs for 18 species of flowering plants spanning nine families. We found that all the genes are highly conserved with no evidence of positive selection. We studied the sequence conservation features of the genes in the context of their known biological function and the strength of the purifying selection acting upon them in relation to their placement within the network. Our results suggest an association between protein length and sequence conservation, evolutionary rates, and functional category. On the other hand, we found no significant correlation between the strength of purifying selection and gene placement. Our results confirm that the studied robust developmental regulatory module has been subjected to strong functional constraints. However, unlike previous studies, our results do not support the notion that network topology plays a major role in constraining evolutionary rates. We speculate that the dynamical functional role of genes within the network and not just its connectivity could play an important role in constraining evolution.

Purifying selection after episodes of recurrent adaptive diversification in fungal pathogens

Tests of selective neutrality based on comparative and population genetic data target different time scales of selection. Investigating both time scales on the same genes has rarely been done and yet can provide insights into histories of selection. In pathogens, such a comparison can elucidate whether the same genes experience recurrent positive selection across specialization events onto novel hosts and selection in the short term, e.g. by coevolution with a current host. Here, we investigated whether the genes showing signs of positive selection (based on dN/dS ratios) between pathogens specialized on different hosts also exhibited footprints of recent positive selection. We sequenced 58 DNA fragments in the model fungal plant pathogen Microbotryum lychnidis-dioicae infecting Silene latifolia. Eleven focal genes were chosen because they showed a significant signal of positive selection in a previous study and putative functions likely involved in host–parasite interactions. In addition, 47 control genes were randomly chosen among genes showing no evidence of positive selection. We used a population genetics approach to search for signatures of recent selection, controlling for population structure and demographic history. We found footprints of purifying selection in all focal genes and of recent positive selection in two of them. Signs of purifying selection were also found at the same eleven genes in a closely related species, Microbotryum silenes-dioicae, infecting Silene dioica. These results suggest that genes experiencing episodes of adaptive diversification during host shifts may subsequently be under strong functional constraint, although some can remain under positive selection in the short term.

Evolutionary constraints in the β-globin cluster: the signature of purifying selection at the δ-globin (HBD) locus and its role in developmental gene regulation.

Human hemoglobins, the oxygen carriers in the blood, are composed by two α-like and two β-like globin monomers. The β-globin gene cluster located at 11p15.5 comprises one pseudogene and five genes whose expression undergoes two critical switches: the embryonic-to-fetal and fetal-to-adult transition. HBD encodes the δ-globin chain of the minor adult hemoglobin (HbA2), which is assumed to be physiologically irrelevant. Paradoxically, reduced diversity levels have been reported for this gene. In this study, we sought a detailed portrait of the genetic variation within the β-globin cluster in a large human population panel from different geographic backgrounds. We resequenced the coding and noncoding regions of the two adult β-globin genes (HBD and HBB) in European and African populations, and analyzed the data from the β-globin cluster (HBE, HBG2, HBG1, HBBP1, HBD, and HBB) in 1,092 individuals representing 14 populations sequenced as part of the 1000 Genomes Project. Additionally, we assessed the diversity levels in nonhuman primates using chimpanzee sequence data provided by the PanMap Project. Comprehensive analyses, based on classic neutrality tests, empirical and haplotype-based studies, revealed that HBD and its neighbor pseudogene HBBP1 have mainly evolved under purifying selection, suggesting that their roles are essential and nonredundant. Moreover, in the light of recent studies on the chromatin conformation of the β-globin cluster, we present evidence sustaining that the strong functional constraints underlying the decreased contemporary diversity at these two regions were not driven by protein function but instead are likely due to a regulatory role in ontogenic switches of gene expression.

Evolutionary analysis of the short-type peptidoglycan-recognition protein gene (PGLYRP1) in primates

Short-type peptidoglycan (PGN)-recognition protein 1 (PGLYRP1), an innate immunity protein that directly breaks down the structure of microbial cell wall PGNs, plays an important role both in antibacterial defenses and several inflammatory diseases. To explore the adaptive evolution of the PGLYRP1 gene in primates and provide insight into the function of this antibacterial protein, we sequenced the entire PGLYRP1 gene from Macaca thibetana and Rhinopithecus roxellana, identified the corresponding sequences from the draft genome of 8 other primates, including humans, and conducted related statistical analyses. Homology analysis showed that the identity of nucleotide and deduced amino acid sequences of PGLYRP1 among 10 primates ranged from 82.0 to 99.0% and 74.5 to 98.5%, respectively. The R value (transition/transversion) and disparity index per site also presented relatively low-base composition biases. Selective pressure analysis for the PGLYRP1 sequences among major primates revealed that both the whole gene and the substructure of PGLYRP1 are under strong purifying selection at similar levels of selective pressure among 6 major primate lineages (human, great ape, lesser ape, Old World monkey, New World monkey, and prosimian monkey). Using the Bayes empirical Bayes procedure, we also detected 2 positively selected codons (121L and 141T sites) that are independent of PGN-binding and PGLYRP-specific regions, implying 2 potential key sites for the functional effect of the PGLYRP1 protein. These results demonstrated that PGLYRP1 was highly conserved at the molecular level and subjected to strong functional constraints during primate evolution.

Characterization and comprehensive analysis of the miiuy croaker TLR2 reveals a direct evidence for intron insert and loss

Toll-like receptor 2 (TLR2) is a member of an ancient pattern recognition receptor family, conserved from insects to mammals and it is best known as a receptor for recognizing conserved components of Gram-positive bacteria. In present study, the genomic structure of TLR2 gene from miiuy croaker was identified and characterized. It comprises twelve exons and eleven introns. The lengths of exons 3 to 10 of miiuy croaker TLR2 and exons 2 to 9 of fugu and pufferfish TLR2 are exactly the same, but most importantly, both of fugu and pufferfish have only eleven exons and ten introns. An intron insert event probably happened on exon 1 of miiuy croaker TLR2 after its divergence from ancestor of zebrafish, and an intron loss event probably happened on those of Tetraodontiformes TLR2 after the divergence with ancestor of miiuy croaker. Our study showed the direct evidence and strongly supported the intron insert and loss on fish TLR2. The pathogen injection experiments indicated that TLR2 might not be an important responder to Gram-negative bacteria in miiuy croaker. Molecular evolutionary analyses indicated TLR2 genes were under strong purifying selection pressure, showing a quite strong functional constraint in both of fish and mammals, despite of their distinct living environment conditions.

Evidence of purifying selection on merozoite surface protein 8 (MSP8) and 10 (MSP10) in Plasmodium spp.

Evidence for natural selection, positive or negative, on gene encoding antigens may indicate variation or functional constraints that are immunologically relevant. Most malaria surface antigens with high genetic diversity have been reported to be under positive-diversifying selection. However, antigens with limited genetic variation are usually ignored in terms of the role that natural selection may have in generating such patterns. We investigated orthologous genes encoding two merozoite proteins, MSP8 and MSP10, among several mammalian Plasmodium spp. These antigens, together with MSP1, are among the few MSPs that have two epidermal growth factor-like domains (EGF) at the C-terminal. Those EGF are relatively conserved (low levels of genetic polymorphism) and have been proposed to act as ligands during the invasion of RBCs. We use several evolutionary genetic methods to detect patterns consistent with natural selection acting on MSP8 and MSP10 orthologs in the human parasites Plasmodium falciparum and P. vivax, as well as closely related malarial species found in non-human primates (NHPs). Overall, these antigens have low polymorphism in the human parasites in comparison with the orthologs from other Plasmodium spp. We found that the MSP10 gene polymorphism in P. falciparum only harbor non-synonymous substitutions, a pattern consistent with a gene under positive selection. Evidence of purifying selection was found on the polymorphism observed in both orthologs from P. cynomolgi, a non-human primate parasite closely related to P. vivax, but it was not conclusive in the human parasite. Yet, using phylogenetic base approaches, we found evidence for purifying selection on both MSP8 and MSP10 in the lineage leading to P. vivax. Such antigens evolving under strong functional constraints could become valuable vaccine candidates. We discuss how comparative approaches could allow detecting patterns consistent with negative selection even when there is low polymorphism in the extant populations.