Contrasting modes of evolution between vertebrate sweet/umami receptor genes and bitter receptor genes

Taste reception is fundamental to diet selection in many animals. The genetic basis underlying the evolution and diversity of taste reception, however, is not well understood. Recent discoveries of T1R sweet/umami receptor genes and T2R bitter receptor genes in humans and mice provided an opportunity to address this question. Here, we report the identification of 20 putatively functional T1R genes and 167 T2R genes from the genome sequences of nine vertebrates, including three fishes, one amphibian, one bird, and four mammals. Our comparative genomic analysis shows that orthologous T1R sequences are relatively conserved in evolution and that the T1R gene repertoire remains virtually constant in size across most vertebrates, except for the loss of the T1R2 sweet receptor gene in the sweet-insensitive chicken and the absence of all T1R genes in the tongueless western clawed frog. In contrast, orthologous T2R sequences are more variable, and the T2R repertoire diverges tremendously among species, from only three functional genes in the chicken to 49 in the frog. These evolutionary patterns suggest the relative constancy in the number and type of sweet and umami tastants encountered by various vertebrates or low binding specificities of T1Rs but a large variation in the number and type of bitter compounds detected by different species. Although the rate of gene duplication is much lower in T1Rs than in T2Rs, signals of positive selection are detected during the functional divergences of paralogous T1Rs, as was previously found among paralogous T2Rs. Thus, functional divergence and specialization of taste receptors generally occurred via adaptive evolution.     

Dynamic evolution of bitter taste receptor genes in vertebrates

Background  

Sensing bitter tastes is crucial for many animals because it can prevent them from ingesting harmful foods. This process is mainly mediated by the bitter taste receptors (T2R), which are largely expressed in the taste buds. Previous studies have identified some T2R gene repertoires, and marked variation in repertoire size has been noted among species. However, the mechanisms underlying the evolution of vertebrate T2R genes remain poorly understood.     

The evolution of pepsinogen C genes in vertebrates: duplication, loss and functional diversification

Background

Aspartic proteases comprise a large group of enzymes involved in peptide proteolysis. This collection includes prominent enzymes globally categorized as pepsins, which are derived from pepsinogen precursors. Pepsins are involved in gastric digestion, a hallmark of vertebrate physiology. An important member among the pepsinogens is pepsinogen C (Pgc). A particular aspect of Pgc is its apparent single copy status, which contrasts with the numerous gene copies found for example in pepsinogen A (Pga). Although gene sequences with similarity to Pgc have been described in some vertebrate groups, no exhaustive evolutionary framework has been considered so far.

Methodology/Principal Findings

By combining phylogenetics and genomic analysis, we find an unexpected Pgc diversity in the vertebrate sub-phylum. We were able to reconstruct gene duplication timings relative to the divergence of major vertebrate clades. Before tetrapod divergence, a single Pgc gene tandemly expanded to produce two gene lineages (Pgbc and Pgc2). These have been differentially retained in various classes. Accordingly, we find Pgc2 in sauropsids, amphibians and marsupials, but not in eutherian mammals. Pgbc was retained in amphibians, but duplicated in the ancestor of amniotes giving rise to Pgb and Pgc1. The latter was retained in mammals and probably in reptiles and marsupials but not in birds. Pgb was kept in all of the amniote clade with independent episodes of loss in some mammalian species. Lineage specific expansions of Pgc2 and Pgbc have also occurred in marsupials and amphibians respectively. We find that teleost and tetrapod Pgc genes reside in distinct genomic regions hinting at a possible translocation.

Conclusions

We conclude that the repertoire of Pgc genes is larger than previously reported, and that tandem duplications have modelled the history of Pgc genes. We hypothesize that gene expansion lead to functional divergence in tetrapods, coincident with the invasion of terrestrial habitats.     

Positive Selection Drives the Evolution of Bat Bitter Taste Receptor Genes

Bitter taste reception is expected to be associated with dietary selection and to prevent animals from ingesting potentially harmful compounds. To investigate the genetic basis of bitter taste reception, we reconfirmed the bitter taste receptor (T2R) genes from cow (herbivore) and dog (carnivore) genome sequences and identified the T2R repertoire from the draft genome of the bat (insectivore) for the first time using an automatic data-mining method. We detected 28 bitter receptor genes from the bat genome, including 9 intact genes, 8 partial but putative functional genes, and 9 pseudogenes. In the phylogenetic analysis, most of the T2R genes from the three species intermingle across the tree, suggesting that some are conserved among mammals with different dietary preferences. Furthermore, one clade of bat-specific genes was detected, possibly implying that the insectivorous mammal could recognize some species-specific bitter tastants. Evolutionary analysis shows strong positive selection was imposed on this bat-specific cluster, indicating that positive selection drives the functional divergence and specialization of the bat bitter taste receptors to adapt diets to the external environment.     

Adaptive diversification of bitter taste receptor genes in Mammalian evolution

The diversity and evolution of bitter taste perception in mammals is not well understood. Recent discoveries of bitter taste receptor (T2R) genes provide an opportunity for a genetic approach to this question. We here report the identification of 10 and 30 putative T2R genes from the draft human and mouse genome sequences, respectively, in addition to the 23 and 6 previously known T2R genes from the two species. A phylogenetic analysis of the T2R genes suggests that they can be classified into three main groups, which are designated A, B, and C. Interestingly, while the one-to-one gene orthology between the human and mouse is common to group B and C genes, group A genes show a pattern of species- or lineage-specific duplication. It is possible that group B and C genes are necessary for detecting bitter tastants common to both humans and mice, whereas group A genes are used for species-specific bitter tastants. The analysis also reveals that phylogenetically closely related T2R genes are close in their chromosomal locations, demonstrating tandem gene duplication as the primary source of new T2Rs. For closely related paralogous genes, a rate of nonsynonymous nucleotide substitution significantly higher than the rate of synonymous substitution was observed in the extracellular regions of T2Rs, which are presumably involved in tastant-binding. This suggests the role of positive selection in the diversification of newly duplicated T2R genes. Because many natural poisonous substances are bitter, we conjecture that the mammalian T2R genes are under diversifying selection for the ability to recognize a diverse array of poisons that the organisms may encounter in exploring new habitats and diets.     

Two families of candidate taste receptors in fishes

Vertebrates receive tastants, such as sugars, amino acids, and nucleotides, via taste bud cells in epithelial tissues. In mammals, two families of G protein-coupled receptors for tastants are expressed in taste bud cells-T1Rs for sweet tastants and umami tastants (l-amino acids) and T2Rs for bitter tastants. Here, we report two families of candidate taste receptors in fish species, fish T1Rs and T2Rs, which show significant identity to mammalian T1Rs and T2Rs, respectively. Fish T1Rs consist of three types: fish T1R1 and T1R3 that show the highest degrees of identity to mammalian T1R1 and T1R3, respectively, and fish T1R2 that shows almost equivalent identity to both mammalian T1R1 and T1R2. Unlike mammalian T1R2, fish T1R2 consists of two or three members in each species. We also identified two fish T2Rs that show low degrees of identity to mammalian T2Rs. In situ hybridization experiments revealed that fish T1R and T2R genes were expressed specifically in taste bud cells, but not in olfactory receptor cells. Fish T1R1 and T1R2 genes were expressed in different subsets of taste bud cells, and fish T1R3 gene was co-expressed with either fish T1R1 or T1R2 gene as in the case of mammals. There were also a significant number of cells expressing fish T1R2 genes only. Fish T2R genes were expressed in different cells from those expressing fish T1R genes. These results suggest that vertebrates commonly have two kinds of taste signaling pathways that are defined by the types of taste receptors expressed in taste receptor cells.     

Evolutionary insights into umami,sweet, and bitter taste receptors in amphibians

Umami and sweet sensations provide animals with important dietary information for detecting and consuming nutrients, whereas bitter sensation helps animals avoid potentially toxic or harmful substances. Enormous progress has been made toward animal sweet/umami taste receptor (Tas1r) and bitter taste receptor (Tas2r). However, information about amphibians is mainly scarce. This study attempted to delineate the repertoire of Tas1r/Tas2r genes by searching for currently available genome sequences in 14 amphibian species. This study identified 16 Tas1r1, 9 Tas1r2, and 9 Tas1r3 genes to be intact and another 17 Tas1r genes to be pseudogenes or absent in the 14 amphibians. According to the functional prediction of Tas1r genes, two species have lost sweet sensation and seven species have lost both umami and sweet sensations. Anurans possessed a large number of intact Tas2rs, ranging from 39 to 178. In contrast, caecilians possessed a contractive bitter taste repertoire, ranging from 4 to 19. Phylogenetic and reconciling analysis revealed that the repertoire of amphibian Tas1rs and Tas2rs was shaped by massive gene duplications and losses. No correlation was found between feeding preferences and the evolution of Tas1rs in amphibians. However, the expansion of Tas2rs may help amphibians adapt to both aquatic and terrestrial habitats. Bitter detection may have played an important role in the evolutionary adaptation of vertebrates in the transition from water to land.     

Lineage-specific loss of function of bitter taste receptor genes in humans and nonhuman primates

Since the process of becoming dead genes or pseudogenes (pseudogenization) is irreversible and can occur rather rapidly under certain environmental circumstances, it is one plausible determinant for characterizing species specificity. To test this evolutionary hypothesis, we analyzed the tempo and mode of duplication and pseudogenization of bitter taste receptor (T2R) genes in humans as well as in 12 nonhuman primates. The results show that primates have accumulated more pseudogenes than mice after their separation from the common ancestor and that lineage-specific pseudogenization becomes more conspicuous in humans than in nonhuman primates. Although positive selection has operated on some amino acids in extracellular domains, functional constraints against T2R genes are more relaxed in primates than in mice and this trend has culminated in the rapid deterioration of the bitter-tasting capability in humans. Since T2R molecules play an important role in avoiding generally bitter toxic and harmful substances, substantial modification of the T2R gene repertoire is likely to reflect different responses to changes in the environment and to result from species-specific food preference during primate evolution.     

Evolutionary diversification of the vertebrate transferrin multi-gene family

In a phylogenetic analysis of vertebrate transferrins (TFs), six major clades (subfamilies) were identified: (a) S, the mammalian serotransferrins; (b) ICA, the mammalian inhibitor of carbonic anhydrase (ICA) homologs; (c) L, the mammalian lactoferrins; (d) O, the ovotransferrins of birds and reptiles; (e) M, the melanotransferrins of bony fishes, amphibians, reptiles, birds, and mammals; and (f) M-like, a newly identified TF subfamily found in bony fishes, amphibians, reptiles, and birds. A phylogenetic tree based on the joint alignment of N-lobes and C-lobes supported the hypothesis that three separate events of internal duplication occurred in vertebrate TFs: (a) in the common ancestor of the M subfamily, (b) in the common ancestor of the M-like subfamily, and (c) in the common ancestor of other vertebrate TFs. The S, ICA, and L subfamilies were found only in placental mammals, and the phylogenetic analysis supported the hypothesis that these three subfamilies arose by gene duplication after the divergence of placental mammals from marsupials. The M-like subfamily was unusual in several respects, including the presence of a uniquely high proportion of clade-specific conserved residues, including distinctive but conserved residues in the sites homologous to those functioning in carbonate binding of human serotransferrin. The M-like family also showed an unusually high proportion of cationic residues in the positively charged region corresponding to human lactoferrampin, suggesting a distinctive role of this region in the M-like subfamily, perhaps in antimicrobial defense.     

Evolutionary Dynamics of the OR Gene Repertoire in Teleost Fishes: Evidence of an Association with Changes in Olfactory Epithelium Shape

Teleost fishes perceive their environment through a range of sensory modalities, among which olfaction often plays an important role. Richness of the olfactory repertoire depends on the diversity of receptors coded by homologous genes classified into four families: OR, TAAR, VR1, and VR2. Herein, we focus on the OR gene repertoire. While independent large contractions of the OR gene repertoire associated with ecological transitions have been found in mammals, little is known about the diversity of the OR gene repertoire and its evolution in teleost fishes, a group that includes more than 34,000 living species. We analyzed genomes of 163 species representing diversity in this large group. We found a large range of variation in the number of functional OR genes, from 15 in the Broad-nose Pipefish Syngnathus typhle and the Ocean Sunfish Mola mola, to 429 in the Zig-zag Eel Mastacembelus armatus. The number of OR genes was higher in species when a multilamellar olfactory rosette was present. Moreover, the number of lamellae was correlated with the richness of the OR gene repertoire. While a slow and balanced birth-and-death process generally drives the evolution of the OR gene repertoire, we inferred several episodes of high rates of gene loss, sometimes followed by large gains in the number of OR genes. These gains coincide with morphological changes of the olfactory organ and suggest a strong functional association between changes in the morphology and the evolution of the OR gene repertoire.     

T2Rs function as bitter taste receptors

Bitter taste perception provides animals with critical protection against ingestion of poisonous compounds. In the accompanying paper, we report the characterization of a large family of putative mammalian taste receptors (T2Rs). Here we use a heterologous expression system to show that specific T2Rs function as bitter taste receptors. A mouse T2R (mT2R-5) responds to the bitter tastant cycloheximide, and a human and a mouse receptor (hT2R-4 and mT2R-8) responded to denatonium and 6-n-propyl-2-thiouracil. Mice strains deficient in their ability to detect cycloheximide have amino acid substitutions in the mT2R-5 gene; these changes render the receptor significantly less responsive to cycloheximide. We also expressed mT2R-5 in insect cells and demonstrate specific tastant-dependent activation of gustducin, a G protein implicated in bitter signaling. Since a single taste receptor cell expresses a large repertoire of T2Rs, these findings provide a plausible explanation for the uniform bitter taste that is evoked by many structurally unrelated toxic compounds.     

Purifying selection and positive selection on the myxovirus resistance gene in mammals and chickens

The myxovirus resistance gene (Mx) expresses antiviral activity in many species, e.g. mouse, human and chicken. It is not clear if the antiviral activity of Mx has evolved in these species to inhibit a set of species-specific pathogens, nor what factors drive Mx evolution in different animal lineages. Therefore, it is important to determine the evolutionary pattern of Mx and positively selected sites which affect the antiviral activity of the Mx gene in mammals and birds. We used sequence comparisons among species to detect positively selected sites by conducting phylogenetic analysis. The two-ratio model was significantly better than the one-ratio model in four species (mouse, rat, chicken and duck, p<0.05). Although selection pressure varied among different lineages, Mx had strong purifying selection in mammals and positive selection in chicken and duck lineages. Relative rate test revealed that Mx evolved faster in chickens than in ducks (Tajima's relative rate test, chi(2)=7.17, p<0.01). In the further analysis using a branch-site model A test, 8 sites were positively selected in the chicken lineage while no positive selection signals were observed for any site in the other lineages. The branch-site model A test had a omega value of 4.374 for the chicken lineage (2Deltal=14.20, d.f.=1, p<0.001). Comparisons of all currently available Mx mRNA sequences showed that these predicted positively selected sites had been fixed in the chicken lineage, suggesting that the chicken Mx gene evolved within the species to resist newly challenging environments. There is an increased selection constraint leading to mammals, while positive selection has acted on the chicken Mx.     

Variations in mitochondrial tRNA gene organization of reptiles as phylogenetic markers

Amplification and sequencing of mitochondrial DNA regions corresponding to three major clusters of transfer RNA genes from a variety of species representing major groups of birds and reptiles revealed some new variations in tRNA gene organization. First, a gene rearrangement from tRNA(His)-tRNA(Ser)(AGY)-tRNA(Leu)(CUN) to tRNA(Ser)(AGY)- tRNA(His)tRNA(Leu)(CUN) occurs in all three crocodilians examined (alligator, caiman, and crocodile). In addition an exceptionally long spacer region between the genes for NADH dehydrogenase subunit 4 and tRNA(Ser)(AGY) is found in caiman. Second, in congruence with a recent finding by Seutin et al., a characteristic stem-and-loop structure for the putative light-strand replication origin located between tRNA(Asn) and tRNA(Cys) genes is absent for all the birds and crocodilians. This stem-and-loop structure is absent in an additional species, the Texas blind snake, whereas the stem-and-loop structure is present in other snakes, lizards, turtles, mammals, and a frog. The disappearance of the stem-and-loop structure in the blind snake most likely occurred independently of that on the lineage leading to birds and crocodilians. Finally, the blind snake has a novel type of tRNA gene arrangement in which the tRNA(Gln) gene moved from one tRNA cluster to another. Sequence substitution rates for the tRNA genes appeared to be somewhat higher in crocodialians than in birds and mammals. As regards the controversial phylogenetic relationship among the Aves, Crocodilia, and Mammalia, a sister group relationship of birds and crocodilians relative to mammals, as suggested from the common loss of the stem-and- loop structure, was supported with statistical significance by molecular phylogenetic analyses using the tRNA gene sequence data.      

The mammalian alphaD-globin gene lineage and a new model for the molecular evolution of alpha-globin gene clusters at the stem of the mammalian radiation

We have explored the evolution of the alpha-globin gene family by comparative sequence and phylogenetic analyses of mammalian alpha-globin genes. Our analyses reveal the existence of a new alpha-globin gene lineage in mammals that is related to the alpha(D)-globin genes of birds, squamates and turtles. The gene is located in the middle of the alpha-globin gene cluster of a marsupial, Sminthopsis macroura and of humans. It exists in a wide variety of additional mammals, including pigs, cows, cats, and dogs, but is a pseudogene in American marsupials. Evolutionary analyses suggest that the gene has generally evolved under purifying selection, indicative of a functional gene. The presence of mRNA products in humans, pigs, and cows also suggest that the gene is expressed and likely to be functional. The analyses support the hypothesis that the alpha(D)-globin gene lineage has an ancient evolutionary origin that predates the divergence of amniotes. The structural similarity of alpha-globin gene clusters of marsupials and humans suggest that an eight gene cluster (5'-zeta2-zeta1-alpha(D)-alpha3-alpha2-alpha1-theta-omega-3'), including seven alpha-like genes and one beta-like globin gene (omega-globin) existed in the common ancestor of all marsupial and eutherian mammals. This basic structure has remained relatively stable in marsupials and in the lineage leading to humans, although omega-globin has been lost from the alpha-globin gene cluster of humans.     

Ferritins in Chordata: Potential evolutionary trajectory marked by discrete selective pressures

Ferritins (FTs) are iron storage proteins that are involved in managing iron‐oxygen balance. In our work, we present a hypothesis on the putative effect of geological changes that have affected the evolution and radiation of ferritin proteins. Based on sequence analysis and phylogeny reconstruction, we hypothesize that two significant factors have been involved in the evolution of ferritin proteins: fluctuations of atmospheric oxygen concentrations, altering redox potential, and changing availability of water rich in bioavailable ferric ions. Fish, ancient amphibians, reptiles, and placental mammals developed the broadest repertoire of singular FTs, attributable to embryonic growth in aquatic environments containing low oxygen levels and abundant forms of soluble iron. In contrast, oviparous land vertebrates, like reptiles and birds, that have developed in high oxygen levels and limited levels of environmental Fe²⁺ exhibit a lower diversity of singular FTs, but display a broad repertoire of subfamilies, particularly notable in early reptiles.     

A novel family of mammalian taste receptors

In mammals, taste perception is a major mode of sensory input. We have identified a novel family of 40-80 human and rodent G protein-coupled receptors expressed in subsets of taste receptor cells of the tongue and palate epithelia. These candidate taste receptors (T2Rs) are organized in the genome in clusters and are genetically linked to loci that influence bitter perception in mice and humans. Notably, a single taste receptor cell expresses a large repertoire of T2Rs, suggesting that each cell may be capable of recognizing multiple tastants. T2Rs are exclusively expressed in taste receptor cells that contain the G protein alpha subunit gustducin, implying that they function as gustducin-linked receptors. In the accompanying paper, we demonstrate that T2Rs couple to gustducin in vitro, and respond to bitter tastants in a functional expression assay.     

灵长类苦味受体基因研究进展

在鲜味、甜味、苦味、咸味和酸味5种味觉形式中,苦味能避免动物摄入有毒有害物质,在动物的生存中发挥着特别重要的作用。苦味味觉的产生依赖于苦味物质与苦味受体的相互作用。苦味受体由苦味受体基因Tas2rs编码,此类基因在不同物种中数量变化较大以适应不同的需求。目前的研究在灵长类中鉴别出了若干苦味受体的配体,并发现有的苦味受体基因所经受的选择压在类群之间、基因之间甚至同一基因不同功能区之间都存在着变化。本文从苦味受体作用的多样性特点,受体与配体的对应关系、受体基因进化模式与食性之间的关系、苦味受体基因的适应性进化方面对灵长类苦味受体基因进行了综述,以期为苦味受体基因在灵长类中的深入研究提供参考。     

Diversification of bitter taste receptor gene family in western chimpanzees

In mammals, bitter taste is mediated by T2R genes, which belong to the large family of seven transmembrane G protein-coupled receptors. Because T2Rs are directly involved in the interaction between mammals and their dietary sources, it is likely that these genes evolved to reflect species' specific diets during mammalian evolution. Here, we investigated the sequences of all 28 putative functional chimpanzee T2R genes (cT2Rs) in 46 western chimpanzees to compare the intraspecies variations in chimpanzees to those already known for all 25 human functional T2R genes (hT2Rs). The numbers of functional genes varied among individuals in western chimpanzees, and most chimpanzees had two or three more functional genes than humans. Similarly to hT2Rs, cT2Rs showed high nucleotide diversity along with a large number of amino acid substitutions. Comparison of the nucleotide substitution patterns in cT2Rs with those in five cT2R pseudogenes and 14 autosomal intergenic noncoding regions among the same individuals revealed that the evolution of cT2R genes was almost identical to that of putative neutral regions with slight but significantly positive Tajima's D values, suggesting that selective constraint on these genes was relaxed with weak balancing selection. These trends have resulted in the occurrence of various divergent alleles of T2Rs within the western chimpanzee populations and in heterozygous individuals who might have the ability to taste a broader range of substances.     

Identification of novel genes for bitter taste receptors in sheep (Ovis aries)

Genetic studies on taste sensitivity, and bitter taste receptors (T2R) in particular, are an essential tool to understand ingestive behavior and its relation to variations of nutritional status occurring in ruminants. In the present study, we conducted a data-mining search to identify T2R candidates in sheep by comparison with the described T2R in cattle and using recently available ovine genome. In sheep, we identified eight orthologs of cattle genes: T2R16, T2R10B, T2R12, T2R3, T2R4, T2R67, T2R13 and T2R5. The in silico predicted genes were then confirmed by PCR and DNA sequencing. The sequencing results showed a 99% to 100% similarity with the in silico predicted sequence. Moreover, we address the chromosomal distribution and compare, in homology and phylogenetic terms, the obtained genes with the known T2R in human, mouse, dog, cattle, horse and pig. The eight novel genes identified map either to ovine chromosome 3 or 4. The phylogenetic data suggest a clustering by receptor type rather than by species for some of the receptors. From the species analyzed, we observed a clear proximity between the two ruminant species, sheep and cattle, in contrast with lower similarities obtained for the comparison of sheep with other mammals. Although further studies are needed to identify the complete T2R repertoire in domestic sheep, our data represent a first step for genetic studies on this field.     

Identification and characterization of human taste receptor genes belonging to the TAS2R family

The sense of taste is a chemosensory system responsible for basic food appraisal. Humans distinguish between five primary tastes: bitter, sweet, sour, salty and umami. The molecular events in the perception of bitter taste are believed to start with the binding of specific water-soluble molecules to G-protein-coupled receptors encoded by the TAS2R/T2R family of taste receptor genes. TAS2R receptors are expressed at the surface of taste receptor cells and are coupled to G proteins and second messenger pathways. We have identified, cloned and characterized 11 new bitter taste receptor genes and four new pseudogenes that belong to the human TAS2R family. Their encoded proteins have between 298 and 333 amino acids and share between 23 and 86% identity with other human TAS2R proteins. Screening of a mono-chromosomal somatic cell hybrid panel to assign the identified bitter taste receptor genes to human chromosomes demonstrated that they are located in chromosomes 7 and 12. Including the 15 sequences identified, the human TAS2R family is composed of 28 full-length genes and 16 pseudogenes. Phylogenetic analyses suggest a classification of the TAS2R genes in five groups that may reflect a specialization in the detection of specific types of bitter chemicals.