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.     

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.     

Local Adaptation of Bitter Taste and Ecological Speciation in a Wild Mammal

Sensory systems are attractive evolutionary models to address how organisms adapt to local environments that can cause ecological speciation. However, tests of these evolutionary models have focused on visual, auditory, and olfactory senses. Here, we show local adaptation of bitter taste receptor genes in two neighboring populations of a wild mammal—the blind mole rat Spalax galili—that show ecological speciation in divergent soil environments. We found that basalt-type bitter receptors showed higher response intensity and sensitivity compared with chalk-type ones using both genetic and cell-based functional analyses. Such functional changes could help animals adapted to basalt soil select plants with less bitterness from diverse local foods, whereas a weaker reception to bitter taste may allow consumption of a greater range of plants for animals inhabiting chalk soil with a scarcity of food supply. Our study shows divergent selection on food resources through local adaptation of bitter receptors, and suggests that taste plays an important yet underappreciated role in speciation.     

Receptor Polymorphism and Genomic Structure Interact to Shape Bitter Taste Perception

The ability to taste bitterness evolved to safeguard most animals, including humans, against potentially toxic substances, thereby leading to food rejection. Nonetheless, bitter perception is subject to individual variations due to the presence of genetic functional polymorphisms in bitter taste receptor (TAS2R) genes, such as the long-known association between genetic polymorphisms in TAS2R38 and bitter taste perception of phenylthiocarbamide. Yet, due to overlaps in specificities across receptors, such associations with a single TAS2R locus are uncommon. Therefore, to investigate more complex associations, we examined taste responses to six structurally diverse compounds (absinthin, amarogentin, cascarillin, grosheimin, quassin, and quinine) in a sample of the Caucasian population. By sequencing all bitter receptor loci, inferring long-range haplotypes, mapping their effects on phenotype variation, and characterizing functionally causal allelic variants, we deciphered at the molecular level how a subjects’ genotype for the whole-family of TAS2R genes shapes variation in bitter taste perception. Within each haplotype block implicated in phenotypic variation, we provided evidence for at least one locus harboring functional polymorphic alleles, e.g. one locus for sensitivity to amarogentin, one of the most bitter natural compounds known, and two loci for sensitivity to grosheimin, one of the bitter compounds of artichoke. Our analyses revealed also, besides simple associations, complex associations of bitterness sensitivity across TAS2R loci. Indeed, even if several putative loci harbored both high- and low-sensitivity alleles, phenotypic variation depended on linkage between these alleles. When sensitive alleles for bitter compounds were maintained in the same linkage phase, genetically driven perceptual differences were obvious, e.g. for grosheimin. On the contrary, when sensitive alleles were in opposite phase, only weak genotype-phenotype associations were seen, e.g. for absinthin, the bitter principle of the beverage absinth. These findings illustrate the extent to which genetic influences on taste are complex, yet arise from both receptor activation patterns and linkage structure among receptor genes.     

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.     

Functional Analyses of Bitter Taste Receptors in Domestic Cats (Felis catus)

Cats are obligate carnivores and under most circumstances eat only animal products. Owing to the pseudogenization of one of two subunits of the sweet receptor gene, they are indifferent to sweeteners, presumably having no need to detect plant-based sugars in their diet. Following this reasoning and a recent report of a positive correlation between the proportion of dietary plants and the number of Tas2r (bitter receptor) genes in vertebrate species, we tested the hypothesis that if bitter perception exists primarily to protect animals from poisonous plant compounds, the genome of the domestic cat (Felis catus) should have lost functional bitter receptors and they should also have reduced bitter receptor function. To test functionality of cat bitter receptors, we expressed cat Tas2R receptors in cell-based assays. We found that they have at least 7 functional receptors with distinct receptive ranges, showing many similarities, along with some differences, with human bitter receptors. To provide a comparative perspective, we compared the cat repertoire of intact receptors with those of a restricted number of members of the order Carnivora, with a range of dietary habits as reported in the literature. The numbers of functional bitter receptors in the terrestrial Carnivora we examined, including omnivorous and herbivorous species, were roughly comparable to that of cats thereby providing no strong support for the hypothesis that a strict meat diet influences bitter receptor number or function. Maintenance of bitter receptor function in terrestrial obligate carnivores may be due to the presence of bitter compounds in vertebrate and invertebrate prey, to the necessary role these receptors play in non-oral perception, or to other unknown factors. We also found that the two aquatic Carnivora species examined had fewer intact bitter receptors. Further comparative studies of factors driving numbers and functions of bitter taste receptors will aid in understanding the forces shaping their repertoire.     

Expression profiles and functional characterization of common carp (Cyprinus carpio) T2Rs

Bitter taste perception is mediated by a family of G protein-coupled receptors (T2Rs) in vertebrates. Common carp (Cyprinus carpio), which has experienced an additional round of whole genome duplication during the course of evolution, has a small number of T2R genes similar to zebrafish, a closely related cyprinid fish species, and their expression pattern at the cellular level or their cognate ligands have not been elucidated yet. Here, we showed through in situ hybridization experiments, that three common carp T2R (ccT2R) genes encoding ccT2R200-1, ccT2R202-1, and ccT2R202-2, were specifically expressed in the subsets of taste receptor cells in the lips and gill rakers. ccT2R200-1 was co-expressed with genes encoding downstream signal transduction molecules, such as PLC-β2 and Gαia. Heterologous expression system revealed that each ccT2R showed narrowly, intermediately, or broadly tuned ligand specificity, as in the case of zebrafish T2Rs. However, ccT2Rs showed different ligand profiles from their orthologous zebrafish T2Rs previously reported. Finally, we identified three ccT2Rs, namely ccT2R200-1, ccT2R200-2, and ccT2R203-1, to be activated by natural bitter compounds, andrographolide and/or picrotoxinin, which elicited no response to zebrafish T2Rs, in a dose-dependent manner. These results suggest that some ccT2Rs may have evolved to function in the oral cavity as taste receptors for natural bitter compounds found in the habitats in a species-specific manner.     

The chromosome-scale assembly of endive (Cichorium endivia) genome provides insights into the sesquiterpenoid biosynthesis

Endive (Cichorium endivia L.) is a leafy vegetable in the Asteraceae family. Sesquiterpene lactones (STLs) in endive leaves bring a bitter taste that varies between varieties. Despite their importance in breeding varieties with unique flavours, sesquiterpenoid biosynthesis pathways in endive are poorly understood. We assembled a chromosome-scale endive genome of 641 Mb with a contig N50 of 5.16 Mb and annotated 46,711 protein-coding genes. Several gene families, especially terpene synthases (TPS) genes, expanded significantly in the C. endivia genome. STLs biosynthesis-related genes and TPS genes in more bitter varieties have shown a higher level of expression, which could be attributed to genomic variations. Our results penetrate the origin and diversity of bitter taste and facilitate the molecular breeding of endive varieties with unique bitter tastes. The high-quality endive assembly would provide a reference genome for studying the evolution and diversity of Asteraceae.     

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.     

Denatonium as a bitter taste receptor agonist damages jejunal epithelial cells of yellow-feathered chickens via inducing apoptosis

The sense of bitter taste is critical for chickens to acquire and select feeds. It is important to understand the roles and mechanisms of bitter taste transduction in chickens. Denatonium is extensively used as a bitter taste receptor agonist to activate bitter taste receptors in recent studies. The objective of this study was to investigate the physiological effects and the potential molecular mechanisms of dietary exposure to a strong bitter taste receptor agonist on the jejunal epithelial cells of yellow-feathered chickens. A total of 240 yellow-feathered chickens were divided into four treatments receiving a normal diet (Control), a low-dose denatonium treatment (Control + 5 mg/kg denatonium), a middle-dose denatonium treatment (Control + 20 mg/kg denatonium) and a high-dose denatonium treatment (Control + 100 mg/kg denatonium) for 56 days, respectively. The results showed that dietary denatonium reduced (P < 0.05) the growth performance of chickens. High-dose denatonium damaged the morphology of the jejunal epithelium and decreased (P < 0.05) the activities of Ca²⁺-ATPase, sucrase and maltase after 56 days of exposure. Meanwhile, high-dose denatonium increased (P < 0.05) mRNA expressions of bitter taste receptors, which resulted in enhanced apoptosis in jejunal epithelial cells after 56 days of exposure. Furthermore, middle-dose and high-dose denatonium exhibited increased (P < 0.05) mRNA level of claudin 2 and decreased (P < 0.05) mRNA level of occludin after 28 days of exposure. Only high-dose denatonium decreased (P < 0.05) mRNA level of occludin after 56 days of exposure. In conclusion, denatonium manifested deleterious effects on the jejunum of chickens in a dose–effect manner via damaging the morphology of the jejunal epithelium, and inducing apoptosis associated with bitter taste receptors. Our data suggest that bitter-tasting feed additives may have side effects on the growth and development of intestines in chickens.     

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。     

Detection of selection signatures in dairy and beef cattle using high-density genomic information

Background

Artificial selection for economically important traits in cattle is expected to have left distinctive selection signatures on the genome. Access to high-density genotypes facilitates the accurate identification of genomic regions that have undergone positive selection. These findings help to better elucidate the mechanisms of selection and to identify candidate genes of interest to breeding programs.

Results

Information on 705 243 autosomal single nucleotide polymorphisms (SNPs) in 3122 dairy and beef male animals from seven cattle breeds (Angus, Belgian Blue, Charolais, Hereford, Holstein-Friesian, Limousin and Simmental) were used to detect selection signatures by applying two complementary methods, integrated haplotype score (iHS) and global fixation index (F_ST). To control for false positive results, we used false discovery rate (FDR) adjustment to calculate adjusted iHS within each breed and the genome-wide significance level was about 0.003. Using the iHS method, 83, 92, 91, 101, 85, 101 and 86 significant genomic regions were detected for Angus, Belgian Blue, Charolais, Hereford, Holstein-Friesian, Limousin and Simmental cattle, respectively. None of these regions was common to all seven breeds. Using the F_ST approach, 704 individual SNPs were detected across breeds. Annotation of the regions of the genome that showed selection signatures revealed several interesting candidate genes i.e. DGAT1, ABCG2, MSTN, CAPN3, FABP3, CHCHD7, PLAG1, JAZF1, PRKG2, ACTC1, TBC1D1, GHR, BMP2, TSG1, LYN, KIT and MC1R that play a role in milk production, reproduction, body size, muscle formation or coat color. Fifty-seven common candidate genes were found by both the iHS and global F_ST methods across the seven breeds. Moreover, many novel genomic regions and genes were detected within the regions that showed selection signatures; for some candidate genes, signatures of positive selection exist in the human genome. Multilevel bioinformatic analyses of the detected candidate genes suggested that the PPAR pathway may have been subjected to positive selection.

Conclusions

This study provides a high-resolution bovine genomic map of positive selection signatures that are either specific to one breed or common to a subset of the seven breeds analyzed. Our results will contribute to the detection of functional candidate genes that have undergone positive selection in future studies.

Electronic supplementary material

The online version of this article (doi:10.1186/s12711-015-0127-3) contains supplementary material, which is available to authorized users.     

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.     

Evolution of opsin genes reveals a functional role of vision in the echolocating little brown bat (Myotis lucifugus)

Echolocating bats are able to orientate, navigate and forage without visual cues. To probe the role of vision in bats, we studied the visual opsin genes from the echolocating little brown bat (Myotis lucifugus). Short-wavelength sensitive (SWS1) opsin, middle/long-wavelength sensitive (M/LWS) opsin and rhodopsin cDNA sequences were identified from the Ensembl database and validated by the sequencing of genomic DNA. We retrieved the published orthologous genes from eleven additional representative species of mammals from GenBank and conducted an evolutionary analysis. We found that the M/LWS opsin and rhodopsin genes were both under strong purifying selection, whereas the SWS1 opsin gene has undergone positive selection at two amino acid sites and one lineage, though the main evolutionary force is still purifying selection. Two-ratio model of the SWS1 opsin gene revealed that the ω ratio for the little brown bat lineage was nearly three times lower than the background ratio, suggesting a much stronger functional constraint. Our relative rate tests show the little brown bat has a lower nonsynonymous substitution rate than those in other mammals (on average 32% lower) for the SWS1 opsin gene. However, no such significant differences were detected for the M/LWS opsin and rhodopsin genes. The results of the relative ratio tests are consistent with that of tests for selection, showing a history of purifying selection on the little brown bat opsin genes. These findings suggest a functional role of vision in the little brown bat despite being nocturnal and using echolocation. We speculate that this echolocating bat may be able to use visual cues to orientate, navigate and forage at night, to discriminate color under moonlight and starlight conditions, or to avoid predation by diurnal raptors.     

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.     

Genetic diversity of bitter taste receptor gene family in Sichuan domestic and Tibetan chicken populations

The sense of bitter taste plays a critical role in animals as it can help them to avoid intake of toxic and harmful substances. Previous research had revealed that chicken has only three bitter taste receptor genes (Tas2r1, Tas2r2 and Tas2r7). To better understand the genetic polymorphisms and importance of bitter taste receptor genes (Tas2rs) in chicken, here, we sequenced Tas2rs of 30 Sichuan domestic chickens and 30 Tibetan chickens. Thirteen single-nucleotide polymorphisms (SNPs) including three nonsynonymous mutations (m.359G >C, m.503C >A and m.583A >G) were detected in Tas2r1 (m. is the abbreviation for mutation); three SNPs were detected in Tas2r2, but none of them were missense mutation; eight SNPs were detected in Tas2r7 including six nonsynonymous substitutions (m.178G >A, m.421A >C, m.787C >T, m.832G >T, m.907A >T and m.943G >A). Tajima’s D neutral test indicates that there is no population expansion in both populations, and the size of the population is relatively stable. All the three networks indicate that red jungle fowls share haplotypes with domestic chickens. In addition, we found that haplotypes H1 and HE1 were positively associated with high-altitude adaptation, whereas haplotypes H4 and HE4 showed a negative correlation with high-altitude adaptation in Tas2rs. Although, chicken has only three Tas2rs, our results showed that both Sichuan domestic chickens and Tibetan chickens have abundant haplotypes in Tas2rs, especially in Tas2r7, which might help chickens to recognize a wide variety of bitter-tasting compounds.     

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.     

The Visayan Warty Pig (Sus cebifrons) Genome Provides Insight Into Chromosome Evolution and Sensory Adaptation in Pigs

It is largely unknown how mammalian genomes evolve under rapid speciation and environmental adaptation. An excellent model for understanding fast evolution is provided by the genus Sus, which diverged relatively recently and lacks postzygotic isolation. Here, we present a high-quality reference genome of the Visayan warty pig, which is specialized to a tropical island environment. Comparing the genome sequences and chromatin contact maps of the Visayan warty pig (Sus cebifrons) and domestic pig (Sus scrofa), we characterized the dynamics of chromosomal structure evolution during Sus speciation, revealing the similar chromosome conformation as the potential biological mechanism of frequent postdivergence hybridization among Suidae. We further investigated the different signatures of adaptive selection and domestication in Visayan warty pig and domestic pig with specific emphasize on the evolution of olfactory and gustatory genes, elucidating higher olfactory diversity in Visayan warty pig and positive and relaxed evolution of bitter and fat taste receptors, respectively, in domestic pig. Our comprehensive evolutionary and comparative genome analyses provide insight into the dynamics of genomes and how these change over relative short evolutionary times, as well as how these genomic differences encode for differences in the phenotypes.