Evolutionary origins of bitter taste receptors in jawed vertebrates.

Taste is a sense that detects information about nutrients and toxins in foods. Of the five basic taste qualities, bitterness is associated with the detection of potentially harmful substances like plant alkaloids. In bony vertebrates, type 2 taste receptors (T2Rs), which are G-protein-coupled receptors (GPCRs), act as bitter taste receptors1,2. In vertebrates, six GPCR gene families are described as chemosensory receptor genes, encoding taste receptor families (T1Rs and T2Rs) and olfactory receptor families (ORs, V1Rs, V2Rs, and TAARs). These families of receptors have been found in all major jawed vertebrate lineages, except for the T2Rs, which are confined to bony vertebrates3. Therefore, T2Rs are believed to have emerged later than the other chemosensory receptor genes in the bony vertebrate lineage. So far, only the genomes of two cartilaginous fish species have been mined for TAS2R genes, which encode T2Rs4. Here, we identified novel T2Rs in elasmobranchs, namely selachimorphs (sharks) and batoids (rays, skates, and their close relatives) by an exhaustive search covering diverse cartilaginous fishes. Using functional and mRNA expression analyses, we demonstrate that their T2Rs are expressed in the oral taste buds and contribute to the detection of bitter compounds. This finding indicates the early origin of T2Rs in the common ancestor of jawed vertebrates.

Color vision evolution in egg-laying mammals: insights from visual photoreceptors and daily activities of Australian echidnas.

Egg-laying mammals (monotremes) are considered "primitive" due to traits such as oviparity, cloaca, and incomplete homeothermy, all of which they share with reptiles. Two groups of monotremes, the terrestrial echidna (Tachyglossidae) and semiaquatic platypus (Ornithorhynchidae), have evolved highly divergent characters since their emergence in the Cenozoic era. These evolutionary differences, notably including distinct electrosensory and chemosensory systems, result from adaptations to species-specific habitat conditions. To date, very few studies have examined the visual adaptation of echidna and platypus. In the present study, we show that echidna and platypus have different light absorption spectra in their dichromatic visual sensory systems at the molecular level. We analyzed absorption spectra of monotreme color opsins, long-wavelength sensitive opsin (LWS) and short-wavelength sensitive opsin 2 (SWS2). The wavelength of maximum absorbance (λmax) in LWS was 570.2 in short-beaked echidna (Tachyglossus aculeatus) and 560.6 nm in platypus (Ornithorhynchus anatinus); in SWS2, λmax was 451.7 and 442.6 nm, respectively. Thus, the spectral range in echidna color vision is ~ 10 nm longer overall than in platypus. Natural selection analysis showed that the molecular evolution of monotreme color opsins is generally functionally conserved, suggesting that these taxa rely on species-specific color vision. In order to understand the usage of color vision in monotremes, we made 24-h behavioral observations of captive echidnas at warm temperatures and analyzed the resultant ethograms. Echidnas showed cathemeral activity and various behavioral repertoires such as feeding, traveling, digging, and self-grooming without light/dark environment selectivity. Halting (careful) behavior is more frequent in dark conditions, which suggests that echidnas may be more dependent on vision during the day and olfaction at night. Color vision functions have contributed to dynamic adaptations and dramatic ecological changes during the ~ 60 million years of divergent monotreme evolution. The ethogram of various day and night behaviors in captive echidnas also contributes information relevant to habitat conservation and animal welfare in this iconic species, which is locally endangered.

Functional Diversity and Evolution of Bitter Taste Receptors in Egg-Laying Mammals.

Egg-laying mammals (monotremes) are a sister clade of therians (placental mammals and marsupials) and a key clade to understand mammalian evolution. They are classified into platypus and echidna, which exhibit distinct ecological features such as habitats and diet. Chemosensory genes, which encode sensory receptors for taste and smell, are believed to adapt to the individual habitats and diet of each mammal. In this study, we focused on the molecular evolution of bitter taste receptors (TAS2Rs) in monotremes. The sense of bitter taste is important to detect potentially harmful substances. We comprehensively surveyed agonists of all TAS2Rs in platypus (Ornithorhynchus anatinus) and short-beaked echidna (Tachyglossus aculeatus) and compared their functions with orthologous TAS2Rs of marsupial and placental mammals (i.e., therians). As results, the agonist screening revealed that the deorphanized monotreme receptors were functionally diversified. Platypus TAS2Rs had broader receptive ranges of agonists than those of echidna TAS2Rs. While platypus consumes a variety of aquatic invertebrates, echidna mainly consumes subterranean social insects (ants and termites) as well as other invertebrates. This result indicates that receptive ranges of TAS2Rs could be associated with feeding habits in monotremes. Furthermore, some orthologous receptors in monotremes and therians responded to ß-glucosides, which are feeding deterrents in plants and insects. These results suggest that the ability to detect ß-glucosides and other substances might be shared and ancestral among mammals.

Functional divergence of the pigmentation gene melanocortin-1 receptor (MC1R) in six endemic Macaca species on Sulawesi Island.

Coat color is often highly variable within and between animal taxa. Among hundreds of pigmentation-related genes, melanocortin-1 receptor (MC1R) plays key roles in regulating the synthesis of the dark eumelanin and the red-yellow pheomelanin. The six species of macaques that inhabit Sulawesi Island diverged rapidly from their common ancestor, M. nemestrina. Unlike most macaques, Sulawesi macaques commonly have a dark coat color, with divergence in shade and color pattern. To clarify the genetic and evolutionary basis for coat color in Sulawesi macaques, we investigated the MC1R sequences and functional properties, including basal cAMP production and α-MSH-induced activity in vitro. We found fixed non-synonymous substitutions in MC1R in each species. Furthermore, we found that six species-specific variants corresponded with variation in agonist-induced and basal activity of MC1R. Inconsistent with the dark coat color, four substitutions independently caused decreases in the basal activity of MC1R in M. hecki, M. nigra, M. tonkeana, and M. ochreata. Selective analysis suggested MC1R of M. nigra and M. nigrescens underwent purifying selection. Overall, our results suggest that fixed differences in MC1R resulted in different functional characteristics and might contribute to divergence in color among the six Sulawesi macaque species.

Noninsect-Based Diet Leads to Structural and Functional Changes of Acidic Chitinase in Carnivora.

Acidic chitinase (Chia) digests the chitin of insects in the omnivorous stomach and the chitinase activity in carnivorous Chia is significantly lower than that of the omnivorous enzyme. However, mechanistic and evolutionary insights into the functional changes in Chia remain unclear. Here we show that a noninsect-based diet has caused structural and functional changes in Chia during the course of evolution in Carnivora. By creating mouse-dog chimeric Chia proteins and modifying the amino acid sequences, we revealed that F214L and A216G substitutions led to the dog enzyme activation. In 31 Carnivora, Chia was present as a pseudogene with stop codons in the open reading frame (ORF) region. Importantly, the Chia proteins of skunk, meerkat, mongoose, and hyena, which are insect-eating species, showed high chitinolytic activity. The cat Chia pseudogene product was still inactive even after ORF restoration. However, the enzyme was activated by matching the number and position of Cys residues to an active form and by introducing five meerkat Chia residues. Mutations affecting the Chia conformation and activity after pseudogenization have accumulated in the common ancestor of Felidae due to functional constraints. Evolutionary analysis indicates that Chia genes are under relaxed selective constraint in species with noninsect-based diets except for Canidae. These results suggest that there are two types of inactivating processes in Carnivora and that dietary changes affect the structure and activity of Chia.

Evolution of the primate glutamate taste sensor from a nucleotide sensor.

Taste perception plays an essential role in food selection. Umami (savory) tastes are sensed by a taste receptor complex, T1R1/T1R3, that detects proteinogenic amino acids.1 High sensitivity to l-glutamate (l-Glu) is a characteristic of human T1R1/T1R3, but the T1R1/T1R3 of other vertebrates does not consistently show this l-Glu response.1,2 Here, we demonstrate that the l-Glu sensitivity of T1R1/T1R3 is a derived state that has evolved repeatedly in large primates that rely on leaves as protein sources, after their divergence from insectivorous ancestors. Receptor expression experiments show that common amino acid substitutions at ligand binding sites that render T1R1/T1R3 sensitive to l-Glu occur independently at least three times in primate evolution. Meanwhile T1R1/T1R3 senses 5'-ribonucleotides as opposed to l-Glu in several mammalian species, including insectivorous primates. Our chemical analysis reveal that l-Glu is one of the major free amino acids in primate diets and that insects, but not leaves, contain large amounts of free 5'-ribonucleotides. Altering the ligand-binding preference of T1R1/T1R3 from 5'-ribonucleotides to l-Glu might promote leaf consumption, overcoming bitter and aversive tastes. Altogether, our results provide insight into the foraging ecology of a diverse mammalian radiation and help reveal how evolution of sensory genes facilitates invasion of new ecological niches.

Lowered sensitivity of bitter taste receptors to ß-glucosides in bamboo lemurs: an instance of parallel and adaptive functional decline in TAS2R16?

Bitter taste facilitates the detection of potentially harmful substances and is perceived via bitter taste receptors (TAS2Rs) expressed on the tongue and oral cavity in vertebrates. In primates, TAS2R16 specifically recognizes ß-glucosides, which are important in cyanogenic plants' use of cyanide as a feeding deterrent. In this study, we performed cell-based functional assays for investigating the sensitivity of TAS2R16 to ß-glucosides in three species of bamboo lemurs (Prolemur simus, Hapalemur aureus and H. griseus), which primarily consume high-cyanide bamboo. TAS2R16 receptors from bamboo lemurs had lower sensitivity to ß-glucosides, including cyanogenic glucosides, than that of the closely related ring-tailed lemur (Lemur catta). Ancestral reconstructions of TAS2R16 for the bamboo-lemur last common ancestor (LCA) and that of the Hapalemur LCA showed an intermediate sensitivity to ß-glucosides between that of the ring-tailed lemurs and bamboo lemurs. Mutagenetic analyses revealed that P. simus and H. griseus had separate species-specific substitutions that led to reduced sensitivity. These results indicate that low sensitivity to ß-glucosides at the cellular level-a potentially adaptive trait for feeding on cyanogenic bamboo-evolved independently after the Prolemur-Hapalemur split in each species.

Response to Drea et al.

We greatly appreciate the critical comments on our paper made by Drea et al. [1]. We would like to emphasize that we are not claiming or giving concrete evidence that the identified compounds are pheromones in our paper. We agree that before we can reasonably conclude that the identified compounds are indeed pheromones, we would at least need to examine whether the responses to the identified compounds are stereotypical and reproducible and exclude the effects of signature differences, such as health, relatedness and genetic quality. To this end, it will be necessary to investigate a broader range of behaviors in the future using a larger number of animals.

Response to Kappeler.

We sincerely appreciate the constructive comments made by Peter Kappeler [1] regarding our paper, "Key male glandular odorants attracting female ring-tailed lemurs" [2]. We largely agree with the points raised in these comments, and believe these should be considered as critical discussion that would enable a more reasonable assessment of our findings.

Key Male Glandular Odorants Attracting Female Ring-Tailed Lemurs.

Among rodents, information about the external world is mainly acquired via the olfactory system, which is one of five sensory modalities. Several semiochemical signals are used for inter- and intraspecies communication [1]. In contrast, primates are generally regarded as vision-oriented mammals, and have been thought to trade their olfactory sensitivity for good sight. However, strepsirrhines have a well-developed olfactory system [2] and a larger repertoire of functional olfactory and vomeronasal receptor genes than haplorhines [3, 4]. Moreover, strepsirrhines are well known for their use of olfactory communication in social behavior. Ring-tailed lemurs are a species of Malagasy strepsirrhines, and use olfactory cues for conspecific communication. Male lemurs mark their scent by spreading volatiles from the antebrachial gland on their wrists. This study combined ethological and chemical approaches to identify a key odorant(s) in antebrachial secretions involved in the sexual communication of lemurs. The results of a behavioral assay indicated that females sniff the males' antebrachial secretions longer during the breeding season than during the nonbreeding season. By examining seasonal changes in volatiles using gas chromatography-mass spectrometry, we found that the secretion of three C12 and C14 aldehydes with a fruity and floral scent significantly increased during the breeding season in a testosterone-dependent manner. Females sniffed for longer at biologically relevant concentrations of two of the aldehydes (12-methyltridecanal and tetradecanal) and were attracted to a mixture of these plus the third aldehyde, dodecanal. Our results suggest that these aldehydes are putative lemur pheromones involved in the attractiveness of males to females during the breeding season.

Evolution of the bitter taste receptor TAS2R38 in colobines.

Bitter taste perception enables the detection of potentially toxic molecules and thus evokes avoidance behavior in vertebrates. It is mediated by bitter taste receptors, TAS2Rs. One of the best-studied TAS2R is TAS2R38. Phenylthiocarbamide (PTC) perception and TAS2R38 receptors vary across primate species, and this variation may be related to variation in dietary preferences. In particular, we previously found that the low sensitivity of TAS2R38s in Asian colobines likely evolved as an adaptation to their leaf-eating behavior. However, it remains unclear whether this low PTC sensitivity is a general characteristic of the subfamily Colobinae, a primate group that feeds predominantly on leaves. We performed genetic analyses, functional assays with mutant proteins, and behavioral analyses to evaluate the general characteristics of TAS2R38 in colobines. We found that PTC sensitivity is lower in TAS2R38s of African colobines than in TAS2R38s of omnivorous macaques. Furthermore, two amino acids shared between Asian and African colobines were responsible for low sensitivity to PTC, suggesting that the last common ancestor of extant colobines had this phenotype. We also detected amino acid differences between TAS2R38s in Asian and African colobines, indicating that they evolved independently after the separation of these groups.

Functional divergence of the bitter receptor TAS2R38 in Sulawesi macaques.

ABSTRACT: Bitter perception is mediated by G protein-coupled receptors TAS2Rs and plays an important role in avoiding the ingestion of toxins by inducing innate avoidance behavior in mammals. One of the best-studied TAS2Rs is TAS2R38, which mediates the perception of the bitterness of synthetic phenylthiocarbamide (PTC). Previous studies of TAS2R38 have suggested that geographical separation enabled the independent divergence of bitter taste perception. The functional divergence of TAS2R38 in allopatric species has not been evaluated. We characterized the function of TAS2R38 in four allopatric species of Sulawesi macaques on Sulawesi Island. We found variation in PTC taste perception both within and across species. In most cases, TAS2R38 was sensitive to PTC, with functional divergence among species. We observed different truncated TAS2R38s that were not responsive to PTC in each species of Macaca nigra and M. nigrescens due to premature stop codons. Some variants of intact TAS2R38 with an amino acid substitution showed low sensitivity to PTC in M. tonkeana. Similarly, this intact TAS2R38 with PTC-low sensitivity has also been found in humans. We detected a shared haplotype in all four Sulawesi macaques, which may be the ancestral haplotype of Sulawesi macaques. In addition to shared haplotypes among Sulawesi macaques, other TAS2R38 haplotypes were species-specific. These results implied that the variation in TAS2R38 might be shaped by geographical patterns and local adaptation. OPEN RESEARCH BADGES: This article has earned an Open Data Badge for making publicly available the digitally-shareable data necessary to reproduce the reported results. The data is available at https://doi.org/10.5061/dryad.908jf3r.

A natural point mutation in the bitter taste receptor TAS2R16 causes inverse agonism of arbutin in lemur gustation.

Bitter taste enables the detection of potentially harmful substances and is mediated by bitter taste receptors, TAS2Rs, in vertebrates. Few antagonists and inverse agonists of TAS2Rs have been identified, especially natural compounds. TAS2R16s in humans, apes and Old World monkeys (Catarrhini, Anthropoidea) recognize ß-glucoside analogues as specific agonists. Here, we investigated responses of TAS2R16 to ß-glucosides in non-anthropoid primates, namely lemurs (Lemuriformes, Strepsirrhini). Salicin acted as an agonist on lemur TAS2R16. Arbutin acted as an agonist in the ring-tailed lemur ( Lemur catta) but as an inverse agonist in black lemur ( Eulemur macaco) and black-and-white ruffed lemur ( Varecia variegata). We identified a strepsirrhine-specific amino acid substitution responsible for the inverse agonism of arbutin. In a food preference test, salicin bitterness was inhibited by arbutin in the black lemur. Structural modelling revealed this locus was important for a rearrangement of the intracellular end of transmembrane helix 7 (TM7). Accordingly, arbutin is the first known natural inverse agonist of TAS2Rs, contributing to our understanding of receptor-ligand interactions and the molecular basis of the unique feeding habit diversification in lemurs. Furthermore, the identification of a causal point mutation suggests that TAS2R can acquire functional changes according to feeding habits and environmental conditions.