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.


| INTRODUC TI ON
Bitter taste perception is important for avoiding the ingestion of toxins by inducing innate avoidance behavior in mammals and is mediated by the G protein-coupled receptors TAS2Rs, expressed in the membranes of sensory cells (Chandrashekar, Hoon, Ryba, & Zuker, 2006;Chandrashekar et al., 2000). TAS2Rs are also expressed in extra-oral cells and may have roles in innate immunity (Lee et al., 2012;Shaw et al., 2018). One of the best-studied TAS2Rs is TAS2R38, which mediates the perception of the bitterness of synthetic phenylthiocarbamide (PTC), propylthiouracil, and natural bitter compounds, such as allyl isothiocyanate, goitrin, limonin, yohimbine, and sinigrin .
Correlations between the divergence of TAS2R38 and variation in PTC sensitivity have been studied previously (Kim et al., 2003;Suzuki et al., 2010;Wooding et al., 2006). Three amino acid sites are responsible for PTC sensitivity in human TAS2R38 (hTAS2R38), at positions 49, 262, and 296. Two common variants are widely distributed worldwide: PAV (Proline 49, Alanine 262, and Valine 296), which encodes the PTC-taster receptor, and AVI (Alanine 49, Valine 262, and Isoleucine 296), which encodes the PTC-non-taster receptor. The less common variants of hTAS2R38, namely AAI, PVI, and AAV, show intermediate sensitivity to PTC (Bufe et al., 2005). Few studies of nonhuman primates have explored the functional divergence of TAS2R38 with respect to PTC sensitivity, including studies of Macaca fuscata and Pan troglodytes (Suzuki et al., 2010;Wooding et al., 2006).
Some studies have suggested that geographical separation enabled the independent divergence of TAS2R38 and bitter taste perception (Campbell et al., 2011;Hayakawa et al., 2012;Suzuki-Hashido et al., 2015). For example, in humans, the frequencies of TAS2R38 haplotypes associated with intermediate bitter taste sensitivity are higher in Africa than in other regions (Campbell et al., 2011).
In some species of primates, such as M. fuscata and P. troglodytes, a .14 RR Note: A trial is defined as the act of a monkey putting an apple slice into its mouth, followed by acceptance or rejection of the slice.
a Probability that the proportion of acceptance in the PTC-treated session is the same as that in the control session.
A notable feature of Sulawesi Island is that it comprises three major tectonic subdivisions, two that originated in the Asian and Australian continental margins and one that emerged by orogeny due to the tectonic collision between two plates (Spakman & Hall, 2010). Accordingly, geological, geographical, and ecological conditions vary within the island, such as soil conditions and the composition of natural vegetation (Whitten & Henderson, 2012). Based on the geographical and ecological variations on Sulawesi Island and the slight differences in food preferences among Sulawesi macaques, we hypothesized that TAS2R38 also diverged among Sulawesi macaque species.
In this study, we performed a behavioral experiment, molecular genetic analysis, and in vitro functional analysis to characterize the divergence of TAS2R38 in four species of Sulawesi macaques. We found that TAS2R38 of Sulawesi macaques shows substantial individual variation in PTC taste perception both within and across species, and that this variation is caused by a variety of species-specific genetic variants. Our analysis of interspecific and intraspecific variation in TAS2R38 in nonhuman primates improves our understanding of the evolution of bitter taste perception.

TA B L E 2 (Continued)
The phenylthiocarbamide was dissolved in assay buffer

| Behavioral experiment
In a behavioral experiment, we evaluated the responses of monkeys

| Genotyping
We determined the nucleotide sequences of TAS2R38 to investigate the genetic basis of PTC-sensitive and PTC-non-sensitive behaviors. to examine the protein function (Bufe et al., 2005). We analyzed noncoding sequences from exome data to evaluate the relatedness among the subject animals. We found no difference between the expected and observed heterozygosity values in both M. tonkeana (p = .36) and M. hecki (p = .06; Bartlett's test). Thus, we conclude that there was no inbreeding among the subjects used in this experiment.

| Functional analysis
We evaluated receptor activity by in vitro functional assays of several TAS2R38 alleles of Sulawesi macaques. We tested the high-frequency allele (haplotype A) and another variant that differed from haplotype A at site 315, one of the sites that was predicted to be responsible for receptor sensitivity (haplotype N). The TAS2R38s showed a significant dose-dependent response to PTC, and the

| Functional divergence of TAS2R38 in Sulawesi macaques
We observed functional divergence of Sulawesi macaque TAS2R38 with respect to responses to PTC. Most Sulawesi macaques are PTC-sensitive, similar to other macaque species (Chiarelli, 1963;Suzuki-Hashido et al., 2015). We also observed PTC-non-sensitive phenotypes which caused by truncated protein in some individuals in two species of Sulawesi macaques, M. nigra and M. nigrescens.
Protein truncations resulting in a lack of sensitivity also exists in other primate species, such as chimpanzees, Japanese macaques, and some species of New World monkey (Suzuki et al., 2010;Wooding, 2012;Wooding et al., 2006). These results confirm the high divergence of TAS2R38.
In addition to the nonsensitive alleles of M. nigra and M. nigrescens, which encode truncated TAS2R38 receptors, we found TAS2R38 alleles in M. tonkeana that express apparently intact TAS2R38 receptors but exhibit very weak responses compared with those of wild-type Sulawesi TAS2R38. Similar intact receptors with low PTC sensitivity have been found in humans (Bufe et al., 2005). suggesting that the nontaster allele enables individual to taste compounds in the fruit (Risso, Sainz, Morini, Tofanelli, & Drayna, 2018).
Thus, we predicted that alleles associated with low sensitivity to PTC in M. tonkeana could detect another ligand. Interestingly, M. tonkeana occasionally consumes Antidesma bunius (Riley, 2007). These feeding behaviors of M. tonkeana are related to the TAS2R38 genotype and geographical distributions of the macaque and plant species.
Sulawesi macaques, which inhabit an island, possess high haplotype diversity, similar to other primates (Campbell et al., 2011;Hayakawa et al., 2012;Suzuki-Hashido et al., 2015). The frequency of each haplotype was also different between species (Table 3). We observed seven amino acid haplotypes in M. hecki, eight in M. nigra, five in M. nigrescens, and nine in M. tonkeana. We analyzed the sequences for signatures of selection, but we did not obtain significance (Table 4). It is difficult to show selection using the number of individuals included in this study. In the near future, we will re-evaluate selection using DNA from more individuals.   (Lee et al., 2012). Thus, TAS2R38 variation may also have a role in the immune response of Sulawesi macaques. We predict that fragmentation played an important role in determining the distribution of genetic variation in TAS2R38 in Sulawesi macaques ( Figure 6).

ACK N OWLED G M ENTS
The authors would like to thank Drs. T. Ueda, T. Misaka, and H.   F I G U R E 6 Overview of TAS2R38 diversity in Sulawesi macaques designed the experiments and wrote the paper and finalized the manuscript. All authors agree to be held accountable for the content in the manuscript and approve the final version.