Complex association between diet and bitter taste receptor gene numbers in vertebrate from an enlarged sampling

Complex association between diet and bitter taste receptor gene numbers in vertebrate from an enlarged sampling | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 26 April 2025 V1 Latest version Share on Complex association between diet and bitter taste receptor gene numbers in vertebrate from an enlarged sampling Authors : Siwen Huang , Wang Hui 0000-0001-9526-2270 , Tuo Kan , Hongling Yu , Xinyue Liang , Yingyi Liang , Zuping Zhou , and Ping Feng 0000-0002-6742-4938 [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.174568689.97009391/v1 383 views 228 downloads Contents Abstract 1 INTRODUCTION 2 MATERIALS AND METHODS 4 DISCUSSION 5 CONCLUSION References Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Bitter taste perception helps animals avoid over-consuming toxic substances via Tas2rs genes-encoded receptors. Currently, most existing studies on the correlation between feeding preference and Tas2r repertoire in animals focus on smaller animal taxonomic groups. With data accumulation in this topic, we reevaluated this on a larger scale. Therefore, we collected the number of Tas2r genes (including intact genes, partial genes, and pseudogenes) and food habits of 227 reported species. Then we reconstructed the species tree with divergent time and performed correlation analysis using phylogenetically independent contrasts (PIC) to control for phylogenetic inertia. The results showed no correlation between diet and intact Tas2r gene number, but a positive correlation between total Tas2r gene number and putative functional Tas2r gene number (intact and partial genes). We conjectured that feeding preference did not affect gene number as a single factor and the discrepancy between the results of some previous and our present studies could be attributed to the divergence time, the species sample size and other factors such as historical contingency. By expanding the sample scope and integrating multi-omics data, this study offers new insights into the relationship between bitter taste perception and feeding adaptation in vertebrates. Complex association between diet and bitter taste receptor gene numbers in vertebrate from an enlarged sampling Siwen Huang 1,2,† , Hui Wang 1,2,† , Tuo Kan 1,2 , Hongling Yu 1,2 , Xinyue Liang 1,2 , Yingyi Liang 1,2 , Zuping Zhou 3 , Ping Feng 1,2* 1 Key Laboratory of Ecology of Rare and Endangered Species and Environmental Protection, Guangxi Normal University, Guilin, Guangxi, China 2 Guangxi Key Laboratory of Rare and Endangered Animal Ecology, Guangxi Normal University, Guilin, Guangxi, China 3 Guangxi Universities Key Laboratory of Stem Cell and Biopharmaceutical Technology, Guangxi Normal University, Guilin, Guangxi, China † Siwen Huang and Hui Wang should be considered joint first author * Correspondence: Ping Feng E-mail address: [email protected] Abstract Bitter taste perception helps animals avoid over-consuming toxic substances via Tas2rs genes-encoded receptors. Currently, most existing studies on the correlation between feeding preference and Tas2r repertoire in animals focus on smaller animal taxonomic groups. With data accumulation in this topic, we reevaluated this on a larger scale. Therefore, we collected the number of Tas2r genes (including intact genes, partial genes, and pseudogenes) and food habits of 227 reported species. Then we reconstructed the species tree with divergent time and performed correlation analysis using phylogenetically independent contrasts (PIC) to control for phylogenetic inertia. The results showed no correlation between diet and intact Tas2r gene number, but a positive correlation between total Tas2r gene number and putative functional Tas2r gene number (intact and partial genes). We conjectured that feeding preference did not affect gene number as a single factor and the discrepancy between the results of some previous and our present studies could be attributed to the divergence time, the species sample size and other factors such as historical contingency. By expanding the sample scope and integrating multi-omics data, this study offers new insights into the relationship between bitter taste perception and feeding adaptation in vertebrates. Keywords: bitter taste receptor, Tas2r gene, diet, vertebrates, PIC 1 INTRODUCTION During the process of tasting food, two senses of animals-smell and taste-are involved. The similarity between smell and taste in detecting chemicals in the environment is the result of convergent evolution or co-constraints (Derby & Caprio, 2024). Unlike smell, taste detects contact-chemicals (Ren & Liu, 2020). In nature, toxic substances are often accompanied by bitter taste experiences (Freeland & Janzen, 1974; Garcia & Hankins, 1975), such as alkaloids (Zagrobelny et al., 2004) and insects’ defensive secretions (Wang & Zhao, 2015), are typical examples of such bitter poisons. To avoid excessive intake of noxious food and increase the chances of survival, animals have adopted several strategies (Sullivan et al., 2008). Among them, developing chemosensors including bitter taste receptor is one of the most important strategies. Bitter taste perception plays a considerable role in food intake and nutrient content absorption, and it happens through the interaction between bitter substances and the bitter taste receptors which encoded by bitter taste receptor genes ( Tas2r s). Bitter receptors, which belong to the G protein-coupled receptors (GPCRs) family, activate downstream signal transduction pathways by binding to specific ligands (various bitter substances), thereby triggering intracellular signal cascade reactions, and finally transmit to taste perception regions to produce bitter sensations (Adler et al., 2000; Chandrashekar et al., 2000; Matsunami et al., 2000). Thus, the Tas2r gene, acting as a ”bitter detector” in the body, is necessary for successful recognition of bitter substances (Chandrashekar et al., 2006; Mueller et al., 2005). A Tas2r gene has a coding sequence about 900bp and no introns, which makes the identification of Tas2r gene easily. Different eating habits exist among animals, as the name implies, carnivorous animals mainly feed on other animals, herbivore on plant tissue and omnivores both. Thus, we wonder whether differences in dietary habits were associated with the Tas2r gene repertoire in different animal taxa. Does it lead to any adaptive changes in the number, type and expression of Tas2r genes? In recent years, with more and more genomes of animals sequenced and published, the identification of Tas2r s becomes prevalent. Researchers have keenly catch this opportunity to solve the questions mentioned above, and they carried out such work on several taxonomic groups including fish, amphibians, reptiles, birds and primates, etc.; and Tas2r genes identified in these species have showed complex evolutionary process with gene expansion or contraction along with phylogenetic branches, through experiencing gene duplication, gene deletion and/or pseudogenization events (Fischer et al., 2005; Hayakawa et al., 2014; Shang et al., 2017b; Shiriagin & Korsching, 2018; Wang & Zhao, 2015; Zhong et al., 2021; Zhong et al., 2017). Several studies have showed that the number of intact Tas2r gene varied among different species, from 0 to 178 (Feng et al., 2014; Zhong et al., 2021; Zhu et al., 2014). It is suggested that Tas2r abundance typically increases the types of detectable toxins, whereas the reduction of Tas2r genes decreases the susceptibility to toxins, perhaps reflecting adaptive strategies for coping with toxin challenges in animals during evolution (Li & Zhang, 2014). For example, amphibians have a large number of Tas2r genes compared to other taxa (Zhong et al., 2021), which can be linked to the various insects in the amphibians’ food composition (Behrens et al., 2014). Insects release toxins like neurotoxins and hemolysin to protect themselves from predation, improving their ability to perceive and respond to toxins(Walker, 2020). The size of Tas2r family is related to the amount of bitter compounds contained in foods. It is well known that giant pandas and red pandas have undergone significant dietary changes in the course of evolution, from carnivorous habits to bamboo as a staple food. Bamboo contains a large number of bitter compounds (Huang et al., 2022), and under long-term dietary adaptive evolution, both panda species contain a higher amount of functional Tas2r than other carnivores (Shan et al., 2018). Plants have much more bitter chemicals because these substances, such as quassin in balsam pear ( Momordica charantia ) and amygdalin in the seeds of apple ( Malus pumila ), can help them to reduce or avoid being eaten by animals (Drewnowski et al., 2001), thus, it is suggested that herbivores possess more Tas2r s than do in carnivores (Li & Zhang, 2014). In different animal groups, researchers have found a link between Tas2r and food preference. As regards to the single group, feeding preference was significantly correlated with the number of functional Tas2r genes consisting of intact and partial genes in birds (Wang & Zhao, 2015); and the repertoire of Tas2r genes in reptiles had a significant positive correlation with the amount of potential toxins in food (Zhong et al., 2017); and herbivores and insectivores in Laurasiatheria mammals had more functional Tas2r genes than did carnivores and omnivores (Liu et al., 2016); besides, data from primate studies also suggest that feeding preference is one of the factors driving the evolution of Tas2rs (Feng et al., 2024). However, the same conclusion was not found in fish, which presumed that the swallowing feeding habit might attenuate the importance of bitter taste perception (Shang et al., 2017b), as it is suggested in cetacean, pinnipedia and pangolin (Liu et al., 2016). In addition, no positive correlation was detected in canids, and this result is attributed to the small sample size (Shang et al., 2017a). In sum, the association between Tas2r s number and diet was investigated in the main taxonomic groups of animals, and in some groups it is a positive relationship, while in others, no significant correlation was detected. However, with small sample sizes (from 18 to 48) and in a single population, these conclusions may be partial or biased, so it remains to be seen whether the conclusions drawn from the above studies can be extended to the whole vertebrate level. In terms of vertebrate groups, Li & Zhang (2014) has investigated the relationship between diet and Tas2r numbers by selecting the samples from fish to mammals, and concluded that diet was one of the key drivers of Tas2r evolution. However, the sample size in this study is small due to the limited number of genome sequenced at that time. Furthermore, in the comparative analyses, the data independence caused by phylogenetic inertia which means more closely-related species tend to share more similar traits (Fisher & Owens, 2004; Harvey & Pagel, 1991) should be considered (Feng et al., 2015). This question is often solved by using phylogenetically independent contrasts (PICs; (Felsenstein, 1985)) analyses implemented in APE (Analyses of Phylogenetics and Evolution) (Paradis et al., 2004) of R package during the process, the divergence time between species should be used. Phylogenetic trees are typically constructed based on genetic data, with branch nodes calibrated using external time markers such as TimeTree databases, fossil records, or geological events to analyze species’ evolutionary relationships more accurately. In the previous research, most of which collected the divergence time from the TimeTree website (http://www.timetree.org/). However, it is found that the divergence time provided in this website varies according to the version, and most divergence time of previous research is based on the TimeTree 3.0, but now, the website has updated to TimeTree 5.0, thus, the divergence time of the previous studies cannot be obtained. Furthermore, if all divergence times of the vertebrate including fishes, amphibians, reptiles, birds, Laurasiatheria mammals, and primates are selected by referring to TimeTree 5.0, some conflicts exist, such as the negative value in divergence time will appear in some species. Considering this situation, we collected the divergence time by referring to the latest literatures estimated the divergence time of species mainly through multi-omics approaches, and we think it better than the divergence time from a changeable website. Thus, in the present study, we collected the number of Tas2r genes identified so far and the feeding preferences of corresponding speces, to examine whether the size of Tas2r s repertoire correlated with dietary at a broader species level, and we performed a PIC analysis by referring to the divergence time estimated by the latest and multi-omics research. 2 MATERIALS AND METHODS The number of Tas2r gene identification up to now was collected as much as possible, and mainly referred to the literature published in recent years, these literatures provide key data support for our study (Feng et al., 2024; Hayakawa et al., 2014; Jiao et al., 2018; Johnson et al., 2018; Li & Zhang, 2014; Liu et al., 2016; Shang et al., 2017a; Shang et al., 2017b; Syed & Korsching, 2014; Wang & Zhao, 2015; Zhong et al., 2021; Zhong et al., 2017; Zhong et al., 2019; Zhou et al., 2021). The species cover taxonomic groups of the vertebrates, including fishes, amphibians, reptiles, birds, and mammals. Among them, the Tas2r number of primates in this study adopted the research results of our team (Feng et al., 2024). The numbers we collected included number of intact genes, partial genes, pseudogenes, and total genes, and the number of total gene is sum of the former three. In the process of identifying and classifying these genes, we found that these papers had some similarities in sequence feature judgment, gene classification and basic logical structure of Tas2r gene analysis process. In detail, an intact gene has a complete open reading frame (ORF), start codon and stop codon, and seven transmembrane domains (validated by TMHMM method (Krogh et al., 2001)). Partial genes are sequences with only start codons or stop codons, often structurally incomplete or functionally deficient. Pseudogenes are similar to gene sequences with normal function, but their ORF terminates prematurely, that is, sequences that terminate the codon prematurely or undergo frameshift mutations, thereby preventing the synthesis of proteins with the original function (Wang & Zhao, 2015; Zhong et al., 2021). When the number of collecting Tas2r genes, we follow the principle of the two. First, if the gene number of a species varies in different researches, we adopt the latest result. This is because with the continuous development of scientific research and technological means, the latest research results often have higher accuracy and reliability in determining the number of genes. Second, if there is research data for a specific group of people, the data will be used first. Otherwise, the research results of Li & Zhang (2014) are adopted. In the event of a conflict between the first principle and the second principle, the provisions of the second principle shall prevail. For example, in Mustela putorius furo , Shang et al. (2017a) and Liu et al. (2016) showed a total of 24 and 20 genes, respectively. Therefore, according to the principles we set, we chose 24 genes as the total number of genes for Mustela putorius furo . At the same time, Liu et al. (2016) studied Laurasiatheria mammals, including canids. In view of this, for the number of Tas2r genes in canids in overlapping species, we prefer to use the data of Shang et al. (2017a), a smaller and more targeted study, as the data source for this paper. In addition, for rodents, studies by Li & Zhang (2014) and Hayakawa et al. (2014) showed species overlap and different numbers of Tas2r genes. According to the second principle, we use the data of Li & Zhang (2014). The number of Tas2r genes can be seen in Figure 1. 2.2 Diet collection and classification After finish the Tas2r number collection, the species used in this study are determined. Next, we searched the literature or database for the diet information of these species. We found that in the previous studies, some of the diet information was from the literature while others were from the Animal Diversity Web (https://animaldiversity.org/). Given that the Animal Diversity Web relies heavily on secondary reports, such as encyclopedias, which may not be accurate, we further searched the literature to confirm the feeding preferences, and a dietary database, Etontrait1.0 (Wilman et al., 2014) with quantitative analysis was also used to judge the diet. We noticed that in the previous research, the criteria for diet classification are different, some used 90% rule as the criteria, which assigned that when more than 90% of food consists with plants tissues, the species will be considered as herbivores (Harestad & Bunnel, 1979), and if 90% of the food component is meat the species are viewed as carnivores; the rest of species are omnivores (Li & Zhang, 2014). Another research defined the diet based on 51% or more of the stomach contents composition (DeGolier et al., 1999; Wang & Zhao, 2015). In this study, we used the 51% rule given that an animal’s diet comprises a series of food items (e.g., plant parts, animal parts, invertebrates, lichen, resin, etc.) and the proportion of between plant item and animal item is not easy to distinguish; and even mammalian herbivores are inclined to feed generally to avoid overwhelming intake of plant secondary compounds (PSC) (Skopec et al., 2015). Thus, we assigned it as an herbivore if the proportion of plant composition is equal to or larger than 51%, and the carnivore was defined in a similar way while the rest were classified as omnivores. It should be noted that for those species that feed on insects, ready-to-eat insectivores, their ingestion of food toxins has similarities to that of herbivores. This is mainly based on two reasons: First, many insects secrete toxins themselves, which increases the amount of toxic substances faced by predators. For example, blister beetles secrete toxic cantharidin when confronted with the danger of preying (Huang et al., 2024); Another reason is that insects mainly feed on plants and they usually sequestrate the toxic plant secondary metabolites to defense against predators (Petschenka & Agrawal, 2016). Milkweed Bugs , for example, have a diet containing cardiac glucosides or colchicine alkaloids, two toxic substances that can effectively boost their defenses (Petschenka et al., 2022). In the present study, our efforts were focused on constructing a phylogenetic tree that encompassed 227 species. The construction was carried out by employing the methods proposed in (Wang & Zhao, 2015). Specifically, this paper takes the phylogenetic tree structure proposed by Delsuc et al. (2018) based on genetic data as the basis for reconstructing phylogenetic trees with divergence time. During this process, with the aim of assigning reasonable divergence time to each branch of the phylogenetic tree in a more comprehensive and precise manner, we extensively referred to the phylogenetic tree related results of many different taxonomic groups, among which were fishes (Hughes et al., 2018), testudinata (Grundler et al., 2022), squamata (Zheng & Wiens, 2016), amphibians (Hime et al., 2021), birds (Jarvis et al., 2014), Laurasiatherian and primates (Alvarez-Carretero et al., 2022; Perelman et al., 2011). These are the taxonomic groups from which we obtained relevant information regarding the divergence time. Besides, the Time Tree database (https://timetree.org/, last accessed October 20, 2022) (Kumar et al., 2022) was used for references if the time from different resources was hard to choose. Finally, based on the published literature, we used the FigTree software (http://tree.bio.ed.ac.uk/software/figtree) to analyze the phylogenetic relationships and divergence time differences among 227 species, and then used it to draw the integrated phylogenetic tree. The final phylogenetic tree was shown in Figure 1. Due to the phylogenetic non-independence caused by the presence of common ancestors among species (Fisher & Owens, 2004), we used phylogenetically independent contrast (PIC) analysis (Paradis et al., 2004) to examine the correlations between the number of Tas2r genes and feeding preference with the guide tree reconstructed above. In total, dietary information was collected for 227 species, as detailed in the Supplementary table S1. It is generally believed that the content of bitter compounds (toxic substances) in plant tissues is higher than that in non-plant tissues. Based on this point of view, the content of bitter compounds faced by different feeding species is inferred, and the feeding information is encoded into diet codes. Then the PIC analysis was conducted between diet code and the number of Tas2r gene. The PIC analysis was calculated using the pic function in the R packet APE (Phylogenetic and Evolutionary analysis) (Paradis et al., 2004), and the resulting Spearman’s rank correlation coefficient (ρ) was then used to assess whether there was a correlation between the number of Tas2r genes and feeding preference. 3 RESULTS 3.1 Characteristics of the Tas2r Gene Repertoire Size To make the data more representative, we collected the number of Tas2r genes in 227 vertebrate species, including 25 fish species, 14 amphibians, 22 reptiles, 48 birds, and 118 mammals. In the detailed classification of mammals, there are 2 species of Monotremata (Prototheria), 4 orders of Marsupialia (Metatheria), 35 species of Primates (Eutheria), 59 species within Laurasiatheria, 10 species of Rodentia, as well as 1 species each of Scandentia, Hyracoidea, Afrosoricida, Sirenia, Cingulata, Pilosa, and 2 species of Lagomorpha. Figure 1 shows the phylogenetic tree of 227 species, which specifically presents the number of Tas2r genes of each species, including the number of intact genes, partial genes and pseudogenes, as well as the corresponding feeding habits of each species. Further details, including feeding references for each species, are provided in Supplementary table S1. We collected a total of 5702.75 Tas2r genes (3772.75 intact genes, 140 partial genes, and 934 pseudogenes). Among these species, as many as 147 species of missing genes, including 25 species of fish, 37 species of Laurasiatheria, 43 species of birds, 19 species of reptiles, 14 species of primates, etc., it can be seen that the deletion of partial genes is common. We found that Tas2r quantity differences in different groups are significant, diversified characteristics (Figure 1). Fish, snakes, terrapin, and Crocodiliformes all have a small number of Tas2r genes, less than 20 in total, although Latimeria chalumnae is an exception, with 80 Tas2r genes. The number of Tas2r genes in amphibians is the largest, and the total number of Tas2r genes ranges from 6~219, and the total number of intact genes ranges from 4~178, but the number of genes varies greatly among different amphibian species. Bullfrog ( Lithobates catesbeianus ), for example, has 178 intact genes, the highest number of all studied species, while Geotrypetes seraphini has only 4 intact genes. Overall, the average number of intact taste receptor genes in mammals is 18.45, which is in the middle of the main groups of vertebrates. Among them, the number of amphibians is extremely prominent, up to 82.57, far more than other groups. In comparison, the average number of fish is only 4.8, which is at a low level in all groups; birds possess an average of 2.42 Tas2r genes, also relatively few; the average number of squamata is 11; Chelonidae had 5.8; The number of crocodiles was 7.25. Among the mammal groups, some insectivores in Laurasiatheria, such as Sorex araneus , have more complete copies of Tas2rs , with a number of 46. In contrast, cetaceans have very few copy numbers, which may be related to the gradual adaptation to an aquatic lifestyle diet (swallowing prey whole) (Lei et al., 2015). In 35 primate species, the total number of Tas2r genes is between 27 and 51. It is worth noting that in proportion Tas2rs pseudogenes, found some interesting situations. For example, the proportion of pseudogenes in whales is extremely high, as high as 90%; Three species of water birds ( Gavia stellata, Aptenodytes forsteri, Pygoscelis adeliae ) also have a special situation, with 100% pseudogenization. However, when looking at most birds and fish, the situation changes, and Tas2rs is hardly pseudogenicized. This phenomenon of receptor pseudogenization may be related to functional redundancy of Tas2r gene, which was reflected in the study of Risso’s reconstruction of complete sequences of hTas2r2 and hTas2r64 (Risso et al., 2017). In the process of evolution, whales are driven by multiple ecological factors, such as the change from phytophagous to carnivorous, the overall feeding mode of swallowing prey, and the living environment with high sodium concentration (Feng et al., 2014), and their Tas2r gene has a reduced need for sensing bitter taste, and gradually loses functional selection pressure and degrades. Similarly, the Tas2r gene is completely pseudogenic in some waterbirds, probably because of their simple diet, while most birds and fish retain the gene intact because some bitter substances in the environment cannot be avoided, highlighting the high coupling of sensory system evolution and ecological strategies. In addition, the proportion of pseudogenes (> 80%) in the obligate representative of Manis pentadactyla is also high, which we predict is because the monogeneity of dietary resources will be pseudogenicized due to loose functional selection (Pinto et al., 2023). Therefore, when gene function and survival fitness are decouple, functional redundancy or lack of environmental requirements will accelerate the pseudogenization process, while persistent ecological threats will strengthen the conservation of functional genes. 3.2 Correlation between diet and the number of Tas2r genes To explore the size of the correlation between diet and Tas2r gene repertoire in the 227 vertebrate species investigated, we coded carnivores as 0, omnivores as 0.5, and herbivores/insectivores as 1 (consistent with the study (Li & Zhang, 2014)). This represents the approximate level of potential toxins that each species may contain. In this section, we note that some species have partial gene numbers, while others do not. So we deal with these situations in two ways. First, we analyzed the intact gene count and diet; Secondly, the putative functional gene number (including intact and partial genes) and diet were used for PIC analysis. Finally, PIC analysis was performed on the total number of Tas2r genes and diet. The PIC results revealed no association between dietary codes and the number of intact Tas2r genes ( ρ = 0.087, P = 0.194; Figure 2A). Notably, the number of Tas2r putative functional genes approached significance with diet ( ρ = 0.135, P = 0.043; Figure 2B), while the total number of Tas2r genes showed a significant positive correlation ( ρ = 0.190, P = 0.004; Figure 2C). 3.3 Differences in phylogenetic tree remodeling In the course of the study, we noticed that compared with the previous study, there were many differences in the topological structure of the divergences time reconstructed phylogenetic trees obtained by multi-omics analysis in this study. In terms of fish phylogeny, taking Fundulus heteroclitus as an example, compared with Shang et al. (2017b), the aggregation sequence of Fundulus heteroclitus with Cyprinodon was different in Hughes et al. (2018), compared with Shang et al. (2017b). Fish such as Oreochromis niloticus and Neolamprologus brichardi were also inconsistently located. In phylogenetic primate trees (Feng et al., 2024; Perelman et al., 2011), Nasalis larvatus , Pygathrix nemaeus and other species have different aggregation relationships in different studies. Phylogenetic differences also existed in Lauroids (Alvarez-Carretero et al., 2022; Liu et al., 2016), the aggregation of species such as Sorex araneus, Condylura cristata , and the clustering relationships of Canis lupus familiaris are also different. Furthermore, at the global vertebrate level (Delsuc et al., 2018; Li & Zhang, 2014), the aggregation order of genera such as Gallus, Anolis, Pelodiscus , and the location of species such as Sus scrofa and Vicugna pacos . The topological structures of Ailuropoda melanoleuca and other animals are different from previous studies. These differences indicate that the understanding of vertebrate phylogenetic relationships is constantly being updated with the deepening of research and the improvement of analytical methods. The topology of phylogenetic trees is often related to the source of data, and the selection of species and different methods of obtaining genetic data may also lead to changes in the topology (Itoigawa, Nakagita, et al., 2024). In addition, Tas2r genes of different species have events such as gene replication and loss during evolution, which will complicate and affect the topology of gene trees (Behrens et al., 2020). 4 DISCUSSION Bitter taste receptor gene family as important vertebrate chemical receptor elements, it can detect bitter substances in food, in the biological diet choice, control the intake of toxic substances and so on play a very key role. Molecular evolution studies have shown that the Tas2r gene family originated from a common ancestor of gnathed vertebrates (Itoigawa, Toda, et al., 2024). In the process of evolution, the functional differentiation of this gene family is mainly achieved through lineage-specific branch expansion, in which the tandem duplication of genes, as the main driving force for the generation of new genes, further promotes the functional differentiation (Shi et al., 2003; Zhou et al., 2009). Phylogenetic analysis (Figure 1) showed that the size of the Tas2r gene family showed obvious group specificity, which may be influenced by gene amplification and loss (Liu et al., 2016). Studies have shown that the Tas2r gene family size of carnivorous animals is generally smaller than that of non-carnivorous taxa, which is related to the rare Tas2r gene replication event in carnivores (Hu & Shi, 2013). In fish populations, the gene family is small and conserved, with the exception of the coelacanth ( Latimeria chalumnae ), which has a ”giant” Tas2r gene repertoire through local tandem chromosome repeats (Syed & Korsching, 2014). Tetrapods showed higher evolutionary plasticity as animals expanded to terrestrial ecosystems, because genealogy-specific gene duplication and loss of the Tas2r gene family mostly occurred in tetrapods (Itoigawa, Nakagita, et al., 2024; Policarpo et al., 2024). For example, some amphibians achieve gene amplification through tandem chromosome duplication (Zhong et al., 2021). In the same way, in bats of the genus Myotis (Jiao et al., 2018), in some birds (e.g., white-throated sparrows ) (Wang & Zhao, 2015), in marsupials koalas (Johnson et al., 2018), and in some primates (Feng et al., 2024), lineage-specific expansion was also found. Gene loss events are not uncommon in mammalian lineages, examples include the Chinese pangolin (Liu et al., 2016), the vampire bat (Ziegler & Behrens, 2021), monotrematous echidnas ( Tachyglossus aculeatus ) (Zhou et al., 2021), and marine mammals (penguins, cetaceans, and pinnipeds) (Feng et al., 2014; Zhu et al., 2014) all observed that the Tas2r gene repertoire showed a shrinking trend. Thus, Tas2r gene loss events seem to be significantly associated with specialized eating. With the advances in genome sequencing and multiomics applied in various research, many Tas2r genes are identified and the divergence time can be estimated in a large scale by utilizing the data from multiomics. Under such a background, we collected the number of Tas2r s, diet, and the divergence time of vertebrates from the recently published research to reevaluate the relationship between numbers of Tas2r and feeding preferences. In contrast with the previous research, which either focused on one group of the vertebrate taxonomic groups, or concentrated on the very limited number of vertebrates, our present study expanded the number of species to 227 and covered the vertebrates with all the groups. The results showed that the total number of Tas2r genes was significantly correlated with diet ( P = 0.004), and the number of putative functional Tas2r genes was also significantly correlated with diet ( P = 0.043), but the number of intact Tas2r genes was not significantly correlated with diet ( P = 0.194) (Figure 2). Although this result acknowledged that Tas2r gene evolution was related to dietary preference in terms of the total number of Tas2r genes and the number of putative functional genes, the intact number of Tas2r genes was not related to diet, which might mean that eating habits did not match the evolution of taste receptor genes. For example, differences in people’s preferences for cilantro are influenced by genetic components ( OR6A2 , a olfactory receptor gene) and chemicals, and dislike of cilantro may stem from variations in OR6A2 , suggesting that there is a complex relationship between genes and taste receptors, rather than a simple one-to-one correspondence (Eriksson et al., 2012). How to explain this result? Several possible reasons can be raised here. First, taste abilities are not important for some species. For example, in whales, penguins and pinnipeds, the complete absence of Tas2r genes could be associated with gulping food (Feng et al., 2014; Jiang et al., 2012; Zhao et al., 2015), and birds (Wang & Zhao, 2015) and snakes (Zhong et al., 2017) swallow directly into the stomach for digestion, without oral chewing. The contraction of Tas2r gene repertoires in these species indicated that the manner of feeding through swallowing makes bitter substances identification unnecessary. Second, food specialization can result in small Tas2r s repertoire. For instance, Chinese pangolin ( Manis pentadactyla ) primarily feeds on termites and ants (Tamang et al., 2022) and Geotrypetes seraphini eats earthworms (Kouete & Blackburn, 2020). Due to the relatively single source of food, the variety and amount of toxins contained are limited, and this narrowly feeding niches make them confront less toxins than do other insectivores and finally result in fewer number of Tas2r s. Third, some species have lost certain organs during the evolutionary process, and taste perceptual capacity has become developed as compensation for such defects. For example, Astyanax mexicanus had expanded its bitter taste receptor gene before losing its visual function during evolution. This dilation is a pre-adaptation mechanism for future visual deficits, and it is speculated that the development of taste perception may compensate for the loss of vision to some extent (Shiriagin & Korsching, 2018). In addition, Astyanax mexicanus used several physiological structural features to compensate for the loss of vision, including a large jaw that accommodated more teeth and a large mouth that increased the opportunity for predation (Powers et al., 2023), all of which may have been intended to better adapt to the cave environment and compensate for the survival disadvantage caused by the loss of vision. Fourth, there is no strict correlation between bitterness and toxicity (Glendinning, 1994; Nissim et al., 2017). Most plants, such as the plant in family Cucurbitaceae (Shang et al., 2014) and citrus fruits ( Citrus spp. ) (Manners, 2007), can produce bitter substances that make them unpalatable for animals to eat, and that substances are often bitter but non-toxic. If animals reject these foods, the nutrients contained will be rejected too. Moreover, animals could use bitter plants as medicines (Huffman, 2003). For example, chimpanzees ( Pan troglodytes ) can use the plants with bitter, such as Vernonia amygdalina , which has medicinal value, to reduce the intestinal parasites (Koshimizu et al., 1994; Ohigashi et al., 1991; Toyang & Verpoorte, 2013). In sum, species could tolerate bitter foods for nutrition or detoxification, indicating that animals are not completely resistant to bitter substances. Fifth, numerous researches have reported bitter taste receptor expression in several extra-oral tissues (Avau & Depoortere, 2016; Kamila & Agnieszka, 2021; Wang et al., 2020). For example, in reproductive system, a great number of Tas2r genes are expressed in mouse testis (Xu et al., 2013) and sperm (Li & Zhou, 2012). Bitter receptors are resident in lymphocytes (Tran et al., 2018), leukocytes (Malki et al., 2015), neutrophils (Kobayashi et al., 2022), and macrophages in other tissues (Gopallawa et al., 2021; Kamila & Agnieszka, 2021) revealed the importance of bitter chemical receptors in immunity. Bitter taste receptors participating in various physiological processes indicated that Tas2r genes have other functions. Thus, selective pressure on Tas2r genes may spring from their multiple and distinct roles in complex systems, rather than just the role of dietary behavior in their gustatory perception. Sixth, various Tas2r -independent pathways contribute to the bitter detection (Banik & Medler, 2022). Caffeine has effects on inositol triphosphate receptor type 3 (IP3R3) and ryanodine receptor (RyR) to transduce a cellular response (Gees et al., 2014; Poole & Tordoff, 2017). The nicotinic acetylcholine receptor responses to nicotine to generate an intracellular response (Ren et al., 2015). As a result, although Tas2r genes play an important role on reacting with bitter compounds, the existence of other pathways to perceive bitterness can reduce the dependence of food toxins on Tas2r genes. Seventh, another factor that had to be taken into consideration was that we coded the toxic substances in food strictly based on diets. However, the toxins in food are more complex. Indospicine is one of the natural toxins in pasture plants which can be accumulated in the tissues of grazing animals and transferred through the food chains to carnivores (Fletcher & Netzel, 2020). Among reptiles, species of Testudoformes feed on worms, insects, fishes, crustaceans as well as marsh plant seeds and leaves occasionally. For instance, according to the Animal Diversity Web (https://animaldiversity.org/), Malaclemys terrapin eats marine crustaceans. Although these foods are meat, they produce pectenotoxins (PTXs), Saxitoxins (STX) and so on that make the food of Malaclemys terrapin with diverse toxins (Espina & Rubiolo, 2008; Leal & Cristiano, 2022). Moreover, other toxins such as Tetrodotoxin (TTX), cephalotoxin and lumbriconeris heteropoda are present in the food of carnivorous marine organisms (Daly, 2004; Gonçalves & Costa, 2021; Newman & Cragg, 2016). As a result, these species have to face more toxins in diets that may be similar with herbivores. However, according to the diet classification rule, the diet code of these marine species was assigned to 0. In addition, most of toxins in cereal-based foods are mycotoxins which do not have a healthy threat to the consumers (Andersen & Thrane, 2006; Kepinska-Pacelik & Biel, 2021; Pleadin et al., 2017), but we still calculated it as 1. This paper also maintains that omnivores have fewer bitter taste receptor genes than herbivores, but a recent study of vertebrate chemoreceptors based on large-scale genomic data suggests that omnivores have higher Tas2r copy numbers than herbivores (Policarpo et al., 2024). It is speculated that omnivorous diet may have played a more important role in the evolution of Tas2r . Similarly, Hou et al. (2024) team, by comparing the number of Tas2r in 8 omnivorous macaques and 2 leaf-eating verthalomonkeys, found that leaf-eating animals were less than omnivorous animals, refuting the simple prediction that common herbivores prefer Tas2r genes. Obviously, it can be seen that the relationship between herbivores and Tas2r gene is relatively complex, and it cannot be simply assumed that herbivores prefer Tas2r gene. As a result, this coding does not adequately account for the diversity and complexity of toxic substances in food, and there is significant uncertainty in accurately assessing the effect of bitter toxic substances in the diet on the number of Tas2r genes. Eighth, the tuning width of Tas2rs was not strictly positively correlated with the size of gene family. The size of the Tas2r gene repertoire is often seen as an important indicator of a species’ bitter taste perception, but studies have shown that even if some species have a small number of functional Tas2r genes, if their receptors have a wide tuning property, it means that they can also recognize a wide range of bitter compounds. For example, chickens ( Meleagris gallopavo ) and turkeys ( Gallus gallus ) with low numbers of Tas2r mentioned in the article have widely tuned receptors that can compensate for the limited number of genes (Behrens et al., 2014; Itoigawa, Nakagita, et al., 2024). Thus, when the dietary niche of a species changes, selection pressure may optimize bitter perception efficiency by adjusting the tuning width of receptors rather than the number of receptors. Ninth, the size of the Tas2rs gene family is influenced by functional redundancy and synergies with other taste receptors in the evolution of dietary adaptation. As far as functional redundancy is concerned, the aforementioned pseudogenization phenomenon can be manifested. Some bitter receptors overlap agonist profiles with others (e.g. hTas2r2/hTas2r64 ) (Lang et al., 2023; Risso et al., 2017), the perception of multiple bitter substances can be compensated by other receptors, which means that even if the diet contains certain toxic substances, species may not need a large number of different Tas2r genes to sense these toxins. In addition, from a synergistic perspective, Tas2r bitter receptors and Tas1r sweet/umami receptors, although structurally different, share conserved intracellular signal transduction pathways (Ahmad & Dalziel, 2020; An et al., 2024; Roper & Chaudhari, 2017). Therefore, the correlation between diet and the number of Tas2r genes is likely to be low, due to the influence of other interacting taste receptors. From the above discussion, it can be inferred that the evolution of bitter taste perception in animals is not only influenced by animal diet, but also may be related to gene replication, functional redundancy, and environmental adaptation. At present, the functions of some genes in Tas2r are not clear, and experiments are needed to verify whether they have actual bitter taste sensing ability. As for the relationship between dietary preference and Tas2r quantity in vertebrate populations, based on the information available so far, more relevant studies and specific analyses are needed to further explore the extent and specific mechanisms of this fit. 5 CONCLUSION In our investigation, the number of vertebrates was expanded in order to examine the relationship between the number of Tas2r genes and diets of species at a macro perspective. Here, we attempted to expand taxonomic groups in vertebrates and examine how diet shaped the chemosensory receptor genes repertoire as one of the environmental factors. The scope of species is far from covering all vertebrate species, and it nevertheless provides a valuable reference for exploring the evolution of taste receptor genes at a more macroscopic level. Diversity dietary characteristics in vertebrates have more complicated impact on the Tas2r genes repertoire extent that genes’ evolution is not just driven by this single factor. AUTHOR CONTRIBUTIONS Siwen Huang: Writing - original draft (equal) , Writing - review & editing (lead). Hui Wang: Writing - original draft (lead) , Investigation (lead), Data curation (lead). Tuo Kan: Investigation (equal), Data curation (equal). Hongling Yu: Investigation (equal). Xinyue Liang: Investigation (equal). Yingyi Liang: Data curation (equal). Zuping Zhou: Writing - review & editing (supporting). Ping Feng: Writing - review & editing (equal), Project administration (lead), Conceptualization (lead). CONFLICT OF INTEREST STATEMENT The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. ACKNOWLEDGEMENTS This work was financed and supported by the National Natural Science Foundation of China (32060111), the Guangxi Natural Science Foundation (2025GXNSFAA069151), the Innovation Project of Guangxi Graduate Education (JGY2022032). We appreciate Yingcan Li and other colleagues for the help and advice. DATA AVAILABILITY STATEMENT All the required data are uploaded as supplementary material. References 1. Adler, E., Hoon, M. A., Mueller, K. L., Chandrashekar, J., Ryba, N. J., Zuker, C. S. (2000). A novel family of mammalian taste receptors. Cell, 100 (6), 693-702. https://doi.org/10.1016/s0092-8674(00)80705-9 Ahmad, R., Dalziel, J. E. (2020). G protein-coupled receptors in taste physiology and pharmacology. Front Pharmacol, 11 , 587664. https://doi.org/10.3389/fphar.2020.587664 Alvarez-Carretero, S., Tamuri, A. U., Battini, M., Nascimento, F. F., Carlisle, E., Asher, R. J., . . . Dos Reis, M. (2022). A species-level timeline of mammal evolution integrating phylogenomic data. Nature, 602 (7896), 263-267. https://doi.org/10.1038/s41586-021-04341-1 An, J.-P., Wang, Y., Munger, S. D., Tang, X. (2024). A review on natural sweeteners, sweet taste modulators and bitter masking compounds: Structure-activity strategies for the discovery of novel taste molecules. Critical Reviews in Food Science and Nutrition , 1-24. https://doi.org/10.1080/10408398.2024.2326012 Andersen, B., Thrane, U. (2006). Food-borne fungi in fruit and cereals and their production of mycotoxins. Advances in experimental medicine and biology, 571 , 137-152. https://doi.org/10.1007/0-387-28391-9_8 Avau, B., Depoortere, I. (2016). The bitter truth about bitter taste receptors: Beyond sensing bitter in the oral cavity. Acta Physiologica, 216 (4), 407-420. https://doi.org/10.1111/apha.12621 Banik, D. D., Medler, K. F., 2022. Taste receptor signaling. In R. K. Palmer & G. Servant (Eds.), Pharmacology of taste (Vol. 275). Cham: Springer. Behrens, M., Di Pizio, A., Redel, U., Meyerhof, W., Korsching, S. I. (2020). At the root of t2r gene evolution: Recognition profiles of coelacanth and zebrafish bitter receptors. Genome Biology and Evolution, 13 (1), evaa264. https://doi.org/10.1093/gbe/evaa264 Behrens, M., Korsching, S. I., Meyerhof, W. (2014). Tuning properties of avian and frog bitter taste receptors dynamically fit gene repertoire sizes. Mol Biol Evol, 31 (12), 3216-3227. https://doi.org/10.1093/molbev/msu254 Chandrashekar, J., Hoon, M. A., Ryba, N. J. P., Zuker, C. S. (2006). The receptors and cells for mammalian taste. Nature, 444 (7117), 288-294. https://doi.org/10.1038/nature05401 Chandrashekar, J., Mueller, K. L., Hoon, M. A., Adler, E., Feng, L., Guo, W., . . . Ryba, N. J. (2000). T2rs function as bitter taste receptors. Cell, 100 (6), 703-711. https://doi.org/10.1016/s0092-8674(00)80706-0 Daly, J. W. (2004). Marine toxins and nonmarine toxins: Convergence or symbiotic organisms? Journal of natural products, 67 (8), 1211-1215. https://doi.org/10.1021/np040016t DeGolier, T. F., Mahoney, S. A., Duke, G. E. (1999). Relationships of avian cecal lengths to food habits, taxonomic position, and intestinal lengths. The Condor, 101 (3), 622-634. https://doi.org/10.2307/1370192 Delsuc, F., Philippe, H., Tsagkogeorga, G., Simion, P., Tilak, M. K., Turon, X., . . . Douzery, E. J. P. (2018). A phylogenomic framework and timescale for comparative studies of tunicates. BMC Biol, 16 (1), 1-14. https://doi.org/10.1186/s12915-018-0499-2 Derby, C. D., Caprio, J. (2024). What are olfaction and gustation, and do all animals have them? Chemical Senses, 49 , bjae009. https://doi.org/10.1093/chemse/bjae009 Drewnowski, A., Henderson, S. A., Barratt-Fornell, A. (2001). Genetic taste markers and food preferences. Drug metabolism and disposition, 29 (4 Pt 2), 535-538. https://doi.org/10.1124/dmd.29.4.535 Eriksson, N., Wu, S., Do, C. B., Kiefer, A. K., Tung, J. Y., Mountain, J. L., . . . Francke, U. (2012). A genetic variant near olfactory receptor genes influences cilantro preference. Flavour, 1 (1), 22. https://doi.org/10.1186/2044-7248-1-22 Espina, B., Rubiolo, J. A. (2008). Marine toxins and the cytoskeleton: Pectenotoxins, unusual macrolides that disrupt actin. The FEBS journal, 275 (24), 6082-6088. https://doi.org/10.1111/j.1742-4658.2008.06714.x Felsenstein, J. (1985). Phylogenies and the comparative method. The American Naturalist, 125 (1), 1-15. https://doi.org/10.1086/284325 Feng, P., Wang, H., Liang, X., Dong, X., Liang, Q., Shu, F., Zhou, Q. (2024). Relationships between bitter taste receptor gene evolution, diet, and gene repertoire in primates. Genome Biology and Evolution, 16 (5), evae104. https://doi.org/10.1093/gbe/evae104 Feng, P., Zhao, H., Lu, X. (2015). Evolution of mitochondrial DNA and its relation to basal metabolic rate. Mitochondrial DNA, 26 (4), 566-571. https://doi.org/10.3109/19401736.2013.873895 Feng, P., Zheng, J., Rossiter, S. J., Wang, D., Zhao, H. (2014). Massive losses of taste receptor genes in toothed and baleen whales. Genome Biology and Evolution, 6 (6), 1254-1265. https://doi.org/10.1093/gbe/evu095 Fischer, A., Gilad, Y., Man, O., Paabo, S. (2005). Evolution of bitter taste receptors in humans and apes. Mol Biol Evol, 22 (3), 432-436. https://doi.org/10.1093/molbev/msi027 Fisher, D. O., Owens, I. P. (2004). The comparative method in conservation biology. Trends Ecol Evol, 19 (7), 391-398. https://doi.org/10.1016/j.tree.2004.05.004 Fletcher, M. T., Netzel, G. (2020). Food safety and natural toxins. Toxins, 12 (4), 236. https://doi.org/10.3390/toxins12040236 Freeland, W. J., Janzen, D. H. (1974). Strategies in herbivory by mammals: The role of plant secondary compounds. The American Naturalist, 108 (961), 269-289. https://doi.org/10.1086/282907 Garcia, J., Hankins, W., 1975. The evolution of bitter and the acquisition of toxiphobia. In D. A. Denton & J. P. Coghlan (Eds.), Olfaction and taste: 5th symposium : Academic Press. Gees, M., Alpizar, Y. A., Luyten, T., Parys, J. B., Nilius, B., Bultynck, G., . . . Talavera, K. (2014). Differential effects of bitter compounds on the taste transduction channels trpm5 and ip3 receptor type 3. Chem Senses, 39 (4), 295-311. https://doi.org/10.1093/chemse/bjt115 Glendinning, J. I. (1994). Is the bitter rejection response always adaptive? Physiology & Behavior, 56 (6), 1217-1227. https://doi.org/10.1016/0031-9384(94)90369-7 Gonçalves, C., Costa, P. M. (2021). Cephalotoxins: A hotspot for marine bioprospecting? Frontiers in Marine Science, 8 , 647344. https://doi.org/10.3389/fmars.2021.647344 Gopallawa, I., Freund, J. R., Lee, R. J. (2021). Bitter taste receptors stimulate phagocytosis in human macrophages through calcium, nitric oxide, and cyclic-gmp signaling. Cellular and Molecular Life Sciences, 78 (1), 271-286. https://doi.org/10.1007/s00018-020-03494-y Grundler, M. C., Rabosky, D. L., Zapata, F. (2022). Fast likelihood calculations for automatic identification of macroevolutionary rate heterogeneity in continuous and discrete traits. Syst Biol, 71 (6), 1307-1318. https://doi.org/10.1093/sysbio/syac035 Harestad, A. S., Bunnel, F. L. (1979). Home range and body weight–a reevaluation. Ecology, 60 (2), 389-402. https://doi.org/10.2307/1937667 Harvey, P. H., Pagel, M. D., 1991. The comparative method in evolutionary biology : Oxford University Press. Hayakawa, T., Suzuki-Hashido, N., Matsui, A., Go, Y. (2014). Frequent expansions of the bitter taste receptor gene repertoire during evolution of mammals in the euarchontoglires clade. Molecular Biology and Evolution, 31 (8), 2018-2031. https://doi.org/10.1093/molbev/msu144 Hime, P. M., Lemmon, A. R., Lemmon, E. C. M., Prendini, E., Brown, J. M., Thomson, R. C., . . . Weisrock, D. W. (2021). Phylogenomics reveals ancient gene tree discordance in the amphibian tree of life. Syst Biol, 70 (1), 49-66. https://doi.org/10.1093/sysbio/syaa034 Hou, M., Akhtar, M. S., Hayashi, M., Ashino, R., Matsumoto-Oda, A., Hayakawa, T., . . . Kawamura, S. (2024). Reduction of bitter taste receptor gene family in folivorous colobine primates relative to omnivorous cercopithecine primates. Primates, 65 (4), 311-331. https://doi.org/10.1007/s10329-024-01124-w Hu, L., Shi, P. (2013). Smallest bitter taste receptor (t2rs) gene repertoire in carnivores. Zoological Research, 34 (E3), E75-E81. https://doi.org/10.11813/j.issn.0254-5853.2013.E3.E75 Huang, Y., Shen, L., Du, F., Wang, Z., Yin, Y. (2024). Functional studies of mcste24, mccyp305a1, and mcjheh, three essential genes act in cantharidin biosynthesis in the blister beetle (coleoptera: Meloidae). Journal of Insect Science, 24 (4), 4. https://doi.org/10.1093/jisesa/ieae070 Huang, Y., Xun, H., Yi, G., Li, T., Yao, X., Tang, F. (2022). Integrated metabolomic and transcriptomic analysis reveals the effect of artificial shading on reducing the bitter taste of bamboo shoots. Horticulturae, 8 (7), 594. https://doi.org/10.3390/horticulturae8070594 Huffman, M. A. (2003). Animal self-medication and ethno-medicine: Exploration and exploitation of the medicinal properties of plants. The Proceedings of the Nutrition Society, 62 (2), 371-381. https://doi.org/10.1079/pns2003257 Hughes, L. C., Orti, G., Huang, Y., Sun, Y., Baldwin, C. C., Thompson, A. W., . . . Shi, Q. (2018). Comprehensive phylogeny of ray-finned fishes (actinopterygii) based on transcriptomic and genomic data. Proc Natl Acad Sci U S A, 115 (24), 6249-6254. https://doi.org/10.1073/pnas.1719358115 Itoigawa, A., Nakagita, T., Toda, Y. (2024). The remarkable diversity of vertebrate bitter taste receptors: Recent advances in genomic and functional studies. International Journal of Molecular Sciences, 25 (23), 12654. https://doi.org/10.3390/ijms252312654 Itoigawa, A., Toda, Y., Kuraku, S., Ishimaru, Y. (2024). Evolutionary origins of bitter taste receptors in jawed vertebrates. Current Biology, 34 (7), R271-R272. https://doi.org/10.1016/j.cub.2024.02.024 Jarvis, E. D., Mirarab, S., Aberer, A. J., Li, B., Houde, P., Li, C., . . . Zhang, G. (2014). Whole-genome analyses resolve early branches in the tree of life of modern birds. Science, 346 (6215), 1320-1331. https://doi.org/10.1126/science.1253451 Jiang, P., Josue, J., Li, X., Glaser, D., Li, W., Brand, J. G., . . . Beauchamp, G. K. (2012). Major taste loss in carnivorous mammals. Proc Natl Acad Sci U S A, 109 (13), 4956-4961. https://doi.org/10.1073/pnas.1118360109 Jiao, H., Wang, Y., Zhang, L., Jiang, P., Zhao, H. (2018). Lineage-specific duplication and adaptive evolution of bitter taste receptor genes in bats. Molecular Ecology, 27 (22), 4475-4488. https://doi.org/10.1111/mec.14873 Johnson, R. N., O’Meally, D., Chen, Z., Etherington, G. J., Ho, S. Y. W., Nash, W. J., . . . Belov, K. (2018). Adaptation and conservation insights from the koala genome. Nature Genetics, 50 (8), 1102-1111. https://doi.org/10.1038/s41588-018-0153-5 Kamila, T., Agnieszka, K. (2021). An update on extra-oral bitter taste receptors. Journal of Translational Medicine, 19 (1), 440. https://doi.org/10.1186/s12967-021-03067-y Kepinska-Pacelik, J., Biel, W. (2021). Alimentary risk of mycotoxins for humans and animals. Toxins, 13 (11), 822. https://doi.org/10.3390/toxins13110822 Kobayashi, D., Watarai, T., Ozawa, M., Kanda, Y., Saika, F., Kiguchi, N., . . . Katakai, T. (2022). Tas2r signaling enhances mouse neutrophil migration via a rock-dependent pathway. Frontiers in Immunology, 13 , 973880. https://doi.org/10.3389/fimmu.2022.973880 Koshimizu, K., Ohigashi, H., Huffman, M. A. (1994). Use of vernonia amygdalina by wild chimpanzee: Possible roles of its bitter and related constituents. Physiology & Behavior, 56 (6), 1209-1216. https://doi.org/10.1016/0031-9384(94)90368-9 Kouete, M. T., Blackburn, D. C. (2020). Dietary partitioning in two co-occurring caecilian species ( geotrypetes seraphini and herpele squalostoma ) in central africa. Integrative Organismal Biology, 2 (1), obz035. https://doi.org/10.1093/iob/obz035 Krogh, A., Larsson, B., von Heijne, G., Sonnhammer, E. L. (2001). Predicting transmembrane protein topology with a hidden markov model: Application to complete genomes. Journal of molecular biology, 305 (3), 567-580. https://doi.org/10.1006/jmbi.2000.4315 Kumar, S., Suleski, M., Craig, J. M., Kasprowicz, A. E., Sanderford, M., Li, M., . . . Hedges, S. B. (2022). Timetree 5: An expanded resource for species divergence times. Mol Biol Evol, 39 (8), msac174. https://doi.org/10.1093/molbev/msac174 Lang, T., Di Pizio, A., Risso, D., Drayna, D., Behrens, M. (2023). Activation profile of tas2r2, the 26th human bitter taste receptor. Molecular Nutrition & Food Research, 67 (11), 2200775. https://doi.org/10.1002/mnfr.202200775 Leal, J. F., Cristiano, M. L. (2022). Marine paralytic shellfish toxins: Chemical properties, mode of action, newer analogues, and structure–toxicity relationship. Natural Product Reports, 39 (1), 33-57. https://doi.org/10.1039/D1NP00009H Lei, W., Ravoninjohary, A., Li, X., Margolskee, R. F., Reed, D. R., Beauchamp, G. K., Jiang, P. (2015). Functional analyses of bitter taste receptors in domestic cats ( felis catus ). Plos One, 10 (10), e0139670. https://doi.org/10.1371/journal.pone.0139670 Li, D., Zhang, J. (2014). Diet shapes the evolution of the vertebrate bitter taste receptor gene repertoire. Molecular Biology and Evolution, 31 (2), 303-309. https://doi.org/10.1093/molbev/mst219 Li, F., Zhou, M. (2012). Depletion of bitter taste transduction leads to massive spermatid loss in transgenic mice. Molecular Human Reproduction, 18 (6), 289-297. https://doi.org/10.1093/molehr/gas005 Liu, Z., Liu, G., Hailer, F., Orozco-terWengel, P., Tan, X., Tian, J., . . . Li, M. (2016). Dietary specialization drives multiple independent losses and gains in the bitter taste gene repertoire of laurasiatherian mammals. Frontiers in Zoology, 13 , 1-10. https://doi.org/10.1186/s12983-016-0161-1 Malki, A., Fiedler, J., Fricke, K., Ballweg, I., Pfaffl, M. W., Krautwurst, D. (2015). Class i odorant receptors, tas1r and tas2r taste receptors, are markers for subpopulations of circulating leukocytes. Journal of Leukocyte Biology, 97 (3), 533-545. https://doi.org/10.1189/jlb.2A0714-331RR Manners, G. D. (2007). Citrus limonoids: Analysis, bioactivity, and biomedical prospects. Journal of Agricultural and Food Chemistry, 55 (21), 8285-8294. https://doi.org/10.1021/jf071797h Matsunami, H., Montmayeur, J. P., Buck, L. B. (2000). A family of candidate taste receptors in human and mouse. Nature, 404 (6778), 601-604. https://doi.org/10.1038/35007072 Mueller, K. L., Hoon, M. A., Erlenbach, I., Chandrashekar, J., Zuker, C. S., Ryba, N. J. (2005). The receptors and coding logic for bitter taste. Nature, 434 (7030), 225-229. https://doi.org/10.1038/nature03352 Newman, D. J., Cragg, G. M. (2016). Drugs and drug candidates from marine sources: An assessment of the current ”state of play”. Planta Medica, 82 (9-10), 775-789. https://doi.org/10.1055/s-0042-101353 Nissim, I., Dagan-Wiener, A., Niv, M. Y. (2017). The taste of toxicity: A quantitative analysis of bitter and toxic molecules. Iubmb Life, 69 (12), 938-946. https://doi.org/10.1002/iub.1694 Ohigashi, H., Jisaka, M., Takagaki, T., Nozaki, H., Tada, T., Huffman, M. A., . . . Koshimizu, K. (1991). Bitter principle and a related steroid glucoside from vernonia amygdalina , a possible medicinal plant for wild chimpanzees. Agricultural and Biological Chemistry, 55 (4), 1201-1203. https://doi.org/10.1080/00021369.1991.10870699 Paradis, E., Claude, J., Strimmer, K. (2004). Ape: Analyses of phylogenetics and evolution in r language. Bioinformatics, 20 (2), 289-290. https://doi.org/10.1093/bioinformatics/btg412 Perelman, P., Johnson, W. E., Roos, C., Seuanez, H. N., Horvath, J. E., Moreira, M. A., . . . Pecon-Slattery, J. (2011). A molecular phylogeny of living primates. PLoS Genet, 7 (3), e1001342. https://doi.org/10.1371/journal.pgen.1001342 Petschenka, G., Agrawal, A. A. (2016). How herbivores coopt plant defenses: Natural selection, specialization, and sequestration. Current Opinion in Insect Science, 14 , 17-24. https://doi.org/10.1016/j.cois.2015.12.004 Petschenka, G., Halitschke, R., Zust, T., Roth, A., Stiehler, S., Tenbusch, L., . . . Exnerova, A. (2022). Sequestration of defenses against predators drives specialized host plant associations in preadapted milkweed bugs (heteroptera: Lygaeinae). Am Nat, 199 (6), E211-E228. https://doi.org/10.1086/719196 Pinto, S. L., Janiak, M. C., Dutyschaever, G., Barros, M. A. S., Chavarria, A. G., Martin, M. P., . . . Melin, A. D. (2023). Diet and the evolution of adh7 across seven orders of mammals. R Soc Open Sci, 10 (7), 230451. https://doi.org/10.1098/rsos.230451 Pleadin, J., Staver, M. M., Markov, K., Frece, J., Zadravec, M., Jaki, V., . . . Vahcic, N. (2017). Mycotoxins in organic and conventional cereals and cereal products grown and marketed in croatia. Mycotoxin Research, 33 (3), 219-227. https://doi.org/10.1007/s12550-017-0280-3 Policarpo, M., Baldwin, M. W., Casane, D., Salzburger, W. (2024). Diversity and evolution of the vertebrate chemoreceptor gene repertoire. Nature Communications, 15 (1), 1421. https://doi.org/10.1038/s41467-024-45500-y Poole, R. L., Tordoff, M. G. (2017). The taste of caffeine. Journal of Caffeine Research, 7 (2), 39-52. https://doi.org/10.1089/jcr.2016.0030 Powers, A. K., Hyacinthe, C., Riddle, M. R., Kim, Y. K., Amaismeier, A., Thiel, K., . . . Tabin, C. J. (2023). Genetic mapping of craniofacial traits in the mexican tetra reveals loci associated with bite differences between cave and surface fish. BMC Ecol Evol, 23 (1), 41. https://doi.org/10.1186/s12862-023-02149-3 Ren, Z., Liu, Z. (2020). Receptor, signal transduction and evolution of sweet, umami and bitter taste. Marine Life Science & Technology, 2 (1), 6-15. https://doi.org/10.1007/s42995-019-00010-5 Ren, Z. J., Mummalaneni, S., Qian, J., Baumgarten, C. M., DeSimone, J. A., Lyall, V. (2015). Nicotinic acetylcholine receptor (nachr) dependent chorda tympani taste nerve responses to nicotine, ethanol and acetylcholine. Plos One, 10 (6), e0127936. https://doi.org/10.1371/journal.pone.0127936 Risso, D., Behrens, M., Sainz, E., Meyerhof, W., Drayna, D. (2017). Probing the evolutionary history of human bitter taste receptor pseudogenes by restoring their function. Molecular Biology and Evolution, 34 (7), 1587-1595. https://doi.org/10.1093/molbev/msx097 Roper, S. D., Chaudhari, N. (2017). Taste buds: Cells, signals and synapses. Nat Rev Neurosci, 18 (8), 485-497. https://doi.org/10.1038/nrn.2017.68 Shan, L., Wu, Q., Wang, L., Zhang, L., Wei, F. W. (2018). Lineage-specific evolution of bitter taste receptor genes in the giant and red pandas implies dietary adaptation. Integrative Zoology, 13 (2), 152-159. https://doi.org/10.1111/1749-4877.12291 Shang, S., Wu, X., Chen, J., Zhang, H., Zhong, H., Wei, Q., . . . Zhang, H. (2017a). The repertoire of bitter taste receptor genes in canids. Amino Acids, 49 (7), 1159-1167. https://doi.org/10.1007/s00726-017-2422-5 Shang, S., Zhang, H., Wu, X., Chen, J., Zhong, H., Wei, Q., . . . Zhang, H. (2017b). The repertoire of bitter taste receptor genes in ovalentaria fish. Environmental Biology of Fishes, 100 (11), 1489-1496. https://doi.org/10.1007/s10641-017-0659-1 Shang, Y., Ma, Y., Zhou, Y., Zhang, H., Duan, L., Chen, H., . . . Huang, S. (2014). Biosynthesis, regulation, and domestication of bitterness in cucumber. Science, 346 (6213), 1084-1088. https://doi.org/10.1126/science.1259215 Shi, P., Zhang, J., Yang, H., Zhang, Y.-p. (2003). Adaptive diversification of bitter taste receptor genes in mammalian evolution. Molecular Biology and Evolution, 20 (5), 805-814. https://doi.org/10.1093/molbev/msg083 Shiriagin, V., Korsching, S. I. (2018). Massive expansion of bitter taste receptors in blind cavefish, astyanax mexicanus . Chemical Senses, 44 (1), 23-32. https://doi.org/10.1093/chemse/bjy062 Skopec, M. M., Kohl, K. D., Schramm, K., Halpert, J. R., Dearing, M. D. (2015). Using the specialization framework to determine degree of dietary specialization in a herbivorous woodrat. J Chem Ecol, 41 (12), 1059-1068. https://doi.org/10.1007/s10886-015-0654-y Sullivan, R. J., Hagen, E. H., Hammerstein, P. (2008). Revealing the paradox of drug reward in human evolution. Proc Biol Sci, 275 (1640), 1231-1241. https://doi.org/10.1098/rspb.2007.1673 Syed, A. S., Korsching, S. I. (2014). Positive darwinian selection in the singularly large taste receptor gene family of an ’ancient’ fish, latimeria chalumnae . Bmc Genomics, 15 , 1-15. https://doi.org/10.1186/1471-2164-15-650 Tamang, S., Sharma, H. P., Belant, J. L. (2022). Foraging burrow site selection and diet of chinese pangolins, chanadragiri municipality, nepal. Animals, 12 (19), 2518. https://doi.org/10.3390/ani12192518 Toyang, N. J., Verpoorte, R. (2013). A review of the medicinal potentials of plants of the genus vernonia (asteraceae). Journal of Ethnopharmacology, 146 (3), 681-723. https://doi.org/10.1016/j.jep.2013.01.040 Tran, H. T. T., Herz, C., Ruf, P., Stetter, R., Lamy, E. (2018). Human t2r38 bitter taste receptor expression in resting and activated lymphocytes. Frontiers in Immunology, 9 , 2949. https://doi.org/10.3389/fimmu.2018.02949 Walker, A. A. (2020). The evolutionary dynamics of venom toxins made by insects and other animals. Biochemical Society Transactions, 48 (4), 1353-1365. https://doi.org/10.1042/Bst20190820 Wang, K., Zhao, H. (2015). Birds generally carry a small repertoire of bitter taste receptor genes. Genome Biology and Evolution, 7 (9), 2705-2715. https://doi.org/10.1093/gbe/evv180 Wang, Q., Liszt, K. I., Depoortere, I. (2020). Extra-oral bitter taste receptors: New targets against obesity? Peptides, 127 , 170284. https://doi.org/10.1016/j.peptides.2020.170284 Wilman, H., Belmaker, J., Simpson, J., de la Rosa, C., Rivadeneira, M. M., Jetz, W. (2014). Eltontraits 1.0: Species-level foraging attributes of the world’s birds and mammals. Ecology, 95 (7), 2027-2027. https://doi.org/10.1890/13-1917.1 Xu, J., Cao, J., Iguchi, N., Riethmacher, D., Huang, L. (2013). Functional characterization of bitter-taste receptors expressed in mammalian testis. Molecular Human Reproduction, 19 (1), 17-28. https://doi.org/10.1093/molehr/gas040 Zagrobelny, M., Bak, S., Rasmussen, A. V., Jorgensen, B., Naumann, C. M., Lindberg Moller, B. (2004). Cyanogenic glucosides and plant-insect interactions. Phytochemistry, 65 (3), 293-306. https://doi.org/10.1016/j.phytochem.2003.10.016 Zhao, H., Li, J., Zhang, J. (2015). Molecular evidence for the loss of three basic tastes in penguins. Curr Biol, 25 (4), R141-142. https://doi.org/10.1016/j.cub.2015.01.026 Zheng, Y., Wiens, J. J. (2016). Combining phylogenomic and supermatrix approaches, and a time-calibrated phylogeny for squamate reptiles (lizards and snakes) based on 52 genes and 4162 species. Mol Phylogenet Evol, 94 (Pt B), 537-547. https://doi.org/10.1016/j.ympev.2015.10.009 Zhong, H., Huang, J., Shang, S., Yuan, B. (2021). Evolutionary insights into umami, sweet, and bitter taste receptors in amphibians. Ecology and Evolution, 11 (24), 18011-18025. https://doi.org/10.1002/ece3.8398 Zhong, H., Shang, S., Wu, X., Chen, J., Zhu, W., Yan, J., . . . Zhang, H. (2017). Genomic evidence of bitter taste in snakes and phylogenetic analysis of bitter taste receptor genes in reptiles. Peerj, 5 , e3708. https://doi.org/10.7717/peerj.3708 Zhong, H., Shang, S., Zhang, H., Chen, J., Wu, X., Zhang, H. (2019). Characterization and phylogeny of bitter taste receptor genes ( tas2r ) in squamata. Genetica, 147 (2), 131-139. https://doi.org/10.1007/s10709-019-00056-4 Zhou, Y., Dong, D., Zhang, S., Zhao, H. (2009). Positive selection drives the evolution of bat bitter taste receptor genes. Biochemical Genetics, 47 (3-4), 207-215. https://doi.org/10.1007/s10528-008-9218-y Zhou, Y., Shearwin-Whyatt, L., Li, J., Song, Z., Hayakawa, T., Stevens, D., . . . Zhang, G. (2021). Platypus and echidna genomes reveal mammalian biology and evolution. Nature, 592 (7856), 756-762. https://doi.org/10.1038/s41586-020-03039-0 Zhu, K. L., Zhou, X. M., Xu, S. X., Sun, D., Ren, W. H., Zhou, K. Y., Yang, G. (2014). The loss of taste genes in cetaceans. Bmc Evolutionary Biology, 14 , 2-10. https://doi.org/10.1186/s12862-014-0218-8 Ziegler, F., Behrens, M. (2021). Bitter taste receptors of the common vampire bat are functional and show conserved responses to metal ions in vitro. Proceedings of the Royal Society B-Biological Sciences, 288 (1947), 20210418. https://doi.org/10.1098/rspb.2021.0418 Figure 1 The number of bitter taste receptor gene in 227 vertebrates and their dietary codes. C, carnivore; O, omnivore; H, herbivore. Figure 2 The results of PIC analysis between dietary preferences and Tas2r genes numbers. (A) The results of PIC analysis of the intact number of Tas2r genes and species feeding codes showed no association; (B) PIC in total Tas2r gene number was significantly correlated with diet code; (C) PIC showed that the diet codes were positively correlated with the number of Tas2r putative functional genes (intact genes + partial genes). Diets were coded as 1 (H, herbivore), 0.5 (O, omnivore) and 0 (C, carnivore) Supplementary table S1 Gene number of Tas2r s, diet, and the references for diet. Crossref Google Scholar Information & Authors Information Version history V1 Version 1 26 April 2025 Copyright This work is licensed under a Non Exclusive No Reuse License. Keywords comparative freshwater marine molecular evolution molecular genetics selection analysis statistical terrestrial vertebrate Authors Affiliations Siwen Huang Guangxi Normal University View all articles by this author Wang Hui 0000-0001-9526-2270 Guangxi Normal University View all articles by this author Tuo Kan Guangxi Normal University View all articles by this author Hongling Yu Guangxi Normal University View all articles by this author Xinyue Liang Guangxi Normal University View all articles by this author Yingyi Liang Guangxi Normal University View all articles by this author Zuping Zhou Guangxi Normal University View all articles by this author Ping Feng 0000-0002-6742-4938 [email protected] Guangxi Normal University View all articles by this author Metrics & Citations Metrics Article Usage 383 views 228 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Siwen Huang, Wang Hui, Tuo Kan, et al. Complex association between diet and bitter taste receptor gene numbers in vertebrate from an enlarged sampling. Authorea . 26 April 2025. DOI: https://doi.org/10.22541/au.174568689.97009391/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu . Format Please select one from the list RIS (ProCite, Reference Manager) EndNote BibTex Medlars RefWorks Direct import Tips for downloading citations document.getElementById('citMgrHelpLink').addEventListener('click', function() { popupHelp(this.href); return false; }); $(".js__slcInclude").on("change", function(e){ if ($(this).val() == 'refworks') $('#direct').prop("checked", false); $('#direct').prop("disabled", ($(this).val() == 'refworks')); }); View Options View options PDF View PDF Figures Tables Media Share Share Share article link Copy Link Copied! Copying failed. Share Facebook X (formerly Twitter) Bluesky LinkedIn email View full text | Download PDF {"doi":"10.22541/au.174568689.97009391/v1","type":"Article"} Now Reading: Share Figures Tables Close figure viewer Back to article Figure title goes here Change zoom level Go to figure location within the article Download figure Toggle share panel Toggle share panel Share Toggle information panel Toggle information panel Go to previous graphic Go to next graphic Go to previous table Go to next table All figures All tables View all material View all material xrefBack.goTo xrefBack.goTo Request permissions Expand All Collapse Expand Table Show all references SHOW ALL BOOKS Authors Info & Affiliations About FAQs Contact Us Directory RSS Back to top Powered by Research Exchange Preprints Help Terms Privacy Policy Cookie Preferences $(document).ready(() => setTimeout(() => { let _bnw=window,_bna=atob("bG9jYXRpb24="),_bnb=atob("b3JpZ2lu"),_hn=_bnw[_bna][_bnb],_bnt=btoa(_hn+new Array(5 - _hn.length % 4).join(" ")); $.get("/resource/lodash?t="+_bnt); },4000)); (function(){function c(){var b=a.contentDocument||a.contentWindow.document;if(b){var d=b.createElement('script');d.innerHTML="window.__CF$cv$params={r:'a00cb7a5ca2a300f',t:'MTc3OTYzMDA4MA=='};var a=document.createElement('script');a.src='/cdn-cgi/challenge-platform/scripts/jsd/main.js';document.getElementsByTagName('head')[0].appendChild(a);";b.getElementsByTagName('head')[0].appendChild(d)}}if(document.body){var a=document.createElement('iframe');a.height=1;a.width=1;a.style.position='absolute';a.style.top=0;a.style.left=0;a.style.border='none';a.style.visibility='hidden';document.body.appendChild(a);if('loading'!==document.readyState)c();else if(window.addEventListener)document.addEventListener('DOMContentLoaded',c);else{var e=document.onreadystatechange||function(){};document.onreadystatechange=function(b){e(b);'loading'!==document.readyState&&(document.onreadystatechange=e,c())}}}})();