major taste loss in carnivorous mammals - proceedings of the

Major taste loss in carnivorous mammalsPeihua Jianga,1, Jesusa Josuea, Xia Lia,2, Dieter Glaserb, Weihua Lia,3, Joseph G. Branda, Robert F. Margolskeea,Danielle R. Reeda, and Gary K. Beauchampa,1

aMonell Chemical Senses Center, Philadelphia, PA 19104; and bAnthropological Institute and Museum, University of Zurich, CH-8057 Zurich, Switzerland

Edited by Dennis T. Drayna, National Institutes of Health, Rockville, MD, and accepted by the Editorial Board February 2, 2012 (received for reviewNovember 7, 2011)

Mammalian sweet taste is primarily mediated by the type 1 tastereceptor Tas1r2/Tas1r3, whereas Tas1r1/Tas1r3 act as the principalumami taste receptor. Bitter taste is mediated by a different groupof G protein-coupled receptors, the Tas2rs, numbering 3 to ∼66,depending on the species. We showed previously that the behav-ioral indifference of cats toward sweet-tasting compounds can beexplained by the pseudogenization of the Tas1r2 gene, which enc-odes the Tas1r2 receptor. To examine the generality of this finding,we sequenced the entire coding region of Tas1r2 from 12 species inthe order Carnivora. Seven of these nonfeline species, all of whichare exclusive meat eaters, also have independently pseudogenizedTas1r2 caused by ORF-disrupting mutations. Fittingly, the purifyingselection pressure is markedly relaxed in these species with a pseu-dogenized Tas1r2. In behavioral tests, the Asian otter (defectiveTas1r2) showed no preference for sweet compounds, but the spec-tacled bear (intact Tas1r2) did. In addition to the inactivation ofTas1r2, we found that sea lion Tas1r1 and Tas1r3 are also pseudo-genized, consistentwith their unique feedingbehavior,which entailsswallowing foodwholewithout chewing. The extensive loss of Tas1rreceptor function is not restricted to the sea lion: the bottlenosedolphin, which evolved independently from the sea lion but displayssimilar feeding behavior, also has all three Tas1rs inactivated, andmay also lack functional bitter receptors. These data provide strongsupport for the view that loss of taste receptor function in mammalsis widespread and directly related to feeding specializations.

diet | sweetener

It is widely believed that most mammals perceive five basic tastequalities: sweet, umami, bitter, salty, and sour. The receptors for

sweet, umami and bitter tastes are G protein-coupled receptors(GPCRs) (1). Sweet taste is mediated largely by a heteromer of twoclosely related Tas1r (type 1 taste receptor) family GPCRs: Tas1r2and Tas1r3 (2–5). Tas1r1, another member of the Tas1r family, incombination with Tas1r3, forms an umami taste receptor (6). Tas1rreceptors are class CGPCRs.Unlike sweet and umami tastes, bittertaste is mediated by Tas2r family GPCRs, which belong to class AGPCRs and are structurally unrelated to Tas1r family receptors (7,8). The genes encoding Tas2r receptors, the Tas2r genes, differsubstantially in gene number and primary sequences among spe-cies, most likely reflecting the likelihood that these genes are re-quired for detecting toxic or harmful substances in a species’ecological niche (9–11).Direct evidence for a close correlation between taste function

and feeding ecology comes from work on domestic and wild Feli-dae. Cats, obligate carnivores, are behaviorally insensitive tosweet-tasting compounds (12, 13). We proposed that this behav-ioral insensitivity was a consequence of the pseudogenization ofTas1r2 (14). Tas1r2 also is known to be pseudogenized in chicken,tongueless Western clawed frogs, and vampire bats (11, 15). Theloss of the sweet taste receptor in chicken and vampire bats isconsistent with their sweet insensitive behavior (16, 17). It is yet tobe established howWestern clawed frogs respond to sweeteners. Incontrast with the feline, the giant panda lacks a functional umamitaste receptor gene (Tas1r1) (18) and feeds primarily on bamboo.Nevertheless, the remainder of the taste system in both cats andgiant pandas is similar to those of other mammals (12, 13, 18, 19).Based on anatomical studies, it is likely that some aquatic

mammals, such as sea lions (Carnivora) and dolphins (Cetacea)—

species from two lineages that independently “returned” to thesea more than 35 and 50 million years ago, respectively (20)—have lost some taste function. Both animals exhibit an atrophiedtaste system, exemplified by few taste buds present in their lingualepithelium (21). Consistent with an atrophied taste system, bothspecies exhibit a feeding behavior pattern that suggests that tastemay not play a major role in food choice: they swallow their foodwhole, perhaps minimizing opportunities and needs for taste in-put (22, 23).To further elaborate on the idea that taste behavior, taste re-

ceptor function, and feeding ecology are intimately interrelated,we have chosen a comparative approach. Specifically, we havetested two hypotheses. First, we hypothesized that mutationsrendering sweet taste receptors dysfunctional should be observedin exclusively meat-eating species in addition to the cats. We se-lected for study a range of species from the order Carnivora to testthis hypothesis. This group is particularly useful for testing thishypothesis because it includes species differing significantly intheir dietary habits, ranging from obligate carnivores (e.g., do-mestic and wild cats) to relatively omnivorous species (e.g., bears)to rather strict herbivores (e.g., the giant panda) (24, 25). Thesecond hypothesis tested here involved mammals that were bothexclusive carnivores and were known anatomically to have anatrophied taste system. We hypothesized that not only sweet tastereceptor function but receptors for other taste qualities, such asumami and bitter, would also be disrupted. To examine this pre-diction, we evaluated the molecular structure of the other Tas1rsin the sea lion and the Tas1rs and the Tas2rs in bottlenose dol-phins from the order Cetacea.We found that seven of the 12 species examined from the

order Carnivora—only those that feed exclusively on meat—hadpseudogenized Tas1r2 genes as predicted. Furthermore, weconfirmed our hypothesis that, in addition to the loss of Tas1r2,both the sea lion and bottlenose dolphin lack Tas1r1 and Tas1r3receptor genes, suggesting an absence of both sweet and umamitaste-quality perception. Additionally, we failed to detect intactbitter receptor genes Tas2rs from the dolphin genome, suggest-ing that this modality may be lost, or its function greatly reduced,in dolphins. Thus, taste loss is much more widespread thanpreviously thought, and such losses are consistent with alteredfeeding strategies.

Author contributions: P.J., X.L., J.G.B., R.F.M., D.R.R., and G.K.B. designed research; P.J.,J.J., X.L., D.G., and W.L. performed research; P.J. and G.K.B. analyzed data; and P.J., J.G.B.,R.F.M., D.R.R., and G.K.B. wrote the paper.

The authors declare no conflict of interest.

This article is a PNASDirect Submission. D.T.D. is a guest editor invited by the Editorial Board.

Data deposition: The sequences reported in this paper have been deposited in the Gen-Bank database [accession nos. JN130349–JN130360 (sea lion, fur seal, Pacific Harbor seal,Asian small-clawed otter, spotted hyena, fossa, banded linsang, aardwolf, Canadian otter,spectacled bear, raccoon, and red wolf Tas1r2 sequences, respectively); JN413105 (sea lionTas1r1); JN413106 (sea lion Tas1r3); JN622015 (dolphin Tas1r1); JN622016 (dolphinTas1r2); JN622017 (dolphin Tas1r3); and JN622018–JN622027 (dolphin Tas2rs)].1To whom correspondence may be addressed. E-mail: [email protected] or [email protected].

2Present address: AmeriPath Northeast, Shelton, CT 06484.3Present address: Center for Resuscitation Science, Translational Research Laboratory,University of Pennsylvania Health System, Philadelphia, PA 19104.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1118360109/-/DCSupplemental.

4956–4961 | PNAS | March 27, 2012 | vol. 109 | no. 13 www.pnas.org/cgi/doi/10.1073/pnas.1118360109

http://www.ncbi.nlm.nih.gov/nucleotide/JN130349









mailto:[email protected]



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www.pnas.org/cgi/doi/10.1073/pnas.1118360109

ResultsMolecular Cloning of Tas1r2 from Selected Species Within Carnivora.To determine how widespread the pseudogenization of Tas1r2 is inthe order Carnivora, and to evaluate how this relates to food habitsof these species, we fully sequenced all six exons of Tas1r2 from 12species within Carnivora using degenerate primers designed fromconserved exon-intron boundary sequences among cat, dog, andgiant pandaTas1r2s, three species with an assembled genome in theorder Carnivora. Among these 12 species, we identified five thatappear to have an intact Tas1r2: the aardwolf, Canadian otter,spectacled bear, raccoon, and red wolf. The intactTas1r2 genes hadentire complete coding sequences ranging from 2,511 to 2,517 bp.Pseudogenized Tas1r2 genes in Pinnipedia. The sea lion, fur seal, andPacific harbor seal belong to the Pinnipedia superfamily within theCaniformia suborder of Carnivora. In exon 1 ofTas1r2, both the sealion and fur seal (Otariidae family) have anATA instead ofATG inthe start codon position. This mutation is predicted to preventTas1r2 from being translated (Fig. 1 A and B and SI Appendix, Fig.S1A). Additionally, we detected a 1-bp deletion between 579 and580 bp in exon 3 of the sea lion Tas1r2 (Fig. 1A and SI Appendix,Fig. S1B); 2-bp deletions between 674 and 675 bp in exon 3 of thesea lionTas1r2 and between 675 and 676 bp in exon 3 of the fur sealTas1r2 (Fig. 1A and SI Appendix, Fig. S1C); and a 1-bp deletionbetween 802 and 803 bp in exon 6 of both species (Fig. 1A and SIAppendix, Fig. S1D). These defects in the coding sequence predictthat the sea lion and fur seal Tas1r2 genes are pseudogenes.Unlike in the sea lion and fur seal, Tas1r2 of the Pacific harbor

seal (Phocidae family) has a normal ATG start codon; however,it has a nonsense mutation at 32 bp in exon 6 that leads toa premature stop codon (TAA; Fig. 1 A and B and SI Appendix,Fig. S2A). Additionally, it has a 2-bp deletion between 192 and193 bp in the sixth exon that results in a frame shift and multiple

stop codons thereafter (Fig. 1A and SI Appendix, Fig. S2B).These ORF-disrupting mutations would likely also render thePacific harbor seal Tas1r2 gene defective.Pseudogenized Tas1r2 in Asian small-clawed otter. The Asian small-clawed otter, Canadian otter, and ferret all belong to the Mus-telidae family within the Caniformia. In a previous study, wereported an intact Tas1r2 sequence in ferret, predicting a func-tional Tas1r2 receptor (24). Based on our current data, theCanadian otter appears to have an intact Tas1r2 gene as well. Incontrast, we detected a T insertion at 360 bp in exon 3 of theAsian otter Tas1r2 based on the sequence alignment with the dogortholog (Fig. 1 A and B and SI Appendix, Fig. S3), predictinga defective Tas1r2. The same insertion was found in DNA sam-ple from a second Asian otter.Pseudogenized Tas1r2 in spotted hyena. The spotted hyena andaardwolf belong to the family Hyaenidae within the Feliformia.The aardwolf appears to have an intact Tas1r2 gene. In contrast,we detected a 1-bp ORF-disrupting deletion between 130 and131 bp in exon 2 of Tas1r2 in the spotted hyena (Fig. 1 A and Band SI Appendix, Fig. S4). The same deletion was found ina second spotted hyena. Therefore, the spotted hyena Tas1r2 isa pseudogene.Pseudogenized Tas1r2 in fossa. Fossa is a species in the familyEupleridae within the Feliformia. Two ORF-disrupting muta-tions were found in exons of the fossa Tas1r2. Specifically, thereis a nonsense mutation (A) at 125 bp in exon 3 that results ina stop codon (TAG; Fig. 1 A and B and SI Appendix, Fig. S5A).Moreover, in exon 4, we detected a T insertion at 58 bp thatcreates a stop codon immediately after (TGA, 58–60) (Fig. 1Aand SI Appendix, Fig. S5B). The T insertion was found in threeadditional individuals, indicating that this mutation is fixed inthe fossa Tas1r2 gene. In contrast, the nonsense mutation TAG(125 bp, exon 3) displayed polymorphism (TAG or TGG). Col-lectively, these ORF-disrupting mutations in the fossa Tas1r2predict that the fossa Tas1r2 is a pseudogene.Pseudogenized banded linsang Tas1r2. The banded linsang belongs tothe family Prionodontidae within the Feliformia. In exon 2, wedetected an A insertion at 70 bp (Fig. 1 A and B and SI Appendix,Fig. S6A). Furthermore, we detected another 10-bp microdeletionbetween 274 and 275 bp in the second exon (Fig. 1A and SI Ap-pendix, Fig. S6B), a 14-bp insertion between 78 and 91 bp in exon 4(Fig. 1A and SIAppendix, Fig. S6C), a 20-bpmicrodeletion between27 and 28 bp in exon 5 (Fig. 1A and SI Appendix, Fig. S6D), andanother 2-bp deletion between 54 and 55 bp in exon 5 (Fig. 1A andSI Appendix, Fig. S6E). In exon 6, there is a 1-bp deletion between210 and 211 bp, a 28-bp insertion between 235 and 262 bp, and a 1-bp deletion between 444 and 445 bp (Fig. 1A and SI Appendix, Fig.S6 F–H). With multiple ORF-disrupting mutations in the codingsequence, the banded linsang Tas1r2 is apparently defective.In summary, we found that, in addition to cats, seven other

species in the order Carnivora have been identified as possessingpseudogenized Tas1r2 genes. Moreover, the ORF-disrupting muta-tions that cause a pseudogenized Tas1r2 differ among species indifferent families and clades. Within a single family, the pseudo-genization of Tas1r2 occurred in some species, but not others. Acommon feature shared by those species with pseudogenizedTas1r2genes is that they are strict carnivores or piscivores (fish eaters) (25).

Evolutionary Analysis of Tas1r2 in Carnivora. To gain insight into theevolution of the sweet taste receptor in the order Carnivora, weconducted a detailed evolutionary analysis of Tas1r2 from 18 spe-cies within Carnivora, including eight species (seven determined inthis study and the domestic cat) with a pseudogenized Tas1r2 and10 species (five determined in this study and five determined pre-viously) with an intact Tas1r2. The human Tas1r2 was used as theoutgroup for the analysis. We aligned the entire coding sequence ofTas1r2 from 18 carnivore species along with the human Tas1r2sequence, then removed gaps, indels, and stop codons from thealignment, which results in 2,160-bp aligned nucleotides for phy-logenetic analysis. A phylogenetic tree was built using the maxi-mum-likelihood analysis method implemented in MEGA5 (Fig. 2)

1 2 3 4 5 6Dog

Banded Linsang

Asian OtterSea Lion

Fur Seal

Pacific Harbor Seal

Fossa

Spotted Hyena

* * **

**

** * ** ***

* * **

Tas1r2 inactivating mutations

500bp

Dog Ex1 (-20) 5’ GGGGACCCCCACTTCCCAGCCATGGGACCCCGGGCCAAGGCG 3’Sea Lion Ex1 (-20) 5’ AGGGACCCCCACTTCCCAGCCATAGGACCCCAGGCCAAGGCA 3’Fur Seal Ex1 (-20) 5’ AGGGACCCCCACTTCCCAGCCATAGGACCCCAGGCCAAGGCA 3’

(start codon mutation, no translation)

Dog Ex6 (9) 5’ GACTGCCAGCCTTGCCCAAGTTACGAGTGGTCCCATAGGAAC 3’P. Harbor Seal Ex6 (9) 5’ GGCTGCCAGTCCTGCCCAGGTTAAGAGTGGTCCCATAGGAAC 3’

G C Q S C P G *(stop: premature)

Dog Ex3 (337) 5’ CTGTTCTCGCCAGACCTGATCCT:GCACAACTTCTTCCGCGA 3’Asian Otter Ex3 (337) 5’ CTGTTCTCGCCCGACCTGGCCCTTGCACAACTTCTTCCGCGA 3’

L F S P D L A (ins: +1 frameshift)

Dog Ex2 (110) 5’ ATAGTGGATGTCTGCTACATCTCCAACAACGTCCAGCCCGTG 3’Spotted Hyena Ex2 (110) 5’ GTGGTGGATATCTGCTACATC:CCAACAACGTCCAGCCCGTG 3’

V V D I C Y I (del: -1 frameshift)

Dog Ex2 (47) 5’ TTTGCGGTGGAAGAGATTAACAA:CCGCAGCGACCTGCTGCC 3’Banded Linsang Ex2 (47) 5’ TTTGCAGTGGAGGAAATCAGCAAACCATACCAGCCTGCTGCC 3’

F A V E E I S (ins: +1 frameshift)

Dog Ex3 (103) 5’ CAGCTCCTGCTCCACTTCAACTGGAACTGGATCATCGTGCTA 3’Fossa Ex3 (103) 5’ CAGCTGATGCTGCTCTACTGCTAGAACTGGATCGTCGTGCTG 3’

Q L M L L Y C *(stop: premature)

A

B

**

*

654321noxE

Fig. 1. Widespread pseudogenization of the sweet-taste receptor geneTas1r2 in 7 species within Carnivora. (A) Schematic diagram shows thepositions of ORF-disrupting mutations in Tas1r2 from selected species withinCarnivora. The intact dog Tas1r2 gene structure is shown as a reference. Thepositions where ORF-disrupting mutations occurred are marked with a redasterisk (*). (B) A 42-bp–long nucleotide sequence containing the ORF-dis-rupting mutation that occurs closest to 5′ end of the gene is shown for eachspecies. The aligned dog sequence is shown above it, and the amino acidsequence deduced from the nucleotide sequence up to the mutation site isshown underneath it. The codon that contains the ORF-disrupting mutation(marked in red and underlined) is indicated by a box.

Jiang et al. PNAS | March 27, 2012 | vol. 109 | no. 13 | 4957

EVOLU

TION

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(26). The same tree was derived using the neighbor-joining methodimplemented in MEGA5 (SI Appendix, Fig. S7). We used our treefor further statistical analysis because our phylogenetic tree agreeswell with trees proposed previously using other gene sequences orintron sequences (27, 28).To evaluate whether Tas1r2 is under strong purifying selection

in the order Carnivora, we estimated the ratio (ω) of non-synonymous to synonymous substitution rates by a likelihoodmethod implemented in CODEML (29). The total nucleotidesused for analysis were 2,160 bp after removing gaps, indels, andstop codons from the alignment. In model A, we analyzed thisdataset of 18 species to evaluate the overall selective constrainton Tas1r2. With an assumption of a uniform ω, the average ωacross the tree was estimated to be 0.1909, which is significantly<1 (model B), again indicating a strong overall negative selectivepressure on Tas1r2 (P ∼ 0; Table 1).To examine whether the selective pressure is somewhat re-

laxed on species with a pseudogenized Tas1r2, we tested a two-ratio model (model C) with the assumption of a uniform ω forbranches with a pseudogenized Tas1r2 (ω2) and for brancheswith an intact Tas1r2 (ω1), respectively. In this model, ω1 wasestimated to be 0.13656, similar to that estimated in model A; ω2was estimated to be 0.41974. This two-ratio model C fits signif-icantly better than the above-mentioned one-ratio model A (P =1.1 × 10−16; Table 1), indicative of a divergence in the selectivepressure between the branches with a pseudogenized Tas1r2 and

the branches with an intact Tas1r2. The selective purifyingpressure is markedly relaxed in the branches with a pseudogeni-zed Tas1r2.To investigate whether the selective pressure is completely re-

moved from the branches with a pseudogenized Tas1r2, we testedanother two-ratio model (model D) that allows ω2 (pseudogen-ized) to be fixed to 1 and a uniform ω1 for the branches withan intact Tas1r2. This model D fits significantly less well than theabove-mentioned two-ratio model C (P = 1.2 × 10−11; Table 1),suggesting that the selective pressure is partially but not com-pletely relaxed. Finally, we tested an alternative model (E) inwhich ω is allowed to vary among branches, this model was foundnot to fit significantly better than a two-ratio model C (P =0.05955).

Behavioral Taste-Testing of the Asian Small-Clawed Otter and SpectacledBear. To investigate the proposed relationship between Tas1r2receptor structure and taste perception and diet, we carried outbehavioral tests on two available carnivore species from amongthose we genotyped. Previously, we had tested sweet preferencesin a number of species, including lesser panda, domestic ferret,haussa genet, meerkat, yellow mongoose, and Asiatic lion (24).With the exception of the Asiatic lion, all these species withinCarnivora appear to have an intact sweet taste receptor andprefer sweet-tasting compounds. Thus, aside from cats, therehave been no previous taste preference studies in carnivoranswith a defective Tas1r2.Here, we tested two Asian small-clawed otters, a species with

defective Tas1r2, and four spectacled bears, which appear tohave an intact sweet-taste receptor, for their preferences fora wide range of sweet-tasting compounds, including both sweetcarbohydrates (natural sugars) and noncaloric sweeteners.We used a two-bowl group test to assess preference for sweet-

tasting compounds. When presented with a simultaneous choicebetween sugar-containing solution and water, the Asian ottersshowed no clear preference for sugars. The preference ratiosrange from 35.4% for sucrose (0.5 M) to 56.5% for galactose (0.8M; Fig. 3). They showed indifference to, or avoidance of, a fewnonnutritive sweeteners tested, including indifference to sucra-lose (45.5% at 5.03 mM) and avoidance to saccharin (20.6% at 6.2mM; Fig. 3), indicating that the taste-test procedure is capable ofdemonstrating discrimination among tastants. The spectacledbears showed strong preferences for natural sugars, with prefer-ence ratios ranging from 83% for glucose (0.5 M) to 100% forgalactose (0.8 M; Fig. 3). They also showed strong preferences forcertain noncaloric sweeteners, including 5.0 mM sucralose(86.2%) and 6.2mMacesulfame-K (92.9%) (Fig. 3). These resultsare congruent with the molecular data from both species.

Sea Lions and Dolphins Lack All Three Tas1r Receptors. Given theatrophied taste system and unique feeding behavior in sea lions,we next investigated what happened to the other two Tas1rs inthe sea lion. Using degenerate primers (SI Appendix, Table S1)and genomic DNA-based PCR, we sequenced the protein-coding

Fig. 2. An evolutionary tree of Tas1r2 gene from 18 species within Carnivora.The evolutionary history is inferred by using the maximum-likelihood methodbased on the Tamura–Nei model (37) implemented in MEGA5 (26). The per-centage of replicate trees in which the associated taxa clustered together in thebootstrap test (2,000 replicates) is shown next to the branches (38). The tree isdrawn to scale, with branch lengths measured in the number of substitutionsper site. Species with a pseudogenized Tas1r2 are marked with a diamond (redand gray depict species characterized in this study or previously, respectively).The human Tas1r2 is used as the outgroup for the analysis.

Table 1. Likelihood ratio tests of selective pressures on carnivore Tas1r2 gene

Models ω (dN/dS) ln L* np† Models compared 2Δ(ln L)‡ P values

A. All branches have the same ω ω = 0.1909 −9,840.81 36B. All branches have the same ω= 1 ω = 1 −10,203.39 35 B vs. A 725.16 ∼0C. The branches with a pseudogenized ω1 = 0.13656Tas1r2 have the ω2, other branches have ω1 ω2 = 0.41974 −9,806.38 37 A vs. C 68.86 1.1 × 10−16

D. The branches with a pseudogenized ω1 = 0.13253Tas1r2 have ω2 = 1, other branches have ω1 ω2 = 1 −9,829.34 36 D vs. C 45.92 1.2 × 10−11

E. Each branch has its own ω Variable ω −9,783.72 69 C vs. E 45.32 0.05955

Dataset: 18 species (all species, after removing gaps and nonsense mutations in pseudogenes).*The natural logarithm of the likelihood value.†No. of parameters.‡Twice the difference in In L between the two models compared.

4958 | www.pnas.org/cgi/doi/10.1073/pnas.1118360109 Jiang et al.




regions of Tas1r exons. By aligning with the dog Tas1r1 sequence,we found that the sea lion Tas1r1 has a 1-bp deletion in exon 2between 53 and 54 bp (Fig. 4A and SI Appendix, Fig. S8A), and an11-bp deletion in exon 6 between 302 and 303 bp (Fig. 4A and SIAppendix, Fig. S8B). Tas1r3 has a 1-bp deletion between 72 and 73bp in exon 4 (Fig. 4A and SI Appendix, Fig. S8C). Only exons 2, 4,and 5 have been sequenced for sea lion Tas1r3. As was the case forTas1r2, sea lion Tas1r1 and Tas1r3 are also pseudogenes.Dolphin is an aquatic mammal that evolved independently from

the sea lion but displays similar feeding behavior and dramaticloss-of-taste system (23). We searched the draft dolphin genome(2.59X) for Tas1r genes using the dog Tas1r amino acid sequences(TBlastN). We found several contigs with sequence similarityto dog Tas1rs (∼70–80% amino acid identity). ABRN01270722contains the first two exons and part of the third exon of dolphinTas1r1, and ABRN01270723 contains exons 4–6 of Tas1r1. Dol-phin intron 2 has a mutation in the donor splice site at the 5′ end(AT instead of GT; Fig. 4B) that would interfere with splicing oftheTas1r1mRNA, a 5-bp deletion in exon 4 that disrupts theORFof dolphin Tas1r1 (Fig. 4B and SI Appendix, Fig. S9A), and a 2-bpdeletion in exon 6 (Fig. 4B). These ORF-disrupting mutationsrender dolphin Tas1r1 nonfunctional.Using the dog Tas1r2 amino acid sequence, we retrieved only

contig ABRN01341268 and determined potential coding sequen-ces of dolphin Tas1r2 exons 1–3 and 5–6. Exon 4 appears to havebeen lost entirely based on using dog, cow, mouse, or humansequences, because queries and in exons 3 and 6 ORF-disruptingmutations were found (Fig. 4B). Specifically, there is a 20-bp ORF-disrupting insertion between 488 and 507 bp in exon 3 (Fig. 4B andSI Appendix, Fig. S9B), and there is a 1-bpORF-disrupting deletionin exon 6 between 378 and 379 bp (Fig. 4B). Thus, dolphin Tas1r2also is a pseudogene.Next, we retrieved two contigs ABRN01316859 (exons 1–3)

and ABRN01316858 (exons 4–6), covering the entire codingsequences of the dolphin Tas1r3 gene. Multiple ORF-disruptingmutations were found throughout the coding region (Fig. 4B). A7-bp deletion between 99 and 100 bp in exon 6 is shown in SIAppendix, Fig. S9C. These data indicate that dolphin Tas1r3 alsois a pseudogene. Like the sea lion, the bottlenose dolphin lacksall three Tas1r receptors.

Pseudogenization of Dolphin Tas2r Receptor Genes. Because all threeTas1r receptor genes are pseudogenized in dolphins, what hap-pened to genes mediating another major taste quality: bitter? Toaddress this question, TBlastN searches were performed on thedolphin genome using 15 dog and 16 cow intact Tas2r genes asqueries. Because Tas2rs are intronless genes, it was relatively easyto identify the dolphin Tas2r genes. The query results revealed only10 pseudogenes, and no intact dolphin Tas2r receptor gene wasfound. The genes are named after their dog or cow orthologs(Tas2r1p,Tas2r2p,Tas2r3p,Tas2r5p,Tas2r16p,Tas2r38p,Tas2r39p,Tas2r60p,Tas2r62ap, andTas2r62bp). Pseudogenization was caused

by ORF-disrupting mutations (including nonsense mutations andframeshift mutations; Fig. 5A andB and SI Appendix, Document 1).Sequence similarities (nucleotide) between dolphin Tas2rs andtheir orthologs in dog and cow are between 75% and 86%. Aneighbor-joining tree was constructed by using aligned dolphin,dog, and cow Tas2r sequences (SI Appendix, Fig. S10) to show theevolutionary relationship among dog, cow, and dolphin bitterreceptors. Despite the relatively low coverage of the dolphin ge-nome assembly, the fact that none of the 10 identified Tas2rreceptors is intact hints that this modality may also be lost, or itsfunction greatly reduced.

DiscussionWidespread Pseudogenization of Tas1r2 in the Order Carnivora. Inthe present study, by examining Tas1r2 receptor genes in addi-tional carnivore species, both closely and distantly related toFelidae, we found that functional loss of Tas1r2 is widespreadand independent in Carnivora. ORF-disrupting mutations (e.g.,stop codons, insertion or deletion of nontriplet nucleotides) weredetected in seven nonfeline carnivore species: the sea lion, furseal, Pacific harbor seal, Asian small-clawed otter, spotted hyena,fossa, and banded linsang, as well as in one cetacean species: thebottlenose dolphin. Looking specifically at the obvious muta-tions, with the exception of the sea lion and fur seal (sisterspecies in the family Otariidae), none of the mutations disruptingthe ORF of Tas1r2 were shared between any two species ofthese seven.The widespread loss of Tas1r2 in species of the order Car-

nivora, the variance in their lineages, and the independentpseudogenizing mutations among species in distinct familiesstrongly support the hypothesis that the loss of Tas1r2 occurredindependently many times during the evolution of the orderCarnivora. It seems most likely that changes in dietary behaviorin the process of becoming obligate carnivores may favor the lossof a functional Tas1r2, arguing for convergent evolution ofpseudogenized Tas1r2. Another case of convergent evolution oftaste was found in the Tas2r38 receptor gene. Mutations in theTas2r38 gene have resulted in independent loss of phenylthio-carbamide sensitivity in some humans, chimpanzees, and ma-caques (30, 31). Notably, the mutations in chimpanzees andmacaques are start codon mutations, resembling the case withsea lion and fur seal Tas1r2s (30, 31).Our evolutionary analysis has provided further evidence to

support the view of convergent evolution of pseudogenizedTas1r2. Similar to many other vertebrate genes, we found thatTas1r2 is under an overall strong purifying selection. During

Fig. 3. Sweet-taste preferences of two genotyped species. Two Asian otterand four spectacled bears were tested behaviorally for their preferences forsweeteners using a two-bowl preference setup. One bowl containedsweetener solution and the other contained plain water. Dashed line indi-cates no preference (50%). Sweeteners were tested at the following con-centrations: fructose (0.8 M), galactose (0.8 M), lactose (0.5 M), maltose (0.7M), sucrose (0.5 M), acesulfame-K (6.0 mM), aspartame (10 mM), neotame(10.5 mM), saccharin (6.2 mM), and sucralose (5.0 mM).

Tas1r1 * * *1 2 4 5 63

Tas1r21 2 3 5 6

* *

500 bp

*12 3 45 6

Tas1r3 ** *****

A

B

1 2 4 5 63

*Tas1r1 *

Tas1r32 45

*

Dolphin Tas1r receptor gene structures

Sea lion Tas1r receptor gene structures

*

Fig. 4. The sea lion and dolphin Tas1r receptor genes are inactivated bypseudogenization. (A) Sea lion Tas1r1 (Upper) and Tas1r3 (Lower) genestructures with boxes representing exons, and lines representing introns.Regions where ORF-disrupting mutations were found are marked with anasterisk. Regions with no sequence coverage are labeled with dashed lines.Only three exons have been sequenced for the sea lion Tas1r3. (B) Schematicdrawings of the dolphin Tas1r1 (Top), Tas1r2 (Middle), and Tas1r3 (Bottom)gene structures. The symbols are the same as in A. The straight line withslashes indicates the intron is not proportionally scaled.


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evolution, several species at separate times may have changedtheir diet to a more carnivorous one. As the animal moved closertoward obligate carnivore it became increasingly likely that lossof the Tas1r2 gene would be well tolerated, reflected by the factthat the relaxation of functional constraint occurred in brancheswith a pseudogenized Tas1r2, although not completely.

Sweet Taste Receptor Genes and Taste Behavior. Cats with a de-fective Tas1r2 gene do not prefer sweet carbohydrates andnoncaloric sweeteners and probably cannot detect them. Is thisalso true for others? We addressed this question in the currentstudy by behavioral taste-testing of the Asian small-clawed otter.Similar to the cat family Felidae, the two Asian otters we testeddid not show a preference for sucrose at a concentration up to800 mM, or for other sugars or artificial sweeteners. These dataare limited in the number of animals tested and the number andkinds of behavioral tests conducted and, consequently, theywarrant caution in interpretation. However, a preference forsweet sugars is a powerful and clear phenotype in most mammalsstudied, so we believe its absence in the Asian otter is striking.Thus, we tentatively conclude that the Asian otter is not capable

of detecting compounds that taste sweet to humans, consistentwith their lack of a functional sweet taste receptor Tas1r2/Tas1r3and their carnivorous diet. In contrast, the spectacled bear, withan intact Tas1r2 gene, shows strong preferences for sugars andsome nonnutritive sweeteners, consistent with the presence ofa functional sweet taste receptor and with their omnivorous diet.

Major Taste Loss in Two Lineages of Aquatic Mammals. In thepresent study, we found that the sweet, umami, and perhapsbitter taste receptor are all pseudogenized in dolphin, and thesweet and umami taste receptors are pseudogenized in sea lions(we have no data on bitter receptor structure in this species).Consistent with the genetic data, anatomical studies of the dol-phin taste system revealed that only few taste bud-like structuresare present in small pits in the root region of dolphin tongues(32). No buds are found in the canonical taste structures, in-cluding fungiform, foliate, and circumvallate papillae (32). Tastebud numbers are greatly reduced in sea lions as well (21). Onlya few studies from two groups were conducted to examine theabilities of dolphins and sea lions to detect sour, bitter, salty, andsweet taste stimuli; umami was not examined in these studies (33,

Tas2r1p

Tas2r39p

Tas2r60p

Tas2r62ap

100 bp

Tas2r2p

Tas2r3p

Tas2r5p

Tas2r16p

Tas2r38p

Tas2r62bp

xx x

x

x

x

Cafa-T2R3 (61) 5’ ATGCTGGGGAATGGTTTCATAGTGTTGGTCAATGGCAGCAGC 3’Dolphin-Tas2r3p (61) 5’ ATGCCGGGGAATAGTTTCATGG-GTTGGTCAATGGTAGCAGC 3’

M P G N S F M (del: -1 frameshift)

Cafa-T2R5 (142) 5’ GTCCTGGGCCTGGCTGTCTGCCGATTTCTCCTGCAGTGTCTG 3’Dolphin-Tas2r5p (142) 5’ GTCCTGGGCCTGGCTGTCTGTTGATTTCTTCTGCAGTGGTTG 3’

V L G L A V C *(stop: premature)

(Dog)Cafa-T2R1 (259) 5’ TTTGTAAATGAATTGGGACTTTGGTTCGCCACATGGCTTGGG 3’Dolphin-Tas2r1p (259) 5’ TTTATAAATGAATCGGGACTTTGATTTGCCACATGGCTCAGC 3’

F I N E S G L *(stop: premature)

Cafa-T2R2 (187) 5’ TTAATGATACAAAGTTTTTTCTCTGTGTTATTTCCACTCTTT 3’Dolphin-Tas2r2p (187) 5’ TTAATGGTAAAG---TTTTTCTC-ATGTTCTTTCCACTCTTT 3’

L M V K F F (del: -1 frameshift)

(Cow)Bota-T2R16 (1) 5’ ATGACAACCAGCCAACTCTCTGTCTTCTTCATGATTATCTAT 3’Dolphin-Tas2r16p (1) 5’ ATGATAACCATCCAACTCT--GTCTTCTTCATGATCATCTAT 3’

M I T I Q L (del: -2 frameshift)

Cafa-T2R38 (460) 5’ ACTGTGCTACTCATGAATAATACAGAATTTAATTCACAAATT 3’Dolphin-Tas2r38p (514) 5’ ACCATGCTACTCATGAATAA--CAATACTCAATTGAGAAACT 3’

T M L L M N (del: -2 framshift)

Cafa-T2R39 (37) 5’ CCATTTGGCATCCTCTCGATTTTAACAATTACAGGCACTGAA 3’Dolphin-Tas2r39p (40) 5’ CCATCTCGCATCATTTTGATGTGAATCGTTATAGGCACCGAA 3’

P S R I I L M *(stop: premature)

Bota-Tas2r60 (187) 5’ GGGGCCTCTCGCTTCTGTCTGCAATGGGTGGTGATTAGTAAG 3’Dolphin-Tas2r60p (187) 5’ GGAGCCCCTAGCTTCTATCTGTGATGGGTGGTGAT----AAG 3’

G A P S F Y L *(stop: premature)

Cafa-T2R62p(148) 5’ TCCCTGGCTGCCTCCTGGTTCTGCCTGCATGGGGTGGCCATC 3’Dolphin-Tas2r62ap(151) 5’ TCCCTGGCCGCCTCCCGGTTCTGACTGCATGGGATGGCCCTC 3’

S L A A S R F *(stop: premature)

Cafa-T2R62p (88) 5’ GTGTTGGGCAGGGAGTGGGTGC-----GACGCCGGACGCTGC 3’Dolphin-Tas2r62bp (88) 5’ GTGCTGAGCCGGACTGGGACGCTGGACGCTGCCGGACGCTGT 3’

V L S R T G T (ins: +2 frameshift)

Dolphin Tas2r receptor gene structuresA

B

Fig. 5. Dolphin Tas2rs are pseudogenes. (A) Schematic diagram shows the premature stop codons (either nonsense mutations or stop codons resulting fromprior frameshift mutations, depicted with a red line) and only the frame-shift mutations (X) before the premature stop codons in dolphin Tas2rs. Tas2rreceptor genes are shown in scale. No start codon is detected for Tas2r62bp. (B) A 42-bp–long nucleotide sequence containing the ORF-disrupting mutationthat occurs closest to 5′ end of the gene is shown for each of the 10 identified dolphin Tas2r receptor genes; the aligned dog sequence is shown above it, andthe amino acid sequence deduced from the nucleotide sequence up to the mutation site is shown underneath it. The codon that contains the ORF-disruptingmutation (marked in red and underlined) is indicated by a box.

4960 | www.pnas.org/cgi/doi/10.1073/pnas.1118360109 Jiang et al.


34). Dolphins could not detect sucrose in one study (33) andshowed reduced sensitivity in another study (34). Furthermore,dolphins detected only elevated levels of quinine (bitter), anda California sea lion showed no response to quinine sulfate butdid respond to elevated levels of quinine monohydrochloridedihydate (0.40 parts per trillion; from 3 to ∼4 orders above hu-man detection threshold) (33, 34). However, these data needto be interpreted cautiously because only a limited number ofanimals were examined. Nevertheless, these behavioral dataagree largely with our molecular data. For instance, no sugardetection (or reduced sensitivity to sugar) correlates well withthe pseudogenization of Tas1r2 and Tas1r3 receptor genes. Theremaining response (if any) to sugar could be due to anotherproperty of sugar solutions, such as osmolarity or viscosity of sugars(13). Dolphins appeared to maintain some response to quinine athigh concentration, but we failed to detect intact Tas2r receptorgenes from the dolphin genome database. One explanation for thisfinding is that due to the low coverage, and therefore incomplete-ness, of the dolphin genome (2.59X), there may exist intact bitterreceptor genes in the genome eluding initial genome sequencecoverage. An alternative explanation is that quinine may becomeirritating at such high concentrations, and therefore elicit irritationresponses (35). Nevertheless, with the reported greatly elevatedthreshold to quinine and the abundance of pseudogenized Tas2rreceptor genes in the available genome database (10 of 10 genes),we predict that bitter taste function of dolphins is also greatly re-duced, if not completely absent.Recently, sweet, umami, and bitter taste receptors have been

implicated in several extraoral functions (36). Pseudogenization ofTas1r receptor genes in dolphins and sea lions and Tas2r receptor

genes in dolphin indicates that these receptors cannot be involvedin extraoral (e.g., gut, pancreas) chemosensation (36) in thesespecies. Thus, to the extent that these extraoral taste receptors arefunctionally significant in rodents and humans, these functionsmust have been assumed by other mechanisms in the species wehave identified here with pseudogenized receptors. What theseother mechanisms are remains to be determined, and further as-sessment of the relationships among taste receptor structure, di-etary choice, and the associated metabolic pathways will lead toa better understanding of the evolution of diet and food choice aswell as their mechanisms.

Materials and MethodsDetails about collection of DNA samples, sequencing Tas1r2 from selectedspecies within Carnivora, sequencing sea lion Tas1r1 and Tas1r3, data miningof the dolphin whole-genome shotgun assembly, evolutionary analysis, andtaste testing as well as associated references are described in SI Appendix,Materials and Methods. Primers for amplification of taste receptors are listedin Table S1. Dataset for evolutionary analysis is provided in Dataset S1.

ACKNOWLEDGMENTS. We thank Dr. Oliver Ryder and Leona Chemnick fromthe Zoological Society of San Diego/Conservation and Research for Endan-gered Species for providing the DNA samples of carnivores, the Naples Zoo atCaribbean Gardens for providing a fossa DNA sample, and Dr. Stephen O’Brien(National Cancer Institute) for providing DNA samples of a southern fur sealand a raccoon; Dr. Robert Zingg (Zoological Garden of Zurich) for allowingaccess to certain individual carnivores and for assistance with behavioral tastetesting; and Dr. Joshua Plotkin for reading the manuscript. This work wasmade possible by National Institutes of Health Grant DC010842 (to P.J.) andinstitutional funds from the Monell Chemical Senses Center.

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http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1118360109/-/DCSupplemental/sd01.rtf

Major taste loss in carnivorous mammals Jiang et al

1

Supporting Information

SI MATERIALS AND METHODS:

Collection of DNA Samples

DNA samples from the aardwolf (Proteles cristatus), Asian small-clawed otter

(Amblonyx cinereus), banded linsang (Prionodon linsang), California sea lion (Zalophus

californianus californianus), Canadian otter (Lontra canadensis), fossa (Cryptoprocta ferox),

Pacific harbor seal (Phoca vitulina richardii), raccoon (Procyon lotor), red wolf (Canis rufus)

spectacled bear (Tremarctos ornatus) and spotted hyena (Crocuta crocuta) were provided by the

Conservation and Research for Endangered Species Program at the San Diego Zoo. DNA

samples of a Southern fur seal (Arctocephalus forsteri) and raccoon (Procyon lotor) were

provided by Dr. Stephen O’Brien (National Cancer Institute). A DNA sample from the

bottlenose dolphin (Tursiops truncates) was provided by Therion International (Saratoga

Springs, New York).

Sequencing Tas1r2 from Selected Species within Carnivora and Sequencing Sea Lion

Tas1r1 and Tas1r3

The design of degenerate primers for Tas1r2 was described previously (1). Genomic

based polymerase chain reaction (PCR) was conducted to amplify all six exons of Tas1r2 from

the 12 carnivore species using degenerate primers designed from conserved exon-intron

boundary sequences. Genomic-based polymerase chain reaction (PCR) was also conducted to

amplify all six exons of Tas1r1 and 3 exons (2, 4, 5) of Tas1r3 from the sea lion using

degenerate primers (Supplementary Table 2) designed from conserved exon-intron boundary

sequences of dog, cat and giant panda. PCR mixtures (50 µl) include 1 µl (50ng/µl) genomic


2

DNA, 5 µl 10 x buffer, 1µl of each primer (primer concentration varies according to degeneracy

of the primer from 10 µm to 80 µm), 0.25 µl Taq polymerase (Roche). The PCR conditions

were: 94 °C for 2min; 35 cycles of 94 °C for 30 s, 66 °C for 45s, 72 °C for 2 min; 72 °C for 10

min; and a 4 °C hold. PCR products were purified and sequenced directly or after being

subcloned into pGEM-Teasy vector at the sequencing facility of the University of Pennsylvania.

Each PCR product was sequenced from both directions to validate the results.

For fragments containing open reading frame mutations, PCR was repeated to confirm

the results using either the original DNA samples or DNA samples from additional animals when

available (3 additional fossaes,1 additional Asian small-clawed otter,1 additional spotted hyena

and 1 additional California sea lion). We assembled the coding sequences of Tas1r2 from all the

selected carnivore species and sea lion Tas1r1 and Tas1r3 using Sequencer 4.8 with the dog

Tas1r2, Tas1r1 and Tas1r3 sequence as references, respectively.

Data-mining of the Dolphin Whole Genome Shotgun (WGS) Assembly

To identify the dolphin Tas1rs, TBlastN searches were conducted on the dolphin genome

database by using the dog T1R1 (xp_546753), T1R2 (xp_855275) and T1R3 (xp_848708) amino

acid sequences as queries. These searches retrieved two contigs (ABRN01270722 and

ABRN01270723) for Tas1r1, a single contig (ABRN01341268) for Tas1r2, and two contigs

(ABRN01316859 and ABRN01316858) for Tas1r3, respectively. We determined the exon-

intron borders of exon 1- 6 of dolphin Tas1rs using Spidey (2) and TBLASTN (3) programs with

dog Tas1r sequences as references. We aligned dolphin Tas1r exon sequences with that of dog

and human using an modified ClustalW (4) program installed in AlignX (www.invitrogen.com).

All the mutations have been confirmed by sequencing Tas1rs amplified from another dolphin

using gene-specific primers.

http://www.invitrogen.com/


3

To identify the dolphin Tas2rs, TBlastN searches were conducted on the dolphin genome

database using the dog and cow Tas2r intact receptors (Genbank accession #: AB249684-

AB249731) as queries (5). AB249725 (bota-T2R56) is a partial sequence of cow Tas2r60

(Genbank accession # xm_002687121, referred to as Bota-Tas2r60). Conversely, each identified

dolphin Tas2r sequences were used to blast Nucleotide collection database (nr/nt) to determine

the similarity toward Tas2rs from other species and then named afterward their dog or cow

orthologs. The Tas2r sequences from dolphin, dog and cow were aligned by ClustalW (4). A

phylogenetic tree was constructed by the Neighbor-Joining (NJ) method implemented in MEGA

5 (6).

Evolutionary Analysis

Nucleotide sequences of Tas1r2 from the selected carnivore species were aligned with

CLUSTALX 1.81 (7), modified with Bioedit 7.04 (8), and confirmed by deduced amino acid

sequence alignment. When allelic variations were found in individuals, we used the allelic

sequence that was the same or similar to that of the reference dog sequence. In addition to the

Tas1r2 sequences from the 12 species reported in this study, we analyzed Tas1r2 sequences from

carnivore species available in NCBI database, including yellow mongoose (FJ356695), cat

(AY819787), giant panda (GL193509), red panda (FJ356693), ferret (FJ356691) and dog

(XM_850182). A consensus phylogenetic tree was built using the maximal likelihood method

implemented in MEGA5 after bootstrapping for 2000 times or the neighbor-joining method after

bootstrapping 10,000 times (9). Nonsynonymous and synonymous nucleotide substitution rates

were calculated using the likelihood method implemented in the CODEML program in PAML

4.1 (10). The sequence data file, configuration files and the result files for the CODEML

analyses were provided (DocS2.rtf). We also used a recent published carnivore phylogeny to test


4

our models using CODEML (11) and conclusions reached using our tree were practically

identical to those using the tree proposed by Yu et al (11).

Taste Testing

The two-bowl preference tests were carried out as previously described (1). One bowl

contained the test compound dissolved in water to a designated solubility, the other bowl

contained an equal volume of water. The test period began at 9:30 am and ended 24 hr later.

After 24 hr, the volume of fluid consumed from each bowl was recorded. Preference ratios were

calculated as the ratio of taste solution intake to total fluid intake x 100%. A “strong” preference

for the test compound is defined here as a preference score greater than 80%. Preferences above

80% are identified by a plus sign. For this study, we were given access to two Asian otters and

four spectacled bears. The animals were born and raised at the Zoological Garden of Zurich in

Germany. They were maintained and tested according to the Monell Chemical Senses Center

animal protocol (Institutional Animal Care and Use Committee No. 1112) and with the

permission and oversight of the director and staff of the zoo.

SI Figure legends:

Fig. S1: Start codon mutation and indel mutations found in the sea lion and fur seal Tas1r2

coding sequences. Shown in panels (A), (B), (C), and (D) are representative DNA

chromatograms and Tas1r2 nucleotide sequences of the sea lion and fur seal aligned with Tas1r2

sequences from the dog. In panel A, the start codon in exon 1 was mutated to ATA in both the

sea lion and fur seal Tas1r2s, preventing the initiation of T1R2 protein translation. In panel B, a

1-bp deletion between 579-580 bp was found in exon 3 of the sea lion Tas1r2. In panel C, a 2-bp

deletion between 674-675 bp (exon 3 of sea lion Tas1r2) and a corresponding 2-bp deletion


5

between 675-676 bp (exon 3 of fur seal Tas1r2) were found. In panel D, a 1-bp deletion was

found between 802-803 in exon 6 of both the sea lion and fur seal Tas1r2s.

Fig. S2: Nonsense and indel mutations found in the pacific harbor seal Tas1r2. Shown in

panels (A) and (B) are representative DNA chromatograms and Tas1r2 nucleotide sequences of

the pacific harbor seal aligned with Tas1r2 nucleotide sequences from the dog. In Panel A, a

premature stop codon TAA (position 32 in exon 6) was found. The deduced amino acid sequence

is shown up to the stop codon. In panel B, a 2-bp deletion was found between 192-193 bp in

exon 6.

Fig. S3. An indel mutation found in the Asian small-clawed otter Tas1r2. Shown are

representative DNA chromatogram and Tas1r2 nucleotide sequences aligned with Tas1r2

sequences from the Canadian otter and dog. A 1-bp insertion was found in position 360 in exon

3. The translated amino acid sequence is shown up to the codon which contains the frameshift

mutation.

Fig. S4. An indel mutation found in the spotted hyena Tas1r2. Shown are representative DNA

chromatogram and Tas1r2 nucleotide sequences aligned with Tas1r2 sequences from the

aardwolf and dog. A 1-bp deletion was found between 130-131 bp of exon 2 of the spotted hyena

Tas1r2. The translated amino acid sequence is shown up to the codon which contains the

frameshift mutation.

Fig. S5. Nonsense and indel mutation found in the Fossa Tas1r2. Shown in panel (A) and (B)

are representative DNA chromatograms and Tas1r2 nucleotide sequences aligned with Tas1r2

sequences from the dog. In panel A, a premature stop codon TAG (Position 125 in exon 3) was

found in the Fossa Tas1r2. Though, polymorphism was observed in this position by sequencing


6

other fossa individuals. The translated amino acid sequence is shown up to the nonsense

mutation. In panel B, a 1-bp insertion at 58 bp was found in exon 4 of the fossa Tas1r2.

Fig. S6: Multiple indel mutations found in the banded linsang Tas1r2. Shown in panel A

through G are chromatograms and nucleotide sequences of the banded linsang Tas1r2 aligned

with the dog coding sequences. In Panel A, a 1-bp insertion was found at 70 bp of exon 2 of the

linsang Tas1r2. The translated amino acid sequence is shown up to the codon which contains the

frameshift mutation. In Panel B, a 10-bp micro-deletion was found between 274-275 bp of exon

2 of the linsang Tas1r2. In Panel C, a 14-bp insertion was found in position between 78-91 bp of

exon 4. In Panel D, a 20-bp deletion was found between 27-28 bp of exon 5. In Panel E, a 2-bp

deletion was found between 54-55 bp of exon 5. In panel F, a 1-bp deletion was found between

210-211 bp of exon 6. In panel G, a 28-bp insertion was found between position 235-262 of exon

6 of the linsang Tas1r2. In panel H, a 1-bp insertion was found between position 444-445 of

exon 6.

Fig. S7. Evolutionary relationships of the order Carnivora

The evolutionary history was inferred using the Neighbor-Joining method (12). The bootstrap

consensus tree inferred from 10000 replicates (13) is taken to represent the evolutionary history

of the taxa analyzed (13). Branches corresponding to partitions reproduced in less than 50%

bootstrap replicates are collapsed. The percentage of replicate trees in which the associated taxa

clustered together in the bootstrap test (10000 replicates) are shown next to the branches (13).

The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary

distances used to infer the phylogenetic tree. The evolutionary distances were computed using

the Maximum Composite Likelihood method (14) and are in the units of the number of base


7

substitutions per site. The rate variation among sites was modeled with a gamma distribution

(shape parameter = 2). The analysis involved 19 nucleotide sequences. Codon positions included

were 1st+2nd+3rd. All positions containing gaps and missing data were eliminated. There were a

total of 2160 positions in the final dataset. Evolutionary analyses were conducted in MEGA5 (9).

Species with a pseudogenized Tas1r2 are marked with a diamond sign (red indicated species

whose Tas1r2 has been characterized in this study, grey indicated species whose Tas1r2 was

reported previously).

Fig. S8. The sea lion Tas1r1 and Tas1r3 genes are inactivated by pseudogenization. A) The

upper half showed a chromatogram trace of the sea lion Tas1r1 exon 2 sequence in which a

mutation was found. The lower half showed the alignment of the above sequence to the

corresponding dog and human Tas1r1 exon 2 sequences, highlighting a 1-bp deletion mutation in

the sea lion Tas1r1 exon 2. B) An additional 11-bp deletion mutation was found in the sea lion

Tas1r1 exon 6. The upper half showed a chromatogram trace of the sea lion Tas1r1 exon 6

sequence in which the mutation was found and the lower half showed the alignment between sea

lion, dog and human Tas1r1 exon 6 sequences. C) The upper half showed a chromatogram trace

of the dolphin Tas1r3 exon 4 and the lower half showed the alignment of the sequence to the

corresponding dog and human sequences, noting a 1-bp deletion mutation in the sea lion Tas1r3.

Fig. S9: The dolphin Tas1r receptor genes are inactivated by pseudogenization. A) The

upper panel showed a representative chromatogram trace of the dolphin Tas1r1 exon 4 sequence

in which a mutation was found (ti: 1417589012). The lower half showed the alignment of the

above sequence to the corresponding dog and human Tas1r1 exon 4 sequences, highlighting a 5-

bp deletion mutation in the dolphin Tas1r1 exon 4. B) The upper part showed a representative

chromatogram trace of the dolphin Tas1r2 exon 3 (ti: 1489305729) and the lower part showed


8

the alignment of the sequence to the corresponding dog and human Tas1r2 exon 3 sequences,

noting an insertion mutation of a 20-bp in the dolphin Tas1r2. C) The upper half showed a

representative chromatogram trace of the dolphin Tas1r3 exon 6 sequence (ti: 1431864905), the

lower part showed the alignment of the sequence to the corresponding dog and human Tas1r3

exon 6 sequences, revealing a deletion mutation of 7-bp in the dolphin Tas1r3 exon 6.

Doc. S1: Alignment of dolphin Tas2r receptor genes to the dog or cow orthologs.

ClustalW alignments of each of 10 dolphin Tas2rs to the ortholog of dog or cow Tasr2s are

shown. Nonsense mutations or premature stop codons that result from frame-shift mutations are

marked in pink. Frameshift mutation (insertion or deletion) are marked in red.

Fig. S10: Evolutionary relationships of dolphin, dog and cow Tas2r receptors

The evolutionary relationship was inferred using the Neighbor-Joining method. The bootstrap

consensus tree inferred from 2000 replicates is taken to represent the evolutionary history of

Tas2r receptor genes analyzed. Branches corresponding to partitions reproduced in less than

50% bootstrap replicates are collapsed. The evolutionary distances were computed using the

Maximum Composite Likelihood method and are in the units of the number of base substitutions

per site. The analysis involved 58 nucleotide sequences (10 dolphin sequences, 19 dog (Cafa)

sequences and 29 cow (Bota) sequences). Evolutionary analyses were conducted in MEGA5 (9).

References

1. Li X, et al. (2009) Analyses of sweet receptor gene (Tas1r2) and preference for sweet stimuli in species of Carnivora. J. Hered. 100 Suppl 1:S90-100.

2. Wheelan SJ, Church DM, & Ostell JM (2001) Spidey: a tool for mRNA-to-genomic alignments. Genome Res 11(11):1952-1957.

3. Johnson M, et al. (2008) NCBI BLAST: a better web interface. Nucleic Acids Res 36(Web Server issue):W5-9.


9

4. Thompson JD, Higgins DG, & Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22(22):4673-4680.

5. Go Y, Satta Y, Takenaka O, & Takahata N (2005) Lineage-specific loss of function of bitter taste receptor genes in humans and nonhuman primates. Genetics 170(1):313-326.

6. Tamura K, et al. (2011) MEGA5: Molecular Evolutionary Genetics Analysis using Maximum Likelihood, Evolutionary Distance, and Maximum Parsimony Methods. Mol Biol Evol.

7. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, & Higgins DG (1997) The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25(24):4876-4882.

8. Hall TA (1999) Bioedit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser 41:95-98.

9. Tamura K, et al. (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28(10):2731-2739.

10. Yang Z (2007) PAML 4: phylogenetic analysis by maximum likelihood. Mol Biol Evol 24(8):1586-1591.

11. Yu L, et al. (2011) Phylogenetic utility of nuclear introns in interfamilial relationships of Caniformia (order Carnivora). Syst Biol 60(2):175-187.

12. Saitou N & Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4(4):406-425.

13. Felsenstein J (1985) Confidence limits on phylogenies: An approach using the bootstrap. Evolution 39:783-791.

14. Tamura K, Nei M, & Kumar S (2004) Prospects for inferring very large phylogenies by using the neighbor-joining method. Proc Natl Acad Sci U S A 101(30):11030-11035.

Dog Ex1 (-20) 5’ GGGGACCCCCACTTCCCAGCCATGGGACCCCGGGCCAAGGCG 3’Sea Lion Ex1 (-20) 5’ AGGGACCCCCACTTCCCAGCCATAGGACCCCAGGCCAAGGCA 3’Fur Seal Ex1 (-20) 5’ AGGGACCCCCACTTCCCAGCCATAGGACCCCAGGCCAAGGCA 3’

(start codon mutation, no translation)

Sea lion

Fur seal

A

Dog Ex3 (559) CCTGAGCCCAACAGGACCAGCCTGGAGGCCACCTGCAACCAGSea Lion Ex3 (559) CCTGCGCCCAACAGGACCAGC:TGGGGGCCACCTGCAACCAG

(1-bp deletion)

B

Fig. S1

Sea lion and fur seal Tas1r2 sequences

10

Dog Ex3 (655) TCCGGCGAGCGCGTGGTCTACAACGTGTACTCGGCTGTCTACSea Lion Ex3 (654) TCTGGTGAGCGCGTGGTCTAG::CATGTACTCGGCTGTCTACFur Seal Ex3 (655) TCTGGTGAGTGCGTGGTCTAG::CGTGTACTCGGTTGTCTAC

(2-bp deletion)

Sea lion

Fur seal

C

Dog Ex6 (780)CTCTTGATCACCGTGCTCAACCTTCTGGGCATCAGCTTTGGCSea Lion Ex6 (780)CTCTTGGTCACCATGCTCAACCC:CTGGGCATCAGCCCGGGCFur Seal Ex6 (780)CTCTTGGTCACCGTGCTCAACCT:CTGGGCATCAGCCCGGGC

(1-bp deletion)

D

Sea lion

Fur seal

Fig. S1 continued

11

Pacific Harbor Seal Tas1r2 sequence

Dog Ex6 (9) 5’ GACTGCCAGCCTTGCCCAAGTTACGAGTGGTCCCATAGGAAC 3’P.H.Seal Ex6 (9) 5’ GGCTGCCAGTCCTGCCCAGGTTAAGAGTGGTCCCATAGGAAC 3’

G C Q S C P G *(stop: pre-mature)

A

Fig. S2

Dog Ex6 (171) TGGAGGCACCTCCACACGCCCGTGGTTCGCTCGGCCGGGGGC P.H.Seal Ex6 (171) TGGAGGCACCTGCACACGCCCG::GTTTGCTCGGCCGGGGGC

(2-bp deletion)

B

12

Dog Ex3 (337) 5’ CTGTTCTCGCCAGACCTGATCCT:GCACAACTTCTTCCGCGAG 3’Otter Ex3 (337) 5’ CTGTTCTCGCCCGACCTGGCCCTTGCACAACTTCTTCCGCGAG 3’

L F S P D L A (1-bp insertion)

Asian Otter Tas1r2 sequence

Fig. S3

Spotted Hyena Tas1r2 sequence

Dog Ex2 (110) 5’ ATAGTGGATGTCTGCTACATCTCCAACAACGTCCAGCCCGTG 3’Hyena Ex2 (110) 5’ GTGGTGGATATCTGCTACATC:CCAACAACGTCCAGCCCGTG 3’

V V D I C Y I (1-bp deletion)

Fig. S4

13

Dog Ex3 (103) 5’ CAGCTCCTGCTCCACTTCAACTGGAACTGGATCATCGTGCTA 3’Fossa Ex3 (103) 5’ CAGCTGATGCTGCTCTACTGCTAGAACTGGATCGTCGTGCTG 3’

Q L M L L Y C *(stop: premature)

Dog Ex4 (37) CTGGGCCACAATGTCTTTTTT:GACAAGCAAGGGGACGTG Fossa Ex4 (37) CTGGGCCACCAGATCTTTTTTTGACAAGCAAGGGGACCTG

(1-bp insertion)

Fig. S5Fossa Tas1r2 sequence

A

B

14

Dog Ex2 (47) 5’ TTTGCGGTGGAAGAGATTAACAA:CCGCAGCGACCTGCTGCC 3’Linsang Ex2 (47) 5’ TTTGCAGTGGAGGAAATCAGCAAACCATACCAGCCTGCTGCC 3’

F A V E E I S (1-bp insertion)

Dog Ex2 (251)GAGTCCACTACTACTGTGGCCCATTTCCTCTCACTCTTCCTCCTTCCACAGLinsang Ex2 (252)GAGTCCACCACAACTGTGGCCCA::::::::::CTCTTCCTCCTTCCACAG

(10-bp deletion)

Dog Ex4 (55) TTTGACAAGCAAGGGGACGTGCT::::::::::::::CATGCCCATGGAGGLinsang Ex4 (55) TTTGACAAGCAAGGGGACCTGCTTGTCCCCTGGAGATCATGCACCTGGAGA

(14-bp insertion)

A

B

C

Fig. S6Banded Linsang Tas1r2 sequence

Dog Ex5(7) TCCATGTGTTCCAAGGACTGCCATCCTGGCCAAAGGAAGAAGCCTGTGGGCALinsang Ex5(7) TCCATGTGTTCCAAAGAC::::::::::::::::::::GAAGCCCATAGCCA

(20-bp deletion)

D

15

Dog Ex5(55) GTGGGCATCCACTCCTGCTGCTTCGAGTGTATTGACTGCCTTLinsang Ex5(35) ATAGCCATCCATCCCTGCTG::TCGAGTGTCTCGACTGCCTT

(2-bp deletion)

Dog Ex6 (189) CCCGTGGTTCGCTCGGCCGGGGGCCCCATGTGCTTCCTGATGLinsang Ex6 (189) CCCATGGTTCGTTCGGCCGGGG:CCCCATGTGCTTCCTGATG

(1-bp deletion)

Dog Ex6(222) TTCCTGATGCTGGT::::::::::::::::::::::::::::GCCGCTGCTLinsang Ex6(221) TTCCTGATGCTGACATAATGATATAATGTTTGCTCAGTGAACGCCGCTGCT

(28-bp insertion)

E

F

G

Dog Ex6 (416) AGGCGCCTCCCGCGCGCCTACGGCTACTGGGTGCGCTGCCACLinsang Ex6 (423) AGGCGCCTCCTGCGAGCCTACG:CTACTGGGTCCGCTACCAC

(1-bp deletion)

H

Fig. S6 continued

16

Fig. S7: Evolutionary Relationships of the order Carnivora

Felidae Cat

Prionodontidae Banded Linsang

Eupleridae Fossa

Yellow Mongoose

Aardwolf

Spotted HyenaHyaenidae

Feliformia

Dog

Red Wolf

Sea Lion

Fur Seal

Pacific Harbor Seal

Pinnipedia

Spectacled Bear

Giant Panda

Red Panda

Raccoon

Ferret

Canadian Otter

Asian Small-Clawed Otter

Musterlidae

Caniformia

Human98

100

100

100

100

93

99

100

100100

100

99

98

58

96

99

0.02

17

Sea Lion Tas1r1 Exon 2 (46)

Dog Tas1r1 Exon 2 (46)

Human Tas1r1 Exon 2 (46)

(1‐bp deletion)

Sea lion Tas1r1 Exon 2





(11‐bp deletion)




(1‐bp deletion)


A

B

C

GAGGTTTG-CATTGAGGAGCGGTTTGGCATTGAGGAGCGGCTTGGGGTTGAGGA

GCTTG-----------CCTCTGCTTGTTGCGCCAAGGCCTCTGCTTGCTACGCCAGGCCCTCT

GCGGGAAC-TGAACATGGGGGGAACGTGAATATGGCGGAAACGTGGACATG

Fig. S8. The Sea Lion Tas1r1 and Tas1r3 Genes

18

B

(20‐bp insertion)

Dolphin Tas1r2 exon 3


(5‐bp deletion)


Dolphin Tas1r1 Exon 4 (80)









A

(7‐bp insertion)

C

GCAGTTAT-----AACTGCCTGGTGGCTATGACATAATTGCCTGGTAGCTATAACATAATTGCCTG

CCCTGGGCATCACCACCCAGAGCAGAGCCC--------------------AGAGTCC--------------------AGAG

CTTCTGCT-------CTGGCCCCGTGCTGCTTATACTGCTGGCTCTCTGCTGCTCCTGCTGCTGAGCCT

Fig. S9 The Dolphin Tas1r Receptor Genes

19

Bo

ta-T

2R

56

Bot

a-T

as2

r60

71

Dol

phin

-Tas

2r60

p

91

Caf

a-T

2R41

Bot

a-T2

R41

96

90

Caf

a-T2

R5

Dol

phin

-Tas

2r5p

Bota-

T2R5p

939950

Dolphin

-Tas

2r16

p

Bota-T2R16

100

Bota-T2R62p

Cafa-T2R62p

Dolphin-Tas2r62ap

Dolphin-Tas2r62bp

4440

5896

34

Cafa-T2R4

Bota-T2R4100

Bota-T2R1

Dolphin-Tas2r1pCafa-T2R1

65

95

31

13

Cafa-T2R40Bota-T2R40p

99

Bota-T2R39Dolphin-Tas2r39p

Cafa-T2R39

75

92

82

34

Dolphin-Tas2r38p

Cafa-T2R

38

100

27

Cafa-T2R

2

Dolphin-T

as2r2p

Bota-T

2R2p

96100

38

Cafa-T

2R7

Ca

fa-T2

R8

p

Bota

-T2R

8p9

85

0

13

Caf

a-T

2R3

Dol

phin

-Tas

2r3p

Bot

a-T

2R3

8499

16

Caf

a-T2

R9p

Bot

a-T2

R11

p

20

17

Caf

a-T2

R12

Bota-

T2R12

100

Bota-T

2R13

Cafa-T2R43Cafa-T2R44p 100

Bota-T2R45pBota-T2R47p50

Bota-T2R46

Bota-T2R43p

Bota-T2R44 6732

46

80

6030

14

Bota-T2R65

Bota-T2R65Bp

83

Cafa-T2R55

Cafa-T2R67

Bota-T2R677934

77

77

Bota-T2R71p

Bota-T2R72p

99

Bota-T2R10Dp

66

Cafa-T2R

10B

ota-T2R10C

Bota-T2R

10B

ota-T2R

10B72

5387

77

Fig. S10: Evolutionary Relationships of Dolphin, Dog and Cow Tas2r receptors

20

Tas1r1

T1R1_EX1_F1 GGCCATGCCAGGCACAGGAC

T1R1_EX1_R1 CCCCTCACTCACCTGTCACAGAGRGT

T1R1_EX2_F1 GCTCTCAGCykGGCTTTCTCyACAG

T1R1_EX2_R1 CCAGCTyACCAsrGGCACCAG

T1R1_EX3_F1 CAGATCAGCTACGAGGCCAGCAG

T1R1_EX3_R1 CTTACCTGCCAGGGGTAGACTCGGT

T1R1_EX4_F1 TATyTCAGCTTCTrGAGCAGATCCGy

T1R1_EX4_R1 TTACCTGGTTGTCCTyyCCGTGCC

T1R1_EX5_F1 CAGAACACCTGTGGCTTCTTGCAGGT

T1R1_EX5_R1 CACTCACCrCTCTTGTTGAGGAAGsTsC

T1R1_EX6_F1 CCTTTyCTTCCAGACCTCCACAGmTGC

T1R1_EX6_R1 TCAGGTGGAGCCGCAGCGCC

Tas1r3

T1R3_EX2_F1 AGGTTCTCGkCyCTbGGCCTGCTC

T1R3_EX2_R1 CACCTGAGGCAyrAGGAAGAAGCTGAA

T1R3_EX4_F1 CAGCTCCTrGArAACATGTACAACd

T1R3_EX4_R1 CTCACCTGsTTCCChGGyGT

T1R3_EX5_F1 CAGCvGCCCGTGTCCCAGTGC

T1R3_EX5_R1 GAGGTGCTCACCTGGGyTGCGCT

Table S1: Degenerate primers used to amplify exons of the sea lion Tas1r1 and Tas1r3.

21

22

Doc. S1: Pairwise alignment of dolphin and dog (cafa) or cow (bota) Tas2r sequences (page 23 – page 35)

Tas1r1 [Cafa-T2R1 (AB249684)]

Dolphin-Tas2r1p ATGCTGGAGTCTCACCTCATTAGCCACCTTTGTTTGGCAGTGATAAAATTTCTCTTTGGG Cafa-T2R1 ATGTTAGAGTTTTACCTTATTATCCATTTTCTTTTCACAGTGATGCAATTTCTCATCGGG *** * **** * **** **** *** ** *** ******* ******** * *** Dolphin-Tas2r1p GTTTTAGTAAATGGCATCATTGTGGTTGTGAATGACACTCACTTGATCAAGCAGAGAAAG Cafa-T2R1 GTTTTAGCAAATGGCATCATTGTGGTGGTGAATGGCACTGAGTTGATCAAGCAGAGAAAG ******* ****************** ******* **** * ****************** Dolphin-Tas2r1p ATGATTCCATTGGATCTCCTTGTTTCCTGCCTGGCGATTTCCAGGATTTGTCTGCAACTA Cafa-T2R1 ATGATTCCCTTGGCTCTCCTTCTTTGCTGTCTGGCGATTTCCAGGATTTGTCTACAATTG ******** **** ******* *** *** *********************** *** * Dolphin-Tas2r1p GCCATCTTCTACGTTAACCTGGCTGTTCTTTCCTTGATTGAATTCCCTCAGCTTGCTGAG Cafa-T2R1 ATCATCTTCTTCATGAATCTGGGTACTCTCTTCTTGATTGAAGTCCCCCTACTTGCTGAT ******** * * ** **** * *** * ********** **** * ******** Dolphin-Tas2r1p AAGTTCGTAATTCTCACATTTATAAATGAATCGGGACTTTGATTTGCCACATGGCTCAGC Cafa-T2R1 AATTTTGTAATTTTCGTGTTTGTAAATGAATTGGGACTTTGGTTCGCCACATGGCTTGGG ** ** ****** ** *** ********* ********* ** *********** * Dolphin-Tas2r1p CTTTTCTACCGTGCCGAGATTGCCACCATTGCTCACCCACACTT---CCGCTTGAAGGTG Cafa-T2R1 GTTTACTACTGTGCCAAGATCGCCCCCATAACTCACTCATTCTTTTTCTGGTTGAAGATA *** **** ***** **** *** **** ***** ** *** * * ****** * Dolphin-Tas2r1p AGGATATCCAAGTTGGTTCCTTGGCTGGCACTTGAGTCCCTGCTATATGCATCCAGCATG Cafa-T2R1 AGGATATCCAAGTGGATGCCATGGCTGATCCTCGGGTCCATGATGTATGCATCCGTCCCT ************* * * ** ****** ** * **** ** * ********* * Dolphin-Tas2r1p GATGTTTTCCACAGCAAACATAGGTGGATATTTTCCAAAGAACACTTCCTGGGCCTTTTC Cafa-T2R1 TCTGTTTTCTGCAGCAAACAGATATGGGTTTATTCCCAAAACGTTTTGTCCAGCCTTTTT ******* ********* * *** * * **** ** * ** ******* Dolphin-Tas2r1p TCCCCAAATGCAACCACCCAATCAAAGAA-TACCTGCTTTACAGTTTGCCTTTCTTTTTG Cafa-T2R1 TCCCCAAACGCAACT--CAAATCAAAGAAACATCTGCTTTACAGATTGCCTTTCTTATTA ******** ***** * ********** * *********** *********** ** Dolphin-Tas2r1p CTGAGTTTCATTGCCATTACTTATCTTCCTTATTTCTTCTCTGCTCTTGATATTTTCTCT Cafa-T2R1 --GGTTATTATTGCCACTGCTTATCTTTCTCGGTTCCACCCTACTTTTGATATTTTCCCT * * * ******* * ******** ** *** * ** ** *********** ** Dolphin-Tas2r1p GGAGAGACACAC-TGACAGATGAGAAACACAGCAACAGGCCCCAGGAGCCCTCGCACATG Cafa-T2R1 GGGGAGACACACCTGGCAGATGAGAAACACAGCAACAGGCCCCAGGGACCCTAGCACAGG ** ********* ** ****************************** **** ***** * Dolphin-Tas2r1p CGTGCACATCAGCACTCTTCTCTCCATCCTGTCCTTTCTGGTCCTCTATCTCTGCCACTC Cafa-T2R1 TGTCCACGTGAGCACGATCCTGTCCGTTCTATCCTTCCTGGTCCTCTGCCTCTCCCACTA ** *** * ***** * ** *** * ** ***** ********** **** ***** Dolphin-Tas2r1p CATGACAGCTGCTTTGCTCTTTTCCCAAATTTTCAACTTTAGAAGCTTCATATTTCTGTT Cafa-T2R1 CATGGCAGCTGCTTTGCTCTCTTTTCAGATCTTTCAGCTCAGAAGCCTCGTCTTTCTGAT **** *************** ** ** ** ** * * ****** ** * ****** * Dolphin-Tas2r1p CTGCATCTTGTGGGTTGGTTCATACCACTCTGGACACTCTATTACCTTAATTTTAGGAAA Cafa-T2R1 CTGTCTCTGGGTGTTTGGGTCCTATCCTTCTGGACACTCTATGATCTTAATTTTAGGAAA *** *** * * **** ** ** * ************** * *************** Dolphin-Tas2r1p TCCTAAAATGAAACAAAATGCAAAGAAATTGCTCCTCCACAGAAAGTGCTGTCAGTGA Cafa-T2R1 TCCTAAATTGAAACAAAATGCAAAGAAGCTCCTCCTCCACGGGAAGTGCTGCCAGTGA ******* ******************* * ********* * ******** ******

23

Tas2r2 [cafa-T2R2 (AB249685)] Dolphin-Tas2r2p ATGGCCTCCTCTTTGTCAGCTCGTCTTCATGTTATCCTCATGTCAGCAGAATTTATCACA Cafa-T2R2 ATGATCTCCTTTTTGTCAGCTCTTCCTCATGTTATTGTTATGTCAGCAGAATTTATCACA *** ***** *********** ** ********* * ********************* Dolphin-Tas2r2p GGGATTACAGTAAATGGATTTCTTATAATCATCGACTGTAATGAATTGGTCAAAAGCAGA Cafa-T2R2 GGGATTACAGTAAATGGATTTCTTATCATCATGAACTGTAAAGAATTGATCAAAAGCAGA ************************** ***** ******* ****** *********** Dolphin-Tas2r2p AAGCTGACACCAATGCATCTCCTTTTCATATGCATAGGGATGTCTAGATTTGGTTTGCAG Cafa-T2R2 AAGCCAACACCAGTGCAACTCCTTTTCATATGTATAGGGATGTCGAGATTTGGTCTGCTC **** ****** **** ************** *********** ********* *** Dolphin-Tas2r2p ATAGTGTTAATGGTAAAG---TTTTTCTC-ATGTTCTTTCCACTCTTTTATAGAGTAAAA Cafa-T2R2 ATGGTGTTAATGATACAAAGTTTTTTCTCTGTGTTATTTCCACTCTTTTATAAGGTAAAC ** ********* ** * ******** **** **************** ***** Dolphin-Tas2r2p ATTTATGGTACAGCGATGAT-TTTTTGGGGATGTTTTTCAGCTCTGTCAGTCTCTGGTTT Cafa-T2R2 ATTTTTGGTACAGCAATGTTGTTCTTTTGGATGTTTTTTAGCTCTGTCAGTTTCTGGTTT **** ********* *** * ** ** ********** ************ ******** Dolphin-Tas2r2p GCCACCTGTCTCTCTGTATTTTACTGCCTCAAGATAACACACTTCACCCAGTACTGTTTT Cafa-T2R2 GCCACCTGCCTTTCTGTATTTTACTGCCTCAAGATAGCAGGCTTCACTCAATCCTGTTTT ******** ** ************************ ** ****** ** * ******* Dolphin-Tas2r2p CTTTGGCTGAAATTCAGGATCTCAAAGTTAATGCCTTGACTGCTTCTGGGAAGCCTGCTG Cafa-T2R2 CTTTGGCTGAAATTCAGGATCTCGAAGTTAATGCCTTGGCTACTTCTGGGAAGTTTGCTG *********************** ************** ** *********** ***** Dolphin-Tas2r2p ACCTCCGTGAGCATTGCAACTCTGTGTGTCAAGGTGGATTACCCTAAAAATGTGGATATT Cafa-T2R2 GCCTCCATGAGCATTGCAGCTCTGTGTATTGAAGCAGATTACCCTAAAAAGGTGGATGAT ***** *********** ******** * * * ************** ****** * Dolphin-Tas2r2p GATGTCCTCAGGGATGCCATGCTAAAGAGGACTAAACTCAAGACAAAGCAGATTAATGAA Cafa-T2R2 GATGCCCTCAAGAATGCCACATTGAAGAGGACTGAACCCAAGATAAGGCAAATTAGTGAA **** ***** * ****** * ********* *** ***** ** *** **** **** Dolphin-Tas2r2p GTGCTTCTTGTCAGCTTGGCATTAATATTTCCTCTGGCCATATCTGTGAGGTGAACTGTT Cafa-T2R2 ATGCTGCTTGTCAACTTGGCATTACTATTTCCTCTAGCCATATTTGTGATGTGCACTTTT **** ******* ********** ********** ******* ***** *** *** ** Dolphin-Tas2r2p ATGTTATTCAGTTCTCTCTATAAACACGCTAATCGGATGCAAAATGGACCTCTTGGTTTT Cafa-T2R2 ATGTTATTCATTTCTCTCTATAAGCACACTCATCGGATGCAAAATGGATCTCATGGTGTT ********** ************ *** ** ***************** *** **** ** Dolphin-Tas2r2p AGAAACGCCAGCACTGAAGCCCATATTAATACATTAAGATCAGTGATAACATTCTTTTGC Cafa-T2R2 AGAAATGCCAGCACAAAAGCCCATATAAATGCATTAAAAACAGTGATAACATTCTTTTGC ***** ******** ********** *** ****** * ******************** Dolphin-Tas2r2p TTCTTTATTTCTTATTTTGCTGCCTTCATGGCAAATATGACATTCAGTATTCCTTATGGG Cafa-T2R2 TTCTTTATTTCTTATTTTGCTGCCTTCATGGCAAATATGACATTCAGTATTCCTTATGGA *********************************************************** Dolphin-Tas2r2p AGTCAGTGCTTCTTTGTGGTGAAGGACATAATGGCAGCATATATCCCTCTGGCCATTCGG Cafa-T2R2 AGTCATTGCTTCTTTGTAGTAAAGGACATAATGGCAGCATTT--CCCTCTGGTCATTCAA ***** *********** ** ******************* * ******** ***** Dolphin-Tas2r2p TTATAATTATCTTGAGTAATTCTCAGTTCCAACAACCAGTCAGGAGACTTCTCTACCTCA Cafa-T2R2 TTATAATCCTCCTGAGTAATTCTAAATACCAACAACCTTTCAGGAGACTTCTCTGCTTCA ******* ** *********** * * ********* *************** * *** Dolphin-Tas2r2p GAAAGAATCAATGA Cafa-T2R2 AAAAGAATCAATGA *************

24

Tas2r3 [Cafa-T2R3 (AB249686)]

Dolphin-Tas2r3p ATGCTGGGACTCACCGAGTGCGGGTTTCTGGTTCTGACTGCCACTCAGTTCATTCTGGGA Cafa-T2R3 ATGTCAGGGCTGGGGAAATCCGTGTTCCTGGTTCTGTCTGTCACTCAGTTCATTCTGGGG *** .**.** . .*.* ** *** *********:*** ******************. Dolphin-Tas2r3p ATGCCGGGGAATAGTTTCATGG-GTTGGTCAATGGTAGCAGCTGGTTCAAGAACAAGAGA Cafa-T2R3 ATGCTGGGGAATGGTTTCATAGTGTTGGTCAATGGCAGCAGCTGGTTCAAGAACAAGACA **** *******.*******.* ************ ********************** * Dolphin-Tas2r3p ACCTCTTTGTCTGACTTCATCATCACTAACCGGGGTCTCTCCAGGATTGTTCTGCTGTGG Cafa-T2R3 GTCTCTTTGTCTGACGTTATCATCACTAACCTGGCTCTCTCCAGGATTGTTCTGCTGTGG . ************* * ************* ** ************************* Dolphin-Tas2r3p ATTCTCTTTTTTTTTTTTTGCGGTACGCGGTCCTCTCACTGTTGCGGCCTCTCCCGTTGC Cafa-T2R3 ATTCTCTTGGTTGATGGTG----------------------------------------- ******** ** :* * Dolphin-Tas2r3p GGAGCACAGGCTCCGGACGCGCAGGCTCAGCAGCCATGGCTCACGGGCCCAGCTGCTCCG Cafa-T2R3 ------------------------------------------------------------ Dolphin-Tas2r3p CGGCATGTGGGATCTTCCCGGACTGCGGCACGAACCCGTGTCCCCTGCATCGGCAGGCGG Cafa-T2R3 ------------------------------------------------------------ Dolphin-Tas2r3p ACTCTCAACCACTGCGCCACCAGGGAAGCCCTCTGCTGTGGATTCTCTTGATTGATGGTG Cafa-T2R3 -------------------------------------------------TTTTAATGGTC :**.***** Dolphin-Tas2r3p TTCTCTTCCAAACTCCACGATGAATAATTTGCAGTCATGCAGATTAGTGATATTTTCTGG Cafa-T2R3 TTCTTTTCCAAAGTACATGATGAAGGG---ACAGTAATGGAAATTATTGATATTTTCTGG **** ******* *.** ****** .. .****.*** *.**** ************* Dolphin-Tas2r3p ACATTTACAAACCATCTGAGCATTTGGCTTGCCACCTGTCTCAGTGTCTTCTACTGCCTG Cafa-T2R3 ACATTTACGAACCACCTGAGCATTTGGCTTGCCACCTGTCTCAGTGTCCTCTACTGCCTG ********.***** ********************************* *********** Dolphin-Tas2r3p AAAGTCGCCAGTTTCTCCCATCCTACGTTCCTCTGGCTCAAGTGGAGAGTTTCCAGGTTG Cafa-T2R3 AAAATTGCCAGTTTCTCCCATCCGACGTTCCTCTGGCTCAAGTGGAGAGTTTCCAGAGTG ***.* ***************** ********************************. ** Dolphin-Tas2r3p GTTGTATGGATGCTGTTGG-TACCCTGCTCTTATCATGTAGCAGTGCCGTCTCTCTGATC Cafa-T2R3 GTCGTACAGATGATTTTGGGTGCACTGCTCTTATCGTGTGCCAGTGCCATGTCTCTGGTC ** *** .****.* **** *.*.***********.***. *******.* ******.** Dolphin-Tas2r3p CATGAATTTAAGATCTAGTCTGTTCTCAGTGGAATTGATGGAACAGGGAATGTGACTGAA Cafa-T2R3 CATGAATTTAAGATCTATTCTATTCTCAGTGGAATTGCTGGTACAGGGAATGTGACCGAG ***************** ***.***************.***:************** **. Dolphin-Tas2r3p CCCTTTAGAAAGAAAAGAAATGAATATAAGCTGATCCATTTTCTTGGCACTCTGTGGGAC Cafa-T2R3 CACTTTAGAAAGAAGAGAAATGACTATAAAGTGGCCCATGTTCTTGGGACTCTGTGGAAC *.************.********.*****. **. **** ******* *********.** Dolphin-Tas2r3p CTCCCTCCCTTAATTGTATCTCTAGCTTCCTACTTTCTGCTCATCCTCTCTCTGGGGAGG Cafa-T2R3 CTCCCTCCCCTAATTGTTTCTCTGGCCTCCTACTTTCTGCTCATCTTCTCCCTGGGAAGG ********* *******:*****.** ****************** **** *****.*** Dolphin-Tas2r3p CGTATGCGGCAGATGCAGCAAAACTTTACCGGCTCCAGATATCCAAGTACTGAGGCCCAA Cafa-T2R3 CACACACAGCAGATGAAGCACAGTGGCACCAGCTCCAGAGATCTGAGCACGGAGGCCCAC *. * .*.*******.****.*. ***.******** *** .** ** ********. Dolphin-Tas2r3p AAGAGGGCCATCAAAATCATCCTTTCCTTCCTCTTTCTCTTCCTACTTTACTTTCTTTTC Cafa-T2R3 CAGAGAGCCATCAAAATCATCGTCTCTTTCCTCTTTCTCTTCCTGCTTTACTTTCTTGCC .****.*************** * ** *****************.************ *

25

Dolphin-Tas2r3p TTTGCAATTTTGACATCCAGTTATTTCT-ACCAGCAACTGAGGTGATTATGATGACTGGA Cafa-T2R3 TTTTTAATTACATCATCCAGTTATTTCATACCAGAAACTGAGATGGTTAAGAGAGTTGGA *** ****: .:**************: *****.*******.**.***:** .. **** Dolphin-Tas2r3p GAAGTAATTACAATGTTATATCCTGCTGGCCGCTCATATATTCTCATTCTGGGAAATAAT Cafa-T2R3 GTAGTTGTTACAATGTTTTACCCTGCCAGCCACTCATTCGTTATCATTCTGGGAAACAAT *:***:.**********:** ***** .***.*****: .**.************* *** Dolphin-Tas2r3p AAGCTGAAGCAGATGTTCATGGAGACGCTTTGGTGTGAGCCTGGTCATCTGAAGCCTGGA Cafa-T2R3 AAGCTGAAGCAGATGTTTACGGAGATGCTGTGCTGTGAGCCTGGTTATCTGAAGCCTGGA ***************** * ***** *** ** ************ ************** Dolphin-Tas2r3p TCCAAGGAACCCGTTTTTCCATAG Cafa-T2R3 TTCAAAAGACCTTTTGCCCCATAA * ***...*** ** *****.

26

Tas2r5 [Cafa-T2R5 (AB249688)] Dolphin-Tas2r5p ATGCTGACTGCTGTCCTAGGACTGTTAATGCGGGTAGCAGTGGCTGAATTTCTCATTGGC Cafa-T2R5 ATGCTGACTGCTGCCCTACCACTGCTGATGGTGGTGGCAGTGGTTGAATTTCTCATTGGC ************* **** **** *.*** ***.******* **************** Dolphin-Tas2r5p CTGGTTGGAAATGGAGTCCTCGTGGTCTGGAGTTTTGGAGAACGGCTCAGAAAATTCAAG Cafa-T2R5 TTGGTGGGAAATGGAGTCCTTATGGTCTGGAGTTTTGGTGAATGGGTCAGAAAATTCAAC **** ************** .****************:*** ** ************* Dolphin-Tas2r5p GGGTCCTCATATAACCTCATTGTCCTGGGCCTGGCTGTCTGTTGATTTCTTCTGCAGTGG Cafa-T2R5 GGGTCCTCATACAACCTCATTGTCCTGGGCCTGGCTGTCTGCCGATTTCTCCTGCAGTGT *********** ***************************** ******* ******** Dolphin-Tas2r5p TTGATTATGGTGGACTTAAGTCTGTTTCCACTTTTCCAGAGCAGCCATTGGCTTCGCTAT Cafa-T2R5 CTGATTATGATGGACTTAAGCCTGTTTCCATTTTTCCAGAGTAGCCGTTGGCTTCACTAT ********.********** ********* ********** ****.********.**** Dolphin-Tas2r5p CTCCATGTCTTCTGGGTCTTAGTAAACCAGACCAGCCTGTGGTTTGCCACTTTTCTCAGT Cafa-T2R5 CTCAGTATCTTCTGGATCCTGGTAAGCCAGGCCAGCCTGTGGTTTGCCACTTTCCTCAGC ***..*.********.** *.****.****.********************** ***** Dolphin-Tas2r5p GTCTTCTACTGCAGGAAGATCATGACCTTTGAACACCCTGTCTACTTGTGGCTGAAGCAG Cafa-T2R5 GTCTTCTACTGCAGGAAGATCATGACCCTTGAACATCCTGTCTGCTTGTGGCTGAAGCAG *************************** ******* *******.**************** Dolphin-Tas2r5p AGGGCCTGTTGTCTTAGTCACTGGTGCCTTCTGGTGTACTTCATGATCAGTTTGTTACTT Cafa-T2R5 AGGGCCTATTGCCTGAGTCTCTGGTGCCTTCTGGTGTACCTCATGATCAGTTTGTTACTT *******.*** ** ****:******************* ******************** Dolphin-Tas2r5p ATAGTCCAGGGTAGCTTAGAGTTCTCCAATCTTTCCCAAGGAAACAGCAGCATTTTATAC Cafa-T2R5 GTAGCACACATTGGCTTAAAGCCCTATAATCCTTCTCAAGGCAACAGCAGCATTCTGTAC .*** .** . *.*****.** **. **** *** *****.************ *.*** Dolphin-Tas2r5p CCCCCTTCAAACTGGCACTGTCTGTATATATTATGGCTCAATACAGGAAGTATAATGCCC Cafa-T2R5 CCCCTTAAAAGCTGGCACTACCTGTATATAGTAAAGCTCAACGCAGGAAGTGGATTGCCT **** *:.**.********. ********* **:.****** .********. *:**** Dolphin-Tas2r5p TTCATGGTGTTGCTTATTTCCTCTGGGATGCAGATTGTCTCTTTGTGTAGACACCGCAGG Cafa-T2R5 CTCATGGTGTTTCTTGTTTCTTCTGGGATGCTGATTGTCTCTTTGTATAGACACCACAAG ********** ***.**** **********:**************.********.**.* Dolphin-Tas2r5p AAGATGAATGTCCATACAGTCGGCAGGAGAGATGCTCAGGCCAAGGCTCACATCACTGTC Cafa-T2R5 AAGATGGAGGTACATACAGCTGGTAGGAGAGATGCTCAGGCCAAGGCTCACATCACTGTA ******.* **.******* ** ***********************************. Dolphin-Tas2r5p CTGAAGTCCTTGGGCTGTTTCCTTATACTTTACATAGTTTACATCCTGGCCAGCCCCTTC Cafa-T2R5 CTGAAGTCCTTGGGCTGCTTCCTTATCCTTCATGTGATTTATATCCTGGCCAGCCCCTTT ***************** ********.*** * .*..**** ***************** Dolphin-Tas2r5p TCCATCACCTCCAGGTCTTTTCCTGCTGCTCTTACCGCTCTCTTCATCTCTGAGACACTC Cafa-T2R5 TCCATTACCTCCAAGTCTT---CTGCTGATCTCCTCGTTGTCTTCATCTCTGAGACAGTC ***** *******.***** ******.*** . ** * ***************** ** Dolphin-Tas2r5p ATGGCTGCCTAGGCTTCTCTTCATTCTGTCATATTGATCATGGGGAATTCCAGGATGAAG Cafa-T2R5 ATGGCTGCCTATCCTTCTCTTCATTCTGTCATTCTGATCCTGGGGAATCCCAGGATGAAG *********** *******************: *****.******** *********** Dolphin-Tas2r5p CAGACTTGTCAGAGAATCCTGTGGAAGACAGTGTACGCTTGGAGAGAAAGGAGTGGTAA Cafa-T2R5 CAGACTTGTCAGAGAATTCTGTGGAAGACAGTGTGTGCTTGGAAATCCTAG-------- ***************** ****************. *******.* ..:.*

27

Tas2r16 [Bota-T2r16 (AB249716)]

Dolphin-Tas2r16p ATGATAACCATCCAACTCT--GTCTTCTTCATGATCATCTATATGCTCAAGCTCTTGACA Bota-T2R16 ATGACAACCAGCCAACTCTCTGTCTTCTTCATGATTATCTATATGCTCGAGTTCTTGATA **** ***** ******** ************** ************ ** ****** * Dolphin-Tas2r16p ATAATTATGCAGAGCAGCTTAACTGTTGTAGTGCTGGGCACAGAGTGGGTAAGTTTCCAA Bota-T2R16 ATAACTGGGCAGAGCAGCCTGATTGTTGTAGCGCTGGGCAGAGACTGGGTGCAGACTCAA **** * ********** * * ******** ******** *** ***** *** Dolphin-Tas2r16p AGGCTATCACCTGTGGAAATGATTCTCACCGGCCTGGGTGTCTGCATGCTTCTGTCAACT Bota-T2R16 AGGCTGCCACCTGCGGACATGATTCTCATCAGCCTGGGCATCT-TTTGCTTCTGTCAACT ***** ****** *** ********** * ******* *** ************** Dolphin-Tas2r16p GTGGTCATCAATGCTGTACAACTTTTGCTCCCACTTCTACCCTAGTTACGAATTTTGGTA Bota-T2R16 GTGGTCATCGATGCTGTACAACTTTTGTTCCCACTTCCACCCTAATTACAATTTTTGGTA ********* ***************** ********* ****** **** * ******** Dolphin-Tas2r16p CTTCAGTATCGTCTGGGAATTTACTAACATTCTTTCATTCTGGTTGACCAGCATGTTTGC Bota-T2R16 TTTCGGGATCATCTGGGAATTTACTAACATCCTTTCCTTCTGGTTGACCAGCTTGCTTGC *** * *** ******************* ***** *************** ** **** Dolphin-Tas2r16p TGTCTTCTACTGTGTCAAAGTCTCCTCCTTCAGCCACCCCATCTTC---TGGCTGAAGTG Bota-T2R16 TGTCTTCTACTGTGTCAAAGTCTCTTTCTTCAGCCACCCCGTCTTCCTCTGGCTGAAGTG ************************ * ************* ***** *********** Dolphin-Tas2r16p GAGAATTGTGAGGTTGGTTCCTTGGCTGTTGCTGGGTTCTCTGCTGACTTCTTGTGTGTC Bota-T2R16 GAGAATTGTGAGATGGGTTCCTCGGCTGTTGCTGGGCTCTCTGCTGATTTCTTGTGTGTC ************ * ******* ************* ********** ************ Dolphin-Tas2r16p TATCATCTTTGCAGCTGTTGGGCATTACAGCAAGATTCAACTAATCTCCATGACGCATTT Bota-T2R16 TACCATATTTCCAGCTACTAGTTATTACATTGATATTCAATTCATCGCCATGAAGCATTT ** *** *** ***** * * ****** * ****** * *** ****** ****** Dolphin-Tas2r16p CCCTAGAAACAGCACCATGACTGAGAGACTTGAGATATTCCTGTGGGATTCTTCCATGTG Bota-T2R16 CCCTAGAAACAGCACCATGCTTGAGAGACTTGAGGCGTTCCTGTGGGATTTTTCCACACT ******************* ************* ************* ***** Dolphin-Tas2r16p TCACAA-GTGGTTGTGTTGATTATTCCTTTCCTCCTGTTCCTGGCCTCCACCGTCTTGCT Bota-T2R16 GCACAAAGTAGTTGTGTTGGTTATTCCTTTCCTCCTGTTCCTGGCCTCCACAGTCTTGCT ***** ** ********* ******************************* ******** Dolphin-Tas2r16p CATGGCCTTATTATTCCAACACCTGAGGCAGATGAAAGATCATCACACCAGCCACTC--- Bota-T2R16 CATGGCCTTATTATCCCGACATCTGAAGCAGATGAAAGACCTTCACACAGGCTGCTCCAA ************** ** *** **** ************ * ****** ** *** Dolphin-Tas2r16p -TCCAGCCTGAAAGCTCACTCTACTGCCCTGAGGTCTCTTGCTGTCTTCCTCATTTTCTT Bota-T2R16 CTCCAGCCCGGAAGCTCACTCTGCCGCCCTGAGGTCCCTTGCCATCGTCCTCATCTTGTT ******* * *********** * *********** ***** ** ******* ** ** Dolphin-Tas2r16p CACCTCTTATTTTCTGACCCTAATAATCTCCATGTGGGGTGTCCTTTTTAATAAGGGGTC Bota-T2R16 CACCTTTTATTTTCTCACCGTGCTCCTCTCCATATTGGATGTCCTATTTAATAAAGAGTC ***** ********* *** * * ******* * ** ****** ******** * *** Dolphin-Tas2r16p CTGGTTCTGGGCCTGGGAAGCTATCATCTGTGCTCTGGTCTCTATTCATTTGACTTCACG Bota-T2R16 CTGGTTCTGGGCCTGGGAAGCTATCATCTATGCATTAGTCTCTATTCATTCTACTTTACT ***************************** *** * ************* **** ** Dolphin-Tas2r16p GATGCTGAGCAGCCCTAAACTGAAAAGGGTTTTAAAGGTAAAGTGCTGGGACCTAGAGGC Bota-T2R16 AATGCTGAGCAGTGTCAAACTGAAAAGAGTTTTAAAGGCAAGGTGCTGGAGCCTAGAAGC *********** *********** ********** ** ******* ****** ** Dolphin-Tas2r16p TGCCTGA Bota-T2R16 TGCCTGA *******

28

Tas2r38 [Cafa-T2R38 (AB249694)] Dolphin-Tas2r38p ATGGTGACTCTGACTGCCACTGTAACTGTGCCCTATGAAGTCAGGAATGCATTTCTGTTC Cafa-T2R38 ------------------------------------------------------ATGTTC ***** Dolphin-Tas2r38p TTTTCAGTCCTGGAGTTTGCAGTAGGGATCCTGGTCAATGCCTTCATTTTCTTGATGAAT Cafa-T2R38 CTTTCAGTACTGGAGCTCGCAGTGGGGATCCTGACCAATGCCTTCATTTTCTTGGTGAAT ******* ****** * ***** ********* ******************* ***** Dolphin-Tas2r38p TTTTGGGTCGTGGTGAGGAGGTGGCCACTGAGCAACTGTGATCTTGTCCTGCTGAATCTC Cafa-T2R38 TTTTGGGATGTGGTGAGGAGGCAGCCACTGAGCAACTGCGATCTTATCCTTCTGAGTCTC ******* ************ *************** ****** **** **** **** Dolphin-Tas2r38p AGCCTCACCTGGCCTTTCCTACACGGGCTGCTCTTTCTGGATGCCATCCAGCTTACCCAC Cafa-T2R38 AGCCTCACTCGACTTTTCCTGCATGGGCTGCTGTTTCTGGATGCCATCCAGCTTACATAC ******** * * ****** ** ******** *********************** ** Dolphin-Tas2r38p TTCCAGTGGGTAAAAGACCCGCTGGGCCTCTGCTACCAGACCACCCTCATGCTCTGGATG Cafa-T2R38 TTCCAGCGGATGAAAGACCCACTGAGCCTCAGCTACCAGACCATCATCATGCTCTGGATG ****** ** * ******** *** ***** ************ * ************** Dolphin-Tas2r38p CTCGTAAATCAAGCTGGCCTCTGGCTCACCACTTGCCTTAGTCTCCTCTACTGCTCCAGG Cafa-T2R38 ATCACAAACCAAGCTGGGCTCTGGCTCACCACCTGTCTCAGTCTTTTCTACTGCTCCAAG ** *** ******** ************** ** ** ***** ************ * Dolphin-Tas2r38p ACTGTCCATTTCTTTCACACCTTCCTCCTCCGCTTGGCAAGCTGGATCTCCAGGAAGATC Cafa-T2R38 ATTGTCCGTTTCTCTCATACCCTCCTTCTCTGCTTGGCAAACTGGGTCTCCAGGAAGGCA * ***** ***** *** *** **** *** ********* **** *********** Dolphin-Tas2r38p CCCCAGATGCTCCTGGGTGCTATTTTTTCCTCCTGTGTCTGCACTGTTCTCTATTTGTGG Cafa-T2R38 CCCCAGATGCTCCTGGGTGCCATGCTTTTCTCTTCTGCCTGCACTCTCCTCTGTTTGGGG ******************** ** *** *** * ** ******* * **** **** ** Dolphin-Tas2r38p GACTTTTTCAATAGATCTCACTTCTCAGTTGCAACCATGCTACTCATGAATAA--CAATA Cafa-T2R38 GACTTCTTTAGTAGATCTGGCTTTGCATTCACAACTGTGCTACTCATGAATAATACAGAA ***** ** * ******* *** ** * **** **************** ** * Dolphin-Tas2r38p CTCAATTGAGAAACTGAGAAAACTCAATTTCTTTCATTCCTTCCTCTTCTGCAGCCTGGG Cafa-T2R38 TTTAATTCACAAATTGT-AAAACTCAATTTCTATTATTCCTCCATCTTCTGTACCCTGGG * **** * *** ** ************** * ****** * ******* * ****** Dolphin-Tas2r38p GTCCACCCCTTCTTTCTTGCTTTTTCTGGTTTCTTCTGGGGTGTTGATTGTCTCCCTGGG Cafa-T2R38 GTCAATCCCTCCTTTCATGTTTTTTCTGGTTTCTTCTGGGGTGCTGATTATCTCTCTGGG *** * **** ***** ** *********************** ***** **** ***** Dolphin-Tas2r38p GAGGCACATGAGGACAAGGAGGGCCAAAACCAGAGACTCTCGGGACCCCAGCCTGGAGGC Cafa-T2R38 AAGGCACATGAGAACAATGAAGGCCAACACCAAAGACTCCGGTGACCCCAGCCTGGAGGC *********** **** ** ****** **** ****** * ***************** Dolphin-Tas2r38p CCACATCAAAGCACTCGGGTCTC--ATCTCTTTCTTCTGCCTGTATGTGGTGTC---CTG Cafa-T2R38 CCATATCAAAGCACTCATATCTCTCATCTCCTTTCTCTGCCTCTATGTGGTGTCATTCTG *** ************ **** ***** ** ******* *********** *** Dolphin-Tas2r38p CGCTGGCTTCATCTCGGTGCCTTTGCTGATGCTGTGGCACAACAAGATCGGGGTCATGGT Cafa-T2R38 TGTTGCCCTTATCTCAGTGCCTTTAACCATGGTGTGGCACAACAAGATCGGGGTAATGAT * ** * * ***** ******** *** ********************** *** * Dolphin-Tas2r38p CTGTGCAGGGATACTGGCAGCCTGCCCCTCGGGGCACACAGTCATCCTGATCTCAGGCAA Cafa-T2R38 CTGTGTAGGGATCCTAGCAGCTTGTCCCTCTATACATGCAGCCATCCTGATCTCAGGCAA ***** ****** ** ***** ** ***** ** *** ****************** Dolphin-Tas2r38p TGCCAAGCTGAAGAGAGCCGTGGAGACCATTCTGCTCCGGGCTCAGAGCAGCCTAAAGGT Cafa-T2R38 TGCCAAGCTGAGGAGAGCTGTGGAGACCATTCTACTCTGGGTTCAGAGCAGCCTTAAGGT *********** ****** ************** *** *** ************ *****

29

Dolphin-Tas2r38p AAGGGCGGACCGCAAGGCAGATCCCAGGATGCCAGATCTATGTTGA Cafa-T2R38 AAGGGCAGGCCACAGGGCAGATCTCAGGACTCCAGATCTATGTTGA ****** * ** ** ******** ***** ***************

30

Tas2r39 [Cafa-T2R39 (AB249695)] Dolphin-Tas2r39p ATGACTGAAACCTGCAATCCCCCAGAAAATCAACTGTCACCATCTCGCATCATTTTGATG Cafa-T2R39 ---ATGGAAACCTGCAATCCCCCAGAAAATGAATTGTCACCATTTGGCATCCTCTCGATT * ************************ ** ********* * ***** * * *** Dolphin-Tas2r39p TGAATCGTTATAGGCACCGAATGCGTCCTTGGTCTCACTGCAAATGGGTTCATTGTGGCT Cafa-T2R39 TTAACAATTACAGGCACTGAATGCATCGTTGGTATCATTGCAAATGGGTTCATCATGGCT * ** *** ****** ****** ** ***** *** *************** ***** Dolphin-Tas2r39p ATAAATACAGCAGGATGGATTCACAACAAGGCAGTTTCCACAAGTGGCAAGATCCTGCTT Cafa-T2R39 ATAAATGCGGCTGAATGGATTAAAAATAAGACAGTTTCCACAAGTGGCAGAGTCCTGTTT ****** * ** * ******* * ** *** ****************** ***** ** Dolphin-Tas2r39p CTCCTGAGCGTATCCGGAAGAGTGCTACAAAGCTTCATGATGCTAGAACTCACCTTCAGT Cafa-T2R39 TTCTTGAGTGCATCCAGAATAGCTCTCCAAAGCTTCACAATGCTAGAAATTACCTTCAGT ** **** * **** *** ** ** ********** ********* * ********* Dolphin-Tas2r39p TCAACATCCCCACACTTTTATAATCAAGACATTCATCGTATATGATACGTTCAAAGGAAG Cafa-T2R39 TCAACATCCCCACGTTTTTATAATGAAGATGTT----ATGTATGACACATTCAAAGTAAG ************* ********* **** ** * ***** ** ******* *** Dolphin-Tas2r39p TTTCATGTTCTTAAATGATTGTAGCCTCTGGTTTGCTGCCTGGCTTAGATTCTTCTACTT Cafa-T2R39 TTTCATGTTCTTAAATCATTGTAGCCTCTGGTTTGCTGCTTGGCTCAGTTTCTTCTACTT **************** ********************** ***** ** *********** Dolphin-Tas2r39p CGTGAAGATGGCGGATTTCTCCTACCCCCTTTTCCTCAAGCCGAAGTAGAGAATTTCTGG Cafa-T2R39 CGTGAAGATTGCTGATTTCTCCCACCCCCTTTTTCTCAAGCTGAAGTGGAGAATTTCCAG ********* ** ********* ********** ******* ***** ********* * Dolphin-Tas2r39p ATGGATGCCCTGGTTTCTGTGACTATCAGTGTTTGTTTCCTTTGGGCCACAGTGTGTTCT Cafa-T2R39 ACTGATGCCCTGGCTTCTGTGGCTTTCAGTGCTTATTTCCTT-GGGCTACAGTATGCTCC * ********** ******* ** ****** ** ******* **** ***** ** ** Dolphin-Tas2r39p TCCTCAAAAACATCTACACTATGCATTGCAACCATCCTTTTTCTAGCCCCTCCTTCAACT Cafa-T2R39 TCTCCAATGACATCTACACTGTGTATTGTAACAATTCTT---CTATCCCCTCTTCCAACT ** *** *********** ** **** *** ** *** *** ****** * ***** Dolphin-Tas2r39p CCACTAAGAAAAATTACTTCACTGAGACCAACGTGATCAGCCTGGTTCTTTTCTTTAACG Cafa-T2R39 CCACTAAGAAAAAATACTTCACTAAGACCAATGTGGTCAACCTGGTTCTTCTCTATAACC ************* ********* ******* *** *** ********** *** **** Dolphin-Tas2r39p TGGGAATCTTCGTTCCTCTGATCACGTTCATCCTACCTGCCACCCTGCTGATCATCTCTC Cafa-T2R39 TGGGGATCTTCATTCCTCTAATCATGTTCATCCTTTCGGCCACCCTGCTGATCATCTCTC **** ****** ******* **** ********* * ********************** Dolphin-Tas2r39p TAAAGAGACACACCCTACACATGGAAAGCAATGCCACTGGTTCCAGGGACCCCAGCATGG Cafa-T2R39 TCAAGAGACATACACTACACATGGAAAGCAATGCCACTGGCTGCAGGGACCCCAGCATGG * ******** ** ************************** * ***************** Dolphin-Tas2r39p AGGCTCATGTGGGGACCATCAAAGCTATCAGCTATTTTCTCATTTTCTAAATTTTCAATG Cafa-T2R39 AGGCTCACATAGGGGCCATCAGAGCGACCAGCTACTTTCTCATTCTCTATATTTTCAATT ******* * *** ****** *** * ****** ********* **** ********* Dolphin-Tas2r39p CAGATGCTCTATTTCTTTCCATGTCCGACATCTTTGATATCAATAGTTCCTAGAATACTT Cafa-T2R39 CAGTTGCTCTATTTCTCTATATGTCCAACATCTTTGATATCAACAGCTCCTGGAATATTT *** ************ * ****** **************** ** **** ***** ** Dolphin-Tas2r39p TGTGCAAAATCATCATGGCTGCCTATCCTGCTGACCACTCCATCCTACTGATACAGGACA Cafa-T2R39 TGTGCAAATTCATCATGGCTGCCTACCCTGCTGGTCACTCCATTCTGCTGATTCAGGACA ******** **************** ******* ******** ** ***** ******* Dolphin-Tas2r39p ACCCTGGGTTGAGAAGAGCCTGGAAGCGGCTTCAGCCTGGAAGCGGAGTTCACCTTTACT Cafa-T2R39 ACCCTGGGTTGAGAAGAGCCTGGAAGCGGCTTCAGCCTCAA------GTTCATTTTTACC ************************************** * ***** *****

31

Dolphin-Tas2r39p TGA--GAGTGGACTCTATGA Cafa-T2R39 TAAAAGAGCAGACTCCATGA * * *** ***** ****

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Tas2r60 [Bota-Tas2r60 (XM_002687121) Dolphin-Tas2r60p ATGAGTGGAGAGGACGTGGTTCCAGGACCTCAGTTGGCTGATAAGATAGCCTTTATCTTT Bota-Tas2r60 ATGAACGGAGGGGACATGGTTCCTGGACCTCAGTTGGTTGATAAGACAGCCCTTGTCTGC **** **** **** ******* ************* ******** **** ** *** Dolphin-Tas2r60p GCTATCATTTTATTCCTTTTGTGCTTGGTGGCAGTGGTGGGTAATGGCTTAATCACCATG Bota-Tas2r60 ATTATTATTTTATTCCTTTTGTTCCTGGTGGCATTGGTAGGTAATGGCTTAATCATCATG *** **************** * ******** **** **************** **** Dolphin-Tas2r60p GCACTGGGCATGGAGTGGTTGCTGCAGAGAACTTTGTCACCCTGCAATAAGTTATTGGTC Bota-Tas2r60 GCACTGGGCAGCGAGTGGCTGCTGCAGAGAACGTTGTCGCCTTGCGATAAGTTATTGGTC ********** ****** ************* ***** ** *** ************** Dolphin-Tas2r60p AGCCTGGGAGCCCCTAGCTTCTATCTGTGATGGGTGGTGAT----AAGAACATTTATATT Bota-Tas2r60 AGCCTGGGGGCCTCTCGCTTCTGTCTGCAATGGGTGGTGATTAGTAAGAACATTTACATT ******** *** ** ****** **** ************ *********** *** Dolphin-Tas2r60p TTCCTGAATCCAATAGCCTTCCCATACAACC--GTATTCCAGTTCCTAGCCTTTCAGTGG Bota-Tas2r60 TTCCTGAATCCCATGGCCTTCCCATACAACCCCGTGTTCCAGCTCCTGGCCGTTCAGTGG *********** ** **************** ** ****** **** *** ******** Dolphin-Tas2r60p GACTTCTTGAATGCTGTCACGTTATGGTTCTCCACCTGGCTCAGTGTCTTCTCCTGTGTG Bota-Tas2r60 GACTTCTGGAACTCTGCAACACTGTGGTTCTCCACCTGGCTCAGTGTCTTCTACTGTGTG ******* *** *** ** * **************************** ******* Dolphin-Tas2r60p AAAATCGCAACCTTCACCCACCCTGTCTTCCTCTGGCTAAAGCAGATAGTGTCTGCGTTG Bota-Tas2r60 AAAATTGCCACCTTCACCCACCCCGTCTTCCTCTGGCTAAAGCGGAATGTATCTGGGTTG ***** ** ************** ******************* ** ** **** **** Dolphin-Tas2r60p GTTCCATGGGTGCTGCTCAGCTCCGTGGGGGTCTCCAGCTTCAGCACCATTCTAGTTTTC Bota-Tas2r60 GTTCCTTGGATGCTACTCAGCTCTCTGGGGTTCTCTACCTTTACCACCATTCTATTTTTC ***** *** **** ******** ***** **** * *** * ********** ***** Dolphin-Tas2r60p ATAGGCAACCGGAGAATAGATCAGAACTATTTAAAGAGGGTTCTGCAACCTTGGAATGTC Bota-Tas2r60 ATAGGCAACCACAGAATGTATCAGAACTATATAAAGAAGGGTCTGCAACCTTGGAATGTC ********** ***** *********** ****** ** ******************* Dolphin-Tas2r60p GCTGGGAATGCTGTGAGAACATATGAGAGACTCTGCTTCTTCCCTTTGAAAATTGTTACC Bota-Tas2r60 ACTAGGAATGCTGTGAGAACATATGAGAGGTTCTGCCTCTTCCCTTTGAGAATTGTTACC ** ************************* ***** ************ ********** Dolphin-Tas2r60p TGGACAGTCCCTACTGTTGTCTTCATCGCTGGCATGGCTTTGCTCATTCCACCTCTGGGA Bota-Tas2r60 TGGACCGTCCCTACTGTTATCTTTATTGTGGGCACAGTTTTGCTCATTACATCTCTGGGA ***** ************ **** ** * **** * ********** ** ******** Dolphin-Tas2r60p AGACACACCAAGCAGGTCTCCCTGTCCATCTCAGGCTCTCACGATCCCAGCACCCAGGCA Bota-Tas2r60 AGACACACCAAGAAGGTCTTCTTCTCCATCTCAGGCTTTCACAGTTCCAGTGCCCAGGCA ************ ****** * * ************* **** * **** ******** Dolphin-Tas2r60p CACATCAAGGCTCTC---------ATCTCCTTTGCTGTCCTCTTTGTTTCCTATTTTCTG Bota-Tas2r60 CACATCAAGGCTCTCTTGGCTTTTATCTCCTTTGCTATCTTCCTCACTTCCTCTTTTCTG *************** ************ ** ** * ***** ******* Dolphin-Tas2r60p TCACTGGTGCTCAGTGCCTCAGGTGTGTTTCCATCACGGGAATTCTG-CACTGGGTGTGG Bota-Tas2r60 TCACTGGTTCTCACTGCCTCAGGTATGTTTCCTTTTGGGGAATTCCGGTTCTGGATATGG ******** **** ********** ******* * ******** * **** * *** Dolphin-Tas2r60p CAGGCTGTGATTTATCTGTGCACAGTAGTCCGCCCCATTGTTCTTTTCTTGAGTAACAGC Bota-Tas2r60 CAGACTGTGATTTATCTGGGTACAGCAATCCACCCCCTTATTCTTCTCTTGAGTAACCGC *** ************** * **** * *** **** ** ***** *********** ** Dolphin-Tas2r60p AGACCGAGAGCTGTGCTAGAGAGGGGCTGCTCCTCAG-GCATGGGGCATCTTGA Bota-Tas2r60 AGGCTGAGAGCTCTGCTAGGGAGGGGCTGCTCCTCAGCACATGGGGCATCTTGA ** * ******* ****** ***************** ***************

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Tas2r62a [Cafa-T2R62p (AB249701)] Dolphin-Tas2r62ap ATGCCCTCCTCACCCATGTTGATCTTCATGGTCATCTTTTTCCTGGAGTTGCTGGCTGCC Cafa-T2R62p ATGTCCTCCTCACCTACATTGATCTTCATGGTCATCTTCTTCCTGGAGTCGTTGGCTGCA *** ********** * ******************** ********** * ******* Dolphin-Tas2r62ap ATGCTGCAGAATGGCTTCATAGTTACTGTGTTGATCAGGGAGTGGGTACAATGCCAGACA Cafa-T2R62p ATGCTGCAGAATGGCTTCATGGTTACTGTGTTGGGCAGGGAGTGGGTGCGACGCCGGACG ******************** ************ ************ * * *** *** Dolphin-Tas2r62ap CTGCTTGCAGGCGACATGATTGCGGCGGCCTCCCTGGCCGCCTCCCGGTTCTGACTGCAT Cafa-T2R62p CTGCCTGCAGGTGACATGATTGTGGC---CTCCCTGGCTGCCTCCTGGTTCTGCCTGCAT **** ****** ********** *** ********* ****** ******* ****** Dolphin-Tas2r62ap GGGATGGCCCTCCTGAACAACCTCGTGGCCTTCTTTGGTTTTGGTTTCAGAATTTACTAT Cafa-T2R62p GGGGTGGCCATCCTGAACAACCTCTTGATCTTCTTTGGTTTTCACTTCGTAAGGGATTAT *** ***** ************** ** ************* *** ** * *** Dolphin-Tas2r62ap TTCAGCATCCCCTAGGACTTCATCAACTCTCTCACTTTCTGGCTTACTGCTTGGCTTGCT Cafa-T2R62p TACAACACCCTCTGGCACTTTGTCAACACTCTCACTCTCTGGCTCACTGCCTGGCTTGCT * ** ** ** ** * **** ***** ******** ******* ***** ********* Dolphin-Tas2r62ap ACATTCTACTGTGTGAAGATCTCATTCTTCTCTCACCCCATCTTCTTTGGGCTGAAGTTG Cafa-T2R62p GTCTTCTACTGTGTGAAGGTCGCCGTCTTCTCTCACCCGGTCTTCTTCTGGCTGAAATGG *************** ** * ************* ******* ******* * * Dolphin-Tas2r62ap AGGATTTCTCGGTCAGTGCCCAGGCTGCTGCTGGGCTCCCTGATCTTATCTGCTCTGGTA Cafa-T2R62p AGGATTTCTCGGTTAGTGCCCAGGCTGCTGCTGGGCTCCCTGGTCTTAGTTGGCCTGACA ************* **************************** ***** ** *** * Dolphin-Tas2r62ap GCCATCCCGTTAGACACTGGGAACACAATTCGTGTGCGGATGGTTGCTGCCCAGAGTTCC Cafa-T2R62p GTCATCTCATCAGCCATTGTGACTGGAATTCTGAAACAGATGATTGCCTCCAAGAGTTCC * **** * * ** ** ** ** ***** * **** **** ** ******** Dolphin-Tas2r62ap CATGGAAACAGCACCCTGGCTGGTAGAACACAGACTGTCTCTTTGTACTTTTTTCTACCT Cafa-T2R62p CAAGGAAACAGCACCTGGGCTGAGAGAGTACAGGCCTTCTATAGGTCTTTTCAAATATTT ** ************ ***** *** **** * *** * ** *** ** * Dolphin-Tas2r62ap CGTGTAATTATTATGCGGTCAATTCCATTTCTCCCGTCCCTGGTGTCCACCCTCTCGTGT Cafa-T2R62p GATGTAATGCTTATGTGGTCAGTTCCATTCCT--CATGGCTTGTGTCCATGCTCTGCTGG ****** ***** ***** ******* ** * * ** ******* **** ** Dolphin-Tas2r62ap TCTCGCTGCGCCGGCATTTGGGGCAGATGAGGGACCATAGACCTGGCCCCGAGTGATCCC Cafa-T2R62p ------------------------------------------------------------ Dolphin-Tas2r62ap AGCACCTGGGCTCACACCGTGGCCCTGAAGTCACTTGCCTTCTTCCTCACCTTCTACCAT Cafa-T2R62p ------------------------------------------------------------ Dolphin-Tas2r62ap CACGTTACCTGTGCCTGATTATCGTTGTTATAAACATCCTAACCCTCTGGAATCACTGGC Cafa-T2R62p ------------------------------------------------------------ Dolphin-Tas2r62ap GCTGGGCCTGGGAAGTGGTGACCTGTGCAGGCATCTGTCGGCACTCCAGCATCTCGGTGC Cafa-T2R62p ------------------------------------------------------------ Dolphin-Tas2r62ap ACGGCAGCCCCAGGCTGAGAAAGGCCCTGATGACGAGGCCTTGGAGAGCCCTGGGCAAGG Cafa-T2R62p ------------------------------------------------------------ Dolphin-Tas2r62ap AGCAGTTTGTCTCATCAGAGTCAGTAACTGCTGTCACTGAACAAGCCCATGGGTGG Cafa-T2R62p --------------------------------------------------------

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Tas2r62b [Cafa-T2R62p (AB 249701)] Dolphin-Tas2r62bp GCGTCCCCCTCACCCACGTGGACCTGCACGGTCACCTTTCTCCTGGAGTCGGTGGCTGCC Cafa-T2R62p ATGTCCTCCTCACCTACATTGATCTTCATGGTCATCTTCTTCCTGGAGTCGTTGGCTGCA **** ******* ** * ** ** ** ***** *** *********** ******* Dolphin-Tas2r62bp AGGCTGCAGAACGGCCTCACAGTCGTCGTGCTGAGCCGGACTGGGACGCTGGACGCTGCC Cafa-T2R62p ATGCTGCAGAATGGCTTCATGGTTACTGTGTTGGGCAGGGAGTGGGTGC-----GACGCC * ********* *** *** ** *** ** ** ** ** ** * *** Dolphin-Tas2r62bp GGACGCTGTGCGCAGGCGACGTGATTGTGGCCCGCCTGGCCGTCTCCCAGTTCTGTCTGC Cafa-T2R62p GGACGCTGCCTGCAGGTGACATGATTGTGGCCTCCCTGGCTGCCTCCTGGTTCTGCCTGC ******** ***** *** *********** ****** * **** ****** **** Dolphin-Tas2r62bp AAGGGATGGCCCTCCAGCGCAACCTCCTGGCTTCCTTTGGTTTTGGTTCCCAATT---TT Cafa-T2R62p ATGGGGTGGCCATCCTGAACAACCTCTTGATCTTCTTTGGTTTTCACTTCGTAAGGGATT * *** ***** *** * ******* ** * ********** * * * ** Dolphin-Tas2r62bp ATTTCAGCATCTCCTGGAGCTTCATCAACACTCTCACTTTCTGGCCGACCAGCTGGCTTG Cafa-T2R62p ATTACAACACCCTCTGGCACTTTGTCAACACTCTCACTCTCTGGCTCACTGCCTGGCTTG *** ** ** * **** *** ************** ****** ** ******** Dolphin-Tas2r62bp CTGTCTTCTACCGTGTGAAGGTAGCATCCTTCTCTCACCCCATCTTCTTCTGGCTGAAGT Cafa-T2R62p CTGTCTTCTACTGTGTGAAGGTCGCCGTCTTCTCTCACCCGGTCTTCTTCTGGCTGAAAT *********** ********** ** ************ **************** * Dolphin-Tas2r62bp GCAGGATTTCTCGGTCAGTGCCCGGGCTGCTGCTGGGCTCCCTGATCCTGTCTGGTCTGA Cafa-T2R62p GGAGGATTTCTCGGTTAGTGCCCAGGCTGCTGCTGGGCTCCCTGGTCTTAGTTGGCCTGA * ************* ******* ******************** ** * *** **** Dolphin-Tas2r62bp CA-TCATC--AGCAGCCACCGGGAAGTCAATTCTTGTGCAGATGGTTGCCACCCAGGGTT Cafa-T2R62p CAGTCATCTCATCAGCCATTGTGACTGGAATTCTGAAACAGATGATTGCCTCCAAGAGTT ** ***** * ****** * ** ****** ****** ***** ** ** *** Dolphin-Tas2r62bp CCCATGGCAACGACACCCTCACGCAATTATCATGCGGTCAGTTCCATTCCTCCTGTTCCT Cafa-T2R62p CCCAAGGAAACAGCACCTGGGCTGAGAGAGTACA-GGCC--TTCTATAGGTCT--TTTCA **** ** *** **** * * * * ** * *** ** ** ** * Dolphin-Tas2r62bp GGTGTCCACCCTCTCGCTCGTGTTCTCGCTGCACCGGCACTTGGGGCAGATGAGGGACCA Cafa-T2R62p AATATTTGATGTAATGCTTATGTGGTCA--GTTCCATTCCTCATGGCTTGTG------TC * * * *** *** ** * ** ** *** ** Dolphin-Tas2r62bp CAGACCCGGCCCGAGTGATCCCAGCACCCGGGCTCACACCGTGGCCCGGAAGTCACTTGC Cafa-T2R62p CATGCTCTGCTGG----------------------------------------------- ** * * ** * Dolphin-Tas2r62bp CTTCTTTTTCATCCTATTTCCTGTGCCTGAGAATTGTCCTTGTGAACATCCCAACCCTCC Cafa-T2R62p ------------------------------------------------------------ Dolphin-Tas2r62bp GGAAGCACCGGCACTGGGAAGCGGTGACCTACGCCGGCATCTGTCTGCACGCCAGCATCT Cafa-T2R62p ------------------------------------------------------------ Dolphin-Tas2r62bp TGGTGCACAGCAGCCCCAAGCCGAGAAGGGCCCCAAAGAAGAGGCTTCGGCGAGCCCTGG Cafa-T2R62p ------------------------------------------------------------ Dolphin-Tas2r62bp GCAAGGAGCAGTTTGTATTGAGTTACCGGTATCAATGA Cafa-T2R62p --------------------------------------

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