![Phylogenetic tree built using MEGA software for the sequenced T2R genes in the different ruminant species.
The evolutionary history was inferred using the Neighbor-Joining method [22]. The bootstrap consensus tree inferred from 500 replicates [21] is taken to represent the evolutionary history of the taxa analyzed [21]. Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed. The evolutionary distances were computed using the JTT matrix-based method [23] and are in the units of the number of base substitutions per site. The analysis involved 55 amino acid sequences. All positions containing gaps and missing data were eliminated. There were a total of 96 positions in the final dataset. Evolutionary analyses were conducted in MEGA6 [20].](https://www.researchgate.net/profile/Vlatka-Cubric-Curik/publication/278028354/figure/fig1/AS:340496221261824@1458192033429/Phylogenetic-tree-built-using-MEGA-software-for-the-sequenced-T2R-genes-in-the-different_Q320.jpg)
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RESEARCH ARTICLE
Sequence Analysis of Bitter Taste Receptor
Gene Repertoires in Different Ruminant
Species
Ana Monteiro Ferreira
1,2
, Andreia Tomás Marques
3¤a
, Mangesh Bhide
4
, Vlatka Cubric-
Curik
5
, Kristin Hollung
6
, Christopher Harold Knight
7
, Katrine Raundrup
8
, John Lippolis
9
,
Mitchell Palmer
10
, Elvira Sales-Baptista
1,11
, Susana Sousa Araújo
2,3¤b
, André Martinho de
Almeida
2,3,12,13¤c
*
1Instituto de Ciências Agrárias e Ambientais Mediterrânicas (ICAAM), Universidade de Évora, 7006–554
Évora, Portugal, 2Plant Cell Biotechnology Laboratory, Instituto de Tecnologia Química e Biológica António
Xavier (ITQB-UNL), Universidade Nova de Lisboa, 2780–157 Oeiras, Portugal, 3Instituto de Investigação
Científica Tropical, 1300–344 Lisboa, Portugal, 4Laboratory of Biomedical Microbiology and Immunology,
University of Veterinary and Pharmacy, 04181 Kosice, Slovakia, 5University of Zagreb, Faculty of
Agriculture, Department of Animal Science, 10000 Zagreb, Croatia, 6NOFIMA, Norwegian Food Research
Institute, N 1430 Aas, Norway, 7Faculty of Health and Medical Sciences, University of Copenhagen, 2200
Copenhagen, Denmark, 8Greenland Institute of Natural Resources, 3900 Nuuk, Greenland, 9National
Animal Disease Center, Ruminant Diseases and Immunology Research Unit, USDA, Ames, IA, 50010,
United States of America, 10 National Animal Disease Center, Bacterial Diseases of Livestock Research
Unit, USDA, Ames, IA, 50010, United States of America, 11 Departamento de Zootecnia, Universidade de
Évora, 7002–554 Évora, Portugal, 12 CIISA—Centro Interdisciplinar de Investigação em Sanidade Animal,
1300–477 Lisboa, Portugal, 13 IBET-Instituto de Biologia Experimental e Tecnológica, 2780–157 Oeiras,
Portugal
¤a Current address: Dipartimento di Scienze Veterinarie e SanitàPubblica, Universitàdegli Studi di 20122,
Milano, Italy
¤b Current address: Plant Biotechnology Laboratory, Department of Biology and Biotechnology 'L.
Spallanzani', 27100 Pavia, Italy
¤c Current address: Ross University School of Veterinary Medicine, PO 334 Basseterre, St. Kitts and Nevis,
West Indies
*adealmeida@rossvet.edu.kn
Abstract
Bitter taste has been extensively studied in mammalian species and is associated with sen-
sitivity to toxins and with food choices that avoid dangerous substances in the diet. At the
molecular level, bitter compounds are sensed by bitter taste receptor proteins (T2R) present
at the surface of taste receptor cells in the gustatory papillae. Our work aims at exploring the
phylogenetic relationships of T2R gene sequences within different ruminant species. To
accomplish this goal, we gathered a collection of ruminant species with different feeding be-
haviors and for which no genome data is available: American bison, chamois, elk, European
bison, fallow deer, goat, moose, mouflon, muskox, red deer, reindeer and white tailed deer.
The herbivores chosen for this study belong to different taxonomic families and habitats,
and hence, exhibit distinct foraging behaviors and diet preferences. We describe the first
partial repertoires of T2R gene sequences for these species obtained by direct sequencing.
We then consider the homology and evolutionary history of these receptors within this
ruminant group, and whether it relates to feeding type classification, using MEGA software.
Our results suggest that phylogenetic proximity of T2R genes corresponds more to the
PLOS ONE | DOI:10.1371/journal.pone.0124933 June 10, 2015 1/11
OPEN ACCESS
Citation: Monteiro Ferreira A, Tomás Marques A,
Bhide M, Cubric-Curik V, Hollung K, Knight CH, et al.
(2015) Sequence Analysis of Bitter Taste Receptor
Gene Repertoires in Different Ruminant Species.
PLoS ONE 10(6): e0124933. doi:10.1371/journal.
pone.0124933
Academic Editor: Hiroaki Matsunami, Duke
University, UNITED STATES
Received: October 16, 2014
Accepted: March 6, 2015
Published: June 10, 2015
Copyright: This is an open access article, free of all
copyright, and may be freely reproduced, distributed,
transmitted, modified, built upon, or otherwise used
by anyone for any lawful purpose. The work is made
available under the Creative Commons CC0 public
domain dedication.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information files.
For genes sequenced described, they can also be
found on GenBank, with accession numbers
KF898049-KF898092.
Funding: The authors acknowledge financial support
from Fundação para a Ciência e a Tecnologia
(Lisboa, Portugal) in the form of the grants SFRH/
BPD/69655/2010 (A. M. Ferreira) and SFRH/BPD/
90916/2012 (A. M. de Almeida) and Research
Contract by the Ciência 2008 program (S. S. Araújo).
The funders had no role in study design, data
traditional taxonomic groups of the species rather than reflecting a categorization by
feeding strategy.
Introduction
The sense of taste is highly relevant for animal survival, as it probably evolved to provide ani-
mals with the ability to differentiate suitable from dangerous foods. There are five basic types
of taste in mammals: sweet, salty, sour, bitter and umami. All act through a complex network
of chemosensory receptors and signal transducers. Bitter taste iswell characterized for humans
at both molecular and genetic levels, but little is known for ruminants, although they have the
anatomical structures for taste perception and they make use of this important taste ability in
their dietary choices [1–4]. Herbivores, ruminants included, have long been known to demon-
strate preferences for different plant species and for parts of plants within species, so in a sense
all herbivores are selective, maximizing nutrient intake and avoiding plant secondary metabo-
lites [5]. According to the predominant type of feed ingested, herbivores can be classified into
three feeding types: grazers (bulk and roughage feeders), browsers (selected diets containing at
least 75% fruit, dicot foliage, and tree and shrub stems and foliage), and intermediate or mixed
feeders (feeders that both browse and graze) [6].
Bitter taste has been extensively studied in various mammals and it is believed that it
evolved to avoid the uptake of toxic substances, however, no strict correlation between bitter-
ness and toxicity is observed [7]). At the molecular level, it is known that taste is sensed by
taste receptor proteins present on the surface of taste receptor cells. Bitter taste receptors, in
particular, are G-protein-coupled receptors (GPCRs) coded by a family of genes, TAS2R, that
contain an average of 300 codons, and which are intronless. These characteristics make them
easy to detect and analyze by DNA sequencing [3]. The repertoire of TAS2R (or T2R) is well
described for several species, and shows rather large differences in gene numbers, from 15
genes in dogs to 54 in frogs, for example [2,3,8]. In the list of best (almost completely) de-
scribed species are human, mouse [9,10], and chicken, consisting of 25, 34, and 3 functional
genes, respectively [11,12]. Only scanty information on ruminant T2R genetics was available
for cattle. Recently our team has reported eight T2R genes for sheep, applying a comparative
genomics approach using cattle T2R data and the recently available sheep genome, followed by
direct sequencing evidence using merino sheep DNA [13]. Using phylogenetic tools, we have
also observed higher sequence conservation between the two ruminant species, sheep and cat-
tle, than when comparing ruminants with other mammals.
In this present study, we have performed a larger sequence analysis to a collection of rumi-
nant species belonging to different taxonomic families and habitats. Differrent foraging behav-
iors and diet preferences are represented within the group: five browsing species (fallow deer,
moose, red deer, reindeer and white tailed deer), five grazing species (American and European
bison, elk, mouflon, and sheep) and three intermediate feeding species (goats, musk ox and
chamois). We aim to explore the phylogenetic relationships of T2R gene sequences within
ruminant species. To achieve this goal, we described the first partial repertoire of T2R gene
sequences for the chosen species and then studied the homology and phylogeny of these recep-
tors within the ruminant group and in relation to previously described T2R sequences of non-
ruminants.
Bitter Taste Receptors in Ruminants
PLOS ONE | DOI:10.1371/journal.pone.0124933 June 10, 2015 2/11
collection and analysis, decision to publish, or
preparation of the manuscript.
Competing Interests: The authors have declared
that no competing interests exist.
Materials and Methods
Sampling and DNA extraction
We analysed samples of the following ruminant species: American bison (Bison bison), cham-
ois (Rupicapra rupicapra), elk (Cervus canadensis), European bison (Bison bonasus), fallow
deer (Dama dama), goat (Capra hircus), moose (Alces alces), mouflon (Ovis ammon musimon),
muskox (Ovibos moschatus), reindeer (Rangifer tarandus), red deer (Cervus elaphus), sheep
(Ovis aries) and white tailed deer (Odocoileus virginianus). Sheep and goat samples were ob-
tained from animals at the University of Évora and the Faculty of Veterinary Medicine of the
University of Lisbon, Portugal, respectively. Reindeer samples were obtained from the Bei-
tostølen region in Norway. Red and fallow deer samples were obtained at the Tapada de Mafra
Natural Reserve (Portugal). European bison samples were obtained from animals from the
Tatra National Park in Slovakia, while chamois and mouflon samples were obtained respective-
ly in the Biokovo Mountain and Sibenik region in Dalmatia, Croatia. Moose samples were
obtained from animals in the Uppsala region in Sweden, muskox samples from the Kangerlus-
suaq region in Western Greenland, and American bison, elk and white tailed deer from the
USDA experimental herd (Ames, IA, USA). DNA from one individual of each species was used
in this study. Genomic DNA was isolated from blood samples, using the Qiagen DNeasy Blood
& Tissue Kit (QIAGEN, Venlo, the Netherlands), according to the instructions by the
manufacturer.
Ethics statement
All the blood samples from which DNA was isolated were obtained during routine health mon-
itoring, by specialized veterinary professionals. No animal experiment was performed; there-
fore, no specific ethical approval was necessary.
PCR and sequencing
Using the Primer3 software version 0.4.0 (http://frodo.wi.mit.edu/), PCR primers (Table 1)
were designed for seven T2R genes (T2R3,T2R4,T2R10,T2R12,T2R13,T2R16,T2R67) that
we have previously found in sheep [13]. Amplification was optimized to be able to use one
primer set only. T2R genes are intronless, so primers were designed on the exon sequence hav-
ing the functional sheep T2R gene sequences as template, in order to amplify most of the cod-
ing sequence of each T2R gene (800–900bp). Oligonucleotides were synthesized by Stabvida
(Stabvida, Caparica, Portugal). PCR reactions using approximately 75ng of DNA were carried
out in a Bio-Rad C1000 Thermal Cycler (Bio-Rad Laboratories, Munich, Germany), using stan-
dard conditions, as previously described [13]. Sheep DNA was used as positive control. PCR
products were loaded on a 1.5% agarose gel to confirm the existence of a unique band with the
Table 1. Sequences of the primer sets used for each T2R gene amplification and sequencing.
Gene Forward Primer Sequence 5'–3' Reverse Primer Sequence 5'–3'
T2R3 AGCAATTTGGGGTTTCTGGT TGGATAAACAGATCCCTTGGA
T2R4 TTTTTCTTCTATCGTTGTCTCTGAAA TGCTTTTGTTTTCAGTTTAGGATG
T2R10 CAGTGGAAGGCCTCCTAATTT TTCTCTTTTCCCCAGCACTT
T2R12 TGGAGAGAACACTGAACAATATACTTA CATCACTTCAGGCTTATTTTTGG
T2R13 TGGCAGATTCTTTGGAAAACA CACAGCACCAAAAGTGAAGC
T2R16 TGTCATAGTGCTGGGCAGAG TCTTTTCAGTTTGGCACTGCT
T2R67 ATGCCATCTGGAATTGAAAA TTTCAGGCGGCAGTTAAGAT
doi:10.1371/journal.pone.0124933.t001
Bitter Taste Receptors in Ruminants
PLOS ONE | DOI:10.1371/journal.pone.0124933 June 10, 2015 3/11
expected size. The PCR products were purified using the QIAquick PCR Purification Kit (Qia-
gen, Venlo, the Netherlands), following the instructions from the manufacturer. The purified
PCR products were then analyzed by direct sequencing (Sanger method) as a purchased service
from Stabvida (Stabvida, Caparica, Portugal) and using the same primer sets as for
PCR reactions.
Sequencing data analysis
Sequencing data were manually checked using Chromas Lite 2.1.1 (free at http://technelysium.
com.au/) for visualization; FASTA files containing the DNA sequences were then used for con-
version to protein sequences, using DNA to protein sequence converter at http://
bioinformatics.picr.man.ac.uk/research/software/tools/sequenceconverter.html, and checked
for ORFs using ORF Finder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html). This application al-
lowed us to check for premature stop codons that would indicate a pseudogene and for protein
domains matching TAS2R protein family. Protein sequences in FASTA format were then used
for multiple sequence alignment by Multiple Sequence Comparison by Log-Expectation (MUS-
CLE), freely available online at http://www.ebi.ac.uk/Tools/msa/muscle (version 3.8.31)
[14,15]. Percent identity matrix was also produced by this software.
Phylogenetic analyses
The MUSCLE data obtained in clustal format were used for phylogenetic analyses using
MEGA version 6 software [16], available at http://www.megasoftware.net. Only protein se-
quences of intact genes were included. Sequences of pseudogenes were excluded, as previously
described by Dong 2009 [3]. Similarly to Dong (2009), we also selected for the neighbor-joining
statistical method of analysis and the bootstrap consensus tree was inferred from 500 replicates
[17,18]. Evolutionary distances were computed using the JTT matrix-based method [19]. For a
second phylogenetic analysis, protein sequences from T2R orthologues already reported for
human and selected non-ruminant animal species of different trophic groups were used. These
animal species were: chimpanzee (Pan troglodytes), dog (Canis lupus), horse (Equus caballus),
mouse (Mus musculus), pig (Sus scrofa) and rabbit (Oryctolagus cuniculus). Bos taurus was in-
cluded as reference ruminant species. The sequences were obtained either from Emsembl data-
base (release 73—September 2013, http://www.ensembl.org), or, when not available, by using
the GenBank to obtain DNA sequences and then convert them to protein sequence. These se-
quences were analyzed together with our sequencing data (converted to protein sequences) as
input to the MEGA 6 software and the same analysis criteria were used as for the first analysis.
Results
T2R gene amplification and sequencing
PCR products of all seven genes analyzed were obtained for sheep, goat and mouflon, whereas,
for the other species a lower number of genes were successfully amplified. PCR fragments ob-
tained were of the expected length for all species using sheep fragments as control. Results of
the PCR amplification of the seven different T2R analyzed in the 13 ruminant species are
shown in Table 2. The sequencing results for each gene and species are shown in S1 Dataset.
These sequences are also deposited in GeneBank (accession numbers KF898049-KF898092).
These sequences were then converted to protein sequences and a percent identity matrix
produced, excluding pseudogenes to this analysis (matrix presented in S1 Table). The identities
are grouped by receptor gene. The primers used in the study were able to amplify sequences
ranging from 81–100% in similarity to the ovine genes, from which the sequences of primers
Bitter Taste Receptors in Ruminants
PLOS ONE | DOI:10.1371/journal.pone.0124933 June 10, 2015 4/11
were designed. For some of the genes we observed 100% matching of the gene in Ovis ammon
musimon to Ovis aries.
Phylogeny
A phylogenetic tree was built with the protein sequences for the obtained intact genes, using
Neighbor-joining statistical method of analysis, with a bootstrap value of 500 (Fig 1). There is
evident clustering by receptor genes. We also observe that some receptors are closer to each
other than others. For instance, T2R4 and T2R16 originate from the same branch, which is sep-
arated from the other branch in the root of two other sub-branches, one for receptors T2R3,
T2R10, T2R67, and another for T2R13 and T2R12. The phylogenetic relations between species
are not constant for every gene, nevertheless, there is a clear trend for phylogenetic distance or
divergence of T2R genes to correspond to the traditional taxonomic groups of the species, rath-
er than to feeding types (grazers, browsers or intermediate feeders). Species of the Bovidae fam-
ily/Caprinae sub-family (sheep, mouflon, muskox, goat and chamois) form a cluster separated
from species of the Bovidae family/Bovinae sub-family (American bison, European bison) and
the Cervidae family (deer, elk, white tailed deer, reindeer, fallow deer and moose). Analyzing
each receptor separately we can see different phylogenetic patterns, with the Cervidade family
closer to the root of the branch, and Bovidae further away. However, some interesting excep-
tions are observed. For T2R10 species of the subfamily Bovinae are closer to the Cervidae fami-
ly than to the Caprinae sub-family of their own family (Bovidae), and for T2R67, there appears
to exist a divergence for each species at a time, not in clusters, albeit keeping the same taxo-
nomic proximities. Finally, O.aries, does not cluster with O.ammon musimon for every gene
even though they are of the same genus. For example, in T2R10, O.aries is closer to C.hircus,
or even to O.moschatus for receptor gene T2R16. Another interesting finding was that for
T2R13 we were only able to find intact genes in the Cervidae samples. We could successfully
amplify and sequence PCR fragments for other species but the resulting sequences have prema-
ture stop codons, indicatig pseudogenezation of this gene for those species.
A second phylogenetic analysis was performed, extending the comparison with the sequenc-
ing data published for the orthologous genes for other animals with even more different feeding
Table 2. Screening of T2R genes.
T2R3 T2R4 T2R10 T2R12 T2R13 T2R16 T2R67
Ovis aries +++ + + + +
Capra hircus +++ + + + +
Rangifer tarandus + + + NP + NP +
Cervus elaphus NP NP + NP + + NP
Dama dama +NPNP+ + NP+
Bison bonasus + + + + NP + NP
Alces alces NP NP NP NP + NP +
Ovis ammon musimon +++ + + + +
Rupicapra rupicapra + + + + NP + NP
Ovibos moschatus +++ + NP+ +
Bison bison NP NP + + + + NP
Cervus canadensis + + NP NP NP + +
Odocoileus virginianus +++ + NP+ +
+ means successful amplification and sequencing.
NP means No PCR/Sequencing Product.
doi:10.1371/journal.pone.0124933.t002
Bitter Taste Receptors in Ruminants
PLOS ONE | DOI:10.1371/journal.pone.0124933 June 10, 2015 5/11
Fig 1. Phylogenetic tree built using MEGA software for the sequenced T2R genes in the different ruminant species. The evolutionary history was
inferred using the Neighbor-Joining method [22]. The bootstrap consensus tree inferred from 500 replicates [21] is taken to represent the evolutionary history
of the taxa analyzed [21]. Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed. The evolutionary distances
were computed using the JTT matrix-based method [23] and are in the units of the number of base substitutions per site. The analysis involved 55 amino acid
sequences. All positions containing gaps and missing data were eliminated. There were a total of 96 positions in the final dataset. Evolutionary analyses
were conducted in MEGA6 [20].
doi:10.1371/journal.pone.0124933.g001
Bitter Taste Receptors in Ruminants
PLOS ONE | DOI:10.1371/journal.pone.0124933 June 10, 2015 6/11
strategies, including non-ruminants and within distant taxa (S1 Fig). S2 Table shows the align-
ment of the 103 sequences used and the 80 residues included in the final analyses, when exclud-
ing gaps and missing data. The correlation of the T2R genes grouping with the taxonomic
relationships was maintained. For example, cattle clustered with American and European
bison at all genes for which the sequencing data was available, confirming also this grouping of
Bovinae at one branch.
Discussion
In the present study a sequence analysis of T2R genes is presented for the first time for a wide
collection of ruminant species. Most of these species have very little genomic data available and
we confirm that the approach herein described and developed using data available from other
sequenced ruminants is a good comparative strategy to find missing genetic information; at
least for features that are coded by considerably conserved genes, such as genes within the bitter
taste receptor family. This is in accordance with previous genetic linkage mapping studies
made with ruminants and humans [20,21]. In addition to obtaining the DNA sequence of sev-
eral T2R we also studied the homology of T2R genes within the ruminant animal group and
compared it to non-ruminant species, in order to study possible correlations with the different
dietary habits of the different species. Our results show more evidence for phylogenetic dis-
tance or divergence of T2R genes corresponding to the traditional taxonomic groups of the spe-
cies, rather than to the feeding types of these ruminants (grazers, browsers or intermediate
feeders).
Novel sequences for T2R genes are presented for the following ruminant species, building
their first partial repertoire of T2R: A.alces,B.bison,B.bonasus,C.hircus,C.canadensis,C.ela-
phus,D.dama,O.virginianus,O.ammon musimon,O.moschatus,R.tarandus and R.rupica-
pra. The protein sequence identities among species for each sequenced receptor ranged from
81 to 100%, confirming evident clustering by receptor gene. The results can be also related to
the fact that the same sheep gene-based primer sets were used for all species. Possibly genes
with lower similarity levels, or with high similarities but containing gaps in the template se-
quence at the annealing point of the primers, could not be amplified by PCR and, therefore,
were not selected for sequencing. We also cannot discard the possibility that some species
might simply not have some receptors, as we know that different animal species have different
number of T2R and/or different proportions of genes/pseudogenes in their T2R repertoires
[2,4]. Interestingly for T2R13 we were only able to find intact genes for the Cervidae samples.
We successfully amplified and sequenced PCR fragments for other species but the resulting se-
quences were pseudogenes. It is well known that some animal species have different number of
T2R and/or different proportions of genes/pseudogenes in their T2R repertoires [2,4]. This re-
sult also suggests that this receptor in particular may not be necessary to function in some envi-
ronments or for some species which are not sensitive to the bitter compounds perceived
through this receptor.
In terms of phylogenetic relations between the sequences obtained, we observed that there is
a correlation between taxonomic classification of the species studied and the obtained receptor
sequences, following the general proximity levels of their genomes, i.e., from a phylogenetic
analysis the divergence of species of the Bovidae family/Caprinae sub-family (sheep, mouflon,
goat, muskox and chamois) from species of the Bovidae family/Bovinae sub-family (American
and European bison) and from the Cervidae family (red deer, reindeer, fallow deer,moose, elk
and white tailed deer) became obvious. However, we could find exceptions. For T2R10, species
of the subfamily Bovinae are closer to the Cervidae family than to the Caprinae sub-family of
the same (Bovidae) family. Also O.aries does not cluster with O.ammon musimon for every
Bitter Taste Receptors in Ruminants
PLOS ONE | DOI:10.1371/journal.pone.0124933 June 10, 2015 7/11
gene: for T2R10 O.aries is closer to C.hircus, for T2R16 to O.moschatus. We propose that
these small alterations might be related to differences in diet or similarities in the environment
and/or available food types, more specifically for the bitter substances that are recognized by
those particular receptors. A database of bitter taste receptors and corresponding ligands has
been built for humans [22]. Such information is not yet available for all these ruminants, thus
further studies are needed to predict what food types are more responsible for these differences.
However, it is noteworthy that recent studies have shown that in humans very similar receptors
may have highly divergent agonist spectra [23,24], whereas dissimilar receptors can have con-
siderable overlaps in their activating bitter compounds [25]. Therefore, sequence similarity
level cannot accurately predict for TAS2R specificity. This might explain why we were not able
to find a correlation between T2R gene similarity and feeding strategies of the different animals
studied. Interestingly, the same correlation of the T2R genes grouping by taxonomic positions
was maintained when previously reported sequences of T2R from mammals other than rumi-
nants were introduced to the analyses. For instance, B.taurus genes, the main reference rumi-
nant species for the known T2R gene repertoire, confirmed our phylogenetic results by
consistently clustering with B.bison and B.bonasus.
As previously referred to in more extensively studied species, there is an ongoing evolution-
ary diversification of T2R receptors [26], where differences found in the T2R gene sequences
and, therefore, protein sequences among species are likely related with the need to adapt to an
environment with a different diet, and consequently food choices. Our study extends this hy-
pothesis to different trophic groups of herbivores. However, we could not find a strong correla-
tion of the sequence similarities with the feeding category of each ruminant (grazers, browsers
or intermediate feeder), at least not stronger than the clustering by taxa. For instance, sheep are
closer to goat in gene sequences than to European bison, but sheep and European bison are
grazers while goat is an intermediate feeder. It is important to keep in mind that feeding types
are indicative of feeding habits and that they can have no relationship with the actual content
of bitter substances in the feeds. Ruminant evolution may be classified taking the physical and
mechanical characteristics of the respective forages into account [27], where intake is related to
relative forestomach capacity, and body weight. This classification is not without exceptions.
For instance, muskox and reindeer cannot be placed into categories with other species. They
have particular feeding habits, as they are adapted to survive and reproduce under the severe
constraints of the Arctic. Muskox prefer a diet of graminoids, sedges and dicots [28] whereas
reindeer prefer lichens [29]. Such different food sources certainly contain different bitter li-
gands for different T2R receptors.
We hypothesize that other genetic differences exist, for example at the level of number of
genes or ratio of pseudogenes/funtional genes, and are not reflected at the level of ortholog
functional genes. The number of (functional) T2R genes seems to be important for taste per-
ception and it has been proposed that carnivores have fewer T2R genes, herbivores an interme-
diate number, and omnivores the largest T2R gene repertoire [30]. The number of taste genes
has also been addressed for other taste receptors in carnivorous mammals. Jiang et al. (2012)
have found pseudogenized TAS1R genes and suggested that T2R losses are consistent with al-
tered feeding strategies when they could not detect intact T2R genes in the dolphin genome
[31]. Sato & Wolsan (2012), on the other hand, recently hypothesized that factors underlying
the pseudogenization of TAS1R1 in pinnipeds may be driven by the specific marine environ-
ment to which these animals are adapted, namely the feeding behavior of swallowing food
whole without mastication (T1R1 + T1R3 receptor is distributed on the tongue and palate),
and the saltiness of sea water (a high concentration of sodium chloride masks umami taste)
[32]. Moreover, Li & Zhang (2014) have also proposed that the number of T2R genes in a spe-
cies correlates with the fraction of plants in its diet, and supported the hypothesis that dietary
Bitter Taste Receptors in Ruminants
PLOS ONE | DOI:10.1371/journal.pone.0124933 June 10, 2015 8/11
toxins are the driving force behind the differences in T2R repertoires among species [33]. How-
ever, we can only speculate on the number of genes as, with the strategy used in this study, only
a portion of the total repertoire of T2R genes of each species is unraveled and compared. For
other taste receptors, it has been shown that function can be obtained using alternative strate-
gies when a certain receptor is absent from the genome. For instance, in 2014 Baldwin and col-
leagues working in hummingbirds demonstrated that the widespread absence from birds of an
essential subunit (T1R2) of the only known vertebrate sweet receptor can be substituted by the
ancestral umami receptor T1R1-T1R3 heterodimer [34]. Behrens et al. (2014) have also ad-
dressed similar problems in birds, demonstrating that the small TAS2R gene repertoire of
chicken and turkey compensates low gene number by large tuning breadths [12].
We demonstrate that using PCR primers from a related species within the same trophic
group is a good strategy to find T2R genes in species for which the genomes are not yet avail-
able. Nevertheless, a strategy using degenerate primers could be used in future works to further
complement the present repertoire. The knowledge of these sequences may also be helpful to
understand the taste perception mechanisms in these animals, particularly if expression studies
will follow. Moreover, data on bitter taste perception in ruminants may have a high impact in
animal nutrition with important consequences for the optimization of feed utilization, hence
contributing to more sustainable and efficient ruminant production systems.
Supporting Information
S1 Dataset. DNA sequences obtained for each T2R gene and species, in FASTA format. Spe-
cies: sheep (Ovis aries), goat (Capra hircus, reindeer (Rangifer tarandus, red deer (Cervus ela-
phus), fallow deer (Dama dama), European bison (Bison bonasus), moose (Alces alces), chamois
(Rupicapra rupicapra), mouflon (Ovis ammon musimon), muskox (Ovibos moschatus), Ameri-
can bison (Bison bison), white tailed deer (Odocoileus virginianus) and elk (Cervus canadensis).
(TXT)
S1 Fig. Phylogenetic tree obtained with the sequencing data of T2R genes on our ruminants
and the orthologous genes for other animals including non-ruminants, using MEGA soft-
ware. Protein sequences were used for building the tree. The evolutionary history was inferred
using the Neighbor-Joining method. The bootstrap consensus tree inferred from 500 replicates
is taken to represent the evolutionary history of the taxa analyzed. Branches corresponding to
partitions reproduced in less than 50% bootstrap replicates are collapsed. The evolutionary dis-
tances were computed using the JTT matrix-based method and are in the units of the number
of amino acid substitutions per site. The analysis involved 103 amino acid sequences. All posi-
tions containing gaps and missing data were eliminated. There were a total of 80 positions in
the final dataset. Evolutionary analyses were conducted in MEGA6.
(PDF)
S1 Table. Percent identity matrix.
(XLS)
S2 Table. Scheme of the alignment of the 103 sequences and the 80 residues included in the
final analyses.
(XLS)
Acknowledgments
The authors also thank J.A. Lopes de Castro, J. Fragoso (University of Évora), M Quaresma
(Faculdade de Medicina Veterinária, Universidade de Lisboa) and J Lérias (Instituto de
Bitter Taste Receptors in Ruminants
PLOS ONE | DOI:10.1371/journal.pone.0124933 June 10, 2015 9/11
Biologia Experimental e Tecnológica) for their assistance in sample collection. The authors
would finally like to thank COST actions FA-1002 Proteomics in Farm Animals (AM Almeida,
M Bhide and K Hollung), FA1308 –Dairycare (AM Almeida and C Knight) and TD1101-RGB
Net (AM Almeida and V Cubric-Curik), financed by the European Science Foundation (Brus-
sels, Belgium) for rendering possible the fruitful scientific relations that led to this manuscript.
Author Contributions
Conceived and designed the experiments: AMF ESB SSA AMA. Performed the experiments:
AMF ATM SSA. Analyzed the data: AMF ATM MB VC KH CK KR JL MP ESB SSA AMA.
Contributed reagents/materials/analysis tools: AMF MB VC KH CK KR JL MP ESB SSA AMA.
Wrote the paper: AMF ATM MB VC KH CK KR JL MP ESB SSA AMA.
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