DISCUSSION
In this study, we showed that two directionally selected nonsynonymous
sites, A52V and Q296H, are associated with decreased sensitivity to
quercitrin in Qinling pandas, which confers upon the pandas a dietary
preference for high-quercitrin bamboo leaves. In agreement with the
hypothesis that polymorphisms in sensory receptor genes may alter
perception by encoding functionally distinct receptor types (Bufe et
al., 2005; Wooding et al., 2006), we add new evidence that genetic
variations in a bitter receptor gene (pTAS2R20 ) are correlated
with differences in bitterness (quercitrin) recognition. Bitter taste
perception provides an important means for animals to detect bitter
compounds in the environments that they occupy. As animals explore new
environments and diets, they may encounter distinct bitterness, which
imposes selective pressure on their bitter taste receptor genes, leading
to individual differences in bitter taste sensitivity (Shi et al., 2003;
Wooding et al., 2006; Li et al., 2013; Shan et al., 2018). The TAS2R
genes of the giant panda are expected to have experienced selective
pressure during the course of the dietary shift of pandas from
carnivorous to omnivorous to herbivorous and, finally, their
specialization for bamboo consumption. Our previous study demonstrated
that the giant and red pandas harbor more putative functional bitter
taste receptors than other carnivores, and several of their TAS2R genes
seem to have experienced selective pressure, as these species are
challenged with many more bitter compounds in diets (Shan et al., 2018).
Although natural selective signatures on several TAS2R genes in the
pandas has been revealed, the functional effects of the selective
pressure on bitter taste perception have not been examined. The present
study has provided a fine example of the effects of directional
selection on giant panda dietary preferences, providing direct evidence
of a close correlation between taste function and the pandas’ feeding
ecology.
pTAS2R20 was shown to be specifically activated by quercitrin, a
flavonoid monomer found in various plants including bamboos that has
previously been identified as the agonist of a bitterness receptor of
the herbivorous insect Papilio hospiton (Sollai et al., 2015).
Studies in humans have suggested that bitter taste receptors are usually
broadly tuned to recognize numerous compounds possessing common chemical
groups responsible for mediating receptor-agonist interactions (Bufe et
al., 2005; Meyerhof et al., 2010). Given that a limited number of bitter
compounds were assayed here and that bamboo produces flavone compounds,
the actual number of dietary agonists for TAS2R20 may be greater.
However, the two directionally selected sites, A52V and Q296H, indeed
conferred TAS2R20 with a significantly decreased sensitivity to
quercitrin, which may reduce aversion to quercitrin in Qinling pandas.
Consequently, although higher quercitrin was contained in the bamboo
leaves from B. fargesii and F. qinlingensis in Qinling
Mountains, Qinling pandas taste the leaves better than other pandas
taste the bamboo leaves from F. denudate and B. faberi in
other areas. Therefore, the two directionally selected nonsynonymous
sites may explain why Qinling pandas prefer to consume more bamboo
leaves than other pandas do.
Recent studies on chemical substances in the giant panda staple food
bamboo
have focused on nutrition and minerals (Christian et al., 2015; Nie et
al., 2015b), but there are few systematic reports of bitter substances.
Thus, we screened agonists for pTAS2R20 from a limited number of common
chemicals found in bamboos and other plants. According to previous
studies indicating that giant pandas are inclined to ingest bamboos that
contain less tannin (Zhao et al., 2001; Hansen et al., 2010), we listed
quercitrin and tannin as important potential agonists of the receptor.
Here, functional expression experiments confirmed that quercitrin is an
agonist of pTAS2R20 that may play a significant role in feeding
preference. Although tannin is the correct agonist, as we previously
conjectured (Zhao et al., 2013), this result is not surprising, since
giant pandas possess numerous bitter receptors, and some of them may
specifically recognize tannic acid.
The giant panda belongs to the order Carnivora (Wei et al., 2014);
nevertheless, it has evolved certain characteristics of herbivores
including a skull, jaw musculature and dentition that are suitable for a
fibrous diet and a pseudothumb that is specifically used for handling
bamboo (Zhang et al., 2007; Hu et al., 2017). Mutations in DUOX2led to a decrease in thyroxine levels, which helped to reduce energy
consumption for adaptation to a low-energy bamboo diet (Nie et al.,
2015a). These studies have emphasized behavioral, physiological,
morphological, and genetic adaptations. However, it is rare to reveal
the molecular basis of feeding adaptation in giant pandas from the
perspective of bitterness receptors
function, with the exception of similar reports on the pseudogenization
of the umami taste receptor gene TAS1R1, which is associated with eating
meat (Hu et al., 2017). This research fills a gap in explaining the
evolution of diet adaptation from the perspective of the molecular
function of bitterness receptors, especially in non-model animals. This
study is also a typical example for linking genome-scan candidate gene
to ecological adaptation at functional verification level.