Evolution Of T2Rs Genes In Bats

Bats belong to the order Chiroptera, which traditionally consists of two major suborders-Megachiroptera and Microchiroptera. Bats are the second largest group in mammals, including～1100 species. The diets of bats have a huge diversity-members of Megachiroptera feed exclusively on fruit, flowers, nectar, pollen and leaves, members of Microchiroptera mainly prey on most orders of insects, such as lepidoptera, hymenoptera, diptera, hemiptera, coleopteran, odonata, orthoptera, homopteran and dermaptera. The bitter taste is mainly responsible for the detection of toxins in food sources, and it is thus very crucial for the survival of animals. The bitter taste genes (T2Rs), which encode bitter taste receptors, were first identified in human and mouse. Afterwards, these genes had been of great interest and were determined in other vertebrates, such as fugu, zebra fish, rat, dog, frog, bird, and opossum. In this work, we examined bitter taste receptor genes in bats, and attempted to find whether natural selection has occurred in these genes due to the dramatic variation of their diets.We first used a bioinformatic method and searched available draft genome sequence of the little brown bat (Myotis lucifugus), and conducted an evolutionary analysis together with the published T2Rs from human, mouse, rat, chicken and frog. We identified nine intact T2R genes (900-996 base pairs in length), eight partial T2R genes (357-876 base pairs) and nine pseudogenes in the little brown bat. The neighbor-joining tree was reconstructed by MEGA4 with protein poisson correction distances, the bootstrap values of this tree were evaluate with 1000 replications. It showed a bat-specific T2R gene cluster in the top of tree. We examined three pair models (M0 versus M3; M1a versus M2a; M7 versus M8) for the bat-specific gene clade and found multiple amino acids under positive selection, suggesting that positive selection drives the functional divergence and specialization of the bat bitter taste receptors to adapt diets to the external environment. To gain a broader picture of T2Rs evolution in bats, we amplified T2R7 gene in 11 bats and analyzed them with 8 additional mammalian T2R7 genes from Genbank. We designed primer pairs in the conserved regions of the published sequences from the little brown bat and other mammals. Polymerase chain reactions (PCRs) were carried out; PCR products were ligated into a PMD19-T vector, and transformed to Top10 E.coli cells. Positive clones were sequenced using ABI 3730 DNA sequencer. Phylogenetic tree was reconstructed in MEGA4 using the Jukes-Cantor method with 1000 bootstrap replicates, it shows a bat monophyletic clade with high bootstrap value. Site-specific models were used to test possible positive selection with three pair models (M1a versus M2a; M8a versus M8); LRTs show significant difference, and suggesting positive selection has shaped the T2R7 evolution in bats. We also found lineage-specific pseudogenization in Megaderma lyra, suggesting loss-of-function of a specific taste ability in this bat. Although we lack information about the complete bat T2Rs repertoire, our results can provide some insights into the dynamics of T2Rs evolution in bats.