Molecular Evolution Of Taste And Trehalase Genes In Bats And Its Significance On The Dietary Adaptation

Chiroptera,commonly known as bats,are the only mammals capable of true flight.Bats,numbering over 1300 species,are the second most speciose group of mammals after rodents.In addition,bats exhibit a huge diversity in diets,including insects,fruits,nectar,mammals,fish,birds and blood.Dietary diversification is often accompanied by adaptive traits in bats,such as sensory perception and digestive systems.This study examined molecular evolution of three diet-related genes(bitter taste receptor genes,sweet taste receptor genes,and the trehalase gene),and attempts to uncover molecular evolutionary mechanism of dietary adaptation in bats.Firstly,we performed an in-depth study to investigate the bitter taste receptor gene family(Tas2rs)in bats.Previous studies have showed that diet shapes the evolution of the vertebrate bitter taste receptor gene repertoire.The proportion of bitter tastants in food items is positively correlated with the number of Tas2 rs.We identified the Tas2 rs from existing genome sequences of 15 bat species,and found that there is no significant difference between Tas2 r gene number in frugivorous and insectivorous bats,although many nocturnal insects release poisonous defensive secretions(likely bitter)that may function to deter insect-eating bats.This finding suggests that diet may play a minor role in bat Tas2 r evolution.Interestingly,number of Tas2 rs in three Myotis species is significantly larger than that in other species,indicative of an expansion.Tas2 r duplication events were inferred to be occur in the common ancestor of Myotis genus after examining their phylogenetic relationships.We then conducted selection tests for three Myotis-specific duplicated genes(Tas2r16,Tas2r18 and Tas2r41).Our results showed that positive selection acts on these genes.We also performed cell-based functional assays of bat Tas2r16.Functional differentiations were apparent among the three copies of Mda_Tas2r16 and between Mda_Tas2r16 copies and the two outgroups,and novel functions also occurred in two duplicates.The genus Myotis is naturally distributed on every continent except Antarctica.Myotis bats are ecologically diversified and can adapt to various habitats.We thus hypothesize that occupation of such ecological diversity in Myotis bats may have driven Tas2 r gene expansion,permitting them to colonize novel habitats.Secondly,we examined the functional evolution of sweet taste receptor genes(Tas1r2/Tas1r3)in bats.Sweet taste receptors are involved in nutrient(sweet substance)sensing.Some carnivorous mammal species have lost their sweet taste receptor genes via pseudogenization because their diets don't include any sweet-tasting compounds.We here present the dataset of bat Tas1r2/Tas1r3,identified from existing genome sequences of 15 bat species and newly sequenced from 3 bat species.Sequence analysis showed that all bat species studied possess intact Tas1r2/Tas1r3,which are free from inactivating mutations.We then conducted selection tests for bat Tas1r2 and Tas1r3,respectively.Our results showed that Tas1r2 evolved under the similar pressure in insectivorous and frugivorous bats,whereas Tas1r3 evolved faster in frugivorous bats than in insectivorous bats.In addition,no signal of positive selection was detected in either gene.We performed cell-based functional assays for sweet taste receptor genes of two frugivorous and one insectivorous bat species.Through the ligand-receptor binding assay,we tested the responses of bat Tas1r2/Tas1r3 to several sweet-tasting compounds.We found that while two frugivorous bat species possess intact and functional Tas1r2/Tas1r3 which can respond to sweet-tasting compounds with different sensitivities,the sweet receptors of insectivorous bat species showed no response to any compounds we tested.These results suggest that diet shapes the functional evolution of sweet taste receptor genes in bats and also highlight the importance of functional confirmation in molecular evolutionary studies.Finally,we studied the trehalase gene(Treh)in bats and mammals.Given that the trehalose is the main blood sugar in insects,insectivorous mammals need trehalase to digest dietary trehalose with high enzyme activities.Previous studies have shown that trehalase activity was largely reduced in bats that have few insects in their diets.In this study,we separately collected the Treh sequence in 16 bat species and 119 mammalian species.While all insectivorous bat species retain a functional Treh that evolved under strong purifying selection,most non-insectivorous bats lost their Treh via pesudogenization.Our enzymatic assays showed that the intestinal trehalase activity is significantly higher in the insectivorous bat species than in the two frugivorous bats,and one frugivorous bat species showed essentially no activity.We also identified a similar partten of Treh evolution across mammals.Widespread losses of Treh were detected in several independent non-insectivorous mammalian lineages.Furthermore,phylogenetic logistic regression analyses revealed a significant positive correlation between the percentage of insects in the diet and retention/loss of Treh.Our study suggested that molecular evolution of Treh in bats and other mammals is strongly associated with diet.