Molecular Evolutionary Studies Of Metabolic Genes In Bats

Bats (Order:Chiroptera) are the second largest mammalian order (after rodents) with-1200species have been identified to date. Bats are divided into the suborders Yinpterochiroptera (bats from the Pteropodidae and the Rhinolophoidea) and Yangochiroptera (all other bats) as supported by recent phylogenetic analyses based on multi-gene datasets. In addition to capacities of echolocation and powered flight, extant bats have evolved diverse food habits including insectivory, carnivory, piscivory, sanguivory, frugivory and nectarivory. Thus, bats are an excellent mammal groups in which to study the effects of diets to genetic evolution of metabolic genes. To investigate whether diets and associated metabolic changes would affect the genetic evolution of metabolic genes in bats, in this study, we conducted molecular evolutionary analyses of four metabolic genes in frugivorous bats, insectivorous bats (including piscivorous, carnivorous and sanguivorous bats) and other mammals.Firstly, to investigate the effect of frugivorous diet to genetic evolution of genes involved in carbohydrate metabolism in fruit-and nectar-eating bats, we sequenced the coding region of the Slc2a4gene (encoding the glucose transporter4protein, GLUT4) from16bat species and studied the molecular evolution of this gene in bats. Our results showed that the Slc2a4gene has undergone a change in selection pressure in Old World fruit bats with11amino acid substitutions detected on the ancestral branch. And these amino acid replacements were biased towards either Serine or Isoleucine, and, of the11changes, six were specific to Old World fruit bats, indicating most of these amino acid changes were more likely to be driven by natural selection rather than to be fixed randomly. However, no such selection pressure change was detected in the New World fruit bats. Our study presents evidence that the Slc2a4gene, which plays a pivot role in transmembrane glucose uptake and glucose homeostasis, has undergone adaptive changes in Old World fruit bats in relation to their special diet.Secondly, to investigate the effect of frugivorous diet and carbohydrate-dominant metabolism to genetic evolution of genes involved in amino acid metabolism in fruit-and nectar-eating bats, we sequenced the coding region of the Tat gene (encoding the tyrosine aminotransferase, TAT) from20bat species and examined the molecular evolution of this gene in bats. Besides, we also conducted biochemical and western blot assays to determine the enzymatic activity and expression levels of TAT in representative bat species. Phylogenetic reconstructions based on Tat gene coding sequences revealed a gene tree in which yinpterochiropteran insectivorous bats are grouped with yangochiropteran insectivorous bats (including the New World fruit bats). The phylogenetic conflict appears to stem from accelerated evolution of the Tat gene in the Old World fruit bats. Indeed, our molecular evolutionary analyses confirmed a change in the selection pressure acting on Tat, which was likely caused by a relaxation of the evolutionary constraints on the Tat gene in the Old World fruit bats. Hepatic TAT activity assays showed that TAT activities in species of the Old World fruit bats are significantly lower than those of insectivorous bats and omnivorous mice, which was not caused by a change in TAT protein levels in the liver. Our study provides evidence that the Tat gene has undergone relaxed evolution in the Old World fruit bats in response to changes in their metabolism. Besides, similar to the Slc2a4gene, no such selection pressure change of the Tat gene was detected in the New World fruit bats.Finally, to investigate the effect of insectivorous diet and protein-dominant metabolism to genetic evolution of genes involved in ammonia detoxification, we sequenced the coding regions of the Cpsl gene (encoding the carbamoyl-phosphate synthase1, CPS1) and the Glul gene (encoding the glutamine synthetase, GS) from17and16bat species, respectively, and determined the molecular evolution of these two genes in bats. Separate phylogenetic reconstructions based on CPS1amino acid sequences and Glul gene coding sequences both revealed a tree in which yinpterochiropteran insectivorous bats are grouped with yangochiropteran insectivorous bats (including the New World fruit bats). For both genes, a large number of parallel amino acid substitutions were detected between branches of yinpterochiropteran and yangochiropteran insectivorous bats. Our results of more explicit tests for convergence showed that, for both genes, the posterior probability of convergence in many branch pairs of yinpterochiropteran and yangochiropteran insectivorous bats are greater than that of divergence. And most of these high convergence probabilities between branches of insectivorous bats were significant when compared observed probabilities against null distribution based on simulations. Our study provide evidence that both the Cpsl gene and the Glul gene have undergone parallel evolution in insectivorous bats in relation to the detoxification of ammonia liberated by the degradation of amino acids.