Evolutionary Study On Vitamin C Synthesis Of Chiropterans

Vitamin C (Vc) (or L-ascorbic acid) is an essential nutrient for all vertebrates, which protects the body against oxidative stress. Lack of vitamin causes the disease scurvy in organism. The vast majority of animals are able to synthesize their own Vc de novo, through a sequence of enzyme-driven steps, which convert glucose to Vc. In reptiles and birds in lower orders the biosynthesis is carried out in the kidneys; in birds in higher orders and mammals it is synthesized in the livers.Loss of Vc synthesis ability is rare in animals. Among the primates that have lost the ability to synthesize Vc are humans and chimpanzees etc., which together make up one of two major primate suborders, the anthropoidea, also called haplorrhini, and the other more primitive primates (strepsirrhini) i.e. lorises and lemurs have maintained the ability to make Vc. Synthesis also does not occur in guinea pigs in the rodent family caviidae, but occurs in other rodents (rats and mice, for example). And a number of species of passerine birds also do not synthesize, but not all of the birds. For the species that do not synthesize Vc de novo, they all lack the L-gulonolactone oxidase (GULO) enzyme, which is required in the last step of Vc synthesis. A non-functional GULO gene which accumulates many harmful mutations leading to pseudogenization is present in the genomes of the guinea pigs and humans.Previous researches suggested that all bats including insect and fruit-eating species have been reported to lose Vc synthesis for lack of GULO in their bodies. However, these studies were focused on New World species and there is a lack of GULO gene information in bats. To verify the loss-of-function of GULO in bats, we designed a series of experiments covering molecular, protein, enzyme and evolutionary rate levels. Our experimental targets include four Old World species:two frugivorous bats [the Leschenault's Rousette(Rousettus leschenaultii) and the Greater Short-nosed Fruit bat(Cynopterus sphinx)] and two insectivorous bats [the Great Roundleaf bat(Hipposideros armiger) and the Greater Horseshoe bat(Rhinolophus ferrumequinum)]. To our surprise, the GULO genes of R. leschenaultii and H. armiger are intact and have regular mRNA expressions. Western blotting ascertains that all four bat species have GULO expression, in which R. leschenaultii and H. armiger have normal expressions however C. sphinx and R. ferrumequinum have weak expressions. Interestingly, Vc activity assay verifies that R. leschenaultii and H. armiger have normal GULO activities but C. sphinx and R. ferrumequinum have no functions. Our evolutionary analysis suggests that GULO genes of R. leschenaultii and H. armiger are under strong purifying selection, correlarating with their gene function, but for the Large Flying Fox(Pteropus vampyrus) (which loses Vc synthesis ability) its gene has been subjected to relaxed selection, which correlates with its loss-of-function.The following large-scale sequencing of bat GULO genes and the evolutionary analyses suggest that loss-of-function of GULO in bats follows the pattern of step-wise accelerated evolution. The whole evolutionary rates of bats are 12 to 54-fold higher than the ancestor of Laurasiatheria species. The GULO of Pteropus bats have the highest evolutionar rate and is close to be neutral evolution, which suggests an early time for their loss-of-function (probably started at around 3 million years ago). Other bat genes exhibit relative low evolutionary rates, suggesting their loss occurred recently.In summary, our results change a 50 year long-hold concept that bats cannot synthesize Vc and most importantly uncover the process of gene pseudogenization and come up with an new concept in evolutionary biology-progressive pseudogenization and uncover the pattern of loss-of-function-step-wise accelerated evolution, which will be of interest not only to scientists working on the biochemical pathways associated with Vc synthesis but also to evolutionary biologist in general who are interested in how adaptations may be lost over time.