Phosphoenolpyruvate carboxykinase 1 gene (Pck1) displays parallel evolution between Old World and New World fruit bats

Abstract

Bats are an ideal mammalian group for exploring adaptations to fasting due to their large variety of diets and because fasting is a regular part of their life cycle. Mammals fed on a carbohydrate-rich diet experience a rapid decrease in blood glucose levels during a fast, thus, the development of mechanisms to resist the consequences of regular fasts, experienced on a daily basis, must have been crucial in the evolution of frugivorous bats. Phosphoenolpyruvate carboxykinase 1 (PEPCK1, encoded by the Pck1 gene) is the rate-limiting enzyme in gluconeogenesis and is largely responsible for the maintenance of glucose homeostasis during fasting in fruit-eating bats. To test whether Pck1 has experienced adaptive evolution in frugivorous bats, we obtained Pck1 coding sequence from 20 species of bats, including five Old World fruit bats (OWFBs) (Pteropodidae) and two New World fruit bats (NWFBs) (Phyllostomidae). Our molecular evolutionary analyses of these sequences revealed that Pck1 was under purifying selection in both Old World and New World fruit bats with no evidence of positive selection detected in either ancestral branch leading to fruit bats. Interestingly, however, six specific amino acid substitutions were detected on the ancestral lineage of OWFBs. In addition, we found considerable evidence for parallel evolution, at the amino acid level, between the PEPCK1 sequences of Old World fruit bats and New World fruit bats. Test for parallel evolution showed that four parallel substitutions (Q276R, R503H, I558V and Q593R) were driven by natural selection. Our study provides evidence that Pck1 underwent parallel evolution between Old World and New World fruit bats, two lineages of mammals that feed on a carbohydrate-rich diet and experience regular periods of fasting as part of their life cycle.

Fig 1. Unconstrained Pck1 Bayesian phylogenetic tree and the species tree.

(A) Unconstrained Bayesian phylogenetic tree based on the Pck1 coding sequences, under the GTR+I+Γ model. Values on the nodes are posterior probabilities. (B) Species tree of the 29 mammals used in this study based on their accepted relationships. Red and green thick lines labeled ‘OWFBs’ and ‘NWFBs’ represent the ancestral branches for the Old World fruit bats and New World fruit bats, respectively. Nonsynonymous amino acid substitutions were mapped onto select branches. 13 sites on the ancestral branch of Old World fruit bats having omega values >1 are shown in blue. Convergent substitutions between OWFBs and NWFBs are shown in orange.

Fig 2. The species tree of 29 mammals with Old World fruit bat specific amino acid replacements identified by ancestral sequence reconstruction using the maximum parsimony method.

(A) A13G, (B) S252G, (C) K349E, (D) F578L, (E) D591V and (F) I608V. Branch lengths are not drawn to scale.

Fig 3. Sliding window analysis of the variation in omega values along Pck1 genes in OWFBs, NWFBs, and insectivorous bats.

Window size and the step size were set to 90bp and 36bp, respectively. Schematic of the PEPCK1 protein domain structure is shown beneath the plot.

Fig 4. Plot of the total posterior probabilities of divergence versus convergence for all pairs of branches in the tree.

Pairwise comparison for the ancestral branch of Old World fruit bats versus Artibeus lituratus, the ancestral branch of NWFBs versus Cynopterus sphinx, ancestral branch of Pteropus versus Leptonycteris yerbabuenae, and the ancestral branch of OWFBs versus Leptonycteris yerbabuenae are highlighted by green, blue, purple and orange, respectively.

Fig 5. Distribution of the six OWFBs-specific amino acid substitutions and four parallel amino acid substitutions occurring between OWFBs and NWFBs in the secondary structure of PEPCK1 protein.

OWFB substitutions are labeled in black, while parallel substitutions are labeled in red within a model of the domain structure of PEPCK1.