Convergent Evolution in Rod Vision Genes Figure 1. Convergent evolution of the CRX gene in bats based on a tree derived from the Bayesian analysis of nucleotide sequences. Numbers and symbols above the branches are the positions and amino acid replacements. Sequences at the internal nodes were reconstructed by the Maximum Likelihood method in PAML. doi:10.1371/journal.pone.0034564.g001 didae, however, do not have laryngeal echolocation, and therefore generally navigate by sight, and thus have larger eyes [7,8,9,10]. While bats from the family Emballonuridae do have laryngeal echolocation, they also have relatively big eyes and are more active at dusk compared to other echolocating bats [14]. The independent development of large eyes may reflect a shared greater functional reliance on rod vision. CRX is a developmental regulatory gene associated with the differentiation of photoreceptor cells involved in rod vision development while SAG is a component of the photo-signaling cascade and functions in the desensitization of the photoactivated transduction cascade. Both genes are involved in distinct but important roles of rod cells. When the evolution of the CRX and SAG protein sequences was examined in bats, we identified two parallel changes in the amino acid sequence of CRX and one parallel amino acid change in SAG that are shared by Emballonuroidea and Pteropodidae. The amino acid sites involved in these parallel changes in these two genes are in general conserved in the sequences for these two genes in other mammals (shown in Figures S1 and S5). Previously we had found convergent evolution in RH1 [5], a rod cell gene directly responsible for the perception of dim light [20]. We hypothesize that these multiple instances of parallel sequence evolution between Emballonuroidea and Pteropodidae bats reflect parallel functional convergence for dim light vision and also reflect the complex genetic mechanisms that are required for this phenotypic convergence. The three instances of parallel evolution in the CRX and SAG genes discussed above were not the only instances of convergent amino acid substitutions observed in these genes in bats. Parallel changes in the CRX gene was observed at amino acid position 259 (G259S, marked in purple in Figure 1) on four divergent branches and at position 260 (A260T, marked in coffee in Figure 1) five times. These multiple changes, which were observed on multiple branches, imply that during the long history of bats, CRX may have been prone to convergence, possibly due to ecological specializations (i.e., to photic environments). Alternatively, since neither of these two sites are well conserved within CRX gene sequences mammals in general, they may simply reflect changes that are tolerated, and these two amino acid states may have no functional difference in bats [5]. Similarly, except the parallel site (I51M) that occurred on Emballonuroidea and Pteropodidae, a few other sites showed parallel changes in other branches, however these sites were also not conserved within mammals and these changes may have no functional consequence. Old World fruit bats (Pteropodidae) do not echolocate but instead rely on their other senses, such as vision, to find food and navigate at night. Although insect-feeding bats rely largely on the key innovation of echolocation to find prey and navigate in close quarters at night, vision has been retained and serves as an important complement to echolocation [14,37,38]. In most echolocating bats, vision is predominantly used only for long- range navigation, where echolocation is less effective [14]. Our analysis found that three rod vision genes (CRX, SAG and RH1) have each experienced strong purifying selection in all bats, reflecting a common need for rod vision in all bats. Bats from two families, Pteropodidae and Emballonuroidea, rely on vision to a greater extent than other bats [14], however, a significant signal PLoS ONE | www.plosone.org 3 April 2012 | Volume 7 | Issue 4 | e34564