In this study, we amplified and sequenced CRX and SAG genes from 38 individuals representing 29 species across the five major groups of bats (Emballonuroidea, Noctilionoidea, Pteropodidae, Rhinolophoidea, and Vespertilionoidea). Similar to previous findings with RH1 [5], we found evidence supporting convergent evolution in both CRX and SAG, providing a rare example of multiple events of convergent evolution occurring in parallel in interrelated genes, suggesting that multiple changes are involved in the network of genes necessary for rod vision to generate the complex molecular and phenotypic convergences. Results The parallel sequence evolution of CRX genes CRX genes were amplified from 38 individuals representing 29 species of bats. The aligned nucleotide sequence was 861 base pairs (bp) in length, of which 287 were variable and generated 83 sites with amino acid variation (Figure S1). The amplified CRX sequence corresponds to bases 25 to 879 of the 897 base coding sequence of the human gene. No insertion/deletion mutations or change that resulted in a stop codon were found in any of the sequences, suggesting that all of the bats have a functional CRX gene. Phylogenetic analyses of the aligned CRX nucleotide sequences (861 bp) with Bayesian, Maximum Likelihood and Neighbor- joining methods resulted in consistent trees that were congruent with the best-supported species tree [29,30] (Figure S2). While Pteropodidae and Emballonuridea are two divergent lineages of bats, the phylogenetic tree generated from amino acid sequence data placed Emballonuroidea and Pteropodidae together (Figure S3). If only nonsynonymous nucleotide changes were used to reconstruct the topology of bats (Figure S4), Pteropodidae was found not to group with Rhinolophoidea (as expected from the nucleotide sequence phylogeny – Figure S2), but instead had a closer relationship with Emballonuroidea and Vespertilionoidea. The bootstrap support values for these relationships in the amino acid and nonsynonymous trees were low, which is most likely due to the small number of nonsynonymous and amino acid substitutions that can be used to reconstruct the topologies. To further examine the evolution of CRX, ancestral CRX sequences, at the internal nodes of the species tree, were reconstructed and the changes that occurred on each lineage were inferred. Parallel changes at amino acid positions 133 (P133A marked in bold black) and 242 (V242M marked in purple) were found in both Pteropodidae and Emballonuroidea (Figure 1). If these two amino acid sites were excluded from the phylogenetic analysis, then the phylogeny was in accord with the nucleotide sequence and the best-supported species tree. Using a statistical test [31] the two branches were shown to contain significantly larger number of parallel evolving sites than expected (P,0.001). Of the two sites that show parallel changes, amino acid 133 was found to be perfectly conserved in all other mammals examined as proline, except in the bat species in Pteropodidae and Emballo- nuridea, while the amino acid site 242 showed greater variation (Figure S1). The maximum likelihood estimate of the average ratio of nonsynonymous to synonymous substitution rate (Ka/Ks) was 0.0754 (M0 model) (Table S1). When Emballonuroidea and Pteropodidae were set as independent foreground lineages, and tested for selection using PAML, we failed to find any signal for positive selection. However, if the two branches were set as a combined foreground lineage, then the branch_site model indicated marginal evidence that these two branches had experienced positive selection (the LRT test statistic, 2l = 3.516, Convergent Evolution in Rod Vision Genes P = 0.06, see Table S1), with the sites 133P (pp = 0.888) and 242V (pp = 0.976) on these lineages being the positively selected sites. The parallel sequence evolution of SAG SAG genes were successfully amplified from the 25 individuals representing 18 species of bats. The amplified SAG sequence corresponds to bases 181 to 951 of the 1218 base coding sequence of the human gene. No insertion/deletion mutations or changes that result in stop codons were found in any of the sequences, suggesting that all bats have a functional SAG gene. The aligned SAG nucleotide sequences were 771 bp in length, including a 3 bp insertion, of which 210 were variable and generated 55 sites with amino acid variations (Figure S5). Phylogenetic trees generated from the nucleotide (771 bp) and amino acid (257 sites) sequences of the aligned SAG gene by multiple methods resulted in trees that were congruent with the best-supported phylogeny of bats generated from other data sources [29,30] (Figure S6). Ancestral SAG sequences for the internal nodes of the species tree were reconstructed and amino acid substitutions were inferred onto each lineage. Pteropodidae and Emballonuroidea were both found to share an I51M amino acid replacement (marked in bold black, Figure 2) at a site that is conserved among examined mammals (Figure S5). The probability that this parallel evolu- tionary change occurred by random on these two branches was significantly rejected (P = 0.011) by a statistical test [31]. Additional parallel replacements were observed between different bat lineages (e.g., T201S, K99R, T95A, and E96D), however they occurred on other branches that had no obvious shared morphological or ecological similarity and as these were sites that are not conserved within mammals, thus the parallel changes at these sites may not have functional importance. The maximum likelihood estimate of the average ratio of the rates of nonsynonymous to synonymous substitution (Ka/Ks) was 0.0933 (M0 model). Both the branch model and branch site model in PAML failed to detect any significant signal for positive selection on the lineages for the common ancestor of all bats, Emballonuroidea bats or Pteropodidae bats (Table S2). Discussion The development of morphological characters is very complex and typically involves a series of genes. Eye development is an example that probably requires the actions of thousands of genes [32]. The fact that large numbers of genes are involved, indicates that the convergent evolution of these complex phenotypic characters, whether vision or echolocation, likely cannot be fully explained by the evolution of a single gene. It would appear that many genes, driven by similar selective pressures, are required for functional convergence. In this study, we tested several genes that are involved in different aspects of rod vision function to determine if similar patterns of evolution occurred to them during the evolution of a morphological character – evolving larger eyes, which may reflect a greater reliance on rod vision. Mammals have two distinct types of photoreceptors, rods and cones, which display important differences in their sensitivity to light intensity and ranges in light wavelength photosensitivity. Rods have a high sensitivity to light and thereby mediate nighttime vision when there are few photons. In contrast, cone photorecep- tors serve for daylight vision when photons are plentiful [33]. In general, nocturnal mammals have relatively larger eyes than diurnal species in order to maximize their visual sensitivity [34,35]. Although bats are nocturnal, most species navigate using laryngeal echolocation, with a limited need for vision, and thus have characteristically small eyes [36]. Bats from the family Pteropo- PLoS ONE | www.plosone.org 2 April 2012 | Volume 7 | Issue 4 | e34564