converted to cDNA and used as template to amplify the CRX and SAG coding sequences. RNA samples were not available for New- World bat species, thus genomic sequences were amplified with exon-specific primers (Table S3 and Table S4). Genes for CRX and SAG were amplified from a total of 38 individuals, representing 29 species of bats, in this study, and were analyzed together with other sequences that were available from GenBank and Ensembl. For the isolation of RNA, 40 bat individuals (listed in Table S5) were sacrificed followed the guidelines of the by-laws on experimentation on animals, and was approved by the Ethics and Experimental Animal Committee of the Kunming Institute of Zoology, Chinese Academy of Sciences, and their eyes were rapidly excised and frozen in liquid nitrogen. Total RNA was isolated from the eyes using the RNAisoTM Plus Kit (Takara, China), and stored at 280uC. cDNA for RT-PCR was generated from 2 mg RNA using the PrimeScriptTM RT-PCR Kit (Takara, China). Total genomic DNA was extracted using a standard 3-step phenol/chloroform extraction method [47]. CRX and SAG genes were amplified from the cDNA or total DNA using gene-specific primers (Tables S3 and S4). PCR amplifications were carried out using the following touchdown program: 95uC 4 min, 20 cycles of 94uC denaturation 1 min, 60–50uC annealing (1 min; 20.5uC/ cycle) or 63uC, 72uC extension 1 min, and finally 15 cycles of 94uC 1 min, 50uC 1 min, 72uC 1 min. PCR products were cleaned using the Watson PCR Purification Kits (Watson BioTechnologies, Shanghai). Each PCR product was sequenced at least three times on an ABI 3730 Sequencer (Applied Biosystems, Foster, CA, USA) using the ABI PRISM BigDye Terminator v3.0. DNA sequences were edited using DNAstar Seqman software (DNASTAR Inc., Madison, WI, USA). The new CRX and SAG sequences were deposited into GenBank (Accession numbers HQ651094–HQ651149, JF831422–JF831446). Phylogenetic and Molecular Evolutionary Analyses For each gene, nucleotide sequences were translated into amino acid sequences and aligned using CLUSTALX 1.81 [46] as a guide for the alignment of the nucleotide sequences for evolutionary analyses. The best fit models for nucleotide and amino-acid substitutions were determined by ModelTest [48] and ProtTest v2.4 [49], respectively, under the Akaike information criterion. The computer algorithm PhyML [50] was used to construct maximum-likelihood (ML) phylogenies of the nucleotide and amino-acid data under their best-fitting models. Bayesian inference (BI) and neighbor-joining (NJ) phylogenies were constructed using MrBayes [51] and MEGA 4 [52], respectively. We used the Li-Wu-Luo method [53] to reconstruct a NJ tree based on synonsymous and nonsynonymous sites. Each site in a codon is allocated to a 0-fold, 2-fold or 4-fold degenerate category. For computing distances, all 0-fold and two-thirds of the 2-fold sites are considered nonsynonymous, whereas one-third of the 2- fold and all of the 4-fold sites are considered synonymous changes. Tests for selection and ancestral sequence reconstruction were carried out using the Codeml program implemented in PAML [54,55]: (1) one-ratio model, which assumes an identical v value for all branches, where v is the ratio of nonsynonymous to synonymous substitution rates; (2) a free-ratio model, assuming an independent v values for each branch, to provide a rough measure of the selective pressure on each branch; (3) two-ratio model and (4) branch-site model were used to determine whether these genes have undergone positive selection on a foreground branch; (5) site models: the neutral model (M1a) estimates two v values (0,v0,1, v1 = 1); the positive selection model (M2a) adds an extra v value to M1a; M8 (b &v model) takes into account the possibility of positively selected (PS) sites; and M8a is the model of M8. Convergent Evolution in Rod Vision Genes Bayes Empirical Bayes (BEB) analysis was used to calculate the Bayesian posterior probability of PS sites. Finally, LRT statistics were calculated between the following model pairs: (1) the two- ratio model vs. the one-ratio model were compared to test whether the v ratio is significantly different from that of other mammals; (2) test 1 (branch-site model vs. site model M1a) and test 2 (branch- site model vs. branch-site model with fixed v1 = 1) for branch-site model [56] were conducted; (3) M1a vs. M2a and M8 vs. M8a were compared to examine possible positive selection sites. In the previous cases, twice the difference in log-likelihood values (2DlnL) between the two models was calculated following a chi-squared (x2) distribution with the degrees of freedom equaling the difference in the number of parameters estimated for the model pairs. Supporting Information Figure S1 Amino acid replacements in the CRX gene sequences of bats. The asterisk is the site of the convergent amino acid replacement P133A. (TIF) Figure S2 Topology based on the nucleotide sequences of CRX. Numbers above the branches are Bayesian posterior probabilities, and numbers below the branches are the ML and NJ bootstrap values. (TIF) Figure S3 Topology based on amino acid sequences of CRX. Numbers above the branches are the Bayesian posterior probabilities. (TIF) Figure S4 NJ tree based on the nonsynonsymous sites of the CRX gene. Numbers above the branches are the NJ bootstrap values. (TIF) Figure S5 Amino acid replacements in the SAG gene sequences of bats. The asterisk is the site of the amino acid replacement I51M. (TIF) Figure S6 Topology of SAG. (A) Topology based on the nucleotide sequences of SAG. Numbers above the branches are the Bayesian posterior probabilities, and below are the ML and NJ bootstrap values. (B) Topology based on amino acid sequences of SAG. Numbers above the branches are the Bayesian posterior probabilities. (TIF) Table S1 Analyses of the selective pressure on the CRX gene of bats. (DOC) Table S2 Analyses of the selective pressure on the SAG gene of bats. (DOC) Table S3 Primers used for amplifying and sequencing CRX genes in bats. (DOC) Table S4 Primers used for amplifying and sequencing SAG genes in bats. (DOC) Table S5 Species and their accession numbers of CRX and SAG genes used in this research. (DOC) PLoS ONE | www.plosone.org 5 April 2012 | Volume 7 | Issue 4 | e34564