Computational protocol: Evolutionary renovation of L/M opsin polymorphism confers a fruit discrimination advantage to ateline New World monkeys

Similar protocols

Protocol publication

[…] Phylogenetic and molecular evolutionary analyses were conducted using MEGA5 (; ). The evolutionary distance (d) in terms of the number of nucleotide substitutions per site for every sequence pair was estimated using substitution models implemented in MEGA5. The L/M opsin genes of primates are known to have been homogenized between alleles by recombination or between paralogs by gene conversion, shortening their apparent evolutionary distances within a species and resulting in clustering of alleles or paralogs by species in reconstructed phylogenetic trees (; ). Nonsynonymous nucleotide differences between alleles or paralogs are less susceptible to the homogenization process because of balancing selection on the alleles or purifying selection on the paralogs against gene conversion, and thus, they have been used to infer antiquity of allelic or paralogous origin of primate L/M opsin genes (; ; ). We thus also evaluated the phylogenetic trees reconstructed only from nonsynonymous differences using synonymous–nonsynonymous substitution models implemented in MEGA5. The ancestral amino acid sequences at every node in the reconstructed trees were inferred by the maximum-likelihood method () implemented in MEGA5. As a mathematical model of amino acid substitution, we used the empirical substitution matrices of Dayhoff and JTT models (; ). The L/M opsin alleles of muriquis (Brachyteles) have been sequenced only partially () and were therefore not included in the phylogenetic analyses. [...] We measured reflectance spectra for 23 fruit species (see Fig. 5 legend for species names) consumed by free-ranging spider monkeys in Santa Rosa Sector of the Área de Conservación Guanacaste, northwest Costa Rica. Although the species consumed by spider monkeys differ by geographical location, major dietary genera at Santa Rosa (such as Ficus, Brosimum, Spondias and Cecropia) are important at other sites, and our sample is a good representation of spider monkey diet that well encapsulates the range of diverse ateline foraging targets (). The chromaticity values, L/(L+M) and S/(L+M), of each specimen were given as QL/(QL+QM) and QS/(QL+QM), respectively (see for definition and calculation of quantum catch Qi). We calculated the chromaticity values using two sets of λmax values of the L/M opsin: one is the expected set from the ‘three-sites’ rule (560 and 550 nm) and the other is the actually observed set via photopigment reconstitution (553 and 538 nm). In both sets, the λ value of the S opsin was assumed to be 432 nm () as in . We also calculated the chromaticity values of the upper and lower sides of leaves separately because those of the two sides sometimes differ dramatically (). Using reflectance spectra of five samples, the mean chromaticity values of fruits and upper and lower sides of leaves were calculated for each species, except for C. peltata for which only two samples were available.Previous studies showed considerable difference of foraging efficiency to ‘conspicuous’ and ‘cryptic’ fruit categories in spider and capuchin monkeys, but the classification relied on human observation and was subjective (; ). We objectively classified fruits into these categories relative to background leaf spectra on the basis of whether a supervised machine-learning algorism, Support Vector Machine (SVM) (), could correctly discriminate fruit from the range of leaf spectral data. The SVM was implemented using LIBSVM () in MATLAB (see for detailed methods).The visual discriminability of conspicuous and cryptic fruits from background leaves was then evaluated by calculating ‘just noticeable difference (JND)’ quantitative perceptual units under bright and dim light conditions. JNDs are well suited for this task because they take into consideration receptor noise and psychological limits of detection (). We calculated JNDs using the mean quantum catch among measured samples for each item (fruit, upper leaf and lower leaf). See Materials and Methods S3 (Supporting information) for more details of the JND calculation. We used the Wilcoxon signed-rank test (2-tailed) to compare JND values between the observed and the expected λmax sets. Statistical analyses were performed by R (ver. 2.14.1). […]

Pipeline specifications

Software tools MEGA, LIBSVM
Applications Miscellaneous, Phylogenetics
Organisms Ateles belzebuth, Lagothrix lagotricha