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A positively selected, Oryzias-specific amino acid change hampers zebrafish sperm compatibility. A Protein sequence alignment of fish Bncr orthologs and predicted ancestral states of Bncr. Fingers 1, 2, and 3 are indicated. Amino acid sites are numbered starting with the first site in the mature protein. Zebrafish-compatible sequences are blue, medaka-compatible sequences are yellow, and dually compatible sequences are green. Red rectangles demarcate sites 15, 45, and 63 which were tested individually and in combination for their role in species specificity, while N-glycosylation sites are marked with a black rectangle (see Fig. 5). The two positively selected sites are highlighted with asterisks. B Phylogenetic tree of the predicted Bncr ancestral states according to fish phylogeny. Tested nodes (A–G) are marked with a closed circle and colored according to compatibility as in (A). Nodes A–D were predicted to have the same sequence and are therefore equivalent. C Comparative medaka/zebrafish IVF data from the tested Bncr ancestral states. Means ± SD are indicated. (Two-tailed Wilcoxon matched-pairs signed rank test with the method of Pratt; ns not significant. D Plot of bias index values derived from the IVF data pairs from nodes E and F in (C). Bias was not calculated for nodes A–D and G for which the average fertilization rates with both sperm were <10%. Median (dashed line) and quartiles (dotted lines) are shown. (Two-tailed Wilcoxon signed rank test vs. theoretical median of 0 with the method of Pratt).
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