Fig. 1. Genomic structure of the csd gene in A

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Fig. 1. Genomic structure of the csd gene in A Fig. 1. Genomic structure of the csd gene in A. mellifera, domain diagram, and conceptual translation of the HVR for pairs of functional heterozygous csd alleles. (A) Coding exons are marked in black; untranslated regions are in white. Translational start and stop sites are indicated. Below, the detailed view of exons 6, 7, and 8 is given. The RS-rich domain (red), the HVR (green), the proline-rich (P) domain (blue), and the PSD are shown. (B) Conceptual translation of the HVR and a portion of the adjacent regions for seven pairs of functional heterozygotus csd alleles. The repetitive region within the HVR and highly conserved amino acid residues flanking the HVR (marked with asterisks) are indicated. From: Nucleotide Variability at Its Limit? Insights into the Number and Evolutionary Dynamics of the Sex-Determining Specificities of the Honey Bee Apis mellifera Mol Biol Evol. 2013;31(2):272-287. doi:10.1093/molbev/mst207 Mol Biol Evol | © The Author 2013. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

Fig. 2. Histograms of differences in the HVR and plot of pairwise amino acid mismatches in the flanking regions. (A) Top: histogram of the total number of differences in the HVR within the 77 pairs of heterozygous functional alleles, including amino acid mismatches and indels. Middle: histogram of the number of amino acid mismatches in the HVR within the 77 pairs. Bottom: histogram of the differences in length in the HVR within the 77 pairs. (B) Scatterplot of the amino acid mismatches in the PSD region versus mismatches in exon 8. The curves represent possible conditions for functional heterozygotes: the convex envelope of the data (blue line), the simple criterion proposed in this paper (green line), and a strict criterion assuming that 5% of the sequenced individuals are actually nonfunctional (red line). From: Nucleotide Variability at Its Limit? Insights into the Number and Evolutionary Dynamics of the Sex-Determining Specificities of the Honey Bee Apis mellifera Mol Biol Evol. 2013;31(2):272-287. doi:10.1093/molbev/mst207 Mol Biol Evol | © The Author 2013. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

Fig. 3. Plot of the mean number of functionally different alleles as a function of the total number of sampled alleles. Each point corresponds to the average of 100 random subsampling processes. (A) The curves correspond to the best fits of coupon-collector models with 1 type of coupons (green), 2 types of coupons (red), and 3 types of coupons (blue). The fits with 2 and 3 types are very similar, and the fits for 4, 5, and 6 types of coupons are indistinguishable from the fit with 3 types. (B) The curves correspond to the best fits of coupon-collector models with 2 (red) and 3 types of coupons (blue). From: Nucleotide Variability at Its Limit? Insights into the Number and Evolutionary Dynamics of the Sex-Determining Specificities of the Honey Bee Apis mellifera Mol Biol Evol. 2013;31(2):272-287. doi:10.1093/molbev/mst207 Mol Biol Evol | © The Author 2013. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

Fig. 7. Codon-by codon analysis of Tajima’s D across A Fig. 7. Codon-by codon analysis of Tajima’s D across A. mellifera csd sequences and occurrence of single amino acids within three Apis species in csd and fem. (A) Nonoverlapping, codon-by-codon sliding-window plot of Tajimas’s D for 107 Kenyan csd sequences. *P < 0.05, **P < 0.01. Black dots indicate signs of positive selection (P < 0.01) based on dN-dS statistics. (B) Underlying amino acids motifs of numbered peaks shown in (A) for csd and fem for A. mellifera, A. cerana, and A. dorsata. (C) Distribution of amino acid motifs given in (B) and their appearance along the csd/fem phylogeny in Apis. From: Nucleotide Variability at Its Limit? Insights into the Number and Evolutionary Dynamics of the Sex-Determining Specificities of the Honey Bee Apis mellifera Mol Biol Evol. 2013;31(2):272-287. doi:10.1093/molbev/mst207 Mol Biol Evol | © The Author 2013. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

Fig. 6. Genealogy of 57 Kenyan csd alleles obtained using the maximum likelihood algorithm. (A) Genealogy based on amino acid divergence, using the Jones–Taylor–Thornton model. (B) Genealogy based on divergence on synonymous sites, using the Pamilo–Bianchi model. Arrows indicate very recently diverged csd alleles harboring no synonymous but numerous amino acid substitutions. From: Nucleotide Variability at Its Limit? Insights into the Number and Evolutionary Dynamics of the Sex-Determining Specificities of the Honey Bee Apis mellifera Mol Biol Evol. 2013;31(2):272-287. doi:10.1093/molbev/mst207 Mol Biol Evol | © The Author 2013. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

Fig. 5. Diagram showing α, the proportion of amino acid substitutions driven by positive selection (white bars, left scale) and ω<sub>a</sub>, the rate of adaptive nonsynonymous substitutions, relative to the rate of synonymous substitutions (gray bars, right scale) calculated in the flanking region of the HVR for groups of csd alleles harboring different length of the repetitive region. In total, 87 csd alleles were grouped into seven sets that contained csd alleles with an amino acid repeat length ranging 6–12 (group 1, n = 13), 13–16 (group 2, n = 15), 17–20 (group 3, n = 22), 21–23 (group 4, n = 14), 24–26 (group 5, n = 11), and 27–33 (group XL, n = 12). From: Nucleotide Variability at Its Limit? Insights into the Number and Evolutionary Dynamics of the Sex-Determining Specificities of the Honey Bee Apis mellifera Mol Biol Evol. 2013;31(2):272-287. doi:10.1093/molbev/mst207 Mol Biol Evol | © The Author 2013. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

Fig. 4. Variation in length and amino acid differences of the HVR Fig. 4. Variation in length and amino acid differences of the HVR. (A) Maximal length variation of the repetitive region (dotted line) within the HVR was detected in the data set. The number of amino acid residues may range from 6 (csd sequence N87-I-1) to 33 (cB1_4). (B) Amino acid variation within the HVR in five sets of csd sequences with otherwise identical flanking regions (nucleotide diversity π = 0 for PSD and exon 8). From: Nucleotide Variability at Its Limit? Insights into the Number and Evolutionary Dynamics of the Sex-Determining Specificities of the Honey Bee Apis mellifera Mol Biol Evol. 2013;31(2):272-287. doi:10.1093/molbev/mst207 Mol Biol Evol | © The Author 2013. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com