Raw data for egg hatch rates

Egg hatch rates of all-female and normal lines

Variable sites in a 658-bp sequence of mitochondrial COI region from Maternally transmitted non-bacterial male killer in <i>Drosophila biauraria</i>

Variable sites of all-female line, normal line, and four injected lines are shown

Model for the parallel loss of the <i>ebony</i> upstream silencer element.

<p>Within the ancestral <i>ebony</i> gene, a silencer element evolved that adopted a repressive role in in the male posterior abdomen. Within the <i>montium</i> subgroup, this silencer’s activity was modified through changes in <i>cis</i> that altered the silencer’s spatial domain of activity. Within <i>D</i>. <i>serrata</i>, this silencer was inactivated, leading to increased <i>ebony</i> expression. In contrast, this silencer was maintained in the species <i>D</i>. <i>auraria</i>, in which intraspecific variation exists for an allele that inactivated this element.</p

Changes in <i>cis</i> to the upstream silencer element of <i>ebony</i> are responsible for a difference in <i>ebony</i> expression between strains.

<p>(A) Comparison of the DNA sequence of the light and dark strains of <i>ebony</i>, as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005279#pgen.1005279.g004" target="_blank">Fig 4A</a>. Schematics of GFP reporter constructs that pinpoint changes to the silencer of <i>ebony</i> are shown above and below the conservation plot. Chimeras appearing below the dark strain are drawn relative to the dark strain due to the presence of a large insertion in this strain. Similarly, chimeras above the light strain are drawn relative to the light strain due to the lack of this repetitive region. A6 midline activity was calculated using the same method described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005279#pgen.1005279.g004" target="_blank">Fig 4</a>. The act (D) + sil (<i>serrata</i>) construct in (A) contains the 1kb silencer region of <i>D</i>. <i>serrata</i> placed in the context of the dark <i>D</i>. <i>auraria</i> strain <i>ebony</i> upstream regulatory region (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005279#pgen.1005279.g006" target="_blank">Fig 6C</a>). (B) Activity of the dark (“PM”) strain <i>ebony</i> upstream regulatory region, containing both activating enhancer and silencer region. Expression is specifically reduced in the dorsal midline. (C) Activity of the light (“00”) strain <i>ebony</i> upstream regulatory region, orthologous to that used for the dark strain. This reporter construct shows a marked increase in midline activity, recapitulating the <i>ebony</i> expression phenotype of this strain. (D) Chimeric reporter containing the light strain activating enhancer fused to the dark strain upstream silencer element. This reporter construct shows midline repression similar to the dark strain. (E) Chimeric reporter containing the dark strain activating enhancer fused to the light strain upstream silencer region, which recapitulates the light strain activity. (F) The CD3 truncation of the light strain reporter drives expression throughout the A6 segment, similar to the same truncation of the dark strain. (G) Graph comparing the relative activity of the Dark and Light strain Full <i>ebony</i> upstream reporter activity in the A4 body segment, a region unaffected by silencer activity. (H) Graph comparing the relative activity of the Dark and Light strain CD3 truncation constructs in the A4 body segment.</p

Intraspecific variation in abdominal pigmentation of <i>D</i>. <i>auraria</i> across Japan.

<p>(A) Map of the Japanese islands, depicting sites (blue dots) from which <i>D</i>. <i>auraria</i> strains in this study were collected. (B-E) The abdominal phenotypes observed in <i>D</i>. <i>auraria</i> range from light (B) to intermediate (C, D), to dark (E).</p

The loss and diversification of body pigmentation in males of the <i>montium</i> subgroup.

<p>Within the <i>montium</i> subgroup, the pigmentation phenotype of males has been reduced from two tergites (as exemplified by <i>D</i>. <i>melanogaster</i>) to one tergite (as in <i>D</i>. <i>auraria</i> and <i>D</i>. <i>jambulina</i>). <i>D</i>. <i>serrata</i> represents a case in which male-specific pigmentation has been lost.</p

<i>D</i>. <i>auraria</i> strains surveyed for the geographic distribution of pigmentation phenotypes.

<p><i>D</i>. <i>auraria</i> strains surveyed for the geographic distribution of pigmentation phenotypes.</p

Parallel inactivation of the <i>ebony</i> upstream silencer in <i>D</i>. <i>serrata</i>.

<p>(A) Expression of <i>ebony</i> in the abdomen of a <i>D</i>. <i>serrata</i> male (UCSD “03” strain). (B) The entire upstream region of <i>D</i>. <i>serrata ebony</i> fused into a GFP reporter construct recapitulates the broad activation of <i>ebony</i> throughout the male posterior abdomen. (C) Chimeric reporter in which a 1 kb segment containing the <i>D</i>. <i>serrata</i> upstream silencer region was placed into the <i>D</i>. <i>auraria</i> dark strain reporter construct. This reporter lacks midline repression, indicating that the <i>D</i>. <i>serrata</i> upstream silencer was inactivated.</p

Localization of a conserved silencer in the <i>D</i>. <i>auraria</i> upstream regulatory region.

<p>(A) Schematic depicting the upstream regulatory region of <i>ebony</i> from a dark <i>D</i>. <i>auraria</i> strain aligned to the orthologous region of <i>D</i>. <i>melanogaster</i>. Gray boxes connecting <i>D</i>. <i>auraria</i> and <i>D</i>. <i>melanogaster</i> sequences indicate the relative position of perfectly conserved stretches. Positions of the minimal abdominal enhancer and silencer are shown above the <i>D</i>. <i>melanogaster</i> sequence are based on [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005279#pgen.1005279.ref018" target="_blank">18</a>]. Position of the <i>D</i>. <i>auraria</i> silencer element is based upon the truncation constructs shown below the conservation plot. Regions experimentally determined to be required for silencer activity are listed in black, while the region between the enhancer and silencer are shown in a gray gradient, as they may contribute to silencing activity. “A6 midline activity” was measured for each truncation construct, in which the fluorescent intensity of the midline was expressed as the percentage of the lateral A6 intensity ± S.E.M. (B) Activity of the dark (“PM”) strain full <i>ebony</i> upstream regulatory region, which recapitulates the midline repression of <i>ebony</i> observed by <i>in situ</i> hybridization (dotted lines). Dashed boxes indicate representative midline and lateral patches used to quantify midline activity. (C-D) The CD1 truncation construct (C), and the CD2 construct (D) both show midline repression similar to that observed in the full <i>ebony</i> upstream region. (E) The CD3 truncation construct shows uniform expression of GFP across the A6 body segment, reflecting the elimination of sequences required for repression.</p