of the DrosDel collection [41,42]. The identity of the amplicons sequenced appears in parentheses (Table S6). The direct target sites duplications of the FRT-bearing TEs are easily identified in the region in which the two subsets of sequences overlap within each alignment. Sequence corresponding to TEs is not shown. (PDF) Figure S9 Average progeny size from seven heterozygotes that carry the ultraconserved region CG15121–CG16894 in its disrupted (INV1, INV2) or intact (REC) form. Data for females and males are shown separately. The INV1, INV2, and REC chromosomes were tested in different combination with 2R standard chromosomes associated with the w2 phenotype (SIM1, REV1, REV2). The resulting progeny from each type of cross among heterozygous individuals were genotyped based on eye-color (the two homozy- gotes have different eye color –red and white- whereas the heterozygotes are orange-eyed) and examined for different parameters. No statistically significant difference was found for the progeny size and sex ratio among the carriers and non-carriers of the disrupted ultraconserved region (ANOVA, P.0.05 in all contrasts) and no deviation from the Mendelian ratios was found either for any heterozygote-by-sex combination analyzed (G-test for goodness of fit, P.0.05 in all contrasts). See Table S9 for further details on the contrasts performed. Error bars indicate 95% CI. (TIF) Figure S10 Relative viability in pairwise competition experi- ments between embryos of different genotypes at three different starting proportions. (A) The relative viability between two competing genotypes was estimated as (n196n2)/(n16n29), where n1 and n2, and n19 and n29, are the number of embryos and imagoes, respectively. The competing genotypes entail one carrying the tested chromosome (in orange, X2: REC, INV1, and INV2) in two possible conditions (heterozygosis, left; homozygosis, right), and the other genotype always in homozy- gosis (X1: SIM1, REV1, and REV2; the tester embryo). The latter 2 always carries the standard arrangement and is invariably w . The tested chromosomes differ in whether they carry the ultracon- served region in its intact (REC) or disrupted form (INV1, INV2). (B) Average relative viability between competing genotypes at three different starting proportions. Values were log2 transformed; departures from zero indicate that the competing genotypes differ in their relative viability. The relative viability can be inferred by comparing the different tested chromosomes to the same tester embryo. Eighteen different comparisons were performed: 3 starting proportions62 genotype conditions for the tested chromosome63 different tester embryos (Table S10). The starting proportions assayed were 1:3, 1:1, and 3:1 (tester embryo : embryo with the tested chromosome), which are indicated in different colors. For each condition of the tested chromosome and starting proportion, seven combinations of competing genotypes were assayed (26367 = 42 in total). With a few exceptions, the tested chromosomes ranked consistently in their relative viability against a particular tester embryo across starting proportions. Only in one of the experiments (starting proportion, 1:1; tester embryo, SIM1/ SIM1; condition of the tested chromosome, heterozygosis), REC shows significantly higher viability than INV1 and INV2, which denotes a detrimental effect associated with the disruption of the ultraconserved region (Table S10). In the remaining experiments, REC exhibited either an intermediate relative viability in relation to INV1 and INV2 or a relative viability indistinguishable from INV1, INV2, or both. One hundred embryos in total were used per competition setting; every competition setting was replicated 10 times. Error bars indicate 95% CI. (TIF) Collinearity Conservation in Eukaryotes Figure S11 Test for differences in fecundity between males with (INV1, INV2) and without (REC) the disrupted ultraconserved region CG15121–CG16894. (A) Progeny size from single-mating experiments between three tester females and the males under scrutiny. The nomenclature of the crosses indicates first the strain of the female and then the strain of the male. No statistically significant differences were found for the crosses examined (Kruskal-Wallis, d.f. = 6; P,0.1348; n = 7–9). After pooling the data however, INV1, INV2, and REC males are shown to differ in progeny size (Kruskal-Wallis, d.f. = 2; P,0.0284; n = 16–25), which is due to differences between INV1 and INV2 males (Steel-Dwass; P,0.0215; n = 16–18; Table S11). (B) Fraction of the progeny sired by red-eyed males in double-mating experiments. For all the crosses, the female genotype is indicated first and the genotypes of the first and second males, which are separated by a comma, are indicated next. Blue, results from SIM1, REV1, and REV2 strains when exposed first to INV1, INV2, or REC males and subsequently to males of their own genotype (direct crosses). Red, results from equivalent experiments in which the order of the males was reversed (reciprocal crosses). A fraction of 0.5 indicates that the sperm of the two males has equivalent fertilization performance. We only considered those days in which progenies sired by the two males were detected. No statistically significant difference was found between INV1, INV2, and REC males irrespective of the order in which they mated (ANOVA; direct crosses: F(2,43) = 0.1159, P,0.8909, n = 10–22; reciprocal crosses: F(2,43) = 0.1152, P,0.8566, n = 14–25; Table S12). Error bars indicate 95% CI. (TIF) Figure S12 Average number of fertilized females after exposure to single males with (INV1, INV2) and without (REC) the disrupted ultraconserved region CG15121–CG16894 during a defined timeframe. Three different timeframes were assayed (1 hr, 3 hr, and 6 hr). Each male was exposed to 10 females of its own strain and 10 males were analyzed per strain and timeframe combination. The exposed females were transferred to individual vials, which were examined for the presence of progeny after 15 days. No differences were found between males with or without the disrupted ultraconserved region CG15121–CG16894 irrespective of the timeframe assayed (Kruskal-Wallis, d.f. = 2; 1 hr, P = 0.1364; 3 hr, P = 0.4838; 6 h, P = 0.7487; n = 10; Table S13). Error bars indicate 95% CI. (TIF) Figure S13 Test for differences in global homeostasis among individuals with (INV1, INV2) and without (REC) the disrupted ultraconserved region CG15121–CG16894 using four proxies. (A) Negative gravitaxis was measured as the average relative height reached in a volumetric cylinder by flies after perturbation. (B) Heat-shock resistance was estimated as the average fraction of flies alive after a heat-shock pulse at 39uC for 30 m. (C) Desiccation resistance was gauged as the average time-to-death of flies under conditions of low humidity. (D) Starvation resistance was assessed as the average time-to-death of flies in the absence to nutrients. The disruption of the ultraconserved region CG15121–CG16894 has no apparent effect for any of the proxies studied in either sex with the exception of the starvation assay in females (Kruskal- Wallis, d.f. = 2; negative gravitaxis: Pmales = 0.688, Pfemales = 0.751, n = 20; heat-shock resistance: Pmales = 0.363, Pfemales = 0.134, n = 5; desiccation resistance: Pmales = 0.964, Pfemales = 0.773, n = 20; starvation resistance: Pmales = 0.822, Pfemales,0.0001, n = 20; Table S15). For this last sex by proxy combination, the statistically significant differences are associated with the higher resistance of the strain INV2 as compared to INV1 and REC (Steel-Dwass; PLoS Genetics | www.plosgenetics.org 11 February 2012 | Volume 8 | Issue 2 | e1002475