DEV030270 Supplementary Material

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• Supplemental Figure S1 -

  Fig. S1. Dynamic early expression of Eba-eve and Eba-h. (A-D) Consecutive stages of Eba-eve expression prior to the onset of cellularization. (E-H) Consecutive stages of Eba-h expression prior to the onset of cellularization. Anterior is towards the left.

• Supplemental Figure S2 -

  Fig. S2. Using CAD and ribosomal 28S DNA sequence alignments, likelihood-based tests reject basal position of Syrphidae within Cyclorrhapha. Maximum likelihood trees were inferred for CAD and ribosomal 28S DNA sequence alignments (Collins and Wiegmann, 2002; Moulton and Wiegmann, 2004) using the full nucleotide alignment (nt) or RY-coding (RY), respectively. Inferences were made by PAUP*(Swofford, 2002) using GTR + Γ₄ + I models for each of the alignments as selected by the Akaike information criterion in MODELTEST (Posada and Crandall, 1998). Sitewise log-likelihood values were computed by PAUP* and P values of AU, SH and RELL BP tests were computed by CONSEL (Shimodaira and Hasegawa, 2001). L, likelihood value; AU, Approximately Unbiased test; SH, Shimodaira-Hasegawa test; RELL BP, Resampling of Estimated Log-Likelihood Bootstrap Percentage. *Statistically significant at the 5% level.

  Collins, K. P. and Wiegmann, B. M. (2002). Phylogenetic relationships of the lower Cyclorrhapha (Diptera: Brachycera) based on 28S rDNA sequences. Insect Syst. Evol. 33, 445-456.

  Moulton, J. K. and Wiegmann, B. M. (2004). Evolution and phylogenetic utility of CAD (rudimentary) among Mesozoic-aged Eremoneuran Diptera (Insecta). Mol. Phylogenet. Evol. 31, 363-378.

  Posada, D. and Crandall, K. A. (1998). MODELTEST: testing the model of DNA substitution. Bioinformatics 14, 817-818.

  Shimodaira, H. and Hasegawa, M. (2001). CONSEL: for assessing the confidence of phylogenetic tree selection. Bioinformatics 17, 1246-1247.

  Swofford, D. L. (2002). PAUP*. Phylogenetic analysis using parsimony (* and other methods). Sunderland, MA: Sinauer Associates.

• Supplemental Figure S3 -

  Fig. S3. Protein alignments. (A) Protein alignment of dipteran Hunchback homologues including sequences from Clogmia albipunctata (Cal, EU589570), Haematopota pluvialis (Hpl, EU589574), Lonchoptera lutea (Llu, EU589577), Platypeza consobrina (Pco, EU589582), Megaselia abdita (Mab, AJ295635), Episyrphus balteatus (Eba, FJ387229) and Drosophila melanogaster (Dme, Y00274). The two zinc-finger domains of the Drosophila sequence are underlined. Asterisks indicate cysteine and histidine residues of the zinc fingers (Tautz et al., 1987). Amino acids that are conserved in at least five sequences are shaded in gray. (B) Protein alignment of dipteran Nanos homologues, including sequences from Chironomus samoensis (Csa, U24697), Anopheles stephensi (Ast, AY583529), Aedes aegypti (Aae, AY583528), Anopheles gambiae (Aga, AY583530), Episyrphus balteatus (FJ387226) and Drosophila melanogaster (M72421). Amino acids that are conserved in at least four sequences are shaded in gray. (C) Protein alignment of dipteran Caudal homologues including sequences from Anopheles gambiae (AF119382), Empis livida (Eli, EU295453), Lonchoptera lutea (EU295452), Megaselia abdita (EU295458), Episyrphus balteatus (FJ387230) and Drosophila melanogaster (NM_057606). Amino acids that are conserved in at least four sequences are shaded in gray. The homeodomain in the Drosophila sequence is underlined. (D) Protein alignment of Orthodenticle homologues, including sequences from Tribolium castaneum (Tca-1, AJ223627; Tca-2, AJ223614), Nasonia vitripennis (Nvi-1, AY684810; Nvi-2, XM_001602624), Episyrphus balteatus (FJ387225) and Drosophila melanogaster (BT011185). Amino acids that are conserved in at least four sequences are shaded in gray, whereas amino acids conserved in fewer than four sequences but between Drosophila and Episyrphus are shaded in red. The homeodomain in the Drosophila sequence is underlined. Dashes denote gaps and numbers refer to the position of the last amino acid in each row.

  Tautz, D., Lehmann, R., Schnürch, H., Schuh, R., Seifert, E., Kienlin, A., Jones, K. and Jäckle, H. (1987). Finger protein of novel structure encoded by hunchback, a second member of the gap class of Drosophila segmentation genes. Nature 327, 383-389.

• Supplemental Figure S4 -

  Fig. S4. Eba-hb expression is absent in ovarian follicles and present in the extra-embryonic anlage. (A,B) Absence of maternal Eba-hb expression. Ovarian follicles from the same pool were divided and stained independently with in situ probes against Eba-hb (A) and Eba-cad (B). Note that Eba-cad transcripts (positive control) are detected in the oocyte (asterisk) and nurse cells (arrow) but not in the somatic follicle cells (arrowhead). (C-E′) At the beginning of gastrulation, extra-embryonic Eba-hb expression is confined to the serosa anlage. Co-expression Eba-zen (C,C′), which served as a marker for prospective serosa cells (Rafiqi et al., 2008), and Eba-hb (D,D′) is shown at the onset of gastrulation in lateral (C,D) and dorsal view (C′,D′). Merged images of C and D or C′ and D′ are shown in E and E′, respectively. Eba-zen expression was false-colored in red and Eba-hb expression was false-colored in green. All embryos are shown with anterior towards the left.

  Rafiqi, A. M., Lemke, S. J., Ferguson, S., Stauber, M. and Schmidt-Ott, U. (2008). Evolutionary origin of the amnioserosa in cyclorrhaphan flies correlates with spatial and temporal expression changes of zen. Proc. Natl. Acad. Sci. USA 105, 234-239.

• Supplemental Figure S5 -

  Fig. S5. Cuticlar phenotypes after ectopic expression of Eba-nos or Eba-cad. (A) Single injection of Eba-nos mRNA at the anterior pole (0% EL). (B) Double injection of Eba-nos mRNA at 0% EL and 20% EL. (C) Single injection of Eba-nos mRNA at the posterior pole (100% EL). (D) Double injection of Eba-cad mRNA at 0% EL and 20% EL. (E) Single injection of Eba-cad mRNA at 100% EL. Each column represents a single cuticle, which has been scored for the markers listed on the left side of each row. Full circles indicate absence of structures, encircled dots indicate affected structures, dots indicate that no difference from wild type was observed. A question mark is used if presence or absence of a structure could not be determined. mt, median tooth; dcp, dorsal cephalopharyngeal plate; vcp, ventral cephalopharyngeal plate; lg, Lateralgräten; aso, antennal sense organ; mso, maxillary sense organ; mh, mouth hooks; nc, neck clasps, Hp, H-piece. T1-T3, thoracic denticle belts; A1-A8, abdominal denticle belts; fk, Filzkörper.

• Supplemental Table S1 - Adobe PDF