Dev Genes Evol (2009) 219:427–435 429 Fig. 1 Alignment and phylogenetic analysis of the Sp8/9 sequence from Oncopeltus. a Alignment of Sp proteins from Drosophila, Oncopeltus, Tribolium, and mouse. Shown is the protein region containing the Btd box motif (blue) and the three zinc finger motifs (red). The sequences of the Sp8/9 proteins including the D-Sp1 protein are almost identical and clearly different from other Sp proteins. b Phylogenetic analysis of all murine Sp factors and Sp factors from Tribolium, Drosophila, and Oncopeltus based on the alignment shown in (a). Shown is the unrooted majority rule consensus computed from 1,000 intermediate trees produced with the quartet puzzling method (Strimmer and von Haeseler 1996). The numbers at the tree edges indicate the reliability values. GenBank accession numbers: Dm_btd NP_511100, D-Sp1 NP_572579, Tc_btd NP_001107792, Tc_Sp8 NP_001034509, Mm_Sp1 NP_038700, Mm_Sp2 NP_084496, Mm_Sp3 NP_035580, Mm_Sp4 NP_033265, Mm_Sp5 NP_071880, Mm_Sp6 NP_112460, Mm_Sp7 NP_569725, Mm_Sp8 NP_796056, Mm_Sp9 NM_001005343. Species abbreviations: Mm Mus musculus, Tc Tribolium castaneum, Of Oncopeltus fasciatus, Dm Drosophila melanogaster In order to further corroborate the orthology of the Sp8/9 fragment isolated from Oncopeltus, we also performed a phylogenetic analysis (Fig. 1b) using the alignment in Fig. 1a in a maximum likelihood analysis with Tree Puzzle (Strimmer and von Haeseler 1996). Most edges in the phylogenetic tree are well supported with reliability values above 95. The Sp8/9 factors from Oncopeltus, Tribolium, and mouse cluster together in a group supported by the maximum reliability value of 100 and with very short edges, indicating that these genes are closely related. This grouping also includes D-Sp1 from Drosophila. These results further support the orthology of the Oncopeltus Sp8/9 gene with the other Sp8/9 genes and also give additional evidence to the previously published conclusion that the Drosophila D-Sp1 gene is actually the Drosophila Sp8/9 homolog (Beermann et al. 2004). Sp8/9 is expressed during appendage development in O. fasciatus Next we examined the expression profile of Sp8/9 in Oncopeltus embryos using whole mount in situ hybridiza- tion. During early stages of germband elongation, strong expression of Sp8/9 is seen in the limb buds of the thoracic