Figtree

Full-length sequences of nodal in N. anomala, and Pitx in O. fusiformis, L. ruber and P. caudatus were identified from RNAseq data of mixed embryonic stages. Protein alignments were constructed with MAFFT v. 7 [51 (link)] and poorly aligned regions were removed with Gblocks v. 0.91b [52 (link)]. RAxML v. 8 [53 (link)] was used to infer gene orthologies (electronic supplementary material, figure S1). Resulting trees were formatted with FigTree and Illustrator CS6 (Adobe). Fixed embryos of N. anomala, O. fusiformis, L. ruber and P. caudatus were used to perform colorimetric whole mount in situ hybridization following previously described protocols [46 (link),49 (link)]. After developing the signal, samples were stored in 70% glycerol and imaged with an Axiocam HRc connected to an Axioscope Ax10 (Zeiss), using bright field Nomarski optics. Images were analysed with Photoshop CS6 (Adobe), and figure plates made with Illustrator CS6 (Adobe). Contrast and brightness were adjusted always to the whole image and not to specific parts of it.

A fragment of foxA and foxQ2, and the full-length sequences of cdx, evx, GATA456-a, gsc, otx, six3/6 and twist-a [GenBank: KT335961–KT335969] were identified from RNAseq data of mixed developmental stages. Protein alignments were constructed with MAFFT v.7 [42 (link)] and poorly aligned regions were removed with Gblocks v.0.91b [43 (link)]. RAxML v.8 [44 (link)] was used to infer gene orthologies (Additional file 2: Figure S1). Resulting trees were formatted with FigTree and Illustrator CS6 (Adobe). Single colorimetric whole mount in situ hybridization was performed as described elsewhere [45 (link)], with the only modification of permeabilizing the samples with proteinase K (10 μg/mL in PTw) for 8 min at RT without shaking. After the whole mount in situ hybridization protocol, embryos were cleared and stored in 70 % glycerol in PTw with a 1:5000 dilution of the nuclear marker DAPI.