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Supplementary Information Supplementary Information Molecular traces of alternative social organization in a termite genome Supplementary Figures Supplementary Figure 1. Coverage depth of assembled genome Supplementary Figure 2. Venn diagram of models predictions for Z. nevadensis protein coding genes. Supplementary Figure 3. Venn diagrams of termite protein coding genes clustered with other arthropod proteins by orthoMCL procedure. Supplementary Figure 4. NCBI taxonomy classification for candidate outgroups. This topology illustrates evolutionary relationships between Neoptera and the three lineages proposed as outgroups to replace the distant and fast evolving D. pulex. Amino acids Nucleotides ML Bayes Supplementary Figure 5. Phylogenetic analyses showing agreement across the obtained topologies. Species names are abbreviated using the first letter of the genus in capital and the three first letters of the species (e.g. ‘Apis’ correspond to A. pisum and not to the bee A. mellifera which abbreviation is ‘Amel’). Supplementary Figure 6. Osiris gene cluster in Z. nevadensis and five reference species. Genes are represented as squares with arrows indicating orientation. Gene names are indicated above while the subfamily classification and gene length are given below. A white cross in the square indicates non-detection of the corresponding domain with the Pfam recommended threshold. Intergenic space is also indicated, with a double large slash to notify genomic sequences larger than 1 Mb. Distinct scaffolds are separated by a vertical line. Supplementary Figure 7. Phylogeny of the Yellow gene family. For the protocol, refer to S5.3, for the mapping of protein IDs to species refer to Supplementary Table 13. Supplementary Figure 8. Phylogenetic analysis of the Zootermopsis nevadensis species tree. Species tree for the eleven different taxa used in the phylogenetic analysis. The numbers above each branch are separated into duplications (D) and selective events (S). Supplementary Figure 9. Phylogeny of the PKD family. For the protocol, refer to Supplementary Notes 3.3, for the mapping of protein IDs to species refer to Supplementary Table 13. Termite proteins and lineage are coloured in blue if differentially expressed in males, in red if differentially expressed in other samples or if not differentially expressed. Supplementary Figure 10. Phylogeny of the monodomain Kelch family. For the protocol refer, to Supplementary Notes 3.3, for the mapping of protein IDs to species refer to Supplementary Table 13. Termite proteins and lineage are coloured in blue if differentially expressed in males, in red otherwise. Supplementary Figure 11. Phylogeny of the multidomain BTB-BACK-Kelch family. For the protocol, refer to Supplementary Notes 3.3, for the mapping of protein IDs to species refer to Supplementary Table 13. Termite proteins and lineage are coloured in blue if differentially expressed in males, in red otherwise when these proteins exhibit the exact tridomain architecture BTB-BACK- Kelch. Light blue and orange are used for proteins having only two of the three required domains and/or additional domains, light blue for proteins differentially expressed in males, orange otherwise. Supplementary Figure 12. Phylogeny of the SINA family. For the protocol, refer to Supplementary Notes 3.3, for the mapping of protein IDs to species refer to Supplementary Table 13. Termite proteins and lineage are coloured in blue if differentially expressed in males, in red otherwise. Supplementary Figure 13. Phylogeny of the alpha tubulin family. For the protocol, refer to Supplementary Notes 3.3, for the mapping of protein IDs to species refer to Supplementary Table 13. Termite proteins and lineage are coloured in blue if differentially expressed in males, in red otherwise. Supplementary Figure 14. Phylogenetic tree of the termite, aphid, and louse ORs. This is a corrected distance tree. The DmOr83b orthologs ZnOrCo, ApOr1, and PhOr1 were declared as the outgroup to root the tree, based on the basal position of this gene in the Or family in analysis of the entire chemoreceptor superfamily in D. melanogaster. The termite, aphid, and louse protein names are highlighted in purple, brown, and blue, respectively, as are the branches leading to them to emphasize gene lineages. Support for major branches is shown above each branch as the percentage of 10,000 bootstrap replications of uncorrected distances followed by Bayesian posterior probabilities. Comments on each gene lineage are on the right. Suffixes after the gene/protein names are: PSE – pseudogene; NTE – N-terminus missing; CTE – C-terminus missing; INT – internal sequence missing; FIX – sequence fixed with raw reads; JOI – gene model joined across scaffolds; multiple suffixes are abbreviated to single letters. Supplementary Figure 15. Phylogenetic tree of the termite, aphid, louse, and select other insect GRs. This is a corrected distance tree and was rooted at the midpoint in the absence of a clear outgroup, an approach that clearly indicates the distinctiveness of the sugar and carbon dioxide receptors, as well as their putative distant relatedness. The termite, aphid, louse, and other insect gene/protein names are highlighted in purple, brown, blue, and red, respectively, as are the branches leading to them to emphasize gene lineages. Bootstrap support levels in percentage of 10,000 replications of uncorrected distance, followed by Bayesian posterior probabilities, are shown above major branches. Comments on major gene lineages are on the right. Suffixes after the gene/protein names are: PSE – pseudogene; NTE – N-terminus missing; CTE – C-terminus missing; INT – internal sequence missing; FIX – sequence fixed with raw reads; JOI – gene model joined across scaffolds; multiple suffixes are abbreviated to single letters. Gene models have been updated in OGSv2.2. Supplementary Figure 16. Phylogenetic tree of the termite, aphid, louse, and D. melanogaster IRs. This is a corrected distance tree and was rooted with the iGluRs and IR25a/8a as the outgroup, based on their highly conserved sequences and ancestral position in the family. The termite, aphid, louse, and Drosophila gene/protein names are highlighted in purple, brown, blue, and red, respectively, as are the branches leading to them to emphasize gene lineages. Bootstrap support levels, expressed as the percentage of 10,000 replications of uncorrected distance analysis, are shown above major branches. Comments on gene lineages are on the right. Suffixes after the gene/protein names are: PSE – pseudogene; NTE – N-terminus missing; CTE – C-terminus missing; INT – internal sequence missing; FIX – sequence fixed with raw reads; multiple suffixes are abbreviated to single letters. Supplementary Figure 17. Sections of olfactory glomeruli. Microphotographs of the dorsal part of the head (a; montage of three sections), the brain (b) and the right antennal lobe (c-e) of Zootermopsis sp. (frontal sections). Brain in (a) highlighted in cyan, antennal lobes in (b) highlighted in magenta; olfactory glomeruli in (c-e) colour coded as follows: cyan - glomeruli only present in upper section (c); green - glomeruli present in upper and middle section; magenta - glomeruli present in middle and lower section; red - glomeruli present only in lower section; orange - glomeruli present in all three sections. Arrow in (d) points at area where two adjacent glomeruli are difficult to discriminate; al - antennal lobe; an - antennal nerve; br - brain; es - esophagus; ey - eye; mbc - mushroom body calyx; mbl - mushroom body lobes; on - optic nerve; ol - optic lobe. Supplementary Figure 18. Phylogeny of the GNBP family. For the protocol, refer to Supplementary Notes 3.3, for the mapping of protein IDs to species refer to Supplementary Table 13. Additional sequences of Isoptera species are labelled “gene name_species initials”. Supplementary Figure 19. Phylogeny of the Vitellogenin family in the reference species and Z. nevadensis. For the protocol, refer to Supplementary Notes 3.3, for the mapping of protein IDs to species refer to Supplementary Table 13. Additional sequences of Isoptera species are labelled “uniprot id-species initials”. Supplementary Figure 20. Phylogenetic trees for four of the Neofem genes. Neofem1 (top-left), Neofem2 (top-right), Neofem3 (bottom-left) and Neofem4 (bottom-right). Genes for the different species are labelled with the initial of the genus, the three first letters of the species and a number. Supplementary Figure 21. Expression levels of sirtuin 6 across castes and life stages. Female reproductive castes show the highest gene expression. Normalized RPKM values have been standardized to 100% across the different life stages and castes. For abbreviations see Supplementary Table 4. Supplementary Figure 22. Expression levels of sirtuin 7 castes and life stages. Female reproductive castes show the highest gene expression. Normalized RPKM values have been standardized to 100% across the different life stages and castes. For abbreviations see Supplementary Table 4. Supplementary Figure 23. Expression levels of KDM4C across castes and life stages. Female reproductive castes show the highest gene expression. Normalized RPKM values have been standardized to 100% across the different life stages and castes. For abbreviations see Supplementary Table 4. Supplementary Figure 24. Gene expression levels of histone acetyl transferases across castes and life stages. Female reproductive castes show the highest gene expression. Normalized RPKM values have been standardized to 100%
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