DOI: 10.5061/dryad.nd1js
title: Data from: Neofunctionalization of embryonic head patterning genes facilitates the positioning of novel traits on the dorsal head of adult beetles

description: The origin and integration of novel traits are fundamental processes
 during the developmental evolution of complex organisms. Yet how novel
 traits integrate into pre-existing contexts remains poorly understood.
 Beetle horns represent a spectacular evolutionary novelty integrated
 within the context of the adult dorsal head, a highly conserved trait
 complex present since the origin of insects. We investigated whether
 otd1/2 and six3, members of a highly conserved gene network that instructs
 the formation of the anterior end of most bilaterians, also play roles in
 patterning more recently evolved traits. Using ablation-based
 fate-mapping, comparative larval RNA interference (RNAi) and transcript
 sequencing, we found that otd1/2, but not six3, play a fundamental role in
 the post-embryonic formation of the adult dorsal head and head horns of
 Onthophagus beetles. By contrast, neither gene appears to pattern the
 adult head of Tribolium flour beetles even though all are expressed in the
 dorsal head epidermis of both Onthophagus and Tribolium. We propose that,
 at least in beetles, the roles of otd genes during post-embryonic
 development are decoupled from their embryonic functions, and that
 potentially non-functional post-embryonic expression in the dorsal head
 facilitated their co-option into a novel horn-patterning network during
 Onthophagus evolution.
description: Onthophagus spp six3 and
 otd gene sequencesSequence and associated
 annotations for O. taurus six3 and otd genes and O. sagittarius six3 and
 otd transcripts. Flat file Genbank format.Onthophagus six3 and otd genes
 and primers.gbInsect SIX protein
 family sequences and tree (data for Figure S1)Aligned sequences and
 RaxML generated phylogram of selected insect SIX
 proteins.InsectSIXtree.nexInsect OTD protein sequences and tree (data for Figure S2)Aligned sequence and FastTree generated phylogram of selected insect orthodenticle proteins.InsectOTDtree.nexR script to reproduce expression plots (Figures S3 and S8)Annotated R script with code to read and plot raw data from qPCR and RNAseq estimations of stage specific and tissue specific levels of six3 and otd transcripts. Requires files "tcas_qPCR_stages.csv", "otau_rnaseq_stages.csv", "osag_rnaseq_stages.csv", "tcas_qpcr_tissues.csv" and "otau_rnaseq_tissues.csv".Expression_plots.RTribolium castaneum qPCR by stageRaw data table for T. castaneum stage-specific qPCR estimation of six3 and otd transcript levels. Use R script in "Expression_plots.R" for analysis and plotting.tcas_qPCR_stages.csvOnthophagus taurus RNAseq by stageRaw data table for O. taurus stage-specific RNAseq estimation of six3 and otd transcript levels. Use R script in "Expression_plots.R" for analysis and plotting.otau_rnaseq_stages.csvOnthophagus sagittarius RNAseq by stageRaw data table for O. sagittarius stage-specific RNAseq estimation of six3 and otd transcript levels. Use R script in "Expression_plots.R" for analysis and plotting.osag_rnaseq_stages.csvTribolium castaneum qPCR by pupal tissueRaw data table for T. castaneum tissue-specific qPCR estimation of six3 and otd transcript levels. Use R script in "Expression_plots.R" for analysis and plotting.tcas_qpcr_tissues.csvOnthophagus taurus RNAseq by pupal tissueRaw data table for O. taurus tissue-specific RNAseq estimation of six3 and otd transcript levels. Use R script in "Expression_plots.R" for analysis and plotting.otau_rnaseq_tissues.csvR script to reproduce RNAi phenotype plots (Figures S4 and S5))Annotated R script with code to read and plot raw data from phenotypic measurements of dsRNA treated beetles . Requires files "tcas_RNAi.csv", "otau_RNAi.csv" and "osag_RNAi.csv".RNAiAnalyses.RTribolium castaneum RNAi phenotype dataPhenotypic measurements from dsRNA injected T. castaneum. For analysis and plotting using the R code in file "RNAiAnalyses.R".tcas_RNAi.csvOnthophagus taurus RNAi phenotype dataPhenotypic measurements from dsRNA injected O. taurus. For analysis and plotting using the R code in file "RNAiAnalyses.R".otau_RNAi.csvOnthophagus sagitarius RNAi phenotype dataPhenotypic measurements from dsRNA injected O. sagittarius. For analysis and plotting using the R code in file "RNAiAnalyses.R".osag_RNAi.csvTribolium castaneum qPCR validation of RNAi KD efficiency (Figure S9)Raw data from qPCR experiments to validate the knockdown efficiency of dsRNA injections in Tribolium castaneum.RNAiknockdownefficiency_qPCR_rawdata.xlsx