Exploring the integration of innovation in the dorsal head of Onthophagus beetles using head compartment-specific RNAseq

The general morphology of the insect head has remained relatively unchanged through more than 400 million years of evolution. Yet throughout this period this same region has also become a hotspot for evolutionary novelty, yielding structures such as the eyestalks of stalk-eyed flies or the cephalic horns of dung beetles. How novelty can be integrated within ancient complex traits without disrupting the function and formation of that trait is a foundational, yet largely unresolved question in developmental and evolutionary biology. Here, we approached this question by performing unique, head compartment specific RNAseq using the heads of Onthophagus taurus beetles, which bear impressive posterior horns in males. We sequenced the transcriptomes in horned males and hornless females from six distinct head compartments covering two major axes of patterning: anterior to posterior (AP) and medial to lateral (ML). Our results provide evidence of differential compartmentalization of the head along both AP and ML axes, and reveal striking parallels between morphological and transcriptomic complexity – that is, head regions with more complex morphologies, such as the posterior region, are more transcriptionally intricate compared to morphologically homogenous regions, such as the anterior of the head. Our findings support the hypothesis that the integration of novel traits within ancestral trait complexes may require the recruitment of additional genes and pathways into the networks instructing within and among compartment development. However, sexual dimorphism in posterior horn development was not paralleled by a corresponding sexual dimorphism in transcriptional complexity, instead hornless females exhibited approximately the same diversity of differentially expressed genes across posterior head compartments than did horn-bearing males. Dorsal head regions of male and female pre-pupae larvae were dissected into six distinct compartments: left and right posterior, medial posterior, left and right anterior, and medial anterior.