Vladimir Jovanović1*, Alex Zemella2, Jasmine Loveland3, Katja Nowick4 and Clemens Küpper2
1Institute for Biology, Freie Universität Berlin, Berlin, Germany;Bioinformatics Solution Center, Freie Universität Berlin, Berlin, Germany
2Max Planck Institute for Biological Intelligence, Seewiesen, Germany
3Max Planck Institute for Biological Intelligence, Seewiesen, Germany; Department of Cognitive and Behavioral Biology, University of Vienna, Vienna, Austria; Messerli Research Institute, University of Veterinary Medicine, Vienna, Austria
4Institute for Biology, Freie Universität Berlin, Berlin, Germany
vladimir.jovanovic [at] fu-berlin.de
Abstract
Alternative reproductive tactics in the ruff (Calidris pugnax) are orchestrated by an autosomal inversion that determines three distinct male morphs: Independents, Satellites, and Faeders. These morphs exhibit nearly discrete differences in size, plumage, aggression, and circulating androgen levels. To resolve the molecular mechanisms driving these complex phenotypes, we employed an integrative bioinformatics framework combining multi-tissue transcriptomics with structural and functional modeling. We performed RNA-seq on eleven tissues, including steroidogenic glands and nine brain nuclei comprising the social behaviour network and mesolimbic reward pathways. Differential gene expression analysis revealed a significant enrichment of morph-differentiating genes within the inverted supergene region. To investigate cis-regulatory effects, we quantified allele-specific expression from RNA-seq data, identifying widespread allelic imbalance in most inversion-linked protein-coding genes across all examined brain areas.
We identified HSD17B2, encoded within the inversion, as the primary driver of morph-specific androgen variation. This enzyme oxidizes testosterone into the less potent androstenedione. HSD17B2 is significantly upregulated in the blood, hypothalamus, and several brain nuclei of Satellites and Faeders, while its expression is nearly absent in Independent blood. This allows the inversion morphs to rapidly metabolize testosterone in the periphery before it reaches the brain.
Using a branch model selection test on related Charadriiform species, we also demonstrated that HSD17B2 is under strong positive selection in the inversion haplotypes. In silico homology modeling and computer simulations of ruff homodimers further revealed that derived isozymes possess higher binding affinities for testosterone and NAD compared to the ancestral variant.
We expected HSD17B2 to likely have a strong pleiotropic effect on the neural expression of several genes across different brain areas, through changes in the circulating sex steroid hormone levels. Therefore, we integrated results from various transcriptomic analyses to characterise the morph-specific neural molecular signatures underlying stereotypical mating tactics of male morphs. Overall, this study demonstrates how structural and regulatory innovations in a single gene can induce modest changes in expression across hundreds of genes in different brain areas and result in a morph-specific mating behaviour.
Keywords: Behavioural genomics, intraspecific selection, supergene