Revealing the mechanisms underlying the breathtaking morphological diversity observed in nature is a major challenge in Biology. It has been established that recurrent mutations in hotspot genes cause the repeated evolution of morphological traits, such as body pigmentation or the gain and loss of structures. To date, however, it remains elusive whether hotspot genes contribute to natural variation in the size and shape of organs. As natural variation in head morphology is pervasive in Drosophila, we studied the molecular and developmental basis of differences in compound eye size and head shape in two closely related Drosophila species. We show differences in the progression of retinal differentiation between species and we applied comparative transcriptomics and chromatin accessibility data to identify the GATA transcription factor Pannier (Pnr) as central factor associated with these differences. Although the genetic manipulation of Pnr affected multiple aspects of dorsal head development, the effect of natural variation is restricted to a subset of the phenotypic space. We present data suggesting that this developmental constraint is caused by the coevolution of expression of pnr and its cofactor u-shaped (ush). We propose that natural variation in expression or function of highly connected developmental regulators with pleiotropic functions is a major driver for morphological evolution and we discuss implications on gene regulatory network evolution. In comparison to previous findings, our data strongly suggest that evolutionary hotspots are not the only contributors to the repeated evolution of eye size and head shape in Drosophila.

Compound eyes in nocturnal or fossorial insects generally express visible light opsins at higher levels than diurnal insects. In this study, we tested whether dorsal (above water) and ventral eyes (below water) of the diurnal four-eyed whirligig beetle Gyretes sericeus Laboulbène, 1853, resemble opsin expression and function of diurnal or nocturnal insect eyes respectively. By immunocytochemistry, we compared expression of green LW-opsin in dorsal and ventral rhabdoms of whirligig beetle ommatidia. Basal rhabdomeres (bR) showed comparable expression levels of LW-opsin in both dorsal and ventral ommatidia. In contrast, the inner proximal (R1p) and distal (R1d) bR showed a weak and narrow expression dorsally, whereas R1p and R1d showed a higher and expanded expression ventrally. To test whether dorso-ventral specialization of ommatidia results in functional differences in light response, we studied the behavior of beetles after selective eye-occlusion experiments. During phototaxis experiments, whirligig beetles typically showed a clear preference for light. Positive phototaxis was mainly disrupted after dorsal eye occlusion suggesting dorsal dominance of light sensing. Together, differences in opsin expression, structure, and function of whirligig beetle eyes suggest dorso-ventral sub-functionalizations resembling eye adaptations of diurnal and nocturnal insects. We discuss how dorsal and ventral eye specializations may have evolved in adephagan beetles.

1The size and shape of an organism is tightly controlled during embryonic and 2 postembryonic development to ensure proper functionality. However, in the light of the 3 breath-taking diversity of body forms observed in nature, developmental processes must have 4 evolved to allow evolutionary changes in adult morphology. Therefore, gene regulatory 5 networks (GRNs) that orchestrate organ development are mostly constrained, but nodes and 6 edges within such networks must change to give rise to morphological divergence. Identifying 7 such tuning nodes remains a major challenge in evolutionary developmental biology. Here, we 8 combined comparative transcriptomics and chromatin accessibility data to study 9 developmental differences leading to natural variation in compound eye size and head shape 10 in the two closely related Drosophila species D. melanogaster and D. mauritiana. We show that 11 variation in expression of the GATA transcription factor Pannier (Pnr) is associated with 12 extensive remodeling of the transcriptomic landscape during head development. Since U-13 shaped (Ush), a co-factor of Pnr, is involved in the same regulatory context, we argue that 14 variation in expression of both factors may be a driver of divergence in head morphology. 15Applying functional genetics and geometric morphometrics we confirmed that manipulation of 16 pnr expression in D. melanogaster largely phenocopies D. mauritiana dorsal head shape and 17 ommatidia number. Therefore, we propose that the regulatory module composed of Pnr and 18 Ush represents a tuning node within the otherwise highly conserved GRN underlying head 19 development in Drosophila. 20 21 developmental GRNs allows gaining new insights into constrained and variable developmental 1 processes, respectively. 2One approach to reveal tuning nodes is first to identify genetic variants associated with 3 morphological diversity and then in a second step to establish regulatory interactions of genes 4 affected by those variants. However, variation in complex morphological traits is influenced by 5 many genetic loci with small effect sizes, which are spread throughout various genomic 6 locations 23-25 . For instance, mandible and craniofacial shape differences between mouse 7 strains are influenced by various loci located on most of the chromosomes [26][27][28][29] . Similarly, 8 studies in Drosophila revealed that loci on several chromosomes contribute to differences in 9 eye size and head shape [30][31][32] . These examples corroborate the complexity of the genetic 10 architecture of such traits and highlight the difficulty to determine individual causative 11 molecular changes. 12As an alternative, we propose to first identify tuning nodes within the GRN governing 13 the development of a variable morphological trait. We assume that variation in the expression 14 or function of putative tuning nodes (e.g. genes coding for transcription factors) directly affects 15 the expression of downstream target genes (i.e. "gene modules" 33 ). Candidate tuning nodes 16 can then ...