Differential recruitment of limb patterning genes during development and diversification of beetle horns

Abstract

The origins of novel complex phenotypes represent one of the most fundamental, yet largely unresolved, issues in evolutionary biology. Here we explore the developmental genetic regulation of beetle horns, a class of traits that lacks obvious homology to traits in other insects. Furthermore, beetle horns are remarkably diverse in their expression, including sexual dimorphisms, male dimorphisms, and interspecific differences in location of horn expression. At the same time, beetle horns share aspects of their development with that of more traditional appendages. We used larval RNA interference-mediated gene function analysis of 3 cardinal insect appendage patterning genes, dachshund, homothorax, and Distal-less, to investigate their role in development and diversification of beetle horns within and between species. Transcript depletion of all 3 patterning genes generated phenotypic effects very similar to those documented in previous studies that focused on general insect development. In addition, we found that Distal-less and homothorax, but not dachshund, regulate horn expression in a species-, sex-, body region-, and body size-dependent manner. Our results demonstrate differential co-option of appendage patterning genes during the evolution and radiation of beetle horns. Furthermore, our results illustrate that regulatory genes whose functions are otherwise highly conserved nevertheless retain the capacity to acquire additional functions, and that little phylogenetic distance appears necessary for the evolution of sex- and species-specific differences in these functions.

Fig. 1.

Diversity and development of beetle horns. (A) Diversity in number, size, location, and shape of horn expression between (i) and within (ii) species of Onthophagus. (B and C) Drosophila model of limb formation (B) compared with the development of a thoracic beetle horn from embryo to adult (C). Cuticle is shown in black, epidermis in blue, including schematic expression domains of the proximodistal patterning genes homothorax (hth, yellow), dachshund (dac, green), and Distal-less (Dll, red). Drosophila legs develop from imaginal discs; epidermal invaginations specified during embryonic development, which grow throughout larval development. Patterning takes place while the disc is a 2-dimensional sheet of tissue, and all disc growth occurs while the disc is invaginated into the body interior. In contrast, beetle horns appear not to be specified during embryonic development. Instead, horns grow from the start as 3-dimensional epidermal outbuddings and all growth is confined to the relatively brief prepupal stage and takes place while the primordium is evaginated into the space between the epidermis and larval cuticle. In addition to a rapid prepupal growth phase, horn expression is also affected at times by a drastic pupal remodeling phase (C1 and C2) during the early pupal stage. During this stage pupal horn primordia are either converted into a future adult structure (C1) or resorbed (C2) via programmed cell death. In the later case, expression of Dll, but not hth or dac, is shifted more posteriorly. (D) Position of dac, hth, and Dll within the basic Drosophila limb patterning network (hh, hedgehog; en, engrailed; dpp, decapentaplegic; wg, wingless; EGFR, epidermal growth factor receptor; al, aristaless; b, bar; bab, bric a brac; exd, extradenticle).

Fig. 2.

Males (Upper) and females (Lower) of O. taurus (A) and O. binodis (B). Pupae are shown on the Left and corresponding adults on the Right. Arrows highlight cases of pupal horn resorption.

Fig. 3.

Larval RNAi-mediated transcript depletion of dachshund (A–C), homothorax (D–F), and Distal-less (G–I). Images illustrate typical phenotypes observed in each experiment compared with wild-type phenotypes. Graphs depict scaling relationships between pupal body size and horn length for thoracic horns (i) and head horns (ii). Pupal body size was measured as thorax width for dac and Dll. Hth RNAi affected thorax shape and we therefore used pupal mass as an estimator of body size. Wild-type is shown in blue and RNAi-treated individuals are shown in red. All data are from male O. taurus except Ii, which were collected from female O. binodis. Sample sizes are given in parentheses.