The origin of novel traits and their subsequent diversification have been central themes in evolutionary biology ever since the discipline's inception over 150 years ago [1
]. Specifically, the genetic, developmental, and ecological mechanisms, and the interactions between them, that allow novel phenotypes and functions to arise from pre-existing variation, continue to represent major frontiers in our understanding of phenotypic diversity. With the advent of modern -omics
approaches, researchers have increasingly departed from a candidate gene or pathway approach and begun to explore organismal development and evolution from a genome, transcriptome, or proteome perspective, focusing in large part on existing genetic model systems such as Drosophila
. However, many key questions in evolutionary biology, including the mechanisms underlying organismal innovation, the role of plasticity in diversification, and the interplay between ecology and developmental evolution, are often difficult to address solely within the confines of classic model systems. Recent efforts have therefore begun to generate genomic and developmental genetic resources for organisms with promise as future model systems in evolutionary developmental biology and ecological genetics (e.g. butterflies: [3
]; honey bees: reviewed in [5
]; red flour beetle: [6
]). Here we present and apply the first genomic resources to advance the study of a class of traits that is both novel and highly diverse in a group of organisms that is ecologically complex and experimentally tractable: beetle horns and horned beetles.
Beetle horns possess many characteristics that make them interesting models for integrating genetic, developmental, and environmental perspectives on the development and evolution of complex, novel traits (reviewed in [7
]). First, beetle horns are major structures, often dominating the phenotype of their bearers. Second, beetle horns function as weapons of sexual selection, thus playing a major role in the behavioral ecology of individuals and populations. Third, beetle horns are inordinately variable, both within and between species, including differences in number, size, shape, and location. Moreover, diversity in horn expression is paralleled by amazing species richness. For instance, the genus Onthophagus
currently contains over 2,400 extant species, making it the most speciose genus in the animal kingdom [8
]. Forth, beetle horns are influenced in their expression by both genetic and environmental factors, ranging from absence of environmental sensitivity to complete determination by nutritional condition. In some cases, both extremes of environmental sensitivity can be found in different horn types expressed by the same individual [10
]. Finally, beetle horns lack any obvious homology to structures in other insects or non-insect arthropods. Beetle horns are not modified antennae or mouthparts, but instead horns were "invented" by beetles in addition to their traditional appendages [11
], and now provide their bearers with an important new function: a weapon used in male-male competition. Beetle horns and horned beetles therefore offer rich opportunities to explore the mechanisms of organismal innovation and diversification.
Beetle horns are rigid outgrowths of the exoskeleton that originate as epidermal outbuddings of the head or prothoracic epithelium. Horns lack joints, muscles, and nervous tissue. Several recent studies have begun to shed light on how beetle horns develop and differentiate during ontogeny [11
] and showed that two developmentally dissociated processes contribute to the final degree of horn expression seen in adults: a prepupal growth phase late in larval development followed by a pupal remodeling phase just prior to the final adult molt (reviewed in [10
]). As such, the development of horns shows many qualitative similarities to the development of traditional appendages, but also exhibits important differences. For instance, prothoracic horn primordia are frequently resorbed during the pupal stage in a sex- and species-specific manner, a phenomenon not usually associated with regular appendages [14
]. Furthermore, earlier studies have also begun to question whether horns that develop in different body regions, such as the head vs
. prothorax, constitute serial homologs, or instead may have evolved and diversified independently of each other [13
Here we present the first steps toward a systematic analysis of the genetic and genomic basis of horn development and diversification in the genus Onthophagus. We first present the results of a comprehensive EST analysis of two normalized cDNA libraries obtained from two disparate developmental stages of Onthophagus taurus: larva and pupa. Second, using microarrays developed from our EST libraries we contrast the transcription profiles of the primordia of developing prothoracic horns, head horns, and legs right after the transition from larva to pupa. We then use these contrasts to identify candidate genes involved in the development and diversification of beetle horns. Furthermore, we examine two basic questions regarding the origin and diversification of horns. (a) Are horns highly simplified versions of more traditional appendages such as legs? If so, transcription profiles of developing horn primordia should largely match those of developing legs. If not, transcription profiles of developing horn primordia should only partly match those of developing legs and also include horn specific transcription signatures. (b) Are different horn types produced in different body regions homonomous, i.e. serial homologs of the same ancestral structure? If so, different horn types should exhibit highly similar transcription profiles. However, if different horn types originated and diversified independently of each other, transcription profiles may be predicted to exhibit important horn-type specific differences. We discuss the significance of our findings in the context of the biology of horned beetles in particular, and the origins and diversification of novel traits in general.