Teiya Kijimoto

DEVELOPMENTAL DECOUPLING OF ALTERNATIVE PHENOTYPES: INSIGHTS FROM THE TRANSCRIPTOMES OF HORN-POLYPHENIC BEETLES

Developmental mechanisms play an important role in determining the costs, limits, and evolutionary consequences of phenotypic plasticity. One issue central to these claims is the hypothesis of developmental decoupling, where alternate morphs result from evolutionarily independent developmental pathways. We address this assumption through a microarray study that tests whether differences in gene expression between alternate morphs are as divergent as those between sexes, a classic example of developmental decoupling. We then examine whether genes with morph‐biased expression are less conserved than genes with shared expression between morphs, as predicted if developmental decoupling relaxes pleiotropic constraints on divergence. We focus on the developing horns and brains of two species of horned beetles with impressive sexual‐ and morph‐dimorphism in the expression of horns and fighting behavior. We find that patterns of gene expression were as divergent between morphs as they were between sexes. However, overall patterns of gene expression were also highly correlated across morphs and sexes. Morph‐biased genes were more evolutionarily divergent, suggesting a role of relaxed pleiotropic constraints or relaxed selection. Together these results suggest that alternate morphs are to some extent developmentally decoupled, and that this decoupling has significant evolutionary consequences. However, alternative morphs may not be as developmentally decoupled as sometimes assumed and such hypotheses of development should be revisited and refined.

Diversification of doublesex function underlies morph-, sex-, and species-specific development of beetle horns.

Sex-specific trait expression is frequently associated with highly variable, condition-dependent expression within sexes and rapid divergence among closely related species. Horned beetles are an excellent example for studying the molecular basis of these phenomena because horn morphology varies markedly among species, between sexes, and among alternative, nutritionally-cued morphs within sexes. In addition, horns lack obvious homology to other insect traits and provide a good opportunity to explore the molecular basis of the rapid diversification of a novel trait within and between species. Here we show that the sex-determination gene doublesex (dsx) underlies important aspects of horn development, including differences between sexes, morphs, and species. In male Onthophagus taurus, dsx transcripts were preferentially expressed in the horns of the large, horned morph, and RNAi-mediated knockdown of dsx dramatically altered male horn allometry by massively reducing horn development in large males, but not in smaller males. Conversely, dsx RNAi induced ectopic, nutrition-sensitive horn development in otherwise hornless females. Finally, in a closely related species (Onthophagus sagittarius) that has recently evolved a rare reversed sexual dimorphism, dsx RNAi revealed reversed as well as novel dsx functions despite an overall conservation of dsx expression. This suggests that rapid evolution of dsx functions has facilitated the transition from a regular sexual dimorphism to a reversed sexual dimorphism in this species. Our findings add beetle horns to existing examples of a close relationship between dsx and sexual trait development, and suggest that dsx function has been coopted to facilitate both the evolution of environmentally-cued intrasexual dimorphisms and rapid species divergences in a novel trait.

Gene discovery in the horned beetle Onthophagus taurus

BackgroundHorned beetles, in particular in the genus Onthophagus, are important models for studies on sexual selection, biological radiations, the origin of novel traits, developmental plasticity, biocontrol, conservation, and forensic biology. Despite their growing prominence as models for studying both basic and applied questions in biology, little genomic or transcriptomic data are available for this genus. We used massively parallel pyrosequencing (Roche 454-FLX platform) to produce a comprehensive EST dataset for the horned beetle Onthophagus taurus. To maximize sequence diversity, we pooled RNA extracted from a normalized library encompassing diverse developmental stages and both sexes.ResultsWe used 454 pyrosequencing to sequence ESTs from all post-embryonic stages of O. taurus. Approximately 1.36 million reads assembled into 50,080 non-redundant sequences encompassing a total of 26.5 Mbp. The non-redundant sequences match over half of the genes in Tribolium castaneum, the most closely related species with a sequenced genome. Analyses of Gene Ontology annotations and biochemical pathways indicate that the O. taurus sequences reflect a wide and representative sampling of biological functions and biochemical processes. An analysis of sequence polymorphisms revealed that SNP frequency was negatively related to overall expression level and the number of tissue types in which a given gene is expressed. The most variable genes were enriched for a limited number of GO annotations whereas the least variable genes were enriched for a wide range of GO terms directly related to fitness.ConclusionsThis study provides the first large-scale EST database for horned beetles, a much-needed resource for advancing the study of these organisms. Furthermore, we identified instances of gene duplications and alternative splicing, useful for future study of gene regulation, and a large number of SNP markers that could be used in population-genetic studies of O. taurus and possibly other horned beetles.

EST and microarray analysis of horn development in Onthophagus beetles.

BackgroundThe origin of novel traits and their subsequent diversification represent central themes in evo-devo and evolutionary ecology. Here we explore the genetic and genomic basis of a class of traits that is both novel and highly diverse, in a group of organisms that is ecologically complex and experimentally tractable: horned beetles.ResultsWe developed two high quality, normalized cDNA libraries for larval and pupal Onthophagus taurus and sequenced 3,488 ESTs that assembled into 451 contigs and 2,330 singletons. We present the annotation and a comparative analysis of the conservation of the sequences. Microarrays developed from the combined libraries were then used to contrast the transcriptome of developing primordia of head horns, prothoracic horns, and legs. Our experiments identify a first comprehensive list of candidate genes for the evolution and diversification of beetle horns. We find that developing horns and legs show many similarities as well as important differences in their transcription profiles, suggesting that the origin of horns was mediated partly, but not entirely, by the recruitment of genes involved in the formation of more traditional appendages such as legs. Furthermore, we find that horns developing from the head and prothorax differ in their transcription profiles to a degree that suggests that head and prothoracic horns are not serial homologs, but instead may have evolved independently from each other.ConclusionWe have laid the foundation for a systematic analysis of the genetic basis of horned beetle development and diversification with the potential to contribute significantly to several major frontiers in evolutionary developmental biology.

Programed cell death shapes the expression of horns within and between species of horned beetles

SUMMARY Holometabolous insects provide an excellent opportunity to study both the properties of development as well as their evolution and diversification across taxa. Here we investigate the developmental basis and evolutionary diversification of secondary trait loss during development in the expression of beetle horns, a novel and highly diverse class of secondary sexual traits. In many species, horn growth during late larval development is followed by a period of dramatic remodeling during the pupal stage, including the complete resorption of horns in many cases. Here we show that programed cell death plays an important and dynamic role in the secondary resorption of pupal horn primordia during pupal development. Surprisingly, the degree of cell death mediated horn resorption depended on species, sex, and body region, suggesting the existence of regulatory mechanisms that can diversify quickly over short phylogenetic distances. More generally, our results illustrate that secondary, differential loss of structures during development can be a powerful mechanism for generating considerable morphological diversity both within and between species.

Emerging model systems in evo-devo: horned beetles and the origins of diversity

SUMMARY Horned beetles and beetle horns are emerging as a model system suited to address fundamental questions in evolutionary developmental biology. Here we briefly review the biology of horned beetles and highlight the unusual opportunities they provide for evo‐devo research. We then summarize recent advances in the development of new approaches and techniques that are now available to scientists interested in working with these organisms. We end by discussing ways to implement and combine these new approaches to explore new frontiers in evo‐devo research previously unavailable to reseachers working outside traditional model organisms.

Beetle horns and horned beetles: emerging models in developmental evolution and ecology

Many important questions in developmental biology increasingly interface with related questions in other biological disciplines such as evolutionary biology and ecology. In this article, we review and summarize recent progress in the development of horned beetles and beetle horns as study systems amenable to the integration of a wide range of approaches, from gene function analysis in the laboratory to population ecological and behavioral studies in the field. Specifically, we focus on three key questions at the current interface of developmental biology, evolutionary biology and ecology: (1) the developmental mechanisms underlying the origin and diversification of novel, complex traits, (2) the relationship between phenotypic diversification and the diversification of genes and transcriptomes, and (3) the role of behavior as a leader or follower in developmental evolution. For each question we discuss how work on horned beetles is contributing to our current understanding of key issues, as well as highlight challenges and opportunities for future studies. WIREs Dev Biol 2013, 2:405–418. doi: 10.1002/wdev.81

The nutritionally responsive transcriptome of the polyphenic beetle Onthophagus taurus and the importance of sexual dimorphism and body region

Developmental responses to nutritional variation represent one of the ecologically most important classes of adaptive plasticity. However, knowledge of genome-wide patterns of nutrition-responsive gene expression is limited. Here, we studied genome-wide transcriptional responses to nutritional variation and their dependency on trait and sex in the beetle Onthophagus taurus. We find that averaged across the transcriptome, nutrition contributes less to overall variation in gene expression than do sex or body region, but that for a modest subset of genes nutrition is by far the most important determinant of expression variation. Furthermore, our results reject the hypothesis that a common machinery may underlie nutrition-sensitive development across body regions. Instead, we find that magnitude (measured by number of differentially expressed contigs), composition (measured by functional enrichment) and evolutionary consequences (measured by patterns of sequence variation) are heavily dependent on exactly which body region is considered and the degree of sexual dimorphism observed on a morphological level. More generally, our findings illustrate that studies into the developmental mechanisms and evolutionary consequences of nutrition-biased gene expression must take into account the dynamics and complexities imposed by other sources of variation in gene expression such as sexual dimorphism and trait type.

Evolutionary and ecological genomics of developmental plasticity: novel approaches and first insights from the study of horned beetles.

Phenotypic plasticity pervades organismal development and physiology where it facilitates an enormous range of adaptive responses to novel or stressful environments. Plasticity also impacts evolutionary processes, reducing the probability of population extinction in the face of environmental changes and sometimes increasing speciation rates in developmentally plastic lineages. Despite the adaptive significance of plasticity, organisms are not infinitely plastic; rather they are constrained in the kinds and ranges of environmental changes to which their body parts, organs, and tissues can respond. Understanding the nature, costs, and limits of developmental plasticity requires insight into (i) the developmental-genetic and genomic mechanisms underlying plastic responses as well as (ii) their interplay with ecological and social conditions. In this chapter we review and summarize recent progress in the development of horned beetles as a study system with which to explore the interactions between changing ecological conditions and plastic, genome-wide responses in gene expression and developmental function. In particular, we focus on plastic responses to nutritional variation, which in horned beetles differ widely as a function of body region, sex, and species. We begin by introducing the study system and summarize the developmental-genetic and genomic tool set currently available for horned beetles. We then present recently developed statistical approaches that can be used to guide the design of multi-factorial genome-wide transcriptional comparisons when circumstances prohibit a fully balanced design. We present an example of such an approach in the horned beetle Onthophagus taurus and end by highlighting the growing opportunities for future ecological-genomic studies in horned beetles.