I: OVERVIEW
II: BEETLE HORNS AND HORNED BEETLES - UNIQUE AND DIVERSE
III
: CURRENT RESEARCH OBJECTIVES
- BEYOND HORNED BEETLES -
I: OVERVIEW
Our research focuses on a central question in biology: how do major phenotypic novelties originate and diversify in nature. In particular we are interested in the ecological, developmental, and genetic mechanisms, and the interactions between them, that drive evolutionary innovation and diversification. To tackle these issues from a variety of perspectives and at different levels of biological organization we use approaches ranging from molecular developmental biology and genomics to quantitative genetics, comparative endocrinology, and behavioral ecology. Below we first provide a brief summary of the biology of our main study organisms: horned beetles in the genus Onthophagus, followed by a summary of our current research foci.
II: BEETLE HORNS AND HORNED BEETLES- UNIQUE AND DIVERSE 
Beetle horns and horned beetles combine several characteristics that make them outstanding models for studying the origin and diversification of novel traits (Moczek 2006a). (1) Beetle horns are large structures, often dominating the phenotype of their bearers. (2) Beetle horns function as weapons in male combat, thus playing a major role in the behavioral ecology of individuals and populations. (3) Beetle horns are inordinately variable within sexes, between sexes, and between species, including differences in number, size, shape and location. (4) Beetle horns are influenced in their expression by both genetic and environmental factors, ranging from absence of environmental sensitivity to complete determination by nutritional conditions. In some cases, both extremes of environmental sensitivity can be found in different horn types expressed by the same individual. (5) Beetle horns lack obvious homology to structures in other insects. In other words, beetle horns are not just modified antennae or mouthparts, instead horns were "invented" by beetles in addition to traditional appendages and now provide their bearers with an important new function: a weapon used in male-male competition. (6) Work in our lab over the past 4 years has substantially increased the experimental tool box available for the most diverse group of horned beetles - the genus Onthophagus. As detailed below, this tool box now allows us to explore molecular, genetic, genomic, hormonal, and behavioral components of horn formation. Combined with the tremendous diversity in horn phenotypes that exists among closely related individuals and species we are now in a position to identify the genetic, developmental, and physiological mechanisms, and the interactions between them, that underlie horns and horn diversity, as well as the ecological components that shape this diversity in natural populations (Moczek et al. 2007).
III: CURRENT RESEARCH OBJECTIVES
Overview: Our main objective is to better understand what needs to come together genetically, developmentally, and ecologically for major complex traits to originate and diversify. We mostly use beetle horns and horned beetles as a microcosm to explore this issue, and detailed below are the current objectives of this effort. Highlighted towards the end of this section are our recent attempts to establish a second group of organisms and class of novel traits - fireflies and their bioluminescent lanterns - to begin exploring which, if any, common themes exist in the mechanics of innovation and diversification in nature.
A: WHICH DEVELOPMENTAL AND GENETIC MECHANISMS UNDERLIE THE FORMATION OF HORNS AND HORN DIVERSITY?
Introduction and background: This objective lies at the heart of the research efforts in our lab, for three reasons. (1) First, beetle horns represent complex, novel traits that lack obvious precursors in other organisms. Evolutionary biologists know remarkably little about how such traits come into being, as well as the developmental and genetic changes that accompany this process. Through this aim we want to identify which genes and developmental pathways are involved in the making of horns, and how their respective contributions have changed as beetles horns and horned beetles have diversified. (2) Secondly, past research on horned beetles, and most other models in Evolutionary Developmental Biology and allied disciplines, has relied solely on comparative gene expression studies of a few, selected candidate genes to explore the developmental regulation and integration of organisms and their parts. In other words, the vast majority of genes relevant to a given trait is likely to go unidentified, and the exact functions of those genes that are identified are only inferred indirectly through expression patterns. By having developed both genomic tools and gene-function-approaches for horned beetles we can now overcome both limitations. These resources have now set the stage to further develop Onthophagus horned beetles into a powerful model system in Evolutionary Developmental Biology. (3) Thirdly, horned beetles provide the rare opportunity to connect micro- and macroevolution of development. Macroevolution of development focuses on the large-scale similarities and differences in developmental regulation observed between distantly related organisms, such as insects and vertebrates, and represents the traditional focus in Evolutionary Developmental Biology. While the contributions of this focus have been absolutely critical, a macro-evolutionary perspective on developmental evolution has been unable to explore the initial causes and conditions required to initiate and shape evolutionary changes in development. Here, a micro-evolutionary perspective, focusing on populations and closely related species, is critical. Onthophagus horned beetles provide substrate for both perspectives, including rapidly evolving populations (less than 50 years of isolation), sister-species (~10,000 years of isolation) and well established species (5-50 Million years of separation). More importantly, all are accessible in nature and can be studied in the lab.
Major findings: (1) To date, our research has shown that the origin of beetle horns was made possible through a combination of pre-existing developmental and genetic mechanisms that have been recruited into a new developmental context (Moczek and Nagy 2005, Moczek et al. 2006a; Moczek 2006b; Moczek and Rose 2009). While the details of these results were often surprising (i.e. combination of genes and pathways, or time and place of their activation) the general result confirms a well-established theme in Evolutionary Developmental Biology: novel traits do not require new genes or developmental pathways to come into being, but instead arise from recruitment of already existing developmental machinery into new contexts. (2) In addition to identifying conserved or putatively novel regulatory properties of interesting genes and pathways, our studies also unearthed tremendous, and surprising, variation in these properties between morphs, sexes, populations and species (Moczek and Nagy 2005, Moczek et al. 2006a; Moczek 2006a,b; Wasik et al. in prep; Moczek and Rose 2009). Combined, these results have three major implications. First, they contradict the common notion that master regulatory genes should be evolutionarily entrenched given their importance in the regulation of basic aspects of animal architecture and thus resistant to the acquisition of novel functions (Davidson and Erwin 2006). Instead, our findings illustrate that regulatory genes whose functions are otherwise highly conserved nevertheless retain the capacity to acquire additional functions. Second, results to date suggest that little phylogenetic distance is necessary for the evolution of sex- and species-specific differences in these functions. Thuis suggests that even master-regulator genes and their interactions can diversify on the level of populations and species with unexpected ease. (3) Third, many of the developmental differences seen between species, such as the presence or absence of horns or horn expression in different body regions, have striking parallels in sexual dimorphisms or male dimorphisms, which raises the possibility that the developmental capacity to generate macroevolutionary differences may originate well within species, between sexes, and - fueled by developmental plasticity - across alternative morphs.
B: WHAT IS THE GENETIC BASIS OF NUTRITION-SENSITIVE DEVELOPMENT? HOW DOES NUTRITION-SENSITIVITY EVOLVE?
Introduction and background: Genetic and environmental inputs are fundamental to the development and integration of all multicellular organisms and their parts, yet the precise genetic mechanisms underlying environment-sensitive development remain very poorly understood. This in turn limits our ability to understand how such interactions evolve and diversify. Onthophagus horned beetles represent a remarkable opportunity to study how an environmental factor of fundamental importance to most organisms, nutrition, affects the formation and diversification of a complex morphological trait, horns.
Onthophagus horned beetles are well known for their conditional, nutrition-dependent, development of horns in males: in many species only male larvae with access to optimal feeding conditions express horns, whereas smaller male larvae remain hornless. In such species, males occur in discrete horned and hornless morphs separated by a sharp size threshold. In contrast, horn development in other species is largely insensitive to nutritional variation. Here, large individuals are simply proportionately enlarged versions of smaller individuals and no alternative morphs exist. In some cases, both extremes of nutritional sensitivity can be found in different horn types present in the same individual.
Our work takes advantage of the existence of nutrition-sensitive and insensitive development in Onthophagus beetles in order to identify on one side genes and developmental pathways that underlie nutrition sensitivity, and on the other explore the behavioral and ecological conditions that shape the evolution of nutrition-sensitivity in nature. Through the recent development of gene-function and genomic resources in our lab Onthophagus beetles now represent a rare model system that permits an integration of evolutionary ecology and developmental genetics.
Major findings: To date, our research has documented the role of quantity and quality of nutrition in horn development (Moczek 1998; Moczek and Emlen 1999) and scaling (Moczek 2001), and the interplay between nutritional quality and maternal provisioning behavior (Moczek 1998. In a series of studies we have then explored the ecological mechanisms that can cause populations to diverge in patterns of resource utilization (Moczek 2003, Moczek and Nijhout 2002). More recently, microarray-based expression analyses have identified the first candidate genes that may link variation in nutrition to variation in trait expression (Kijimoto et al., in review). Many of those genes, such as those involved in insulin signaling, also play a major role in how humans interact with nutritional variation. Experiments are now on their way to explore the evolutionary history and developmental function of these genes in the origin and diversification of nutrition-sensitive development in Onthophagus horned beetles.
C: WHAT ARE THE INTERACTIONS BETWEEN GENETIC AND HORMONAL REGULATORS DURING HORN DEVELOPMENT AND EVOLUTION?
Introduction and major findings: Interactions between genetic and hormonal (endocrine) regulators play a pivotal role in the development of most multicellular organisms including humans. Hormonal signals modulate the activation or repression of genes, often in response to environmental conditions, and genes and their products affect when, where, and how much hormones are produced, released or metabolized. However, degree and nature of these interactions remain poorly understood. Onthophagus horned beetles provide a rewarding microcosm in which to study the interplay between genetic and hormonal regulators of development, and the role of environmental conditions (such as nutrition, see above) in this interplay.
For example, we have shown that a common insect hormone, juvenile hormone (JH) is likely to play important roles in several aspects of horn development in male beetles. Specifically , male larvae in many species develop into alternative horned (aggressive fighter) or hornless (non-aggressive sneaker) adults depending on larval nutrition, and juvenile hormone appears to mediate between nutritional conditions experienced by larvae, and the final horn phenotype expressed by adults (Moczek and Nijhout 2002). Furthermore, we have documented heritable differences in the timing and degree of sensitivity to juvenile hormone between populations of horned beetles. This suggested that evolutionary changes in juvenile hormone action provide developmental mechanism by which beetle horn formation could begin to diversify in natural populations (Moczek and Nijhout 2002). More recently, two undergraduate researchers in our lab showed that juvenile hormone also regulates the expression of sexual dimorphism in horn shape and size, however, this 
time independent of nutritional conditions (Moczek et al. 2007). These findings support a role of juvenile hormone in the development and diversification of horns, and highlight juvenile hormone as a critical mediator between nutritional variation experienced by larvae and differences in horn formation present in adults. However, the genetic and developmental processes that juvenile hormone interacts with are entirely unknown. Similarly, if and how these interactions change of evolutionary time scales, and the corresponding causes and consequences, are completely unclear.
Our current research therefore focuses on identifying genes and developmental pathways whose activation or repression during horn development is sensitive to JH, and to characterize their function via RNAinterference. Moreover, we want to identify the level of conservation or lability in hormone-gene interactions over evolutionary time scales, with special emphasis on those genes that we have identified previously to also play a role in nutrition-sensitive development. For example, if species differ in the level nutritional sensitivity, is this brought about by evolutionary changes in the identity of hormonal target genes, changes in the nature of interactions between hormone and target gene (e.g. activational vs. repressive) or are do evolutionary changes in nutritional-sensitivity generated by mechanisms outside hormone target gene interactions.
D: HOW DO ECOLOGICAL AND SOCIAL CONDITIONS INFLUENCE EVOLUTION OF DEVELOPMENTAL PROCESSES?
Introduction and background: The horns of beetles function as weapons in male combat over females. Not all males fight, however, and especially in species with alternative horned and hornless male morphs, smaller males frequently rely on non-aggressive sneaking behavior to gain access to females. While horns improve fighting success, they impede sneaking abilities, hence horned and hornless males morphs represent alternative solutions to the same problem: gaining access to females in the face of competition.
The horns of beetles therefore have a rich behavioral and social context. Documenting use and consequences of horn possession as well as the existence and success of alternative reproductive tactics presented our first entryway into the biology of horned beetles (Moczek 1998, 1999, 2002, 2003; Moczek and Emlen 1999, 2000) and the present research objectives now permits us to reconnect to this starting point. Specifically, given our increasing insights into the genetic, developmental, and hormonal mechanisms that underlie the formation and diversification of horns, our goal is now to understand (i) how these mechanisms and the interactions between them evolve in natural populations and (ii) how the ecological and social conditions within which horns and their bearers function shape evolutionary changes in horn development.
Major findings: To date, our research allowed us to characterize the behavioral and ecological context of beetle horns in a fair amount of detail. Most importantly, we have been able to document the existence of alternative reproductive tactics (Moczek and Emlen 2000), measure the costs and benefits of horn possession (Moczek and Emlen 2000; Madewell and Moczek 2006), and explore the role of paternal and maternal behavior in offspring development and performance (Moczek 1998, 1999). Most recently, an undergraduate researcher in our lab has been able to document alternative reproductive tactics in female beetles as well, which has important implications for our understanding of the evolution of female horns in some species. As a second step, we have begun to take advantage of several geographically isolated populations of horned beetles to explore the behavioral and social mechanisms that bring about divergences in horn formation on the level of populations (Moczek et al. 2002; Moczek 2003, Moczek and Nijhout 2003), the hormonal mechanisms that mediate such divergences (Moczek and Nijhout 2002), and more recently, the degree to which these divergences reflect divergences normally seen between species (Pizzo et al. 2008). We further intensified our integration of behavioral and social aspects of horned beetle biology on one side, and developmental genetic and physiological aspects on the other, through the study of trade-offs during development and evolution. Trade-offs can occur when the enlargement of one structure, organ, or body part is only feasible at the expense of another. Trade-offs arising during development (if, for example, organs are limited by a finite amount of resources to sustain their growth) also have the potential to bias evolutionary outcomes. If natural selection favors the enlargement of a given organ, this may be only feasible developmentally through the corresponding reduction of another. Along these lines, we have recently documented that the formation of long horns during the development of large males may result in reduced muscle development and compromises the later ability to effectively thermoregulate in the face of temperature fluctuations (Shepherd et al. 2008). Similarly, we found that the development of horns trades off with the development of primary sexual traits such as the copulatory organ: populations or species whose social conditions force them to invest more into horns end up investing less into copulatory organs and vice versa (Parzer and Moczek 2008). This later trade-off is particularly intriguing as changes in copulatory organ size in insects are generally thought to play a major role in the formation of new species, and we are currently investigating the developmental and hormonal underpinnings of this trade-off as well as its behavioral consequences.
With the recent addition of genomic and gene-function approaches we are now in a position to take it one step further and indentify and examine key genes and developmental pathways that link behavioral, developmental, and endocrine components of the biology of horned beetles. For example, recent RNAinterference-mediated gene knockdown identified a subset of genes whose effects appear to be most pronounced in horns and copulatory organs, suggesting candidate genes that may underlie the trade-offs we have observed in natural populations and species.
E: BEYOND HORNED BEETLES: DEVELOPMENT OF FIREFLIES AS A MODEL SYSTEM FOR STUDYING THE EVOLUTION OF COMPLEX TRAITS
Introduction and background: Our research into the biology of beetle horns and horned beetles has begun to establish horned beetles as a rewarding microcosm for exploring the mechanisms that underlie innovation and diversification in nature. As we are learning more about this system, it is important to begin exploring other groups of organisms and trait classes to determine if and how common themes exist in how nature evolves complex new traits. Over the past year 
graduate student Matt Stansbury and I have begun to develop local and regional firefly species into a possible model system. Fireflies are beetles famous for their nightly bioluminescent displays, produced by an organ, called the lantern, which is unique to these organisms and unlike anything known from other insects. This organ is diverse within and between species, and much like the horns of horned beetles represent an evolutionary novelty that fireflies somehow invented during their evolutionary history. Our goal is to explore the genetic and developmental origins of firefly lanterns, and the ecological conditions that have shaped their diversity.
Major findings and productivity to date: This is a project in its early stages, but one which holds terrific promise. So far we have been able to establish, rear and experimentally manipulate firefly larvae and adults in the lab, and have begun to clone the first candidate genes from their tissues. We have also completed the first expression analyses, which suggest that at least some of the genes used normally in the patterning of appendages also pattern the development of the firefly lantern. In addition, we have developed successful RNAinterference protocols for Photuris fireflies and are currently using them to examine the functions of selected candidate genes in the development and evolution of firefly lanterns.
