The process of innovation in evolution has captivatedevolutionary biologists ever since the inception of the dis-cipline (Raff 1996; West-Eberhard 2003). What does ittake genetically, developmentally, or ecologically fornovel traits to arise and diversify in nature? Is the origin ofnovel traits underlain by processes separate from thosethat govern quantitative changes in preexisting traits or isinnovation merely an extrapolation of diversification overtime (Erwin 2000; Davidson and Erwin 2006; Moczek2008)? In this chapter, I propose that beetle horns andhorned beetles offer unusual opportunities to integrategenetic, developmental, and ecological mechanisms into aholistic understanding of how novel complex traits origi-nate and diversify during both development and evolution.Specifically, I highlight and synthesize recent advances inour understanding of the genetic, developmental, and eco-logical origins of horns and horn diversity, as well as theirconsequences for diversification and radiation of hornedbeetles. I begin with a brief review of the basic biology andnatural history of horned beetles.

A BRIEF NATURAL HISTORY OF BEETLEHORNS AND HORNED BEETLES

Beetle horns unite several characteristics that makethem outstanding models for exploring the origin anddiversification of novel traits (Moczek 2005). First, bee-tle horns are massive, solid, three-dimensional out-growths that often severely transform the shape ofwhoever bears them (Fig. 1). Second, beetle horns func-tion as weapons in male competition over breeding oppor-tunities, thereby defining the behavioral ecology ofindividuals and populations. Third, beetle horns are inor-dinately diverse (Figs. 1 and 2A). Horns differ in size,shape, number, and location of expression, and much ofthis variation can be found not only among species, butalso between, and oftentimes within, sexes. Finally, andmost importantly, beetle horns are unique structures in thesense that they lack clear homology with other traits in

insects. Horns are not modified mouthparts or legs;instead, they exist alongside these structures in bodyregions in which insects normally do not produce any out-growths (Moczek 2005, 2006a). Hence, we can look athorns as an example of an evolutionary novelty thathorned beetles invented at some point during their historyand which has since fueled one of the most impressiveradiations of secondary sexual traits in the animal king-dom (Arrow 1951; Balthasar 1963; Emlen et al. 2007).Here, I explore the mechanisms that have mediated theinitial origin and subsequent diversification of horns.Specifically, I argue that the origin and diversification ofhorns was made possible through (1) widespread coop-tion of preexisting developmental mechanisms into newdevelopmental contexts, (2) exaptation of preexistingstructures that originally performed unrelated functions,and (3) trade-offs arising during development that bias,and possibly accelerate, patterns of diversification.However, before introducing any of these mechanisms,we must first briefly review the basic biology of horndevelopment and formation.

THE ONTOGENY OF HORNS

The horns of beetles first become discernible duringthe last larval stage as the animal nears the larval-to-pupalmolt (Fig. 2B) (for review, see Moczek 2006a; Moczekand Rose 2009). At this stage, selected regions of the epi-dermis detach from the larval cuticle and proliferateunderneath. The resulting tissue is thrown into folds as itis trapped underneath the larval cuticle, but it expandsonce the animal molts to the pupal stage. It is at this stagethat horns become visible externally for the first time.This period of prepupal horn growth is then followed bya period of pupal remodeling of horn primordia. Duringthe pupal stage, pupal horns undergo at times substantialremodeling in both size and shape, including, in somecases, the complete resorption of horns before the adultmolt. After the competition of the pupal-to-adult molt,

On the Origins of Novelty and Diversity in Development and Evolution: A Case Study on Beetle Horns

A.P. MOCZEKDepartment of Biology, Indiana University, Bloomington, Indiana 47405

Correspondence: armin@indiana.edu

The origin of novel features continues to represent a major frontier in evolutionary biology. What are the genetic, develop-mental, and ecological processes that mediate not just the modification of preexisting traits, but the origin of novel traits thatlack obvious homology with other structures? In this chapter, I highlight a class of traits and organisms that are emerging asnew models for exploring the mechanisms of innovation and diversification in nature: beetle horns and horned beetles. Here,I review recent significant findings and their contributions to current frontiers in evolutionary developmental biology.

Cold Spring Harbor Symposia on Quantitative Biology,Volume LXXIV. ©2009 Cold Spring Harbor Laboratory Press 978-087969870-6 289

Figure 1. Examples of horned beetles illustratingdiversity and magnitude of horn expression inadult beetles. (Clockwise from top left) Phanaeusimperator, Eupatorus gracilicornis, Onthophaguswatanabei, Golofa claviger, and Trypoxylus(Allomyrina) dichotoma.

Figure 2. Diversity and development of beetle horns. (A) Diversity in number, size, location, and shape of horn expression between(A1) and within (A2) species of Onthophagus. (B) Drosophilamodel of limb formation compared to (C) the development of a thoracicbeetle horn from embryo to adult. (Black) Cuticle, (blue) epidermis, including schematic expression domains of the proximo/distalpatterning genes homothorax (hth, yellow), dachshund (dac, green), and Distal-less (Dll, red). Drosophila legs develop from imagi-nal discs, epidermal invaginations specified during embryonic development, which grow throughout larval development. Patterningtakes place while the disc is a two-dimensional sheet of tissue, and all disc growth occurs while the disc is invaginated into the bodyinterior. In contrast, beetle horns appear not to be specified during embryonic development. Instead, horns grow from the start as three-dimensional epidermal outbuddings, and all growth is confined to the relatively brief prepupal stage and takes place while the pri-mordium is evaginated into the space between epidermis and larval cuticle. In addition to a rapid prepupal growth phase, hornexpression is also affected by an at times drastic pupal remodeling phase (C1, C2) during the early pupal stage. During this stage, pupalhorn primordia are either converted into a future adult structure (C1) or resorbed (C2) via programmed cell death (PCD). In the lattercase, expression of Dll, but not hth or dac, is shifted more posteriorly. (D) Position of dac, hth, and Dll within the basic Drosophilalimb 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). (Modified from Moczek and Rose 2009.)

horns have then attained their final adult size and shape.The horns of adult beetles are thus the product of (1) a pre-pupal growth phase followed by a (2) pupal remodelingphase, and therefore develop in many ways similar to tra-ditional appendages in other holometabolous insects(Svácha 1992). However, more substantial differencesexist when compared to appendage formation in the beststudied insect: Drosophila. Here, appendages developfrom imaginal discs, which represents a highly derivedmode of appendage formation absent in the majority ofinsect orders (Kojima 2004). Imaginal discs are epidermalinvaginations specified during embryonic developmentthat grow throughout larval development. More over,many important patterning steps take place while the discis a two-dimensional sheet of tissue, and most disc growthoccurs while the disc is invaginated into the body interior(Fig. 3A). Beetle horns differ in that they appear not to bespecified during embryonic development, grow rightaway as three-dimensional epidermal outbuddings, havetheir growth confined to the relatively brief period justpreceding the larval-to-pupal molt, and as they grow,evaginate into the space between epidermis and larvalcuticle (see Fig. 2) (Moczek 2006a). Un for tun ately, mostof our understanding of insect appendage formationcomes from studies of imaginal disc development inDrosophila, and as such, the Drosophila model of limb

development represents our best starting point to beginexploring the regulation of horn growth and differentia-tion.

THE ORIGIN OF NEW THROUGH COOPTION OF THE OLD. I: THE REGULATION OF HORN GROWTH

Beetle horns can be thought of, at least in some ways,as simplified appendages. Alhough they lack muscles,nerves, or joints, they are three-dimensional outgrowthsof epidermal origin with clearly defined proximodistal,mediolateral, and anteroposterior axes. Recent studiesshow that the similarities do not end on the surface butthat much of the underlying developmental machineryused in the making of traditional appendages such as legsand antennae has been redeployed in the development andevolution of horns. The first clues came from a series of expression studies

which showed that several cardinal appendage patterninggenes known to have important roles in establishing theproximodistal axis of insect appendages (Distal-less [Dll],aristaless [al], dachshund [dac], homothorax [hth],extradenticle [exd]) were expressed during horn formation(Moczek and Nagy 2005; Moczek et al. 2006). Recentgene function analyses using RNA interference (RNAi)-

ORIGIN AND DIVERSIFICATION OF BEETLE HORNS 291

Figure 3. Larval RNAi-mediated transcript depletion of dachshund (A–C), homothorax (D–F), and Distal-less (G–I). Images illus-trate typical phenotypes observed in each experiment compared to wild-type phenotypes. Graphs depict scaling relationships betweenpupal body size and horn length for thoracic horns (i) and head horns (ii). Pupal body size was measured as thorax width for dac andDll. hthRNAi affected thorax shape and we therefore used pupal mass as an estimator of body size. (Blue) Wild type, (red) RNAi-treated individuals. All data are from male O. taurus except I(i), which were collected from female O. binodis. Sample sizes are givenin parentheses. (Modified from Moczek and Rose 2009.)

mediated transcript depletion further refined our insightsinto the roles of a subset of these genes. Specifically,Moczek and Rose (2009) examined the function of dac,hth, and Dll during horn development in Onthophagusbeetles, a focal genus of horned beetles for evo-devo stud-ies. Independent of any involvement in horn development,larval RNAi-mediated transcript depletion of all three pat-terning genes generated phenotypic effects identical orsimilar to those documented by previous studies in othertaxa, such as loss or fusion of distal and medial leg andantennal regions in the case of Dll (Prpic et al. 2001;Angelini and Kaufman 2004; Koijma 2004) or acceleratedeye differentiation and ectopic wing tissue formation onthe first thoracic segment in the case of hth (Ryoo et al.1999; Yao et al. 1999; Bessa et al. 2002). These resultsdocumented that all three patterning genes exhibited con-servation of function with respect to the patterning of tra-ditional appendages as well as the general feasibility oflarval RNAi in Onthophagus beetles. In addition, however,this study yielded many surprising insights into the func-tional regulation of horn development, including unex-pected twists that would have been undetectable using atraditional gene expression approach. For instance, despitebeing widely expressed throughout prepupal horn primor-dia in Onthophagus (Moczek et al. 2006), dac did notappear to have any obvious role in the regulation of size,shape, or identity of horns. Instead, dacRNAi individualsexpressed thoracic and head horns of precisely the samesize and overall shape as control animals despite severedac knockdown phenotypes elsewhere in the same indi-viduals. In contrast, hthRNAi had a dramatic effect onhorn expression, but only affected horns produced by theprothorax. Head horns expressed by the same individualswere completely unaffected, despite severe effects onother head appendages such as antennae. The results ofDllRNAi only added to the complexity. Unlike hth,DllRNAi affected the expression of both head and thoracichorns but not in the same individuals or even species. In O.taurus, head horn expression was only affected in largemales otherwise determined to express a full set of headhorns, whereas horn expression in small- and medium-sized males was unaffected. Similarly unaffected was theexpression of pupal thoracic horns in both males andfemales regardless of body size. In the congener O. bin-odis, however, DllRNAi affected the expression of tho-racic horns in both males and females, although the effectwas strongest in large individuals. Combined, these resultsillustrate that Onthophagus Dll and hth, but not dac, alterhorn expression in a sex-, body-region-, and body-size-specific manner and that even closely related species candiverge rather substantially in aspects of this regulation. More generally, these results suggest that horn develop-

ment evolved via differential recruitment of at least someproximodistal axis patterning genes normally involved inthe formation of traditional appendages. On one hand,these results are not surprising because they confirm ageneral theme in the evolution of novel traits: New mor-phologies do not require new genes or developmentalpathways and instead may arise by recruiting existingdevelopmental mechanisms into new contexts (Shubin et

al. 2009). On the other hand, these results also highlightedan unexpected degree of evolutionary lability, rangingfrom the absence of patterning function (dac) to pattern-ing function in selected horn types only (hth, Dll) to func-tion in one size class, sex, or species but not another (Dll).Combined, these data suggest that different horn types,and even the same horn type in different species, may beregulated at least in part by different pathways and that dif-ferent horn types may therefore have experienced distinct,and possibly independent, evolutionary histories. Similarconclusions emerge when we take a closer look at the reg-ulation of the second developmental period relevant toadult horn expression: pupal remodeling.

THE ORIGIN OF NEW THROUGH COOPTION OF THE OLD. II: THE

REGULATION OF PUPAL REMODELING

During the pupal stage, horns are sculpted into theirfinal adult shape. As such, pupal remodeling of horns isnot unusual; instead, all pupal appendages and bodyregions of holometabolous insects undergo at least somedegree of sculpting during the pupal stage (Cullen andMcCall 2004). What is unusual, however, is the oftenextreme nature of pupal horn remodeling, especially withrespect to horns emanating from the thorax (Moczek2006b). Here, horn remodeling is so extreme that it oftenresults in the complete resorption of pupal horn primor-dia, causing fully horned pupae to molt into thorax horn-less adults lacking any signs of the previous existence ofa thoracic horn primordium (Fig. 4). Moreover, pupalhorn resorption may or may not affect both sexes equally.For instance, of 19 Onthophagus species studied thus far,three species used female-specific resorption of thoracichorn tissue to generate sexual dimorphism (only malesremained horned) and one species used male-specificresorption of thoracic horn tissue, leaving females as theonly sex with thoracic horns. The remaining 15 speciesused the same process to remove thoracic horn primordiain both sexes. In at least one of these, O. taurus, pupal tho-racic horn resorption actually eliminated a pronouncedsexual dimorphism in thoracic horns evident in pupae butnot in the resulting adults (Moczek et al. 2006). Com -bined, these data suggest that pupal horn resorption isincredibly widespread at least in Onthophagus, but at thesame time, it is evolutionarily labile with respect to thesex in which it operates (Fig. 4). Recent work nowstrongly implicates programmed cell death (PCD) in theresorption of horn primordial tissue. PCD mediates the coordinated destruction of cells and

their content (Potten and Wilson 2004). As such, PCD usesa complex cascade of developmental and cellular processes.Despite this apparent complexity, PCD is an ancient physi-ological process used by all metazoan organisms to elimi-nate cells during development (Potten and Wilson 2004).Recent work has now shown that primordial horn epidermisfated to be resorbed undergoes premature PCD during thefirst 48 hours of the pupal stage (Kijimoto et al. 2009).Relying on two different biochemical assays, the same studythen showed that PCD is considerably more frequent among

292 MOCZEK

horn primordial cells of transient horns compared to indi-viduals whose pupal horns persist and become convertedinto an adult structure. Comparisons across species suggestthat the exact position and timing of PCD-mediated hornremodeling can differ remarkably from one species to thenext. Combined, the regulation of pupal remodeling there-fore reveals many of the same features highlighted above forthe regulation of horn growth. On one hand, pupal remodel-ing and resorption of horns appears to rely on a preexistingdevelopmental machinery that became recruited into a newdevelopmental context. On the other hand, remarkable vari-ation exists within and among species regarding when,where, and how much remodeling and resorption of hornsoccurs. By extension, this variation suggests the existenceof rapidly evolving modifier mechanisms that regulatespecies-, sex-, and body-region-specific resorption ofhorns. The identity and nature of these modifier mecha-nisms are currently being investigated.It is important to realize that this is clearly just the

beginning of a more-detailed analysis of the developmen-tal regulation and diversification of beetle horns. Therecent development of Onthophagus expressed sequencetag (EST) libraries, microarrays, and transcriptome pro-files has now rapidly increased the number and diversityof genes and pathways implicated in the regulation ofhorns and horn diversity (Kijimoto et al. 2009). Althoughmuch work clearly lies ahead before we achieve a satis-factory understanding of the origins of horn development,it appears that the most critical resources are now avail-able to place this goal within reach.

EXAPTATION AND THE ORIGIN OF ADULT THORACIC HORNS

PCD-mediated resorption of pupal thoracic horn tissueis ubiquitous among Onthophagus species, raising thequestion as to the adaptive significance, if any, of such tran-sient horn expression. Why expend all this energy to builda conspicuous pupal outgrowth if seemingly all that hap-pens to it is its subsequent removal through PCD? Ex per i -mental approaches have now revealed that pupal horns,regardless of whether they are resorbed or converted intoan adult structure, have a crucial role during the larval-to-pupal molt and especially the removal of the larval headcapsule (Moczek et al. 2006). Unlike in larval-to-larval andpupal-to-adult molts, larvae that molt into pupae have very

little muscle tissue left that could aid in the shedding of thelarval cuticle. Instead, animals rely on peristaltic contrac-tions to pump hemolymph and the swallowing of air toinflate selected body regions and to force old cuticle to rup-ture. This suffices for the removal of the thoracic andabdominal cuticles of larval scarab beetles that are highlymembranous and paper-thin. However, shedding the larvalhead capsule poses much greater challenges because it iscomposed of extremely thick cuticle. During larval life,this robust cuticle provides important attachment points forthe powerful jaw muscles of fiber-feeding scarab larvae,such as Onthophagus. Histological studies have nowshown that during Onthophagus’ prepupal stage, thoracichorn primordia force themselves into the space vacatedbetween the larval head capsule and corresponding epider-mis, fill with hemolymph, and expand. This expansionforces the larval head capsule to fracture along preexistinglines of weakness. As a consequence, as the larval headmolts into a pupal head, the first pupal structure visiblefrom the outside is not a part of the head, but the thoracichorn primordium as it bursts through the head capsule.When the precursor cells that would normally give rise tothoracic horn primordia are removed before the prepupalstage, the resulting pupae not only lacked a thoracic horn,but also failed to shed their larval head capsule (Moczek etal. 2006). Replicating this approach across Onthophagusspecies as well as outside the genus showed that this puta-tive dual function of thoracic horn primordia appears to beunique to onthophagine beetles. Phylogenetic analysessuggested that the pupal molting function of horns mayhave preceded the horns-as-a-weapon function of the adultcounterparts and that ancestrally, pupal horns may havealways been resorbed before the adult molt (Moczek et al.2006). If correct, this would explain why prepupal thoracichorn growth has been maintained in so many Onthophagusspecies even though the resulting pupal horns are not usedto form a functional structure in the adult.These results also raise the possibility that the first ori-

gin of adult horns could have involved a simple failure toremove otherwise pupal-specific projections via PCD.Anecdotal evidence suggests that such events occur in nat-ural populations frequently enough to be detected by ento-mologists (see, e.g., Paulian 1945; Ziani 1994; Ballerio1999). Although such a failure would have resulted in anadult outgrowth that at first would have been rather small,behavioral studies have shown that even very small

ORIGIN AND DIVERSIFICATION OF BEETLE HORNS 293

Figure 4. Pupal horn remodeling is common in the genus Onthophagus yet variable among species. Males (top) and females (bottom)of four Onthophagus species. Pupae are shown on the left and corresponding adults on the right. (A) O. nigriventris, (B) O. binodis,(C) O. sagittarius, (D) O. taurus. Arrows highlight cases of pupal horn resorption. (Modified from Moczek 2009.)

increases in horn length are sufficient to bring about sig-nificant increases in fighting success and fitness (Emlen1997; Moczek and Emlen 2000). Behavioral studies havealso shown that fighting behavior is widespread amongbeetles, occurring well outside horned taxa, and that pos-session of horns is not a prerequisite for fighting.Combined, this may have created a selective environmentin which the first pupal horn that failed to be removedbefore the pupal-to-adult molt could have provided animmediate fitness advantage. Thoracic beetle horns maythus be a good example of a novelty that arose via exapta-tion from traits originally selected for providing a very dif-ferent function at a different stage of development.However, it is important to keep in mind that none of thesearguments holds up for other horn types such as headhorns. Head horns, at least in Onthophagus, undergo littleto no remodeling, and morphological differences amongadults are already largely established in the precedingpupal stage (Moczek 2007). These basic differences fur-ther underscore the likely evolutionary and developmentalindependence already highlighted above that characterizesdifferent types of horns and most likely different lineagesof horned beetles (Emlen et al. 2007). At the same time,the presumed origin of adult thoracic horns from ancestralmolting devices vividly illustrates the crooked routes thatdevelopmental evolution can take as it generates what inthe end may be perceived as an evolutionary novelty. Thesame complexity in the interactions among development,morphology, and ecology emerges when our emphasis isshifted away from the origin and diversification of hornsand toward the diversification of horned beetles, as the lastsection of this chapter hopes to illustrate.

TRADE-OFFS DURING DEVELOPMENT ANDEVOLUTION OF HORNED BEETLES

In an important study, Kazuo Kawano (2002) showedthat two species of giant southeast Asian rhinoceros beetles(genus Chalcosoma) had diverged in both relative hornsizes and copulatory organ sizes and that this divergencewas more pronounced among sympatric (overlapping)populations than among allopatric (separated) populations.His findings were fully consistent with reproductive char-acter displacement reinforced in sympatry but not allopa-try. What was intriguing, however, was the observation thatthe species which had evolved relatively longer horns hadalso evolved relatively shorter copulatory organs, i.e., malehorn copulatory organ sizes had coevolved in oppositedirections or antagonistically. Later experimental work onO. taurus (Moczek and Nijhout 2004) suggested that thisantagonistic coevolution may not have been a coincidence.In this study, surgical removal of the genital primordia dur-ing larval development resulted in males lacking a copula-tory organ but they had disproportionately longer horns,suggesting that there may indeed be a connection betweenhow horns and copulatory organs developed, and thereforepossibly how they evolved. This was particularly intriguingbecause changes in male copulatory organs are thought tobe closely associated with the origin of reproductive isola-tion and thus speciation (Eberhard 1985). In fact, copula-

tory organ morphology is often the only way to distinguishcryptic species, suggesting that whatever mechanism isable to influence how copulatory organs develop in a pop-ulation may have immediate repercussions for that popula-tion’s ability to interbreed with others. A recent study examining both within and between

species covariation in horn versus copulatory organ invest-ment provides the strongest evidence to date suggestingexactly that kind of interaction between horn and copula-tory organ evolution (Parzer and Moczek 2008). Spe cif i -cally, this study focused first on three rapidly divergingexotic O. taurus populations. These populations wereintroduced from their native Mediterranean range to theeastern United States as well as to eastern and westernAustralia <50 years ago and since then had evolved sig-nificant differences in male horn investment. Ecologicalstudies are consistent with the hypothesis that this diversi-fication was driven by selection acting directly on hornexpression, rather than other traits. As a result, present-daywestern Australian males grow the relatively shortesthorns of any population, whereas eastern United Statesmales grow the relatively longest, with the other two pop-ulations (eastern Australia and Mediterranean) beingintermediate (Moczek 2003; Moczek and Nijhout 2003).The study showed that across these four populations, therewas a perfect negative correlation between relative invest-ment into horns and copulatory organ size (Fig. 5a,b).Western Australian males invested the least in horns but byfar the most in copulatory organ size, whereas the rela-tionship was reversed for eastern United States males, andintermediate for males from the other two populations.As a second step, the study applied the same approach to

nine different Onthophagus species, and the same highlysignificant negative correlation between relative invest-ment into horns and copulatory organ size emerged (Fig.5c). Importantly, the greatest differences observed betweenpopulations were similar in nature and magnitude to someof the differences detected between species. Combined,these results had three important implications. First, theysuggest that copulatory organ size may, under certain cir-cumstances, diverge as a by-product of evolutionarychanges occurring in horns. Second, the resulting signa-tures of antagonistic coevolution detectable during bothvery recent divergences between populations and macro -evolutionary divergence between species suggested thatthis tradeoff can bias evolutionary trajectories over a rangeof phylogenetic distances. Third, and most remarkable,given the significance of copulatory organ morphology forthe evolution of reproductive isolation, these findings raisethe possibility that horn diversification may promote spe-ciation as a by-product. If correct, this might help toexplain how the genus Onthophagus, famous for its dra-matic diversity in patterns of horn expression, was able toradiate into more than 2400 extant species, making it themost speciose genus in the animal kingdom (Arrow 1951).

CONCLUSIONS

I have argued here that the origin and diversification ofhorns were mediated by widespread cooption of preexist-

294 MOCZEK

ing developmental mechanisms into a new spatiotempo-ral context, exaptation of preexisting structures that orig-inally performed unrelated functions, and trade-offsarising during development that bias, and possibly accel-erate, patterns of diversification. Beetle horns and hornedbeetles emerge as a rich microcosm within which toexplore the mechanisms underlying organismal innova-tion and diversification. Combining ecological richnessand morphological exuberance with increasing accessi-bility to genetic and developmental manipulation along-side the development of genomic resources, hornedbeetles emerge as an increasingly powerful model systemin evo-devo and eco-devo research.

ACKNOWLEDGMENTS

I thank Cold Spring Harbor Laboratories for organizinga fantastic and memorable conference. Research presentedhere was supported by National Science Foundation grantsIOS 0445661 and IOS 0718522 to A.P.M.

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