When ontogeny reveals what phylogeny hides: gain and loss of horns during development and evolution of horned beetles

How ecological, developmental and genetic mechanisms interact in the genesis and subsequent diversification of morphological novelties is unknown for the vast majority of traits and organisms. Here we explore the ecological, developmental, and genetic underpinnings of a class of traits that is both novel and highly diverse: beetle horns. Specifically, we focus on the origin and diversification of a particular horn type, those protruding from the pronotum, in the genus Onthophagus, a particularly speciose and morphologically diverse genus of horned beetles. We begin by documenting immature development of nine Onthophagus species and show that all of these species express pronotal horns in a developmentally transient fashion in at least one or both sexes. Similar to species that retain their horns to adulthood, transient horns grow during late larval development and are clearly visible in pupae. However, unlike species that express horns as adults, transient horns are resorbed during pupal development. In a large number of species this mechanisms allows fully horned pupae to molt into entirely hornless adults. Consequently, far more Onthophagus species appear to possess the ability to develop pronotal horns than is indicated by their adult phenotypes. We use our data to expand a recent phylogeny of the genus Onthophagus to explore how the widespread existence of developmentally transient horns alters our understanding of the origin and dynamics of morphological innovation and diversification in this genus. We find that including transient horn development into the phylogeny dramatically reduces the number of independent origins required to explain extant diversity patters and suggest that pronotal horns may have originated only a few times, or possibly only once, during early Onthophagus evolution. We then propose a new and previously undescribed function for pronotal horns during immature development. We provide histological as well as experimental data that illustrate that pronotal horns are crucial for successful ecdysis of the larval head capsule during the larval-to-pupal molt, and that this molting function appears to be unique to the genus Onthophagus and absent in the other scarabaeine genera. We discuss how this additional function may help explain the existence and maintenance of developmentally transient horns, and how at least some horn types of adult beetles may have evolved as exaptations from pupal structures originally evolved to perform an unrelated function.     

Beetle horns are regulated by the Hox gene,Sex combs reduced,in a species‐ and sex‐specific manner

Discovering the mechanisms that underlie the origin of novel features represents a major frontier in developmental and evolutionary biology. Here we begin to characterize the role of the Hox gene Sex combs reduced (Scr) during the development and evolution of a morphologically novel trait: beetle horns. Beetle horns develop as epidermal outgrowths from the prothorax and/or head, and size and location vary dramatically across species and between sexes. Using both comparative gene expression and larval RNA interference in two species of the horned beetle genus Onthophagus, we show that Scr functions in patterning adult labial mouthpart identity and suppressing wing development in the prothorax. At the same time, however, our results illustrate that Scr has acquired, within its ancestral domain of expression, additional new functions including the regulation of prepupal growth and pupal remodeling of pronotal horn primordia. Furthermore, comparative analyses of our results across both Onthophagus species, which differ in location of horn development (thoracic horns vs. thoracic and head horns) as well as patterns of sexual dimorphism (traditional vs. reversed sexual dimorphism), reveal surprising differences in exactly when, where, and to what degree Scr regulates horn formation in different sexes. These observations suggest that the interactions between Scr and its targets in the regulation of horn development can diversify quickly over remarkably short phylogenetic distances. More generally, our results suggest that the Hox complex can play an integral role in the development and evolution of novel complex traits while maintaining traditional patterning responsibilities.     

Pupal remodeling and the evolution and development of alternative male morphologies in horned beetles

Background  

How novel morphological traits originate and diversify represents a major frontier in evolutionary biology. Horned beetles are emerging as an increasingly popular model system to explore the genetic, developmental, and ecological mechanisms, as well as the interplay between them, in the genesis of novelty and diversity. The horns of beetles originate during a rapid growth phase during the prepupal stage of larval development. Differential growth during this period is either implicitly or explicitly assumed to be the sole mechanism underlying differences in horn expression within and between species. Here I focus on male horn dimorphisms, a phenomenon at the center of many studies in behavioral ecology and evolutionary development, and quantify the relative contributions of a previously ignored developmental process, pupal remodeling, to the expression of male dimorphism in three horned beetle species.     

Horn quality and postmortem sperm parameters in Spanish ibex (Capra pyrenaica hispanica)

The horns are secondary sexual characteristics used by males of many ungulate species for intra-sexual fights during the rut. Thus, the dominant males with most developed horns are naturally selected for reproduction. Several studies have suggested that the quality of the horn, in many wild ruminants, may be correlated with semen quality. The aim of the present study was to determine whether inter-individual differences in levels of horn asymmetry and horn size are related to differences in sperm quality in a wild population of Spanish ibex by the assay of epididymal spermatozoa collected postmortem. In order to test this hypothesis we collected morphometric horns data from a total of 59 mature males (9-15 years of age) that were legally hunted during rutting season. The testicles were recovered, and the collection of epididymal spermatozoa was done at different times after death (2-60 h). The percentage of motile spermatozoa, motility rate, plasma membrane integrity, sperm viability, sperm morphology, and acrosome integrity were evaluated. Our findings showed that viable epididymal spermatozoa may be retrieved from dead animals many hours after death. However, sperm parameters were affected by the elapsed time between the death of the animal and spermatozoa collection. The study revealed that the horn quality was firstly associated with sperm motility.     

Juvenile hormone mediates sexual dimorphism in horned beetles

The causes and consequences of sexual dimorphism are major themes in biology. Here we explore the endocrine regulation of sexual dimorphism in horned beetles. Specifically, we explore the role of juvenile hormone (JH) in regulating horn expression in females of two species with regular sexual dimorphism for pronotal horns (females have much shorter horns than males) and a third species with a rare reversed sexual dimorphism for both pronotal and head horns (females have much larger horns in both body regions compared with males). Applications of the JH analog methoprene caused females of the two more typical species to grow significantly shorter pronotal horns than control females, whereas no consistent effect on pronotal horn development was detected in the third, sex-reversed species. Instead, females in this species showed an unexpected and significant increase in head horn expression in response to methoprene treatment. Lastly, horn shape was also affected in females of one of the regularly sexually dimorphic species, but in the opposite direction than horn length. Although methoprene exerted a feminizing effect on female horn length in this species, it significantly masculinized horn shape by inducing a peculiar shape change observed naturally only in males. Our results suggest that JH influences both overall size and shape of female horns, but does so flexibly and as a function of species, sex and horn location. We use our results to review current models on the role of endocrine mechanisms in development and evolution of horned beetle diversity.     

Linear and threshold-dependent expression of secondary sexual traits in the same individual: insights from a horned beetle (Coleoptera: Scarabaeidae)

Elaborate horns or horn‐like structures in male scarab beetles commonly scale with body size either (a) in a linear fashion with horn size increasing relatively faster than body size or (b) in a threshold‐dependent, sigmoid fashion; that is, males smaller than a certain critical body size develop no or only rudimentary horns, whereas males larger than the threshold size express fully developed horns. The development of linear vs. sigmoid scaling relationships is thought to require fundamentally different regulatory mechanisms. Here we show that such disparate regulatory mechanisms may co‐occur in the same individual. Large males of the south‐east Asian Onthophagus (Proagoderus) watanabei (Ochi & Kon) (Scarabaeidae, Onthophagini) develop a pair of long, curved head horns as well as a single thoracic horn. We show that unlike paired head horns in a large number of Onthophagus species, in O. watanabei the relationship between head horns and body size is best explained by a linear model. Large males develop disproportionately longer horns than small males, but the difference in relative horn sizes across the range of body sizes is small compared to other Onthophagus species. However, the scaling relationship between the thoracic horn and body size is best explained by a strongly sigmoid model. Only males above a certain body size threshold express a thoracic horn and males smaller than this threshold express no horn at all. We found a significant positive correlation between head and thoracic horn length residuals, contrary to what would be expected if a resource allocation tradeoff during larval development would influence the length of both horn types. Our results suggest that the scaling relationship between body size and horn length, and the developmental regulation underlying these scaling relationships, may be quite different for different horns, even though these horns may develop in the same individual. We discuss our results in the context of the developmental biology of secondary sexual traits in beetles. © 2004 The Linnean Society of London, Biological Journal of the Linnean Society, 2004, 83 , 473–480.     

A negative association between horn length and survival in a weakly dimorphic ungulate

While all models of sexual selection assume that the development and expression of enlarged secondary sexual traits are costly, males with larger ornaments or weapons generally show greater survival or longevity. These studies have mostly been performed in species with high sexual size dimorphism, subject to intense sexual selection. Here, we examined the relationships between horn growth and several survival metrics in the weakly dimorphic Pyrenean chamois (Rupicapra pyrenaica). In this unhunted population living at high density, males and females were able to grow long horns without any apparent costs in terms of longevity. However, we found a negative relationship between horn growth and survival during prime age in males. This association reduces the potential evolutionary consequences of trophy hunting in male chamois. We also found that females with long horns tended to have lower survival at old ages. Our results illustrate the contrasting conclusions that may be drawn when different survival metrics are used in analyses. The ability to detect trade‐off between the expression of male secondary sexual traits and survival may depend more on environmental conditions experienced by the population than on the strength of sexual selection.     

Hormonal control of male horn length dimorphism in the dung beetle Onthophagus taurus (Coleoptera: Scarabaeidae)

Male dung beetles (Onthophagus taurus) facultatively produce a pair of horns that extend from the base of the head: males growing larger than a threshold body size develop long horns, whereas males that do not achieve this size grow only rudimentary horns or no horns at all. Here we characterize the postembryonic development of these beetles, and begin to explore the hormonal regulation of horn growth. Using radioimmune assays to compare the ecdysteroid titers of horned males, hornless males, and females, we identify a small pulse of ecdysteroid which is present in both hornless males and females, but not in horned males. In addition, we identify a brief period near the end of the final (third) larval instar when topical applications of the juvenile hormone analog methoprene can switch the morphology of developing males. Small, normally hornless, males receiving methoprene during this sensitive period were induced to produce horns in 80% of the cases. We summarize this information in two models for the hormonal control of male dimorphism in horn length.     

Conservation, innovation, and the evolution of horned beetle diversity

Beetle horns represent an evolutionary novelty exhibiting remarkable diversity above and below the species level. Here, we show that four typical appendage patterning genes, extradenticle (exd), homothorax (hth), dachshund (dac), and Distal-less (Dll) are expressed in the context of the development of sexually dimorphic thoracic horns in three Onthophagus species. At least two of these genes, Dll and hth, exhibited expression patterns consistent with a conservation of patterning function during horn development relative to their known roles in the development of insect legs. exd, hth, and dac expression patterns during horn development were largely invariable across species or sexes within species. In contrast, Dll expression was far more discrete and exhibited consistent differences between sexes and species. Most importantly, differences in location and domain size of Dll expression tightly correlated with the degree to which prepupal horn primordia were retained or resorbed before the final adult molt. Our results lend further support to the hypothesis that the origin of beetle horns relied, at least in part, on the redeployment of already existing developmental mechanisms, such as appendage patterning processes and that changes in the exact location and domain size of Dll expression may represent important modifier mechanisms that modulate horn expression in different species or sexes. If correct, this would imply that certain components of genetic basis of horn development may be able to diversify rapidly within lineages and largely independent of phylogenetic distance. We present a first model that integrates presently available data on the genetic regulation of horn development and diversity.     

Environmental and evolutionary effects on horn growth of male bighorn sheep

The development of male secondary sexual characters such as antlers or horns has substantial biological and socio‐economic importance because in many species these traits affect male fitness positively through sexual selection and negatively through trophy hunting. Both environmental conditions and selective hunting can affect horn growth but their relative importance remains unexplored. We first examined how a large‐scale climate index, the Pacific Decadal Oscillation (PDO), local weather and population density influenced both absolute and relative annual horn growth from birth to three years of male bighorn sheep Ovis canadensis over 42 years. We then examined the relative influence of environmental conditions and evolution mainly driven by trophy hunting on male horn length at three years of age. Horn growth was positively influenced by low population density and warm spring temperature, suggesting that ongoing climate change should lead to larger horns. Seasonal values of PDO were highly correlated. Horn growth increased with PDO in spring or summer at low density, but was weak at high density regardless of PDO. The interaction between population density and PDO in spring or summer accounted for a similar proportion of the observed annual variation in horn growth (32% or 37%) as did the additive effects of spring temperature and density (34%). When environmental conditions deteriorated, males allocated relatively more resources to summer mass gain than to horn growth, suggesting a conservative strategy favoring maintenance of condition over allocation to secondary sexual characters. Population density explained 27% of the variation in horn length, while evolutionary effects explained 9% of the variance. Thus, our study underlines the importance of both evolution and phenotypic plasticity on the development of a secondary sexual trait.     

<Emphasis Type="Italic">Decapentaplegic</Emphasis> (<Emphasis Type="Italic">dpp</Emphasis>) regulates the growth of a morphological novelty,beetle horns

Studies focusing on the development of morphological novelties suggest that patterning genes underlying traditional appendage development (i.e. mouthparts, legs, and wings) also play important roles in patterning novel morphological structures. In this study, we examine whether the expression and function of a member of the TGF-β signaling pathway, decapentaplegic (dpp), promotes development of a morphologically novel structure: beetle horns. Beetle horns are complex secondary sexual structures that develop in the head and/or prothorax, lack obvious homology to other insect outgrowths, and vary remarkably between species and sexes. We studied dpp expression through in situ hybridization, performed functional analyses with RNA interference, and gathered allometric measurements to determine the role of dpp during both pronotal and head horn development in both sexes of two morphologically dissimilar species in the Onthophagus genus, Onthophagus binodis and Onthophagus sagittarius. Our findings show that in addition to affecting growth and patterning of traditional appendages, dpp regulates beetle horn growth and remodeling.     

Trade-off between horns and other functional traits in two <Emphasis Type="Italic">Onthophagus</Emphasis> species (Scarabaeidae,Coleoptera)

Beetle horns are extraordinarily diversified secondary sexual structures used for mate choice and male–male combat. Due to an interaction of nutritional, hormonal and genetic factors, their polyphenic development is metabolically expensive and occurs in the virtually closed system of the pre-pupal stage, after the developing larva has stopped feeding. Previous studies showed the occurrence of resource competition resulting in a trade-off between horns and other morphological structures. These studies also revealed functional associations between autoecology and horns, as a function of their physical location (i.e. head versus pronotum), and suggested that constraints imposed by trade-offs on adult morphology may have profound evolutionary consequences, such as ecological and reproductive isolation. In this study, we compared trade-off patterns between horns and other functional traits (eyes, antennae, legs, head, epipharynges and genitalia) in two congeneric species bearing horns located in the same anatomical area, but with different morphologies. Specifically, we considered Onthophagus taurus, characterised by a pair of long, lunated cephalic horns, and Onthophagus fracticornis, expressing a single cephalic horn. We demonstrated that, even when horns are located in the same physical position on the insect’s body, differences in horn morphology can bring about differences in how functional traits respond to horn investment. These differences are interpretable in the light of the hierarchy of functions carried out by these structures and their component parts in each species.     

Proximate determination of male horn dimorphism in the beetle Onthophagus taurus (Coleoptera: Scarabaeidae)

The existence of discrete phenotypic variation within one sex poses interesting questions regarding how such intrasexual polymorphisms are produced and modified during the course of evolution. Approaching these kinds of questions requires insights into the genetic architecture underlying a polymorphism and an understanding of the proximate mechanisms determining phenotype expression. Here we explore the genetic underpinnings and proximate factors influencing the expression of beetle horns – a dramatic sexually selected trait exhibiting intramale dimorphism in many species. Two relatively discrete male morphs are present in natural populations of the dung beetle Onthophagus taurus (Scarabaeidae, Onthophagini). Males exceeding a critical body size develop a pair of long, curved horns on their heads, while those smaller than this critical body size remain essentially hornless. We present results from laboratory breeding experiments designed to assess the relative importance of inherited and environmental factors as determinants of male morphology. Using father–son regressions, our findings demonstrate that horn length and body size of male progeny are not predicted from paternal morphology. Instead, natural variation in an environmental factor, the amount of food available to larvae, determined both the body sizes exhibited by males as adults and the presence or absence of horns. The nonlinear scaling relationship between the body size and horn length of males bred in the laboratory did not differ from the pattern of variation present in natural populations, suggesting that nutritional conditions account for variation in male morphology in natural populations as well. We discuss our results by extending ideas proposed to explain the evolution of conditional expression of alternative phenotypes in physically heterogeneous environments toward incorporating facultative expression of secondary sexual traits. We use this synthesis to begin characterizing the potential origin and subsequent evolution of facultative horn expression in onthophagine beetles.     

Hormonal control of male horn length dimorphism in Onthophagus taurus (Coleoptera: Scarabaeidae): a second critical period of sensitivity to juvenile hormone

Male dung beetles (Onthophagus taurus) facultatively produce a pair of horns that extend from the base of the head: males larger than a threshold body size develop long horns, whereas males that do not achieve this size develop only rudimentary horns or no horns at all. Using topical applications of methoprene, we identified a sensitive period during the feeding stage of third (final) instar larvae when application of methoprene shifted the threshold body size for horn expression. Male larvae that received methoprene at this time delayed horn production until they attained a larger threshold body size than acetone-treated control larvae. This new sensitive period occurs earlier than a sensitive period previously reported for male horn regulation, and it coincides with a morph-specific pulse of ecdysteroid secretion described for this species. It appears that male horn expression is influenced by endocrine events at two different periods of larval development. We incorporate these results into an expanded model for the endocrine regulation of male horn expression.     

Modes of cell death in the pupal perivisceral fat body tissue of the silkworm Bombyx mori L.

Cell death is a scheduled event during animal development and tissue turnover. Here, we affirm the presence of two major pathways of programmed cell death (PCD), viz. apoptotic and autophagic cell death, in the disintegrated pupal perivisceral (PV) fat body during pupal-adult metamorphosis. The acridine orange (a vital stain for apoptosis) staining pattern and DNA fragmentation assay have revealed the exact day (6th day of the pupal stage) of disintegration in the PV fat body as represented by chromatin condensation and DNA laddering. Electron microscopy and scanning electron microscopy have demonstrated the presence of cytoplasmic budding and giant autophagic vacuoles and the low numbers of mitochondria, all of which are attributes of autophagic cell death. Immunoblot analysis of proteosomal subunits 20S and 26S has established the involvement of proteolytic activity during PCD of PV tissue. Lysosomal participation during the PCD of PV tissues has been confirmed by the elevated level of the marker enzyme, acid phosphatase, which is distinct on day 6 of the pupal period. The results of the present study have thus ascertained the co-existence of both autophagic and apoptotic cell death in PV fat body tissue.     

The Evolution of Positive Allometry of Weaponry in Horned Lizards (Phrynosoma)

The allometry of weapons and other conspicuous structures has long fascinated biologists. Recently, a debate has arisen about the roles of sexual and natural selection in driving the allometry of structures, with some authors suggesting that positive allometry is exclusively the result of sexual selection. Although some studies, often focusing on weapons, support this hypothesis, others have shown that many naturally selected structures also exhibit positive allometry. We study the allometry of the cranial horns in 14 species of horned lizards (Phrynosoma). These horns are purely defensive weapons and so are under natural, as opposed to sexual, selection. In almost all cases, the length of these horns is positively allometric through ontogeny (intraspecifically) and through evolution (interspecifically). Our findings demonstrate that positive allometry can be the product of natural selection, suggesting that the function of structures may dictate allometry and not the type of selection. For example, it is possible that weapons tend to be positively allometric. Our findings also suggest that longer horns may arise through the prolongation of horn growth, and that the horns that are most effective at defense from predators attacking a lizard from above are integrated with one another. Finally, we find that species with unusual horn morphologies have diverged from other species in their horn allometries, indicating that the evolution of morphological diversity can be mediated through the evolution of allometry.     

Variation in cross‐sectional horn shape within and among rhinoceros beetle species

Sexual selection has equipped male rhinoceros beetles with large horns on their head and prothorax to aid in battle over access to females. Horns are used to pry and dislodge opponents from resource sites that attract females, so an optimal horn should be able both to withstand the high stresses imposed during fights, and to resist deflection in response to these loads. We examined the cross‐sectional morphology of horns using micro‐computed tomography scanning to determine how horn structure changes with horn length to withstand the different fighting loads. Specifically, we measured the second moment of area of horns within and among rhinoceros beetle species to assess whether changes in cross‐sectional morphology accompany changes in body size in order to maintain high strength and stiffness during fights. We find that the second moment of area of horns increases with body size both intra‐specifically and inter‐specifically, and that these relationships closely fit those predicted if horns have been selected to be strong and stiff fighting structures. Our results therefore support the hypothesis that rhinoceros beetle horns are structurally adapted for combat.