Eye development influences horn size but not patterning in horned beetles.

Understanding the origin of novel morphological traits is a long-standing objective in evolutionary developmental biology. We explored the developmental genetic mechanisms that underpin the formation of a textbook example of evolutionary novelties, the cephalic horns of beetles. Previous work has implicated the gene regulatory networks associated with compound eye and ocellar development in horn formation and suggested that horns and compound eyes may influence each other's sizes. Therefore, we investigated the functional significance of genes central to visual system formation in the initiation, patterning, and size determination of head horns across three horned beetle species. We find that while the downregulation of canonical eye patterning genes reliably reduces or eliminates compound eye formation, it does not alter the position or shape of head horns yet does result in an increase in relative horn length. We discuss the implications of our results for our understanding of the genesis of cephalic horns in particular and evolutionary novelties in general.

Histone deacetylases regulate organ-specific growth in a horned beetle.

BACKGROUND: Nutrient availability is among the most widespread means by which environmental variability affects developmental outcomes. Because almost all cells within an individual organism share the same genome, structure-specific growth responses must result from changes in gene regulation. Earlier work suggested that histone deacetylases (HDACs) may serve as epigenetic regulators linking nutritional conditions to trait-specific development. Here we expand on this work by assessing the function of diverse HDACs in the structure-specific growth of both sex-shared and sex-specific traits including evolutionarily novel structures in the horned dung beetle Onthophagus taurus. RESULTS: We identified five HDAC members whose downregulation yielded highly variable mortality depending on which HDAC member was targeted. We then show that HDAC1, 3, and 4 operate in both a gene- and trait-specific manner in the regulation of nutrition-responsiveness of appendage size and shape. Specifically, HDAC 1, 3, or 4 knockdown diminished wing size similarly while leg development was differentially affected by RNAi targeting HDAC3 and HDAC4. In addition, depletion of HDAC3 transcript resulted in a more rounded shape of genitalia at the pupal stage and decreased the length of adult aedeagus across all body sizes. Most importantly, we find that HDAC3 and HDAC4 pattern the morphology and regulate the scaling of evolutionarily novel head and thoracic horns as a function of nutritional variation. CONCLUSION: Collectively, our results suggest that both functional overlap and division of labor among HDAC members contribute to morphological diversification of both conventional and recently evolved appendages. More generally, our work raises the possibility that HDAC-mediated scaling relationships and their evolution may underpin morphological diversification within and across insect species broadly.

Vertically inherited microbiota and environment modifying behaviours conceal genetic variation in dung beetle life history.

Diverse organisms actively manipulate their (sym)biotic and physical environment in ways that feed back on their own development. However, the degree to which these processes affect microevolution remains poorly understood. The gazelle dung beetle both physically modifies its ontogenetic environment and structures its biotic interactions through vertical symbiont transmission. By experimentally eliminating (i) physical environmental modifications and (ii) the vertical inheritance of microbes, we assess how environment modifying behaviour and microbiome transmission shape heritable variation and evolutionary potential. We found that depriving larvae of symbionts and environment modifying behaviours increased additive genetic variance and heritability for development time but not body size. This suggests that larvae's ability to manipulate their environment has the potential to modify heritable variation and to facilitate the accumulation of cryptic genetic variation. This cryptic variation may become released and selectable when organisms encounter environments that are less amenable to organismal manipulation or restructuring. Our findings also suggest that intact microbiomes, which are commonly thought to increase genetic variation of their hosts, may instead reduce and conceal heritable variation. More broadly, our findings highlight that the ability of organisms to actively manipulate their environment may affect the potential of populations to evolve when encountering novel, stressful conditions.

Plasticity, symbionts and niche construction interact in shaping dung beetle development and evolution.

Developmental plasticity is an important product of evolutionary processes, allowing organisms to maintain high fitness in the face of environmental perturbations. Once evolved, plasticity also has the potential to influence subsequent evolutionary outcomes, for example, by shaping phenotypic variation visible to selection and facilitating the emergence of novel trait variants. Furthermore, organisms may not just respond to environmental conditions through plasticity but may also actively modify the abiotic and (sym)biotic environments to which they themselves respond, causing plasticity to interact in complex ways with niche construction. Here, we explore developmental mechanisms and evolutionary consequences of plasticity in horned dung beetles. First, we discuss how post-invasion evolution of plasticity in an introduced Onthophagus species facilitated rapid range expansion and concurrent local adaptation of life history and morphology to novel climatic conditions. Second, we discuss how, in addition to plastically responding to variation in nutritional conditions, dung beetles engage in behaviors that modify the environment that they themselves respond to during later development. We document that these environment-modifying behaviors mask heritable variation for life history traits within populations, thereby shielding genetic variants from selection. Such cryptic genetic variation may be released and become selectable when these behaviors are compromised. Together, this work documents the complex interactions between plasticity, symbionts and niche construction, and highlights the usefulness of an integrative Eco-Evo-Devo framework to study the varied mechanisms and consequences of plasticity in development and evolution.

Genome evolution and divergence in cis-regulatory architecture is associated with condition-responsive development in horned dung beetles.

Phenotypic plasticity is thought to be an important driver of diversification and adaptation to environmental variation, yet the genomic mechanisms mediating plastic trait development and evolution remain poorly understood. The Scarabaeinae, or true dung beetles, are a species-rich clade of insects recognized for their highly diversified nutrition-responsive development including that of cephalic horns-evolutionarily novel, secondary sexual weapons that exhibit remarkable intra- and interspecific variation. Here, we investigate the evolutionary basis for horns as well as other key dung beetle traits via comparative genomic and developmental assays. We begin by presenting chromosome-level genome assemblies of three dung beetle species in the tribe Onthophagini (> 2500 extant species) including Onthophagus taurus, O. sagittarius, and Digitonthophagus gazella. Comparing these assemblies to those of seven other species across the order Coleoptera identifies evolutionary changes in coding sequence associated with metabolic regulation of plasticity and metamorphosis. We then contrast chromatin accessibility in developing head horn tissues of high- and low-nutrition O. taurus males and females and identify distinct cis-regulatory architectures underlying nutrition- compared to sex-responsive development, including a large proportion of recently evolved regulatory elements sensitive to horn morph determination. Binding motifs of known and new candidate transcription factors are enriched in these nutrition-responsive open chromatin regions. Our work highlights the importance of chromatin state regulation in mediating the development and evolution of plastic traits, demonstrates gene networks are highly evolvable transducers of environmental and genetic signals, and provides new reference-quality genomes for three species that will bolster future developmental, ecological, and evolutionary studies of this insect group.

Sexually transmitted mutualist nematodes shape host growth across dung beetle species.

Many symbionts are sexually transmitted and impact their host's development, ecology, and evolution. While the significance of symbionts that cause sexually transmitted diseases (STDs) is relatively well understood, the prevalence and potential significance of the sexual transmission of mutualists remain elusive. Here, we study the effects of sexually transmitted mutualist nematodes on their dung beetle hosts. Symbiotic Diplogastrellus monhysteroides nematodes are present on the genitalia of male and female Onthophagus beetles and are horizontally transmitted during mating and vertically passed on to offspring during oviposition. A previous study indicates that the presence of nematodes benefits larval development and life history in a single host species, Onthophagus taurus. However, Diplogastrellus nematodes can be found in association with a variety of beetle species. Here, we replicate these previous experiments, assess whether the beneficial effects extend to other host species, and test whether nematode-mediated effects differ between male and female host beetles. Rearing three relatively distantly related dung beetle species with and without nematodes, we find that the presence of nematodes benefits body size, but not development time or survival across all three species. Likewise, we found no difference in the benefit of nematodes to male compared to female beetles. These findings highlight the role of sexually transmitted mutualists in the evolution and ecology of dung beetles.

Utilizing geometric morphometrics to investigate gene function during organ growth: Insights through the study of beetle horn shape allometry.

Static allometry is a major component of morphological variation. Much of the literature on the development of allometry investigates how functional perturbations of diverse pathways affect the relationship between trait size and body size. Often, this is done with the explicit objective to identify developmental mechanisms that enable the sensing of organ size and the regulation of relative growth. However, changes in relative trait size can also be brought about by a range of other distinctly different developmental processes, such as changes in patterning or tissue folding, yet standard univariate biometric approaches are usually unable to distinguish among alternative explanations. Here, we utilize geometric morphometrics to investigate the degree to which functional genetic manipulations known to affect the size of dung beetle horns also recapitulate the effect of horn shape allometry. We reasoned that the knockdown phenotypes of pathways governing relative growth should closely resemble shape variation induced by natural allometric variation. In contrast, we predicted that if genes primarily affect alternative developmental processes, knockdown effects should align poorly with shape allometry. We find that the knockdown effects of several genes (e.g., doublesex, Foxo) indeed closely aligned with shape allometry, indicating that their corresponding pathways may indeed function primarily in the regulation of relative trait growth. In contrast, other knockdown effects (e.g., Distal-less, dachs) failed to align with allometry, implicating these pathways in potentially scaling-independent processes. Our findings moderate the interpretation of studies focusing on trait length and highlight the usefulness of multivariate approaches to study allometry and phenotypic plasticity.

Vertically inherited microbiota and environment-modifying behaviors indirectly shape the exaggeration of secondary sexual traits in the gazelle dung beetle.

Many organisms actively manipulate the environment in ways that feed back on their own development, a process referred to as developmental niche construction. Yet, the role that constructed biotic and abiotic environments play in shaping phenotypic variation and its evolution is insufficiently understood. Here, we assess whether environmental modifications made by developing dung beetles impact the environment-sensitive expression of secondary sexual traits. Gazelle dung beetles both physically modify their ontogenetic environment and structure their biotic interactions through the vertical inheritance of microbial symbionts. By experimentally eliminating (i) physical environmental modifications and (ii) the vertical inheritance of microbes, we assess the degree to which (sym)biotic and physical environmental modifications shape the exaggeration of several traits varying in their degree and direction of sexual dimorphism. We expected the experimental reduction of a larva's ability to shape its environment to affect trait size and scaling, especially for traits that are sexually dimorphic and environmentally plastic. We find that compromised developmental niche construction indeed shapes sexual dimorphism in overall body size and the absolute sizes of male-limited exaggerated head horns, the strongly sexually dimorphic fore tibia length and width, as well as the weakly dimorphic elytron length and width. This suggests that environmental modifications affect sex-specific phenotypic variation in functional traits. However, most of these effects can be attributed to nutrition-dependent plasticity in size and non-isometric trait scaling rather than body-size-independent effects on the developmental regulation of trait size. Our findings suggest that the reciprocal relationship between developing organisms, their symbionts, and their environment can have considerable impacts on sexual dimorphism and functional morphology.

Assessing the evolutionary lability of insulin signalling in the regulation of nutritional plasticity across traits and species of horned dung beetles.

Nutrition-dependent growth of sexual traits is a major contributor to phenotypic diversity, and a large body of research documents insulin signalling as a major regulator of nutritional plasticity. However, findings across studies raise the possibility that the role of individual components within the insulin signalling pathway diverges in function among traits and taxa. Here, we use RNAi-mediated transcript depletion in the gazelle dung beetle to investigate the functions of forkhead box O (Foxo) and two paralogs of the insulin receptor (InR1 and InR2) in shaping nutritional plasticity in polyphenic male head horns, exaggerated fore legs, and weakly nutrition-responsive genitalia. Our functional genetic manipulations led to three main findings: FoxoRNAi reduced the length of exaggerated head horns in large males, while neither InR1 nor InR2 knock-downs resulted in measurable horn phenotypes. These results are similar to those documented previously for another dung beetle (Onthophagus taurus), but in stark contrast to findings in rhinoceros beetles. Secondly, knockdown of Foxo, InR1, and InR2 led to an increase in the intercept or slope of the scaling relationship of genitalia size. These findings are in contrast even to results documented previously for O. taurus. Lastly, while FoxoRNAi reduces male forelegs in D. gazella and O. taurus, the effects of InR1 and InR2 knockdowns diverged across dung beetle species. Our results add to the growing body of literature indicating that despite insulin signalling's conserved role as a regulator of nutritional plasticity, the functions of its components may diversify among traits and species, potentially fuelling the evolution of scaling relationships.

Gene regulatory networks underlying the development and evolution of plasticity in horned beetles.

Horned beetles have emerged as a powerful study system with which to investigate the developmental mechanisms underlying environment-responsive development and its evolution. We begin by reviewing key advances in our understanding of the diverse roles played by transcription factors, endocrine regulators, and signal transduction pathways in the regulation of horned beetle plasticity. We then explore recent efforts aimed at understanding how such condition-specific expression may be regulated in the first place, as well as how the differential expression of master regulators may instruct conditional expression of downstream target genes. Here, we focus on the significance of chromatin remodeling as a powerful but thus far understudied mechanism able to facilitate trait-, sex-, and species-specific responses to environmental conditions.

Promises and limits of an agency perspective in evolutionary developmental biology.

An agent-based perspective in the study of complex systems is well established in diverse disciplines, yet is only beginning to be applied to evolutionary developmental biology. In this essay, we begin by defining agency and associated terminology formally. We then explore the assumptions and predictions of an agency perspective, apply these to select processes and key concept areas relevant to practitioners of evolutionary developmental biology, and consider the potential epistemic roles that an agency perspective might play in evo devo. Throughout, we discuss evidence supportive of agential dynamics in biological systems relevant to evo devo and explore where agency thinking may enrich the explanatory reach of research efforts in evolutionary developmental biology.

Allometric plasticity and the evolution of environment-by-environment (E×E) interactions during a rapid range expansion of a dung beetle.

Plastic responses to environmental conditions may themselves depend on other environmental conditions, but how such environment-by-environment (E×E) interactions may impact evolution remains unclear. We investigate how temperature shapes the nutritional polyphenism in horn length in a beetle and test whether "allometric plasticity" (a form of E×E) predicts latitudinal differentiation during a rapid range expansion. Rearing populations under common garden conditions demonstrates that increased temperatures reduce the body size threshold separating two male morphs in all populations but also that the magnitude of temperature-dependent changes in allometry diverged across recently established populations. Furthermore, we found a latitudinal increase in the threshold in the species' exotic range at one of the temperatures, suggesting that allometric plasticity in response to temperature may predict evolved clinal differences. Our findings demonstrate that E×E interactions can be similar in magnitude to G×E interactions and that allometric plasticity and its evolution may impact population's responses to environmental changes.

Evaluating old truths: Final adult size in holometabolous insects is set by the end of larval development.

For centuries, it has been understood that the final size of adult holometabolous insects is determined by the end of the larval stage, and that once they transform to adults, holometabolous insects do not grow. Despite this, no previous study has directly tested these "old truths" across holometabolous insects. Here, we demonstrate that final adult size is set at the end of the last larval stage in species representing each of the four orders of holometabolous insects: the fruit fly Drosophila melanogaster (Diptera), the tobacco hornworm Manduca sexta (Lepidoptera), the dung beetle Onthophagus taurus (Coleoptera), and the Florida carpenter ant Camponotus floridanus (Hymenoptera). Furthermore, in both D. melanogaster and C. floridanus, we show that the size of adult individuals fluctuates but does not significantly change. Therefore, our study finally confirms these two basic assumptions in the biology of insects, which have for centuries served as the foundation for studies of insect growth, size, and allometry.

The transcription factor Zfh1 acts as a wing-morph switch in planthoppers.

Insect wing polyphenism is characterized by its ability to produce two or more distinct wing morphs from a single genotype in response to changing environments. However, the molecular basis of this phenomenon remains poorly understood. Here, we identified a zinc finger homeodomain transcription factor Zfh1 that acts as an upstream regulator for the development of long-winged (LW) or shorted-winged (SW) morphs in planthoppers. Knockdown of Zfh1 directs SW-destined nymphs to develop into LW morphs by down-regulating the transcriptional level of FoxO, a prominent downstream effector of the insulin/IGF signaling (IIS) pathway. The balance between transcriptional regulation via the Zfh1-FoxO cascade and post-translational regulation via the IIS-FoxO cascade provides a flexible regulatory mechanism for the development of alternative wing morphs. These findings help us understand how phenotypic diversity is generated by altering the activity of conserved proteins, and provide an extended framework for the evolution of wing morphological diversity in insects.

Developmental bias in the evolution and plasticity of beetle horn shape.

The degree to which developmental systems bias the phenotypic effects of environmental and genetic variation, and how these biases affect evolution, is subject to much debate. Here, we assess whether developmental variability in beetle horn shape aligns with the phenotypic effects of plasticity and evolutionary divergence, yielding three salient results. First, we find that most pathways previously shown to regulate horn length also affect shape. Second, we find that the phenotypic effects of manipulating divergent developmental pathways are correlated with each other as well as multivariate fluctuating asymmetry-a measure of developmental variability. Third, these effects further aligned with thermal plasticity, population differences and macroevolutionary divergence between sister taxa and more distantly related species. Collectively, our results support the hypothesis that changes in horn shape-whether brought about by environmentally plastic responses, functional manipulations or evolutionary divergences-converge along 'developmental lines of least resistance', i.e. are biased by the developmental system underpinning horn shape.

Bridging the explanatory gaps: What can we learn from a biological agency perspective?

We begin this article by delineating the explanatory gaps left by prevailing gene-focused approaches in our understanding of phenotype determination, inheritance, and the origin of novel traits. We aim not to diminish the value of these approaches but to highlight where their implementation, despite best efforts, has encountered persistent limitations. We then discuss how each of these explanatory gaps can be addressed by expanding research foci to take into account biological agency-the capacity of living systems at various levels to participate in their own development, maintenance, and function by regulating their structures and activities in response to conditions they encounter. Here we aim to define formally what agency and agents are and-just as importantly-what they are not, emphasizing that agency is an empirical property connoting neither intention nor consciousness. Lastly, we discuss how incorporating agency helps to bridge explanatory gaps left by conventional approaches, highlight scientific fields in which implicit agency approaches are already proving valuable, and assess the opportunities and challenges of more systematically incorporating biological agency into research programs.

Evolutionary and plastic variation in larval growth and digestion reveal the complex underpinnings of size and age at maturation in dung beetles.

Age and size at maturity are key life-history components, yet the proximate underpinnings that mediate intra- and interspecific variation in life history remain poorly understood. We studied the proximate underpinnings of species differences and nutritionally plastic variation in adult size and development time in four species of dung beetles. Specifically, we investigated how variation in insect growth mediates adult size variation, tested whether fast juvenile growth trades-off with developmental stability in adult morphology and quantified plastic responses of digestive systems to variation in food quality. Contrary to the common size-development time trade-off, the largest species exhibited by far the shortest development time. Correspondingly, species diverged strongly in the shape of growth trajectories. Nutritionally plastic adjustments to growth were qualitatively similar between species but differed in magnitude. Although we expected rapid growth to induce developmental costs, neither instantaneous growth rates nor the duration of larval growth were related to developmental stability in the adult. This renders the putative costs of rapid growth enigmatic. We further found that larvae that encounter a challenging diet develop a larger midgut and digest more slowly than animals reared on a more nutritious diet. These data are consistent with the hypothesis that larvae invest into a more effective digestive system when exposed to low-quality nutrition, but suggest that species may diverge readily in their reliance on these mechanisms. More generally, our data highlight the complex, and often hidden, relationships between immature growth and age and size at maturation even in ecologically similar species.

Doublesex mediates species-, sex-, environment- and trait-specific exaggeration of size and shape.

Context-dependent trait exaggeration is a major contributor to phenotypic diversity. However, the genetic modifiers instructing development across multiple contexts remain largely unknown. We use the arthropod tibia, a hotspot for segmental differentiation, as a paradigm to assess the developmental mechanisms underlying the context-dependent structural exaggeration of size and shape through nutritional plasticity, sexual dimorphism and segmental differentiation. Using an RNAseq approach in the sexually dimorphic and male-polyphenic dung beetle Digitonthophagus gazella, we find that only a small portion (3.7%) of all transcripts covary positively in expression level with trait size across contexts. However, RNAi-mediated knockdown of the conserved sex-determination gene doublesex suggests that it functions as a context-dependent master mediator of trait exaggeration in D. gazella as well as the closely related dung beetle Onthophagus taurus. Taken together, our findings suggest (i) that the gene networks associated with trait exaggeration are highly dependent on the precise developmental context, (ii) that doublesex differentially shapes morphological exaggeration depending on developmental contexts and (iii) that this context-specificity of dsx-mediated trait exaggeration may diversify rapidly. This mechanism may contribute to the resolution of conflict arising from environment-dependent antagonistic selection among sexes and divergent developmental contexts in a wide range of animals.

Signals of selection beyond bottlenecks between exotic populations of the bull-headed dung beetle, Onthophagus taurus.

Colonization of new environments can lead to population bottlenecks and rapid phenotypic evolution that could be due to neutral and selective processes. Exotic populations of the bull-headed dung beetle (Onthophagus taurus) have differentiated in opposite directions from native beetles in male horn-to-body size allometry and female fecundity. Here we test for genetic and transcriptional differences among two exotic and one native O. taurus populations after three generations in common garden conditions. We sequenced RNA from 24 individuals for each of the three populations including both sexes, and spanning four developmental stages for the two exotic, differentiated populations. Identifying 270,400 high-quality single nucleotide polymorphisms, we revealed a strong signal of genetic differentiation between the three populations, and evidence of recent bottlenecks within and an excess of outlier loci between exotic populations. Differences in gene expression between populations were greatest in prepupae and early adult life stages, stages during which differences in male horn development and female fecundity manifest. Finally, genes differentially expressed between exotic populations also had greater genetic differentiation and performed functions related to chitin biosynthesis and nutrient sensing, possibly underlying allometry and fecundity trait divergences. Our results suggest that beyond bottlenecks, recent introductions have led to genetic and transcriptional differences in genes correlated with observed phenotypic differences.