Fundamental Patterns of Structural Evolution Revealed by Chromosome-Length Genomes of Cactophilic Drosophila.

A thorough understanding of adaptation and speciation requires model organisms with both a history of ecological and phenotypic study as well as a complete set of genomic resources. In particular, high-quality genome assemblies of ecological model organisms are needed to assess the evolution of genome structure and its role in adaptation and speciation. Here, we generate new genomes of cactophilic Drosophila, a crucial model clade for understanding speciation and ecological adaptation in xeric environments. We generated chromosome-level genome assemblies and complete annotations for seven populations across Drosophila mojavensis, Drosophila arizonae, and Drosophila navojoa. We use these data first to establish the most robust phylogeny for this clade to date, and to assess patterns of molecular evolution across the phylogeny, showing concordance with a priori hypotheses regarding adaptive genes in this system. We then show that structural evolution occurs at constant rate across the phylogeny, varies by chromosome, and is correlated with molecular evolution. These results advance the understanding of the D. mojavensis clade by demonstrating core evolutionary genetic patterns and integrating those patterns to generate new gene-level hypotheses regarding adaptation. Our data are presented in a new public database (cactusflybase.arizona.edu), providing one of the most in-depth resources for the analysis of inter- and intraspecific evolutionary genomic data. Furthermore, we anticipate that the patterns of structural evolution identified here will serve as a baseline for future comparative studies to identify the factors that influence the evolution of genome structure across taxa.

Novel genetic basis of resistance to Bt toxin Cry1Ac in Helicoverpa zea.

Crops genetically engineered to produce insecticidal proteins from the bacterium Bacillus thuringiensis have advanced pest management, but their benefits are diminished when pests evolve resistance. Elucidating the genetic basis of pest resistance to Bacillus thuringiensis toxins can improve resistance monitoring, resistance management, and the design of new insecticides. Here, we investigated the genetic basis of resistance to Bacillus thuringiensis toxin Cry1Ac in the lepidopteran Helicoverpa zea, one of the most damaging crop pests in the United States. To facilitate this research, we built the first chromosome-level genome assembly for this species, which has 31 chromosomes containing 375 Mb and 15,482 predicted proteins. Using a genome-wide association study, fine-scale mapping, and RNA-seq, we identified a 250-kb quantitative trait locus on chromosome 13 that was strongly associated with resistance in a strain of Helicoverpa zea that had been selected for resistance in the field and lab. The mutation in this quantitative trait locus contributed to but was not sufficient for resistance, which implies alleles in more than one gene contributed to resistance. This quantitative trait locus contains no genes with a previously reported role in resistance or susceptibility to Bacillus thuringiensis toxins. However, in resistant insects, this quantitative trait locus has a premature stop codon in a kinesin gene, which is a primary candidate as a mutation contributing to resistance. We found no changes in gene sequence or expression consistently associated with resistance for 11 genes previously implicated in lepidopteran resistance to Cry1Ac. Thus, the results reveal a novel and polygenic basis of resistance.

Contributions of cis- and trans-Regulatory Evolution to Transcriptomic Divergence across Populations in the Drosophila mojavensis Larval Brain.

Natural selection on gene expression was originally predicted to result primarily in cis- rather than trans-regulatory evolution, due to the expectation of reduced pleiotropy. Despite this, numerous studies have ascribed recent evolutionary divergence in gene expression predominantly to trans-regulation. Performing RNA-seq on single isofemale lines from genetically distinct populations of the cactophilic fly Drosophila mojavensis and their F1 hybrids, we recapitulated this pattern in both larval brains and whole bodies. However, we demonstrate that improving the measurement of brain expression divergence between populations by using seven additional genotypes considerably reduces the estimate of trans-regulatory contributions to expression evolution. We argue that the finding of trans-regulatory predominance can result from biases due to environmental variation in expression or other sources of noise, and that cis-regulation is likely a greater contributor to transcriptional evolution across D. mojavensis populations. Lastly, we merge these lines of data to identify several previously hypothesized and intriguing novel candidate genes, and suggest that the integration of regulatory and population-level transcriptomic data can provide useful filters for the identification of potentially adaptive genes.

From phenotype to genotype: the precursor hypothesis predicts genetic influences that facilitate transitions in social behavior.

Parental care is expected to be one of the key evolutionary precursors to advanced social behavior. This suggests that there could be common genetic underpinnings to both parental care and sociality. However, little is known of the genetics underlying care. Here, we suggest that ethological predictions of behavioral precursors to care along with a genetic toolkit for behavior provide testable hypotheses and a defined approach to investigating genetics of sociality. We call this the 'precursor hypothesis'.

Assessing the Architecture of Drosophila mojavensis Locomotor Evolution with Bulk Segregant Analysis.

Behavior is frequently predicted to be especially important for evolution in novel environments. If these predictions are accurate, there might be particular patterns of genetic architecture associated with recently diverged behaviors. Specifically, it has been predicted that behaviors linked to population divergence should be underpinned by a few genes of relatively large effect, compared to architectures of intrapopulation behavioral variation, which is considered to be highly polygenic. More mapping studies of behavioral variation between recently diverged populations are needed to continue assessing the generality of these predictions. Here, we used a bulk segregant mapping approach to dissect the genetic architecture of a locomotor trait that has evolved between two populations of the cactophilic fly Drosophila mojavensis We created an F8 mapping population of 1,500 individuals from advanced intercross lines and sequenced the 10% of individuals with the highest and lowest levels of locomotor activity. Using three alternative statistical approaches, we found strong evidence for two relatively large-effect QTL that is localized in a region homologous to a region of densely packed behavior loci in Drosophila melanogaster, suggesting that clustering of behavior genes may display relatively deep evolutionary conservation. Broadly, our data are most consistent with a polygenic architecture, though with several loci explaining a high proportion of variation in comparison to similar behavioral traits. We further note the presence of several antagonistic QTL linked to locomotion and discuss these results in light of theories regarding behavioral evolution and the effect size and direction of QTL for diverging traits in general.

Changes of gene expression but not cytosine methylation are associated with male parental care reflecting behavioural state, social context and individual flexibility.

Behaviour is often a front line response to changing environments. Recent studies show behavioural changes are associated with changes of gene expression; however, these studies have primarily focused on discrete behavioural states. We build on these studies by addressing additional contexts that produce qualitatively similar behavioural changes. We measured levels of gene expression and cytosine methylation, which is hypothesized to regulate the transcriptional architecture of behavioural transitions, within the brain during male parental care of the burying beetle Nicrophorus vespilloides in a factorial design. Male parenting is a suitably plastic behaviour because although male N. vespilloides typically do not provide direct care (i.e. feed offspring) when females are present, levels of feeding by a male equivalent to the female can be induced by removing the female. We examined three different factors: behavioural state (caring versus non-caring), social context (with or without a female mate) and individual flexibility (if a male switched to direct care after his mate was removed). The greatest number of differentially expressed genes were associated with behavioural state, followed by social context and individual flexibility. Cytosine methylation was not associated with changes of gene expression in any of the factors. Our results suggest a hierarchical association between gene expression and the different factors, but that this process is not controlled by cytosine methylation. Our results further suggest that the extent a behaviour is transient plays an underappreciated role in determining its underpinning molecular mechanisms.

Behavioral evolution accompanying host shifts in cactophilic Drosophila larvae.

For plant utilizing insects, the shift to a novel host is generally accompanied by a complex set of phenotypic adaptations. Many such adaptations arise in response to differences in plant chemistry, competitive environment, or abiotic conditions. One less well-understood factor in the evolution of phytophagous insects is the selective environment provided by plant shape and volume. Does the physical structure of a new plant host favor certain phenotypes? Here, we use cactophilic Drosophila, which have colonized the necrotic tissues of cacti with dramatically different shapes and volumes, to examine this question. Specifically, we analyzed two behavioral traits in larvae, pupation height, and activity that we predicted might be related to the ability to utilize variably shaped hosts. We found that populations of D. mojavensis living on lengthy columnar or barrel cactus hosts have greater activity and pupate higher in a laboratory environment than populations living on small and flat prickly pear cactus cladodes. Crosses between the most phenotypically extreme populations suggest that the genetic architectures of these behaviors are distinct. A comparison of activity in additional cactophilic species that are specialized on small and large cactus hosts shows a consistent trend. Thus, we suggest that greater motility and an associated tendency to pupate higher in the laboratory are potential larval adaptations for life on a large plant where space is more abundant and resources may be more sparsely distributed.

Variation in mandible development and its relationship to dependence on parents across burying beetles.

BACKGROUND: In species with parental care, there is striking variation in offspring dependence at birth, ranging from feeding independence to complete dependency on parents for nutrition. Frequently, highly dependent offspring further evolve reductions or alterations of morphological traits that would otherwise promote self-sufficiency. Here, we examine evidence for morphological evolution associated with dependence in burying beetles (Nicrophorus spp.), in which dependence upon parents appears to have several independent origins. In many species, precocial first instar larvae can survive without parenting, but several altricial species die at this stage on their own. We focused specifically on the mandibles, which are expected to be related to feeding ability and therefore independence from parents. RESULTS: We find no evidence that the size of the mandible is related to dependence on parents. However, we do find a developmental and phylogenetic correlation between independence and the presence of serrations on the inner edge of the mandible. Mandibles of independent species bear serrations at hatching, whereas dependent species hatch with smooth mandibles, only developing serrations in the second instar when these larvae gain the ability to survive on their own. Phylogenetic evidence suggests that serrations coincide with independence repeatedly. We note a single exception to this trend, a beetle with a serrated mandible that cannot survive without parents. However, this exception occurs in a species that has recently evolved the loss of independence. CONCLUSIONS: We argue that the absence of mandible serrations occurs due to alternative selection pressures incurred in larvae dependent upon parents to survive. We suggest that this may have led to a variable function for mandibles, perhaps related to increased competitive ability among siblings or increased efficiency in receiving nutrition from parents. Furthermore, we propose that the phylogenetic pattern we see is consistent with the long-held evolutionary hypothesis that evolutionary change in behavior and physiology precede morphological change.

Duplication and Sub/Neofunctionalization of Malvolio, an Insect Homolog of Nramp, in the Subsocial Beetle Nicrophorus vespilloides.

With growing numbers of sequenced genomes, increasing numbers of duplicate genes are being uncovered. Here we examine Malvolio, a gene in the natural resistance-associated macrophage protein (Nramp) family, that has been duplicated in the subsocial beetle, Nicrophorus vespilloides, which exhibits advanced parental behavior. There is only one copy of Mvl in honey bees and Drosophila, whereas in vertebrates there are two copies that are subfunctionalized. We first compared amino acid sequences for Drosophila, beetles, mice, and humans. We found a high level of conservation between the different species, although there was greater variation in the C-terminal regions. A phylogenetic analysis across multiple insect orders suggested that Mvl has undergone several independent duplications. To examine the potential for different functions where it has been duplicated, we quantified expression levels of Mvl1 and Mvl2 in eight tissues in N. vespilloides We found that while Mvl1 was expressed ubiquitously, albeit at varying levels, expression of Mvl2 was limited to brain and midgut. Because Mvl has been implicated in behavior, we examined expression during different behavioral states that reflected differences in opportunity for social interactions and expression of parental care behaviors. We found differing expression patterns for the two copies, with Mvl1 increasing in expression during resource preparation and feeding offspring, and Mvl2 decreasing in these same states. Given these patterns of expression, along with the protein analysis, we suggest that Mvl in N. vespilloides has experienced sub/neofunctionalization following its duplication, and may be evolving differing and tissue-specific roles in behavior and physiology.

Relating quantitative variation within a behavior to variation in transcription.

Many studies have shown that variation in transcription is associated with changes in behavioral state, or with variation within a state, but little has been done to address if the same genes are involved in both. Here, we investigate the transcriptional basis of variation in parental provisioning using two species of burying beetle, Nicrophorus orbicollis and Nicrophorus vespilloides. We used RNA-seq to compare transcription in parents that provided high amounts of provisioning behavior versus low amounts in males and females of each species. We found no overarching transcriptional patterns distinguishing high from low caring parents, and no informative transcripts that displayed particularly large expression differences in either sex. However, we did find subtler gene expression differences between high and low provisioning parents that are consistent across both sexes and species. Furthermore, we show that transcripts previously implicated in transitioning into parental care in N. vespilloides had high variance in the levels of transcription and were unusually likely to display differential expression between high and low provisioning parents. Thus, quantitative behavioral variation appears to reflect many transcriptional differences of small effect. Furthermore, the same transcripts required for the transition between behavioral states are also related to variation within a behavioral state.

Biparental care is predominant and beneficial to parents in the burying beetle Nicrophorus orbicollis (Coleoptera: Silphidae).

Parenting strategies can be flexible within a species, and may have varying fitness effects. Understanding this flexibility and its fitness consequences is important for understanding why parenting strategies evolve. Here, we investigate the fitness consequences of flexible parenting in the burying beetle Nicrophorus orbicollis, a species known for its advanced provisioning behaviour of regurgitated vertebrate carrion to offspring by both sexes. We show that even when a parent is freely allowed to abandon the carcass at any point in time, biparental post-hatching care is the most common pattern of care adopted in N. orbicollis. Furthermore, two parents together raised more offspring than single parents of either sex, showing that the presence of the male can directly influences parental fitness even in the absence of competitors. This contrasts with studies in other species of burying beetle, where biparental families do not differ in offspring number. This may explain why biparental care is more common in N. orbicollis than in other burying beetles. We suggest how fitness benefits of two parents may play a role in the evolution and maintenance of flexible biparental care in N. orbicollis.

Difference in parenting in two species of burying beetle, Nicrophorus orbicollis and Nicrophorus vespilloides.

Burying beetles (Nicrophorus) are model parents among insects, with all studied species known to regurgitate flesh from vertebrate carcasses to their offspring. However, most studies focus on a very few species, yet the interpretation of the function and importance of care is typically generalized to all burying beetles. Here we characterize subtle variation within and between individuals and sexes, and how this variation differs between two species of burying beetle. We find that Nicrophorus orbicollis exhibits low variance, with a normal distribution of parental care provided during peak care periods. In N. vespilloides, however, the distribution is more uniform as values of care are spread across the possible phenotypic spectrum. This suggests that there is stabilizing selection on care in N. orbicollis, but relaxed or disruptive selection in N. vespilloides. Although repeatability was similar between both species, transitions from other care behaviors into feeding were more common in N. orbicollis than N. vespilloides. Thus, while parenting is coarsely similar across the genus, variation in its expression should not be extrapolated to all Nicrophorus. We suggest that subtle variation both within and among species merits greater attention, and could inform us about the factors that lead to different distributions of care.

The Genome and Methylome of a Beetle with Complex Social Behavior, Nicrophorus vespilloides (Coleoptera: Silphidae).

Testing for conserved and novel mechanisms underlying phenotypic evolution requires a diversity of genomes available for comparison spanning multiple independent lineages. For example, complex social behavior in insects has been investigated primarily with eusocial lineages, nearly all of which are Hymenoptera. If conserved genomic influences on sociality do exist, we need data from a wider range of taxa that also vary in their levels of sociality. Here, we present the assembled and annotated genome of the subsocial beetle Nicrophorus vespilloides, a species long used to investigate evolutionary questions of complex social behavior. We used this genome to address two questions. First, do aspects of life history, such as using a carcass to breed, predict overlap in gene models more strongly than phylogeny? We found that the overlap in gene models was similar between N. vespilloides and all other insect groups regardless of life history. Second, like other insects with highly developed social behavior but unlike other beetles, does N. vespilloides have DNA methylation? We found strong evidence for an active DNA methylation system. The distribution of methylation was similar to other insects with exons having the most methylated CpGs. Methylation status appears highly conserved; 85% of the methylated genes in N. vespilloides are also methylated in the hymentopteran Nasonia vitripennis. The addition of this genome adds a coleopteran resource to answer questions about the evolution and mechanistic basis of sociality and to address questions about the potential role of methylation in social behavior.

Male age mediates reproductive investment and response to paternity assurance.

Theory predicts that male response to reduced paternity will depend on male state and interactions between the sexes. If there is little chance of reproducing again, then males should invest heavily in current offspring, regardless of their share in paternity. We tested this by manipulating male age and paternity assurance in the burying beetle Nicrophorus vespilloides. We found older males invested more in both mating effort and parental effort than younger males. Furthermore, male age, a component of male state, mediated male response to perceived paternity. Older males provided more prenatal care, whereas younger males provided less prenatal care, when perceived paternity was low. Adjustments in male care, however, did not influence selection acting indirectly on parents, through offspring performance. This is because females adjusted their care in response to the age of their partner, providing less care when paired with older males than younger males. As a result offspring, performance did not differ between treatments. Our study shows, for the first time, that a male state variable is an important modifier of paternity-parental care trade-offs and highlights the importance of social interactions between males and females during care in determining male response to perceived paternity.

Morphological correlates of a combat performance trait in the forked fungus beetle, Bolitotherus cornutus.

Combat traits are thought to have arisen due to intense male-male competition for access to females. While large and elaborate weapons used in attacking other males have often been the focus of sexual selection studies, defensive traits (both morphological and performance) have received less attention. However, if defensive traits help males restrict access to females, their role in the process of sexual selection could also be important. Here we examine the morphological correlates of grip strength, a defensive combat trait involved in mate guarding, in the tenebrionid beetle Bolitotherus cornutus. We found that grip strength was repeatable and differed between the sexes. However, these differences in performance were largely explained by body size and a non-additive interaction between size and leg length that differed between males and females. Our results suggest that leg size and body size interact as part of an integrated suite of defensive combat traits.