The evolutionary trajectory of drosophilid walking.

Neural circuits must both execute the behavioral repertoire of individuals and account for behavioral variation across species. Understanding how this variation emerges over evolutionary time requires large-scale phylogenetic comparisons of behavioral repertoires. Here, we describe the evolution of walking in fruit flies by capturing high-resolution, unconstrained movement from 13 species and 15 strains of drosophilids. We find that walking can be captured in a universal behavior space, the structure of which is evolutionarily conserved. However, the occurrence of and transitions between specific movements have evolved rapidly, resulting in repeated convergent evolution in the temporal structure of locomotion. Moreover, a meta-analysis demonstrates that many behaviors evolve more rapidly than other traits. Thus, the architecture and physiology of locomotor circuits can execute precise individual movements in one species and simultaneously support rapid evolutionary changes in the temporal ordering of these modular elements across clades.

cis-Regulatory changes in locomotor genes are associated with the evolution of burrowing behavior.

How evolution modifies complex, innate behaviors is largely unknown. Divergence in many morphological traits, and some behaviors, is linked to cis-regulatory changes in gene expression. Given this, we compare brain gene expression of two interfertile sister species of Peromyscus mice that show large and heritable differences in burrowing behavior. Species-level differential expression and allele-specific expression in F1 hybrids indicate a preponderance of cis-regulatory divergence, including many genes whose cis-regulation is affected by burrowing behavior. Genes related to locomotor coordination show the strongest signals of lineage-specific selection on burrowing-induced cis-regulatory changes. Furthermore, genetic markers closest to these candidate genes associate with variation in burrow shape in a genetic cross, suggesting an enrichment for loci affecting burrowing behavior near these candidate locomotor genes. Our results provide insight into how cis-regulated gene expression can depend on behavioral context and how this dynamic regulatory divergence between species may contribute to behavioral evolution.

Mechanosensory input during circuit formation shapes Drosophila motor behavior through patterned spontaneous network activity.

Neural activity sculpts circuit wiring in many animals. In vertebrates, patterned spontaneous network activity (PaSNA) generates sensory maps and establishes local circuits.1-3 However, it remains unclear how PaSNA might shape neuronal circuits and behavior in invertebrates. Previous work in the developing Drosophila embryo discovered intrinsic muscle activity that did not require synaptic transmission, and hence was myogenic, preceding PaSNA.4-6 These studies, however, monitored muscle movement, not neural activity, and were therefore unable to observe how myogenic activity might relate to subsequent neural network engagement. Here we use calcium imaging to directly record neural activity and characterize the emergence of PaSNA. We demonstrate that the spatiotemporal properties of PaSNA are highly stereotyped across embryos, arguing for genetic programming. Neural activity begins well before it becomes patterned, emerging during the myogenic stage. Remarkably, inhibition of mechanosensory input, as well as inhibition of muscle contractions, results in premature and excessive PaSNA, demonstrating that muscle movement serves as a brake on this process. Finally, transient mechanosensory inhibition during PaSNA, followed by quantitative modeling of larval behavior, shows that mechanosensory modulation during development is required for proper larval foraging. This work provides a foundation for using the Drosophila embryo to study the role of PaSNA in circuit formation, provides mechanistic insight into how PaSNA is entrained by motor activity, and demonstrates that spontaneous network activity is essential for locomotor behavior. These studies argue that sensory feedback during the earliest stages of circuit formation can sculpt locomotor behaviors through innate motor learning.

Modular genetic control of social status in a cichlid fish.

Social hierarchies are ubiquitous in social species and profoundly influence physiology and behavior. Androgens like testosterone have been strongly linked to social status, yet the molecular mechanisms regulating social status are not known. The African cichlid fish Astatotilapia burtoni is a powerful model species for elucidating the role of androgens in social status given their rich social hierarchy and genetic tractability. Dominant A. burtoni males possess large testes and bright coloration and perform aggressive and reproductive behaviors while nondominant males do not. Social status in A. burtoni is in flux, however, as males alter their status depending on the social environment. Due to a teleost-specific whole-genome duplication, A. burtoni possess two androgen receptor (AR) paralogs, ARα and ARß, providing a unique opportunity to disentangle the role of gene duplication in the evolution of social systems. Here, we used CRISPR/Cas9 gene editing to generate AR mutant A. burtoni and performed a suite of experiments to interrogate the mechanistic basis of social dominance. We find that ARß, but not ARα, is required for testes growth and bright coloration, while ARα, but not ARß, is required for the performance of reproductive behavior and aggressive displays. Both receptors are required to reduce flees from females and either AR is sufficient for attacking males. Thus, social status in A. burtoni is inordinately dissociable and under the modular control of two AR paralogs. This type of nonredundancy may be important in facilitating social plasticity in A. burtoni and other species whose social status relies on social experience.

Gene expression variation in the brains of harvester ant foragers is associated with collective behavior.

Natural selection on collective behavior acts on variation among colonies in behavior that is associated with reproductive success. In the red harvester ant (Pogonomyrmex barbatus), variation among colonies in the collective regulation of foraging in response to humidity is associated with colony reproductive success. We used RNA-seq to examine gene expression in the brains of foragers in a natural setting. We find that colonies differ in the expression of neurophysiologically-relevant genes in forager brains, and a fraction of these gene expression differences are associated with two colony traits: sensitivity of foraging activity to humidity, and forager brain dopamine to serotonin ratio. Loci that were correlated with colony behavioral differences were enriched in neurotransmitter receptor signaling & metabolic functions, tended to be more central to coexpression networks, and are evolving under higher protein-coding sequence constraint. Natural selection may shape colony foraging behavior through variation in gene expression.

Behavioral evolution contributes to hindbrain diversification among Lake Malawi cichlid fish.

The evolutionary diversification of animal behavior is often associated with changes in the structure and function of nervous systems. Such evolutionary changes arise either through alterations of individual neural components ("mosaically") or through scaling of the whole brain ("concertedly"). Here we show that the evolution of a courtship behavior in Malawi cichlid fish is associated with rapid, extensive, and specific diversification of orosensory, gustatory centers in the hindbrain. We find that hindbrain volume varies significantly between species that build pit (depression) compared to castle (mound) type bowers and that this trait is evolving rapidly among castle-building species. Molecular analyses of neural activity via immediate early gene expression indicate a functional role for hindbrain structures during bower building. Finally, comparisons of bower building species in neighboring Lake Tanganyika suggest parallel patterns of neural diversification to those in Lake Malawi. Our results suggest that mosaic brain evolution via alterations to individual brain structures is more extensive and predictable than previously appreciated.

Hormonal regulation of social ascent and temporal patterns of behavior in an African cichlid.

For many species, social rank determines which individuals perform certain social behaviors and when. Higher ranking or dominant (DOM) individuals maintain status through aggressive interactions and perform courtship behaviors while non-dominant (ND) individuals do not. In some species ND individuals ascend (ASC) in social rank when the opportunity arises. Many important questions related to the mechanistic basis of social ascent remain to be answered. We probed whether androgen signaling regulates social ascent in male Astatotilapia burtoni, an African cichlid whose social hierarchy can be readily controlled in the laboratory. As expected, androgen receptor (AR) antagonism abolished reproductive behavior during social ascent. However, we discovered multiple AR- and status-dependent temporal behavioral patterns that typify social ascent and dominance. AR antagonism in ASC males increased the time between successive behaviors compared to DOM males. Socially ascending males, independent of AR activation, were more likely than DOM males to follow aggressive displays with another aggressive display. Further analyses revealed differences in the sequencing of aggressive and courtship behaviors, wherein DOM males were more likely than ASC males to follow male-directed aggression with courtship displays. Strikingly, this difference was driven mostly by ASC males taking longer to transition from aggression to courtship, suggesting ASC males can perform certain DOM-typical temporal behavioral patterns. Our results indicate androgen signaling is necessary for social ascent and hormonal signaling and social experience may shape the full suite of DOM-typical behavioral patterns.

Behavior-dependent cis regulation reveals genes and pathways associated with bower building in cichlid fishes.

Many behaviors are associated with heritable genetic variation [Kendler and Greenspan (2006) Am J Psychiatry 163:1683-1694]. Genetic mapping has revealed genomic regions or, in a few cases, specific genes explaining part of this variation [Bendesky and Bargmann (2011) Nat Rev Gen 12:809-820]. However, the genetic basis of behavioral evolution remains unclear. Here we investigate the evolution of an innate extended phenotype, bower building, among cichlid fishes of Lake Malawi. Males build bowers of two types, pits or castles, to attract females for mating. We performed comparative genome-wide analyses of 20 bower-building species and found that these phenotypes have evolved multiple times with thousands of genetic variants strongly associated with this behavior, suggesting a polygenic architecture. Remarkably, F1 hybrids of a pit-digging and a castle-building species perform sequential construction of first a pit and then a castle bower. Analysis of brain gene expression in these hybrids showed that genes near behavior-associated variants display behavior-dependent allele-specific expression with preferential expression of the pit-digging species allele during pit digging and of the castle-building species allele during castle building. These genes are highly enriched for functions related to neurodevelopment and neural plasticity. Our results suggest that natural behaviors are associated with complex genetic architectures that alter behavior via cis-regulatory differences whose effects on gene expression are specific to the behavior itself.

Assessing the Genetic Landscape of Animal Behavior.

Although most animal behaviors are associated with some form of heritable genetic variation, we do not yet understand how genes sculpt behavior across evolution, either directly or indirectly. To address this, I here compile a data set comprised of over 1000 genomic loci representing a spectrum of behavioral variation across animal taxa. Comparative analyses reveal that courtship and feeding behaviors are associated with genomic regions of significantly greater effect than other traits, on average threefold greater than other behaviors. Investigations of whole-genome sequencing and phenotypic data for 87 behavioral traits from the Drosophila Genetics Reference Panel indicate that courtship and feeding behaviors have significantly greater genetic contributions and that, in general, behavioral traits overlap little in individual base pairs but increasingly interact at the levels of genes and traits. These results provide evidence that different types of behavior are associated with variable genetic bases and suggest that, across animal evolution, the genetic landscape of behavior is more rugged, yet predictable, than previously thought.

Cis-regulatory evolution in prokaryotes revealed by interspecific archaeal hybrids.

The study of allele-specific expression (ASE) in interspecific hybrids has played a central role in our understanding of a wide range of phenomena, including genomic imprinting, X-chromosome inactivation, and cis-regulatory evolution. However across the hundreds of studies of hybrid ASE, all have been restricted to sexually reproducing eukaryotes, leaving a major gap in our understanding of the genomic patterns of cis-regulatory evolution in prokaryotes. Here we introduce a method to generate stable hybrids between two species of halophilic archaea, and measure genome-wide ASE in these hybrids with RNA-seq. We found that over half of all genes have significant ASE, and that genes encoding kinases show evidence of lineage-specific selection on their cis-regulation. This pattern of polygenic selection suggested species-specific adaptation to low phosphate conditions, which we confirmed with growth experiments. Altogether, our work extends the study of ASE to archaea, and suggests that cis-regulation can evolve under polygenic lineage-specific selection in prokaryotes.