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Lake Malawi cichlid fishes exhibit extensive divergence in form and function built from a relatively small number of genetic changes. We compared the genomes of rock- and sand-dwelling species and asked which genetic variants differed among the groups. We found that 96% of differentiated variants reside in non-coding sequence but these non-coding diverged variants are evolutionarily conserved. Genome regions near differentiated variants are enriched for craniofacial, neural and behavioral categories. Following leads from genome sequence, we used rock- vs. sand-species and their hybrids to (i) delineate the push–pull roles of BMP signaling and irx1b in the specification of forebrain territories during gastrulation and (ii) reveal striking context-dependent brain gene expression during adult social behavior. Our results demonstrate how divergent genome sequences can predict differences in key evolutionary traits. We highlight the promise of evolutionary reverse genetics—the inference of phenotypic divergence from unbiased genome sequencing and then empirical validation in natural populations.

Lake Malawi cichlid fishes exhibit extensive divergence in form and function among closely related species separated by a relatively small number of genetic changes. During the past million years, hundreds of species have diversified along an ecological axis in rock vs. sand habitats. We compared the genomes of rock- and sand-dwelling species and asked which genetic variants in which genes differed among the groups. We found that 96% of differentiated variants reside in non-coding sequence but these non-coding diverged variants are evolutionarily conserved. The majority of divergent coding variants are missense and/or loss of function. Regions near differentiated variants are enriched for craniofacial, neural and behavioral functional categories. To follow up experimentally, we used rock- vs. sand-species and their hybrids to (i) clarify the push-pull roles of BMP signaling andirx1bin the specification of forebrain territories during gastrulation and (ii) reveal striking context-dependent brain gene expression during adult social behavior. Our results suggest compelling ties between early brain development and adult behavior and highlight the promise of evolutionary reverse genetics – the identification of functional variants from genome sequencing in natural populations.

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.

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.

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 (“conceitedly”). Here we show that the evolution of a specific courtship behavior in Malawi cichlid fish, the construction of mating nests known as bowers, 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 hindbrain features evolve rapidly and independently of phylogeny among castle-building species. Using immediate early gene expression, we confirmed a functional role for hindbrain structures during bower building. Comparisons of bower building species in neighboring Lake Tanganyika show patterns of neural diversification parallel 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.

Significance We do not fully understand how behavior evolves. Here we investigate the genomic basis of bower building among Lake Malawi cichlid fishes. Males construct bowers of two major types, pits and castles, to attract females in mating displays. Thousands of genetic variants are strongly associated with divergence in bower behavior. Remarkably, F1 hybrids of pit-digging and castle-building species perform sequential construction of first pit and then castle bowers. Analysis of brain gene expression in hybrids showed behavior-dependent allele-specific expression with preferential expression of pit-digging alleles during pit digging and castle-building alleles during castle building. Our results suggest that behaviors evolve via complex genetic architectures featuring cis-regulatory differences whose effects on gene expression are specific and context dependent., 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.

Despite considerable research, we still know little about the proximate and ultimate causes behind behavioral evolution. This is partly because understanding the forces acting on behavioral phenotypes requires the study of species-rich clades with extensive variation in behavioral traits, of which we have few current examples. In this paper, we introduce the bower-building cichlids of the Lake Malawi adaptive radiation, a lineage with over 100 species, each possessing a distinct male extended phenotype used to signal reproductive fitness. Extended phenotypes are useful units of analysis for the study of behavior since they are static structures that can be precisely measured within populations. To this end we recognize two core types of bowers - mounds (“castles”) and depressions (“pits”). We employ an established framework for the study of adaptive radiations to ask how traits related to other stages of radiations, macrohabitat and feeding morphology, are associated with the evolution of pit and castle phenotypes. We demonstrate that pits and castles are evolutionarily labile traits and have been derived numerous times in multiple Malawi genera. Using public ecological and phenotypic data sets we find significant and correlated differences in macrohabitat (depth), sensory ability (opsin expression), and feeding style (jaw morphology and biomechanics) between pit-digging and castle-building species. Phylogeny-corrected comparisons also show significant differences in several measures of jaw morphology while indicating non-significant differences in depth. Finally, using laboratory observations we assay courtship behaviors in a pit-digging (Copadichromis virginalis) and a castle-building species (Mchenga conophoros). Together, these results show that traits at multiple biological levels act to regulate the evolution of a courtship behavior within natural populations.