The Ancestral Modulation Hypothesis: Predicting Mechanistic Control of Sexually Heteromorphic Traits Using Evolutionary History.

AbstractAcross the animal kingdom there are myriad forms within a sex across, and even within, species, rendering concepts of universal sex traits moot. The mechanisms that regulate the development of these trait differences are varied, although in vertebrates, common pathways involve gonadal steroid hormones. Gonadal steroids are often associated with heteromorphic trait development, where the steroid found at higher circulating levels is the one involved in trait development for that sex. Occasionally, there are situations in which a gonadal steroid associated with heteromorphic trait development in one sex is involved in heteromorphic or monomorphic trait development in another sex. We propose a verbal hypothesis, the ancestral modulation hypothesis (AMH), that uses the evolutionary history of the trait-particularly which sex ancestrally possessed higher trait values-to predict the regulatory pathway that governs trait expression. The AMH predicts that the genomic architecture appears first to resolve sexual conflict in an initially monomorphic trait. This architecture takes advantage of existing sex-biased signals, the gonadal steroid pathway, to generate trait heteromorphism. In cases where the other sex experiences evolutionary pressure for the new phenotype, that sex will co-opt the existing architecture by altering its signal to match that of the original high-trait-value sex. We describe the integrated levels needed to produce this pattern and what the expected outcomes will be given the evolutionary history of the trait. We present this framework as a testable hypothesis for the scientific community to investigate and to create further engagement and analysis of both ultimate and proximate approaches to sexual heteromorphism.

The social environment alters neural responses to a lipopolysaccharide challenge.

Sick animals display drastic changes in their behavioral patterns, including decreased activity, decreased food and water intake, and decreased interest in social interactions. These behaviors, collectively called "sickness behaviors", can be socially modulated. For example, when provided with mating opportunities, males of several species show reduced sickness behaviors. While the behavior is known to change, how the social environment affects neural molecular responses to sickness is not known. Here, we used a species, the zebra finch, Taeniopygia guttata, where males have been shown to decrease sickness behaviors when presented with novel females. Using this paradigm, we obtained samples from three brain regions (the hypothalamus, the bed nucleus of the stria terminalis, and the nucleus taeniae) from lipopolysaccharide (LPS) or control treated males housed under four different social environments. Manipulation of the social environment rapidly changed the strength and co-expression patterns of the neural molecular responses to the immune challenge in all brain regions tested, therefore suggesting that the social environment plays a significant role in determining the neural responses to an infection. In particular, brains of males paired with a novel female showed muted immune responses to LPS, as well as altered synaptic signaling. Neural metabolic activity in response to the LPS challenge was also affected by the social environment. Our results provide new insights into the effects of the social environment on brain responses to an infection, thereby improving our understanding of how the social environment can affect health.

Transcriptomic changes associated with maternal care in the brain of mouthbrooding cichlid Astatotilapia burtoni reflect adaptation to self-induced metabolic stress.

Parental care in Astatotilapia burtoni entails females protecting eggs and developing fry in a specialized buccal cavity in the mouth. During this mouthbrooding behavior, which can last 2-3 weeks, mothers undergo voluntary fasting accompanied by loss of body mass and major metabolic changes. Following release of fry, females resume normal feeding behavior and quickly recover body mass as they become reproductively active once again. In order to investigate the molecular underpinnings of such dramatic behavioral and metabolic changes, we sequenced whole-brain transcriptomes from females at four time points throughout their reproductive cycle: 2 days after the start of mouthbrooding, 14 days after the start of mouthbrooding, 2 days after the release of fry and 14 days after the release of fry. Differential expression analysis and clustering of expression profiles revealed a number of neuropeptides and hormones, including the strong candidate gene neurotensin, suggesting that molecular mechanisms underlying parental behaviors may be common across vertebrates despite de novo evolution of parental care in these lineages. In addition, oxygen transport pathways were found to be dramatically downregulated, particularly later in the mouthbrooding stage, while certain neuroprotective pathways were upregulated, possibly to mitigate negative consequences of metabolic depression brought about by fasting. Our results offer new insights into the evolution of parental behavior as well as revealing candidate genes that would be of interest for the study of hypoxic ischemia and eating disorders.

Correspondence of aCGH and long-read genome assembly for detection of copy number differences: A proof-of-concept with cichlid genomes.

Copy number variation is an important source of genetic variation, yet data are often lacking due to technical limitations for detection given the current genome assemblies. Our goal is to demonstrate the extent to which an array-based platform (aCGH) can identify genomic loci that are collapsed in genome assemblies that were built with short-read technology. Taking advantage of two cichlid species for which genome assemblies based on Illumina and PacBio are available, we show that inter-species aCGH log2 hybridization ratios correlate more strongly with inferred copy number differences based on PacBio-built genome assemblies than based on Illumina-built genome assemblies. With regard to inter-species copy number differences of specific genes identified by each platform, the set identified by aCGH intersects to a greater extent with the set identified by PacBio than with the set identified by Illumina. Gene function, according to Gene Ontology analysis, did not substantially differ among platforms, and platforms converged on functions associated with adaptive phenotypes. The results of the current study further demonstrate that aCGH is an effective platform for identifying copy number variable sequences, particularly those collapsed in short read genome assemblies.

Epigenetic Regulation and Environmental Sex Determination in Cichlid Fishes.

Studying environmental sex determination (ESD) in cichlids provides a phylogenetic and comparative approach to understand the evolution of the underlying mechanisms, their impact on the evolution of the overlying systems, and the neuroethology of life history strategies. Natural selection normally favors parents who invest equally in the development of male and female offspring, but evolution may favor deviations from this 50:50 ratio when environmental conditions produce an advantage for doing so. Many species of cichlids demonstrate ESD in response to water chemistry (temperature, pH, and oxygen concentration). The relative strengths of and the exact interactions between these factors vary between congeners, demonstrating genetic variation in sensitivity. The presence of sizable proportions of the less common sex towards the environmental extremes in most species strongly suggests the presence of some genetic sex-determining loci acting in parallel with the ESD factors. Sex determination and differentiation in these species does not seem to result in the organization of a final and irreversible sexual fate, so much as a life-long ongoing battle between competing male- and female-determining genetic and hormonal networks governed by epigenetic factors. We discuss what is and is not known about the epigenetic mechanism behind the differentiation of both gonads and sex differences in the brain. Beyond the well-studied tilapia species, the 2 best-studied dwarf cichlid systems showing ESD are the South American genus Apistogramma and the West African genus Pelvicachromis. Both species demonstrate male morphs with alternative reproductive tactics. We discuss the further neuroethology opportunities such systems provide to the study of epigenetics of alternative life history strategies and other behavioral variation.

Epigenetic regulation of gonadal and brain aromatase expression in a cichlid fish with environmental sex determination.

A fit animal must develop testes or ovaries, with brain and physiology to match. In species with alternative male morphs this coordination of development across tissues operates within sexes as well as between. For Pelvicachromis pulcher, an African cichlid in which early pH exposure influences both sex and alternative male morph, we sequence both copies of aromatase (cyp19a1), a key gene for sex determination. We analyze gene expression and epigenetic state, comparing gonad and brain tissue from females, alternative male morphs, and fry. Relative to brain, we find elevated expression of the A-copy in the ovaries but not testes. Methylation analysis suggests strong epigenetic regulation, with one region specifying sex and another specifying tissue. We find elevated brain expression of the B-copy with no sex or male morph differences. B-copy methylation follows that of the A-copy rather than corresponding to B-copy expression. In 30-day old fry, we see elevated B-copy expression in the head, but we do not see the expected elevated A-copy expression in the trunk that would reflect ovarian development. Interestingly, the A-copy epialleles that distinguish ovaries from testes are among the most explanatory patterns for variation among fry, suggesting epigenetic marking of sex prior to differentiation and thus laying the groundwork for mechanistic studies of epigenetic regulation of sex and morph differentiation.

The Genomic Substrate for Adaptive Radiation: Copy Number Variation across 12 Tribes of African Cichlid Species.

The initial sequencing of five cichlid genomes revealed an accumulation of genetic variation, including extensive copy number variation in cichlid lineages particularly those that have undergone dramatic evolutionary radiation. Gene duplication has the potential to generate substantial molecular substrate for the origin of evolutionary novelty. We use array-based comparative heterologous genomic hybridization to identify copy number variation events (CNVEs) for 168 samples representing 53 cichlid species including the 5 species for which full genome sequence is available. We identify an average of 50-100 CNVEs per individual. For those species represented by multiple samples, we identify 150-200 total CNVEs suggesting a substantial amount of intraspecific variation. For these species, only ∼10% of the detected CNVEs are fixed. Hierarchical clustering of species according to CNVE data recapitulates phylogenetic relationships fairly well at both the tribe and radiation level. Although CNVEs are detected on all linkage groups, they tend to cluster in "hotspots" and are likely to contain and be flanked by transposable elements. Furthermore, we show that CNVEs impact functional categories of genes with potential roles in adaptive phenotypes that could reasonably promote divergence and speciation in the cichlid clade. These data contribute to a more complete understanding of the molecular basis for adaptive natural selection, speciation, and evolutionary radiation.

The intestinal environment as an evolutionary adaptation to mouthbrooding in the Astatotilapia burtoni cichlid.

Many of the various parental care strategies displayed by animals are accompanied by a significant reduction in food intake that imposes a substantial energy trade-off. Mouthbrooding, as seen in several species of fish in which the parent holds the developing eggs and fry in the buccal cavity, represents an extreme example of reduced food intake during parental investment and is accompanied by a range of physiological adaptations. In this study we use 16S sequencing to characterize the gut microbiota of female Astatotilapia burtoni cichlid fish throughout the obligatory phase of self-induced starvation during the brooding cycle in comparison to stage-matched females that have been denied food for the same duration. In addition to a reduction of gut epithelial turnover, we find a dramatic reduction in species diversity in brooding stages that recovers upon release of fry and refeeding that is not seen in females that are simply starved. Based on overall species diversity as well as differential abundance of specific bacterial taxa, we suggest that rather than reflecting a simple deprivation of caloric intake, the gut microbiota is more strongly influenced by physiological changes specific to mouthbrooding including the reduced epithelial turnover and possible production of antimicrobial agents.

Gene expression signatures of mating system evolution.

The diversity of mating systems among animals is astounding. Importantly, similar mating systems have evolved even across distantly related taxa. However, our understanding of the mechanisms underlying these convergently evolved phenotypes is limited. Here, we examine on a genomic scale the neuromolecular basis of social organization in cichlids of the tribe Ectodini from Lake Tanganyika. Using field-collected males and females of four closely related species representing two independent evolutionary transitions from polygyny to monogamy, we take a comparative transcriptomic approach to test the hypothesis that these independent transitions have recruited similar gene sets. Our results demonstrate that while lineage and species exert a strong influence on neural gene expression profiles, social phenotype can also drive gene expression evolution. Specifically, 331 genes (∼6% of those assayed) were associated with monogamous mating systems independent of species or sex. Among these genes, we find a strong bias (4:1 ratio) toward genes with increased expression in monogamous individuals. A highly conserved nonapeptide system known to be involved in the regulation of social behavior across animals was not associated with mating system in our analysis. Overall, our findings suggest deep molecular homologies underlying the convergent or parallel evolution of monogamy in different cichlid lineages of Ectodini.

Dissecting the Transcriptional Patterns of Social Dominance across Teleosts.

In many species, under varying ecological conditions, social interactions among individuals result in the formation of dominance hierarchies. Despite general similarities, there are robust differences among dominance hierarchies across species, populations, environments, life stages, sexes, and individuals. Understanding the proximate mechanisms underlying the variation is an important step toward understanding the evolution of social behavior. However, physiological changes associated with dominance, such as gonadal maturation and somatic growth, often complicate efforts to identify the specific underlying mechanisms. Traditional gene expression analyses are useful for generating candidate gene lists, but are biased by choice of significance cut-offs and difficult to use for between-study comparisons. In contrast, complementary analysis tools allow one to both test a priori hypotheses and generate new hypotheses. Here we employ a meta-analysis of high-throughput expression profiling experiments to investigate the gene expression patterns that underlie mechanisms and evolution of behavioral social phenotypes. Specifically, we use a collection of datasets on social dominance in fish across social contexts, sex, and species. Using experimental manipulation to produce female dominance hierarchies in the cichlid Astatotilapia burtoni, heralded as a genomic model of social dominance, we generate gene lists, and assess molecular gene modules. In the dominant female gene expression profile, we demonstrate a strong pattern of up-regulation of genes previously identified as having male-biased expression and furthermore, compare expression biases between male and female dominance phenotypes. Using a threshold-free approach to identify correlation throughout ranked gene lists, we query previously published datasets associated with maternal behavior, alternative reproductive tactics, cooperative breeding, and sex-role reversal to describe correlations among these various neural gene expression profiles associated with different instances of social dominance. These complementary approaches capitalize on the high-throughput gene expression profiling from similar behavioral phenotypes in order to address the mechanisms associated with social dominance behavioral phenotypes.

Gene duplication in an African cichlid adaptive radiation.

BACKGROUND: Gene duplication is a source of evolutionary innovation and can contribute to the divergence of lineages; however, the relative importance of this process remains to be determined. The explosive divergence of the African cichlid adaptive radiations provides both a model for studying the general role of gene duplication in the divergence of lineages and also an exciting foray into the identification of genomic features that underlie the dramatic phenotypic and ecological diversification in this particular lineage. We present the first genome-wide study of gene duplication in African cichlid fishes, identifying gene duplicates in three species belonging to the Lake Malawi adaptive radiation (Metriaclima estherae, Protomelas similis, Rhamphochromis "chilingali") and one closely related species from a non-radiated riverine lineage (Astatotilapia tweddlei). RESULTS: Using Astatotilapia burtoni as reference, microarray comparative genomic hybridization analysis of 5689 genes reveals 134 duplicated genes among the four cichlid species tested. Between 51 and 55 genes were identified as duplicated in each of the three species from the Lake Malawi radiation, representing a 38%-49% increase in number of duplicated genes relative to the non-radiated lineage (37 genes). Duplicated genes include several that are involved in immune response, ATP metabolism and detoxification. CONCLUSIONS: These results contribute to our understanding of the abundance and type of gene duplicates present in cichlid fish lineages. The duplicated genes identified in this study provide candidates for the analysis of functional relevance with regard to phenotype and divergence. Comparative sequence analysis of gene duplicates can address the role of positive selection and adaptive evolution by gene duplication, while further study across the phylogenetic range of cichlid radiations (and more generally in other adaptive radiations) will determine whether the patterns of gene duplication seen in this study consistently accompany rapid radiation.

Characterization of the OmyY1 Region on the Rainbow Trout Y Chromosome.

We characterized the male-specific region on the Y chromosome of rainbow trout, which contains both sdY (the sex-determining gene) and the male-specific genetic marker, OmyY1. Several clones containing the OmyY1 marker were screened from a BAC library from a YY clonal line and found to be part of an 800 kb BAC contig. Using fluorescence in situ hybridization (FISH), these clones were localized to the end of the short arm of the Y chromosome in rainbow trout, with an additional signal on the end of the X chromosome in many cells. We sequenced a minimum tiling path of these clones using Illumina and 454 pyrosequencing. The region is rich in transposons and rDNA, but also appears to contain several single-copy protein-coding genes. Most of these genes are also found on the X chromosome; and in several cases sex-specific SNPs in these genes were identified between the male (YY) and female (XX) homozygous clonal lines. Additional genes were identified by hybridization of the BACs to the cGRASP salmonid 4x44K oligo microarray. By BLASTn evaluations using hypothetical transcripts of OmyY1-linked candidate genes as query against several EST databases, we conclude at least 12 of these candidate genes are likely functional, and expressed.

Females of an African cichlid fish display male-typical social dominance behavior and elevated androgens in the absence of males.

Social environment can affect the expression of sex-typical behavior in both males and females. Males of the African cichlid species Astatotilapia burtoni have long served as a model system to study the neural, endocrine, and molecular basis of socially plastic dominance behavior. Here we show that in all-female communities of A. burtoni, some individuals acquire a male-typical dominance phenotype, including aggressive territorial defense, distinctive color patterns, and courtship behavior. Furthermore, dominant females have higher levels of circulating androgens than either subordinate females or females in mixed-sex communities. These male-typical traits do not involve sex change, nor do the social phenotypes in all-female communities differ in relative ovarian size, suggesting that factors other than gonadal physiology underlie much of the observed variation. In contrast to the well-studied situation in males, dominant and subordinate females do not differ in the rate of somatic growth. Dominant females are not any more likely than subordinates to spawn with an introduced male, although they do so sooner. These results extend the well known extraordinary behavioral plasticity of A. burtoni to the females of this species and provide a foundation for uncovering the neural and molecular basis of social dominance behavior while controlling for factors such as sex, gonadal state and growth.

Comparative gene expression profiles for highly similar aggressive phenotypes in male and female cichlid fishes (Julidochromis).

Julidochromis marlieri and Julidochromis transcriptus are two closely related Tanganyikan cichlids that have evolved different behavior and mating strategies since they diverged from their common ancestor. While J. transcriptus follows the ancestral pattern of male dominance, male-biased sexual size dimorphism and territoriality, the pattern is reversed in J. marlieri. In J. marlieri, females show all of these behavioral and morphological characteristics. This raises the question of whether female J. marlieri achieve the dominant phenotype by expressing the same genes as J. transcriptus males or whether novel brain gene expression patterns have evolved to produce a similar behavioral phenotype in the females of J. marlieri. This study used cDNA microarrays to investigate whether female J. marlieri and male J. transcriptus show conserved or divergent patterns of brain gene expression. Analysis of microarray data in both species showed certain gene expression patterns associated with sex role independent of gonadal sex and, to a lesser extent, gene expression patterns associated with sex independent of sex role. In general, these data suggest that while there has been substantial divergence in gene expression patterns between J. transcriptus and J. marlieri, we can detect a highly significant overlap for a core set of genes related to aggression in both species. These results suggest that the proximate mechanisms regulating aggressive behavior in J. transcriptus and J. marlieri may be shared.

Ecological genomics--changing perspectives on Darwin's basic concerns.

Ecological Genomics is an interdisciplinary field that seeks to understand the genetic and physiological basis of species interactions for evolutionary inferences. At the 7th annual Ecological Genomics Symposium, November 13-15, 2009, members of the Ecological Genomics program at Kansas State University invited 13 speakers and 56 poster presentations.

A critical assessment of cross-species detection of gene duplicates using comparative genomic hybridization.

BACKGROUND: Comparison of genomic DNA among closely related strains or species is a powerful approach for identifying variation in evolutionary processes. One potent source of genomic variation is gene duplication, which is prevalent among individuals and species. Array comparative genomic hybridization (aCGH) has been successfully utilized to detect this variation among lineages. Here, beyond the demonstration that gene duplicates among species can be quantified with aCGH, we consider the effect of sequence divergence on the ability to detect gene duplicates. RESULTS: Using the X chromosome genomic content difference between male D. melanogaster and female D. yakuba and D. simulans, we describe a decrease in the ability to accurately measure genomic content (copy number) for orthologs that are only 90% identical. We demonstrate that genome characteristics (e.g. chromatin environment and non-orthologous sequence similarity) can also affect the ability to accurately measure genomic content. We describe a normalization strategy and statistical criteria to be used for the identification of gene duplicates among any species group for which an array platform is available from a closely related species. CONCLUSIONS: Array CGH can be used to effectively identify gene duplication and genome content; however, certain biases are present due to sequence divergence and other genome characteristics resulting from the divergence between lineages. Highly conserved gene duplicates will be more readily recovered by aCGH. Duplicates that have been retained for a selective advantage due to directional selection acting on many loci in one or both gene copies are likely to be under-represented. The results of this study should inform the interpretation of both previously published and future work that employs this powerful technique.

Using comparative genomic hybridization to survey genomic sequence divergence across species: a proof-of-concept from Drosophila.

BACKGROUND: Genome-wide analysis of sequence divergence among species offers profound insights into the evolutionary processes that shape lineages. When full-genome sequencing is not feasible for a broad comparative study, we propose the use of array-based comparative genomic hybridization (aCGH) in order to identify orthologous genes with high sequence divergence. Here we discuss experimental design, statistical power, success rate, sources of variation and potential confounding factors. We used a spotted PCR product microarray platform from Drosophila melanogaster to assess sequence divergence on a gene-by-gene basis in three fully sequenced heterologous species (D. sechellia, D. simulans, and D. yakuba). Because complete genome assemblies are available for these species this study presents a powerful test for the use of aCGH as a tool to measure sequence divergence. RESULTS: We found a consistent and linear relationship between hybridization ratio and sequence divergence of the sample to the platform species. At higher levels of sequence divergence (< 92% sequence identity to D. melanogaster) approximately 84% of features had significantly less hybridization to the array in the heterologous species than the platform species, and thus could be identified as "diverged". At lower levels of divergence (>or= 97% identity), only 13% of genes were identified as diverged. While approximately 40% of the variation in hybridization ratio can be accounted for by variation in sequence identity of the heterologous sample relative to D. melanogaster, other individual characteristics of the DNA sequences, such as GC content, also contribute to variation in hybridization ratio, as does technical variation. CONCLUSIONS: Here we demonstrate that aCGH can accurately be used as a proxy to estimate genome-wide divergence, thus providing an efficient way to evaluate how evolutionary processes and genomic architecture can shape species diversity in non-model systems. Given the increased number of species for which microarray platforms are available, comparative studies can be conducted for many interesting lineages in order to identify highly diverged genes that may be the target of natural selection.

Interspecific profiling of gene expression informed by comparative genomic hybridization: A review and a novel approach in African cichlid fishes.

Modern genomic approaches have facilitated great progress in our understanding of the molecular and genetic underpinnings of ecological and evolutionary processes. Analysis of gene expression through heterologous hybridization in particular has enabled genome-scale studies in many ecologically and evolutionarily interesting species. However, these studies have been hampered by the difficulty of comparing-on a common array platform-gene-expression profiles across species due to sequence divergence altering the dynamics of hybridization. All too often, comparisons of expression profiles across species were limited to contrasting lists of gene or even of just functional categories. Here we review these issues and propose a novel solution. Exploiting the diverse cichlid lineages of East Africa as our model-system, we then present results from an experimental case study that compares the neural gene-expression profiles of males and females of two species that differ in mating system. Using a single microarray platform that contains genes from one species, Astatotilapia burtoni, we conducted a total of 16 direct comparisons for neural gene-expression level between individual males and females from a pair of sister species, the polygynous Enantiopus melanogenys and the monogamous Xenotilapia flavipinnis. Next, we conducted a meta-analysis with previously published data from two different intra-specific expression studies to determine whether sex-specific neural gene expression is more closely associated with behavioral phenotype than it is with gonadal sex. Our results indicate that the gene expression profiles are species-specific to a large extent, as relatively few genes show conserved expression patterns associated with either sex. Finally, we describe how competitive genomic DNA hybridizations between the two focal species allow us to assess the degree to which divergence of sequences biases the results. We propose a masking technique that correlates interspecific expression ratios obtained with cDNA with hybridization ratios obtained with genomic DNA for the same set of species and determines threshold sequence divergence to reduce false positives. Our approach should be applicable to a wide range of interesting questions related to the evolution and ecology of gene expression.