Allele-specific cis-regulatory methylation of the gene for vasoactive intestinal peptide in white-throated sparrows.

White-throated sparrows (Zonotrichia albicollis) offer a unique opportunity to connect genotype with behavioral phenotype. In this species, a rearrangement of the second chromosome is linked with territorial aggression; birds with a copy of this "supergene" rearrangement are more aggressive than those without it. The supergene has captured the gene VIP, which encodes vasoactive intestinal peptide, a neuromodulator that drives aggression in other songbirds. In white-throated sparrows, VIP expression is higher in the anterior hypothalamus of birds with the supergene than those without it, and expression of VIP in this region predicts the level of territorial aggression regardless of genotype. Here, we aimed to identify epigenetic mechanisms that could contribute to differential expression of VIP both in breeding adults, which exhibit morph differences in territorial aggression, and in nestlings, before territorial behavior develops. We extracted and bisulfite-converted DNA from samples of the hypothalamus in wild-caught adults and nestlings and used high-throughput sequencing to measure DNA methylation of a region upstream of the VIP start site. We found that the allele inside the supergene was less methylated than the alternative allele in both adults and nestlings. The differential methylation was attributed primarily to CpG sites that were shared between the alleles, not to polymorphic sites, which suggests that epigenetic regulation is occurring independently of the genetic differentiation within the supergene. This work represents an initial step toward understanding how epigenetic differentiation inside chromosomal inversions leads to the development of alternative behavioral phenotypes.

Inside the supergene of the bird with four sexes.

The white-throated sparrow (Zonotrichia albicollis) offers unique opportunities to understand the adaptive value of supergenes, particularly their role in alternative phenotypes. In this species, alternative plumage morphs segregate with a nonrecombining segment of chromosome 2, which has been called a 'supergene'. The species mates disassortatively with respect to the supergene; that is, each breeding pair consists of one individual with it and one without it. This species has therefore been called the "bird with four sexes". The supergene segregates with a behavioral phenotype; birds with it are more aggressive and less parental than birds without it. Here, we review our efforts to identify the genes inside the supergene that are responsible for the behavioral polymorphism. The gene ESR1, which encodes estrogen receptor α, differs between the morphs and predicts both territorial and parental behavior. Variation in the regulatory regions of ESR1 causes an imbalance in expression of the two alleles, and the degree to which this imbalance favors the supergene allele predicts territorial singing. In heterozygotes, knockdown of ESR1 causes a phenotypic switch, from more aggressive to less aggressive. We recently showed that another gene important for social behavior, vasoactive intestinal peptide (VIP), is differentially expressed between the morphs and predicts territorial singing. We hypothesize that ESR1 and VIP contribute to behavior in a coordinated way and could represent co-adapted alleles. Because the supergene contains more than 1000 individual genes, this species provides rich possibilities for discovering alleles that work together to mediate life-history trade-offs and maximize the fitness of alternative complex phenotypes.

Vasoactive intestinal peptide as a mediator of the effects of a supergene on social behaviour.

Supergenes, or linked groups of alleles that are inherited together, present excellent opportunities to understand gene-behaviour relationships. In white-throated sparrows (Zonotrichia albicollis), a supergene on the second chromosome associates with a more aggressive and less parental phenotype. This supergene includes the gene for vasoactive intestinal peptide (VIP), a neuropeptide known to play a causal role in both aggression and parental behaviour. Here, using a free-living population, we compared the levels of VIP mRNA between birds with and without the supergene. We focused on the anterior hypothalamus and infundibular region, two brain regions containing VIP neurons known to play a causal role in aggression and parental behaviour, respectively. First, we show that the supergene enhances VIP expression in the anterior hypothalamus and that expression positively predicts vocal aggression independently of genotype in both sexes. Next, we show that the supergene reduces VIP expression in the infundibular region, which suggests reduced secretion of prolactin, a pro-parental hormone. Thus, the patterns of VIP expression in these two regions are consistent with the enhanced aggression and reduced parental behaviour of birds with the supergene allele. Our results illustrate mechanisms by which elements of genomic architecture, such as supergenes, can contribute to the evolution of alternative behavioural phenotypes.

Metabolic Rates Predict Baseline Corticosterone and Reproductive Output in a Free-Living Passerine.

Organisms continuously face environmental fluctuations, and allocation of metabolic investment to meet changing energetic demands is fundamental to survival and reproductive success. Glucocorticoid (GC) hormones (e.g., corticosterone [CORT]) play an important role in energy acquisition and allocation in the face of environmental challenges, partly through mediation of energy metabolism. Although GCs and metabolic rate are expected to covary, surprisingly few empirical studies have demonstrated such relationships, especially in wild animals. Moreover, studies testing for associations between GCs and fitness generally do not account for among-individual differences in energy expenditure or energy allocation. We measured CORT (baseline and stress-induced) and metabolic traits (resting metabolic rate [RMR], cold-induced VO2max [Msum], and aerobic scope [the difference between Msum and RMR]) in female tree swallows (Tachycineta bicolor) during chick-rearing, and tested for their associations with several variables of reproductive performance. We found a positive relationship between RMR and baseline CORT, but no consistent associations between stress-induced CORT (SI-CORT) and Msum. This suggests that while baseline CORT may be a good indicator of an individual's baseline metabolic investment, SI-CORT responses are not associated with aerobic scope or the upper limits of aerobic performance. Furthermore, we found that metabolic traits were associated with reproductive performance: females with higher reproductive output showed higher Msum, and also tended to show higher RMR. Overall, these results suggest that metabolic traits are better predictors of reproductive output in tree swallows than CORT concentrations. They further point to the maximal aerobic capacity being higher in females investing more heavily in a current reproductive event, but whether this association reflects trade-offs between current and future reproductive efforts remains to be tested.

Los organismos se enfrentan continuamente a fluctuaciones ambientales, y la distribución de la inversión metabólica es fundamental para la supervivencia y el éxito reproductivo ante demandas energéticas cambiantes. Las hormonas glucocorticoides (GC), como la corticosterona (CORT), juegan un papel importante en los procesos de adquisición y distribución de energía durante los desafíos ambientales, en parte a través de su papel mediador del metabolismo energético. Aunque es esperable que los GCs y la tasa metabólica covaríen, muy pocos estudios empíricos han demostrado esta relación, especialmente en animales salvajes. Además, los estudios que han testado la asociación entre GCs y eficacia biológica normalmente no tienen en cuenta las diferencias en gasto o distribución energética existentes entre individuos. En este estudio, medimos CORT (basal e inducida por estrés) y parámetros metabólicos [tasa metabólica en reposo (RMR), VO2max inducido por frío (Msum) y el alcance metabólico (la diferencia entre Msum y RMR)] en hembras de golondrina bicolor (Tachycineta bicolor) durante el período de crianza de los pollos, y testamos sus asociaciones con varios parámetros reproductivos. Encontramos una relación positiva entre RMR y CORT basal, pero ninguna asociación consistente entre CORT inducida por el estrés y Msum. Esto sugiere que mientras que la CORT basal sería un buen indicador individual del gasto metabólico base, las respuestas de CORT inducidas por estrés no están asociadas con el alcance metabólico o con los límites superiores del rendimiento aeróbico. Encontramos, además, una asociación entre parámetros metabólicos y reproducción: las hembras con mayor éxito reproductivo mostraron mayor Msum, y una tendencia a mostrar mayor RMR. A nivel general, estos resultados sugieren que los parámetros metabólicos son mejores predictores del éxito reproductivo que los niveles de CORT en la golondrina bicolor. También apuntan a que la capacidad aeróbica máxima es mayor en aquellas hembras que invierten más intensamente en el evento reproductivo actual, pero queda por investigar si esta asociación refleja compromisos entre esfuerzo reproductivo actual y futuro.