Brent M. Horton

Estrogen receptor α polymorphism in a species with alternative behavioral phenotypes

Significance In this series of studies, we provide a rare illustration of how a chromosomal polymorphism has affected overt social behavior in a vertebrate. White-throated sparrows occur in two alternative phenotypes, or morphs, distinguished by a chromosomal rearrangement. That the morphs differ in territorial and parental behavior has been known for decades, but how the rearrangement affects behavior is not understood. Here we show that genetic differentiation between the morphs affects the transcription of a gene well known to be involved in social behavior. We then show that in a free-living population, the neural expression of this gene predicts both territorial and parental behavior. We hypothesize that this mechanism has played a causal role in the evolution of alternative life-history strategies. The evolution of behavior relies on changes at the level of the genome; yet the ability to attribute a behavioral change to a specific, naturally occurring genetic change is rare in vertebrates. In the white-throated sparrow (Zonotrichia albicollis), a chromosomal polymorphism (ZAL2/2m) is known to segregate with a behavioral phenotype. Individuals with the ZAL2m haplotype engage in more territorial aggression and less parental behavior than individuals without it. These behaviors are thought to be mediated by sensitivity to sex steroids, and the chromosomal rearrangement underlying the polymorphism has captured a prime candidate gene: estrogen receptor 1 (ESR1), which encodes estrogen receptor α (ERα). We therefore hypothesized that the behavioral effects of the ZAL2m rearrangement are mediated by polymorphism in ESR1. We report here that (i) the ESR1 promoter region contains fixed polymorphisms distinguishing the ZAL2m and ZAL2 alleles; (ii); those polymorphisms regulate transcription efficiency in vitro and therefore potentially do the same in vivo (iii); the local expression of ERα in the brain depends strongly on genotype in a free-living population; and (iv) ERα expression in the medial amygdala and medial preoptic area may fully mediate the effects of genotype on territorial aggression and parenting, respectively. Thus, our study provides a rare glimpse of how a chromosomal polymorphism has affected the brain and social behavior in a vertebrate. Our results suggest that in this species, differentiation of ESR1 has played a causal role in the evolution of phenotypes with alternative life-history strategies.

Morph Matters: Aggression Bias in a Polymorphic Sparrow

In species with discrete morphs exhibiting alternative behavioral strategies, individuals may vary their aggressive behavior in competitive encounters according to the phenotype of their opponent. Such aggression bias has been documented in multiple polymorphic species evolving under negative frequency-dependent selection, but it has not been well-studied under other selection regimes. We investigated this phenomenon in white-throated sparrows (Zonotrichia albicollis), a passerine with plumage polychromatism maintained by disassortative mating. The two distinct color morphs differ with respect to reproductive strategy in that white-striped birds invest more in territorial aggression than tan-striped birds. Whether territorial aggression in this species is biased according to the morph of an intruder is less understood. We found that during peak territorial and mating activity, both color morphs and sexes can exhibit aggression bias, but whether they do so depends on the strategy (morph) of the intruder. During simulated territorial intrusions, resident white-striped males and tan-striped females, which represent the opposite ends of a continuum from high to low territorial aggression, altered their territorial responses according to intruder morph. Tan-striped males and white-striped females, which represent the middle of the continuum, did not show a bias. We propose that because of the disassortative mating system and morph differences in reproductive strategy, the fitness risks of intrusions vary according to the morphs of the resident and the intruder, and that aggression bias is an attuned response to varying threats to fitness.

New insights into the hormonal and behavioural correlates of polymorphism in white-throated sparrows, Zonotrichia albicollis.

The white-throated sparrow is a promising model for behavioural neuroendocrinology and genetics because behaviour and endocrine function may be linked to a chromosomal rearrangement that determines plumage colour. The notion that the two colour morphs, tan-striped (TS) and white-striped (WS), differ predictably in aggression and parenting has been widely accepted, despite conflicting evidence. It is also hypothesized that morph-typic behaviour is hormone mediated, yet no field study has measured sex steroids and behaviour in the same birds. Here, we re-evaluate the TS and WS phenotypes, describe the conditions under which they differ and investigate relationships between sex steroids and behaviour. We report that (1) during territorial intrusions, WS males were more aggressive than TS birds, but this difference was restricted to singing; WS males sang more than TS males but showed identical levels of physical aggression. WS females sang more than TS females and were also more physically aggressive. (2) TS males provisioned young more frequently than did WS males, but only during first broods. The parental strategy of WS males was flexible, and during replacement broods, WS and TS males provisioned at equal rates. (3) Consistent with previous studies, we detected no morph difference in female provisioning. (4) Plasma testosterone and dihydrotestosterone were higher in WS males than in TS males during periods of peak territorial defence and during first broods; within breeding stages, male androgen levels were positively correlated with singing and negatively correlated with provisioning. Plasma oestradiol levels were higher in WS females than in TS females and higher during peak territorial defence; oestradiol levels tended to be positively correlated with singing. Overall, our results refine the TS and WS phenotypes, show that behavioural differences between them are restricted to periods with relatively high mating opportunity, and demonstrate an association between sex steroids and morph-typic behaviour. These results will inform future studies of this promising model.

Proximity data-loggers increase the quantity and quality of social network data

Social network analysis is an ideal quantitative tool for advancing our understanding of complex social behaviour. However, this approach is often limited by the challenges of accurately characterizing social structure and measuring network heterogeneity. Technological advances have facilitated the study of social networks, but to date, all such work has focused on large vertebrates. Here, we provide proof of concept for using proximity data-logging to quantify the frequency of social interactions, construct weighted networks and characterize variation in the social behaviour of a lek-breeding bird, the wire-tailed manakin, Pipra filicauda. Our results highlight how this approach can ameliorate the challenges of social network data collection and analysis by concurrently improving data quality and quantity.

Evaluation of reference genes for quantitative real-time PCR in the brain, pituitary, and gonads of songbirds.

Quantitative real-time PCR (qPCR) is becoming a popular tool for the quantification of gene expression in the brain and endocrine tissues of songbirds. Accurate analysis of qPCR data relies on the selection of appropriate reference genes for normalization, yet few papers on songbirds contain evidence of reference gene validation. Here, we evaluated the expression of ten potential reference genes (18S, ACTB, GAPDH, HMBS, HPRT, PPIA, RPL4, RPL32, TFRC, and UBC) in brain, pituitary, ovary, and testis in two species of songbirds: zebra finch and white-throated sparrow. We used two algorithms, geNorm and NormFinder, to assess the stability of these reference genes in our samples. We found that the suitability of some of the most popular reference genes for target gene normalization in mammals, such as 18S, depended highly on tissue type. Thus, they are not the best choices for brain and gonad in these songbirds. In contrast, we identified alternative genes, such as HPRT, RPL4 and PPIA, that were highly stable in brain, pituitary, and gonad in these species. Our results suggest that the validation of reference genes in mammals does not necessarily extrapolate to other taxonomic groups. For researchers wishing to identify and evaluate suitable reference genes for qPCR in songbirds, our results should serve as a starting point and should help increase the power and utility of songbird models in behavioral neuroendocrinology.

Movements and survival of black-footed ferrets associated with an experimental translocation in South Dakota

Black-footed ferrets (Mustela nigripes) apparently were extirpated from all native habitats by 1987, and their repatriation requires a combination of captive breeding, reintroductions, and translocations among sites. Improvements in survival rates of released ferrets have resulted from experience in quasi-natural environments during their rearing. Reestablishment of a self-sustaining wild population by 1999 provided the 1st opportunity to initiate new populations by translocating wild-born individuals. Using radiotelemetry, we compared behaviors and survival of 18 translocated wild-born ferrets and 18 pen-experienced captive-born ferrets after their release into a prairie dog colony not occupied previously by ferrets. Translocated wild-born ferrets moved significantly less and had significantly higher short-term survival rates than their captive-born counterparts. Using mark–recapture methods, we also assessed potential impacts to the established donor population of removing 37% of its estimated annual production of kits. Annual survival rates for 30 ferret kits remaining at the donor subcomplex were higher than rates for 54 ferret kits at the control subcomplex (unmanipulated) for males (+82%) and females (+32%). Minimum survival of translocated kits did not differ significantly from survival of those at the control subcomplex. Direct translocation of young, wild-born ferrets from site to site appears to be an efficient method to establish new populations.

Genes located in a chromosomal inversion are correlated with territorial song in white-throated sparrows

The genome of the white‐throated sparrow (Zonotrichia albicollis) contains an inversion polymorphism on chromosome 2 that is linked to predictable variation in a suite of phenotypic traits including plumage color, aggression and parental behavior. Differences in gene expression between the two color morphs, which represent the two common inversion genotypes (ZAL2/ZAL2 and ZAL2/ZAL2m), may therefore advance our understanding of the molecular underpinnings of these phenotypes. To identify genes that are differentially expressed between the two morphs and correlated with behavior, we quantified gene expression and terrirorial aggression, including song, in a population of free‐living white‐throated sparrows. We analyzed gene expression in two brain regions, the medial amygdala (MeA) and hypothalamus. Both regions are part of a ‘social behavior network’, which is rich in steroid hormone receptors and previously linked with territorial behavior. Using weighted gene co‐expression network analyses, we identified modules of genes that were correlated with both morph and singing behavior. The majority of these genes were located within the inversion, showing the profound effect of the inversion on the expression of genes captured by the rearrangement. These modules were enriched with genes related to retinoic acid signaling and basic cellular functioning. In the MeA, the most prominent pathways were those related to steroid hormone receptor activity. Within these pathways, the only gene encoding such a receptor was ESR1 (estrogen receptor 1), a gene previously shown to predict song rate in this species. The set of candidate genes we identified may mediate the effects of a chromosomal inversion on territorial behavior.

Hormonal Regulation of Vasotocin Receptor mRNA in a Seasonally Breeding Songbird

Behaviors associated with breeding are seasonally modulated in a variety of species. These changes in behavior are mediated by sex steroids, levels of which likewise vary with season. The effects of androgens on behaviors associated with breeding may in turn be partly mediated by the nonapeptides vasopressin (VP) and oxytocin (OT) in mammals, and vasotocin (VT) in birds. The effects of testosterone (T) on production of these neuropeptides have been well-studied; however, the regulation of VT receptors by T is not well understood. In this study, we investigated steroid-dependent regulation of VT receptor (VTR) mRNA in a seasonally breeding songbird, the white-throated sparrow (Zonotrichia albicollis). We focused on VTR subtypes that have been most strongly implicated in social behavior: V1a and oxytocin-like receptor (OTR). Using in situ hybridization, we show that T-treatment of non-breeding males altered V1a and OTR mRNA expression in several regions associated with seasonal reproductive behaviors. For example, T-treatment increased V1a mRNA expression in the medial preoptic area, bed nucleus of the stria terminalis, and ventromedial hypothalamus. T-treatment also affected both V1a and OTR mRNA expression in nuclei of the song system; some of these effects depended on the presence or absence of a chromosomal rearrangement that affects singing behavior, plasma T, and VT immunolabeling in this species. Overall, our results strengthen evidence that VT helps mediate the behavioral effects of T in songbirds, and suggest that the chromosomal rearrangement in this species may affect the sensitivity of the VT system to seasonal changes in T.

Divergent selection on bill morphology contributes to nonrandom mating between swamp sparrow subspecies

Traits subject to ecologically based divergent selection that also affect nonrandom mating (i.e. ‘magic traits’) may hasten incipient speciation. In this study, we investigated whether nonrandom mating results from ecological divergence between swamp sparrow, Melospiza georgiana , subspecies. As a by-product of adaptive divergence in bill morphology between populations of swamp sparrows, there is a corresponding divergence in features of song shown to be salient to females, potentially providing a mechanism of reproductive isolation. Thus, female mating preferences for song could result in prezygotic isolation that prevents gene flow between subspecies. In this study we tested the hypothesis that female preferences for song provide a mechanism of reproductive isolation that limits gene flow between subspecies. Using copulation solicitation assays, we found that female coastal plain swamp sparrows showed a significant preference for consubspecific songs over heterosubspecific songs. We further tested whether mating preferences in coastal populations explain observed differences in song between subspecies. We found evidence that mating preferences in coastal females may explain the evolution of some acoustic features of song. We suggest that the bill may be an example of a ‘magic trait’ that contributes to continued divergence of swamp sparrow subspecies.

Estrogen Receptor Alpha as a Mediator of Life-History Trade-offs

Trade-offs between competitive and parental strategies often are mediated by sex steroids. The mechanisms underlying steroid signaling and metabolism may therefore serve as targets of disruptive selection that leads to alternative behavioral phenotypes. White-throated sparrows exhibit two color morphs that differ in both competitive and parental behavior; white-striped (WS) birds engage in more territorial singing, whereas tan-striped (TS) birds provision nestlings more often. Although WS birds have higher levels of plasma testosterone (T) and estradiol than do TS birds, experimental equalization of these hormones does not abolish morph differences in singing. Neural sensitivity to sex steroids may differ between the morphs because the gene for estrogen receptor alpha (ERα) has been captured by a chromosomal rearrangement found only in the WS birds. We recently showed that expression of this gene differs between the morphs and may drive the behavioral polymorphism. First, the ERα promoter region contains fixed polymorphisms that affect transcription efficiency in vitro. Second, in a free-living population, local expression of ERα depends strongly on morph and predicts both territorial singing and parental provisioning. Differential ERα expression is particularly striking in the medial amygdala; WS birds have three times more ERα mRNA than do TS birds. This difference persists during the non-breeding season and is unaffected by exogenous T treatment. Finally, preliminary data generated by RNA-seq confirm that ERα expression in MeA is both differentially expressed and correlated with territorial singing. Together, these results suggest that ERα may be a target of disruptive selection that leads to alternative behavioral strategies. Our future directions include a more detailed analysis of the ERα promoter regions to determine the molecular basis of differential expression as well as gene network analyses to identify genes connected to ERα.