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 (ZAL2/2 m ) is known to segregate with a behavioral phenotype. Individuals with the ZAL2 m 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 ZAL2 m rearrangement are mediated by polymorphism in ESR1. We report here that (i) the ESR1 promoter region contains fixed polymorphisms distinguishing the ZAL2 m 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 freeliving 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.estradiol | testosterone | morph | reproductive tactics B ecause the genetic basis of social behavior is complex and involves many genes throughout the genome, it has been difficult to link behavior with specific genes-particularly in vertebrates. The white-throated sparrow (Zonotrichia albicollis), a common North American songbird, represents a unique model for connecting genetic sequence with behavior (1). This species occurs in two plumage morphs (Fig. 1) that segregate absolutely with a rearranged chromosome 2 called ZAL2 m (2). ZAL2 m consists of at least two pericentric inversions (3) and is present in all white-striped (WS) individuals but never in tan-striped (TS) individuals. The rearrangement occurs equally in males and females, and almost all breeding pairs consist of one individual with and one without it (4). This disassortative mating system is the mechanism by which balancing selection acts to maintain ZAL2/2 m in the population, and consequently, homozygosity for ZAL2 m is rare (4,5). Recombination between ZAL2 and ZAL2 m is profoundly suppressed (3), and this genetic isolation has resulted in the differentiation of alternative chromosome types (6).Genetic differentiation of ZAL2/2 m is associated with a behavioral phenotype (1, 4); WS males engage in more territorial aggression and mate-finding than do TS males. In contrast, TS males show more nestling provisioning and mate guarding....

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.

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), are therefore of potential interest toward understanding the molecular underpinnings of these phenotypes. To identify genes that are differentially expressed between the two morphs and correlated with behavior, we quantified both behavior and brain gene expression in a population of free-living white-throated sparrows. We quantified behavioral responses to simulated territorial intrusions (STIs) early during the breeding season. In the same birds, we then performed a transcriptome-wide analysis of gene expression in two regions, the medial amygdala 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 network analyses, we identified modules of genes that were correlated with both morph and STI-induced singing behavior. The majority of these genes were located within the inversion, demonstrating the profound effect the inversion has on the expression of genes captured by the rearrangement. Gene pathway analyses revealed that in the medial amygdala, 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 alpha). Our results thus suggest that ESR1 and related genes are important for behavioral differences between the morphs.

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.

The white-throated sparrow is rapidly becoming an important model in the genetics of social behavior because of a chromosomal rearrangement that segregates with a behavioral phenotype. Within a population, 50 % of individuals are heterozygous for a rearranged chromosome 2 (ZAL2m). These birds sing more and are more aggressive than the other 50 %, who lack the rearrangement. A disassortative mating system, in which heterozygotes almost never interbreed, ensures that ZAL2m/2m homozygotes are extremely rare. Here, we provide the first systematic characterization of such a homozygote, a hatch-year female. Her plumage was atypical of her age and sex, resembling that of an adult male. She was extremely vocal and aggressive, dominating her opponents in behavioral tests. Her phenotype was thus an exaggerated version of a typical ZAL2/2m heterozygote, supporting the hypothesis that alleles inside the ZAL2m rearrangement confer high aggression and further emphasizing this species’ value as a model of social behavior.

Table A1. Behavioral phenotypes of male wire-tailed manakins. Grand means for each status 8 class were calculated from n = 4,669 daily measures of 180 control individuals. Behavioral phenotypes of territory-holding males were calculated using pings and interactions received on 10 the male's own territory, whereas floater behaviors included any territory visited by the floater male. Note that for effort, one ping corresponds to approximately 0.33 minutes of lek attendance. 12Thus, the average effort of floater and territorial males is about 49 and 195 minutes per day, respectively. 14 Behavioral phenotypeFloater mean (SE) Territory-holder mean (SE) Effort (pings per day) 147 (24) 586 (36) Strength (interactions per day) 10.1 (1.6) 5.1 (0.5) Degree (partners per day) 2.6 (0.2) 1.4 (0.1) 16 Table A2. Behavioral correlations among-and within-individuals. Each cell gives the 18 median posterior estimate of the correlation coefficient (ranging from -1 to 1), followed by the [95% central range], derived from a multivariate analysis of n = 4,669 daily measures of 180 20 control individuals. The analysis accounts for status, field season (fixed effects), and ID (random effect). 22 Effort Strength Degree Effort --0.77 [0.69, 0.84] 0.72 [0.63, 0.80] Strength --0.95 [0.93, 0.97] Degree --Effort Strength Degree Effort --0.66 [0.64, 0.68] 0.63 [0.61, 0.65] Strength --0.90 [0.89, 0.90] Degree --24

18variation (2-8%) in social behavior within each status class. Collectively, our findings show that 32 the hormonal control of cooperation depends on a male's social status. We propose that the status-dependent reorganization of hormone-regulatory pathways can facilitate stable cooperative 34 partnerships, and thus provide direct fitness benefits for males. 36

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 songbird: 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 songbirds, our results should serve as a starting point and should help increase the power and utility of songbird models in behavioral neuroendocrinology.