A supergene-linked estrogen receptor drives alternative phenotypes in a polymorphic songbird Jennifer R. Merritta,1, Kathleen E. Grogana, Wendy M. Zinzow-Kramera, Dan Sunb, Eric A. Ortlundc, Soojin V. Yib, and Donna L. Maneya aDepartment of Psychology, Emory University, Atlanta, GA 30322; bSchool of Biological Sciences, Georgia Institute of Technology, Atlanta, GA 30332; and cDepartment of Biochemistry, Emory University, Atlanta, GA 30322 Edited by Gene E. Robinson, University of Illinois at Urbana–Champaign, Urbana, IL, and approved July 8, 2020 (received for review June 3, 2020) Behavioral evolution relies on genetic changes, yet few behaviors tan-striped (TS) morph are homozygous for the standard ar- can be traced to specific genetic sequences in vertebrates. Here we rangement, ZAL2 (13, 14) (Fig. 1A). The rearrangement is provide experimental evidence showing that differentiation of a maintained in the population because of the species’ unique dis- single gene has contributed to the evolution of divergent behav- assortative mating system; nearly every breeding pair consists of ioral phenotypes in the white-throated sparrow, a common back- one individual of each morph (15). Because almost all WS birds yard songbird. In this species, a series of chromosomal inversions are heterozygous for ZAL2m (15, 16), this mating system keeps has formed a supergene that segregates with an aggressive phe- ZAL2m in a near-constant state of heterozygosity (Fig. 1B), pro- notype. The supergene has captured ESR1, the gene that encodes foundly suppressing recombination and causing it to differentiate estrogen receptor α (ERα); as a result, this gene is accumulating from ZAL2 (15, 17). changes that now distinguish the supergene allele from the stan- The rearranged region of ZAL2m in white-throated sparrows dard allele. Our results show that in birds of the more aggressive can be considered a supergene because it harbors a discrete set phenotype, ERα knockdown caused a phenotypic change to that of coinherited, coevolving genes that influence a suite of traits. of the less aggressive phenotype. We next showed that in a free- This supergene dictates not only plumage coloration, but also living population, aggression is predicted by allelic imbalance fa- levels of territorial aggression. In both field and laboratory cis voring the supergene allele. Finally, we identified -regulatory studies, WS birds have been shown to be more aggressive than features, both genetic and epigenetic, that explain the allelic im- TS birds. In free-living, breeding populations, for example, WS NEUROSCIENCE balance. This work provides a rare illustration of how genotypic birds of both sexes respond to simulated territorial intrusion divergence has led to behavioral phenotypic divergence in a (STI) with higher levels of aggression than their TS counterparts vertebrate. (18, 19). ZAL2m/ZAL2m homozygotes are quite rare (15, 20); those that have been described were extraordinarily aggressive chromosomal inversion | songbird | social behavior (16, 18). Thus, the ZAL2m rearrangement is associated with aggression in a dose-dependent manner. here is no doubt that many social behaviors have a genetic Territorial aggression in songbirds has been strongly linked to Tbasis. They are heritable, are acted on by natural selection, circulating levels of gonadal steroid hormones (21, 22). In white- and they evolve (1). Nevertheless, few genetic sequences have throated sparrows, WS birds have higher plasma levels of tes- been linked directly to social behaviors in vertebrates. Most tosterone and estradiol (E2) than TS birds (19); however, this behavioral phenotypes are polygenic, and social behavior itself is difference does not explain the morph difference in aggression. flexibly expressed depending on context. This complexity, to- gether with the many levels of biological organization separating Significance a gene sequence from a social behavior, make it difficult to completely understand why and how natural genotypic variation contributes to behavioral phenotypes (2–5). The white-throated sparrow, a common North American song- The most promising animal models for identifying genetic bird, is a valuable model in behavioral genetics because of a targets of behavioral evolution are those with well-documented chromosomal rearrangement that segregates with a behavioral genetic variation linked to clear behavioral phenotypes. These phenotype. Birds with the rearrangement are more aggressive organisms include Microtus voles (6) and Peromyscus mice (7), in than those without it. Here we show that genetic differentiation which genetic variation in the vasopressin system has been of a single gene inside the rearrangement changes how that causally linked with variation in affiliative and parental behavior, gene is regulated, driving higher levels of expression in birds with the aggressive phenotype and altering behavior. By ex- respectively. Other promising models include organisms in which perimentally reducing the expression of this one gene, we were a behavioral phenotype is linked to large-scale changes in ge- able to change the phenotype of the more aggressive birds to nomic architecture. In fire ants (Solenopsis invicta), for example, the less aggressive phenotype. These results contribute to a the social structure of colonies segregates with a chromosomal greater understanding of how social behaviors can be encoded inversion inside which recombination is suppressed, leading in the genome and how they evolve. to the formation of a “supergene,” a group of genes that are “ ” locked together and inherited as a unit (8). Similarly, in ruffs Author contributions: J.R.M., K.E.G., W.M.Z.-K., E.A.O., S.V.Y., and D.L.M. designed re- (Philomachus pugnax), a chromosomal inversion appears to search; J.R.M., K.E.G., W.M.Z.-K., D.S., E.A.O., S.V.Y., and D.L.M. performed research; mediate a number of alternative reproductive strategies (9, 10), J.R.M., K.E.G., W.M.Z.-K., D.S., E.A.O., S.V.Y., and D.L.M. analyzed data; and J.R.M. and although the causal genes have not been identified. D.L.M. wrote the paper. Decades before the discovery of the chromosomal inversions The authors declare no competing interest. in fire ants and ruffs, Thorneycroft (11, 12) described a rear- This article is a PNAS Direct Submission. rangement of the second chromosome in white-throated sparrows Published under the PNAS license. (Zonotrichia albicollis), a common North American songbird. This 1To whom correspondence may be addressed. Email: [email protected]. m rearrangement, which has been called ZAL2 (“m” for meta- This article contains supporting information online at https://www.pnas.org/lookup/suppl/ centric) segregates with a plumage morph. White-striped (WS) doi:10.1073/pnas.2011347117/-/DCSupplemental. m birds of both sexes have a copy of ZAL2 , whereas birds of the First published August 17, 2020. www.pnas.org/cgi/doi/10.1073/pnas.2011347117 PNAS | September 1, 2020 | vol. 117 | no. 35 | 21673–21680 Downloaded by guest on September 29, 2021 A B WS birds, but not TS birds, are sensitive to the effects of E2 on aggression (23). To test whether this morph-specific sensitivity can be explained by the morph difference in ESR1 expression in TnA, we knocked down ESR1 expression in TnA using antisense oligonucleotides (Fig. 2A). Our rationale for this approach was that if we knocked down ESR1 expression in TnA to a TS-like level in WS birds, then the behavioral response to E2 in those WS birds would be TS-like (low response), and the morph dif- ference in aggression would be abolished. This prediction was supported by our findings. In animals treated with scrambled (control) oligonucleotides, an oral bolus dose of E2 enhanced aggression toward a same-morph conspe- cific (Fig. 2 B, C, and F). This effect was observed in WS birds Fig. 1. Polymorphism in white-throated sparrows. (A) White-throated but not in TS birds (morph × treatment interaction, P < 0.01; SI sparrows occur in two morphs: a more aggressive white-striped (WS) Appendix, Table S2), consistent with previous findings (23, 24). morph and a less aggressive tan-striped (TS) morph. WS birds are hetero- m In other words, the morph with naturally higher expression of zygous for a rearrangement of chromosome 2, known as ZAL2 , which ESR1 in TnA responded to exogenous E2, but the morph with functions as a supergene. Note that we follow conventional nomenclature for avian chromosomes, numbering them from largest to smallest (13). low expression did not. When the morph difference in ESR1 Chromosome 2 in white-throated sparrows corresponds to chromosome 3 in expression in TnA was then abolished via administration of an- chickens (14). (B) Nearly all breeding pairs consist of one TS (ZAL2/ZAL2) bird tisense oligonucleotides (Fig. 2A), the E2-induced aggression in and one WS (ZAL2/ZAL2m) bird. As a result, approximately 50% of the off- WS birds was indistinguishable from that in TS birds (Fig. 2 C spring are ZAL2/ZAL2m heterozygotes and thus WS, and the rest are ZAL2/ and F). Thus, WS-typical levels of ESR1 expression in TnA were ZAL2 homozygotes and thus TS. (Photo courtesy of Jennifer Merritt.) necessary for E2 to facilitate aggression (SI Appendix, Tables S1–S3). To provide further support for the explanatory power of ESR1, Even when plasma testosterone or E2 is experimentally equal- we next tested for a correlation between the aggressive behavior ized, WS birds are still more aggressive (23, 24). This finding in the behavioral trials described above (Fig. 2B) and the level of suggests that WS birds are more sensitive than TS birds to the ESR1 expression in TnA. At 24 h after the second behavioral behavioral effects of these hormones, perhaps because of dif- trial, brains were collected and mRNA was extracted from TnA ferential expression of a steroid hormone receptor. One of the in each bird. Even when our analysis was limited to the control genes inside the supergene is ESR1 (14), which encodes estrogen animals and “misses”—in other words, animals not receiving receptor α (ERα).