Inside the supergene of the bird with four sexes

Abstract

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.

Keywords: Alternative phenotypes; Coadaptation; Estrogen receptor alpha; Inversion polymorphism; Life-history strategy; Parental behavior; Social behavior; Song; Territorial aggression; Vasoactive intestinal peptide.

Figure 1.

Polymorphism in white-throated sparrows. (A) Males and females occur in two plumage morphs, tan-striped (TS) and white-striped (WS). (B) WS birds of both sexes respond to simulated territorial intrusions with more vocal aggression (song rate is plotted here) than do TS birds. (C) TS males provision young in the nest at a higher rate (trips per hr) than do WS males. Photo by Jennifer Merritt. Data are replotted from Horton et al. (2014a) and Zinzow-Kramer et al. (2015).

Figure 2.

The white-striped phenotype in white-throated sparrows is linked to a rearrangement of chromosome 2. Note that we follow conventional nomenclature for avian chromosomes, numbering them from largest to smallest (Ladjali-Mohammedi et al., 1999). Chromosome 2 in white-throated sparrows corresponds to chromosome 3 in chickens (Thomas et al., 2008). (A) Zebra finch BAC clones 55A1 and 5K13 both map to the long arm of ZAL2, but because of a series of inversions, they map to opposite arms of ZAL2^m. (B) Fluorescent in situ hybridization shows the locations of the two clones (red and green) on ZAL2 and ZAL2^m. Tan-striped (TS) birds have two copies of ZAL2 and white-striped (WS) birds have one copy of ZAL2 and one of ZAL2^m. ZAL2^m/ZAL2^m homozygotes, or superwhite (SW) birds, are rare. The three birds shown in (C) are hatch-year females; at this age, the plumage is usually duller than in adults (compare to Fig. 1A). Nonetheless, this SW bird showed striking bright plumage even as a hatch year female. This bird also showed high levels of aggression (D), performing more displacements in one-on-one behavioral trials than TS or WS birds matched with same-morph opponents. Map of chromosome two (A) redrawn from Thomas et al. (2008). Photos in (B) and (C) reprinted from Horton et al. (2013) with permission. Data in (D) redrawn from Horton et al. (2013).

Figure 3.

Genetic divergence between the ZAL2 and ZAL2^m chromosomes in the white-throated sparrow. Fixation index (F_ST) shown in (A) indicates a high degree of population differentiation (suppression of recombination) between the two chromosomes inside the rearrangement. Pairwise nucleotide divergence (d_xy) shown in (B) indicates significant genetic differentiation between the two chromosomes within the rearrangement compared with the rest of the genome. F_ST and d_xy were measured in 10-kb non-overlapping windows and were significantly higher in scaffolds within the rearrangement than in those outside it (Mann–Whitney U test, ***P < 0.001; NS, not significant). Reprinted from Sun et al. (2018) with permission.

Figure 4.

Expression of ESR1 mediates morph differences in behavior in white-throated sparrows. (A) Expression of ESR1 mRNA in the rostral portion of the medial preoptic area (rPOM) differs by morph and (B) predicts parental provisioning behavior (male data are shown). Provisioning rate is the number of trips made to the nest to feed nestlings per hour on post-hatch day seven during the first nest of the season. (C) ESR1 expression in nucleus taeniae of the amygdala (TnA) differs by morph and (D) predicts the number of songs produced by males in response to a 10-min simulated territorial intrusion early in the breeding season. mRNA expression in (A – D) was measured using in situ hybridization. In (A) and (C), values are normalized to the series mean within sex. Scatterplots in (B) and (D) show residual scores after controlling for the effects of morph, plasma testosterone, and plasma estradiol (E2). (E) ESR1 is alternatively spliced. Dark blue or red regions are cis-regulatory elements (CREs); transcribed regions are light colors. Black lines within CREs represent 42 fixed differences distinguishing ZAL2 from ZAL2^m. Lollipops represent CpG sites. Stacked circles represent transcription factors that are expressed in TnA and for which a binding site is disrupted by a fixed difference. (F) Allelic imbalance in rPOM and TnA of free-living heterozygous (WS) adults during the breeding season. In the bar graphs, each column represents the relative expression of ZAL2 (blue) and ZAL2^m (red). The white dashed line represents a null ratio of 0.5. (G) Behavioral responses of free-living adult males to STI were predicted by the degree of allelic imbalance in TnA. (H) ESR1 knockdown eliminated the morph difference in E2-induced aggression in laboratory-housed birds. Data show the extent to which an oral dose of E2 increased attacks directed toward a conspecific, compared with baseline. * p < 0.05. Data are replotted from Horton et al. (2014b) and Merritt et al. (2020).

Figure 5.

Expression of VIP mRNA in the anterior hypothalamus differs between the morphs (A, B) and predicts territorial singing (C, D) in white-throated sparrows. mRNA expression was measured using in situ hybridization. In (A) and (B), values are normalized to the series mean. (C) and (D) show residual scores after controlling for the effects of morph and plasma steroid hormones (testosterone for males, estradiol for females). Song rate is the number of songs produced by the resident in response to a 10-min simulated territorial intrusion. All data are from birds sampled early in the breeding season. *p < 0.05. Data replotted from Horton et al. (2020).

Figure 6.

Negative correlations between ESR1 and VIP expression in the anterior hypothalamus (A) and nucleus taeniae (B) of free-living white-throated sparrows (n = 79). mRNA was labeled using in situ hybridization in alternate brain sections (see Horton et al., 2014b; 2020). The analysis includes birds of both sexes, sampled throughout the breeding season. Data are normalized to the series mean for each gene in each region. Correlations are significant when morphs are pooled but nonsignificant within morph. Trendlines are shown for each morph separately.