A supergene determines highly divergent male reproductive morphs in the ruff

Abstract

Three strikingly different alternative male mating morphs (aggressive 'independents', semicooperative 'satellites' and female-mimic 'faeders') coexist as a balanced polymorphism in the ruff, Philomachus pugnax, a lek-breeding wading bird. Major differences in body size, ornamentation, and aggressive and mating behaviors are inherited as an autosomal polymorphism. We show that development into satellites and faeders is determined by a supergene consisting of divergent alternative, dominant and non-recombining haplotypes of an inversion on chromosome 11, which contains 125 predicted genes. Independents are homozygous for the ancestral sequence. One breakpoint of the inversion disrupts the essential CENP-N gene (encoding centromere protein N), and pedigree analysis confirms the lethality of homozygosity for the inversion. We describe new differences in behavior, testis size and steroid metabolism among morphs and identify polymorphic genes within the inversion that are likely to contribute to the differences among morphs in reproductive traits.

Figure 1

Comparison of of Independent, Satellite, and Faeder ruff males. a, Representative breeding plumages. b, Body size distributions (see Methods). c, Comparisons of morph behavioural profiles for Aggression (“Aggr.” – black), Display (“Disp.” – red), Proximity (“Prox.” – brown), and Alert stance (“Alert” – green). Morph-specific boxplots are plotted for each behaviour from the means of individual male ruffs, standardized across the sample population by subtracting the global mean and dividing by s.d.. Morphs differed in their distributions of behaviour (MANOVA on individual bird mean values; Wilks’s Lambda = 0.33, approximate F_6,44 = 5.37, P < 0.0003). d, Residuals of the logarithm of testes volume index corrected for date differ among morphs (F_2,37 = 6.31, P = 0.0045). Testes of Independents (n = 29) are substantially smaller than those of Satellites (n = 4, Tukey-adjusted P = 0.006) and Faeders (n = 6, P = 0.02). Boxplots indicate median (bold line), 25% and 75% quartiles (box) and ranges (whiskers). e, f, Seasonal patterns of circulating steroid concentrations in blood plasma of male ruffs: e, androstenedione (A4), f, testosterone plus dihydrotestosterone (T+dHT). Points are daily means±s.d. Independents (n = 11: red, jittered −1 day; Satellites (n = 9): blue; Faeders (n = 2): black, jittered +1 day. Independents have higher date-specific levels of T than Satellites (F_1,97 = 10.01, P = 0.002), and Satellites higher levels of A4 than Independents (F_1,97 = 17.03, P < 0.0001; Supplementary Table 1); Faeders appear similar to Satellites, but were not tested statistically due to n = 2.

Figure 2

Genetic mapping and genome-wide association analysis identify the genomic region determining ruff reproductive morphs. a, The ruff linkage mapping pedigree. Paternal links are shown as red lines and maternal links as blue lines. b, Linkage maps (in centiMorgans) of the inversion region on ruff chromosome 11 support the presence of the inversion polymorphism, indicated by an ~70% shorter linkage map when only Independents are considered. SNPs on the same contig share the same colour. c, Association between markers and morphs based on 41 unrelated males (10 Faeder, 10 Satellite, 21 Independent). Alternating shades of grey indicate different contigs, ordered based on synteny with the chicken genome. The top panel shows −log₁₀P-values of association across the entire ruff genome; middle panel (Faeder) and bottom (Satellite) show blow ups of the associated peak (0.703–0.713 Gbp, indicated by dashed lines in the top panel) for comparisons with independent. Red and blue dots indicate genome-wide significance (P < 0.05, 1000 permutations) for the Faeder and Satellite loci, respectively.

Figure 3

Sequence divergence among morph-determining haplotypes. a, Divergence (D_xy) among morphs across the inversion (4.4 Mbp) and flanking regions. Vertical dotted lines refer to the two inversion breakpoints. The gene CENP-N crosses the breakpoint located on Contig 3357.Each line is staggered by 15 kb to make each line visible. b, c, d, Evolutionary relationships among the three morphs. Maximum-likelihood trees showing the relationship among the sequences from two resequenced Satellites, two Faeders, and the non-inverted reference (Independent 1) and a resequenced Independent (Independent 2) for different regions within and around the inversion: b, adjacent to the inverted region (Contig 3913: 140,000–150,000), c, within regions of the inversion exhibiting high divergence from the reference (Contig 3357: 280,000–290,000), d, within regions of the inversion where Satellites show low divergence from Independents (Contig 3208). e, f, g, h, Divergence patterns between Faeder and Satellite across regions containing candidate loci involved in steroid hormone metabolism (PLCG2, SDR42E1, HSD17B2 and CYB5B), sperm motility (GAS8) and pigmentation (MC1R), plus also transcription factors expressed in the gonads (ZFPM1) and in proximity to MC1R showing Satellite-specific divergence (TCF25). Arrows indicate the presence of morph-specific deletions affecting the coding region of PLCG2 (see Supplementary Fig. 6) and deletions in noncoding regions surrounding HSD17B2. Dots indicate the presence of morph-specific nucleotide substitutions (Satellite – blue; Faeder – red).