A single gene in the shorebird species known as ruffs controls the blood concentrations of testosterone in males, and this in turn affects their appearances and behavior.
Ruffs, Calidris pugnax, are medium-sized Eurasian sandpipers that get their common name from the large ruff of feathers that adorn the males’ necks. When raised, this feathery ruff is integral to the males’ ostentatious lekking displays that are intended to attract the attention of the females.
Leks are small areas where groups of males display to watching females and where strong female mate choices are exerted. Leks confer indirect benefits to males and reduced costs to females.
In short, leks are like comparison window shopping for your future childrens’ father. Especially because the males are not choosy with whom they mate with — whereas females most certainly are.
“Females provide all incubation and parental care, so males are just trying to mate with as many females as possible,” said one of the study’s co-authors, David Lank, a research fellow and adjunct professor of biology at Simon Fraser University. “It is a highly polygynous species. Males are half again as large as females … except for the [smaller] female mimics.”
Male ruffs have three distinct morphs
Male ruffs have three distinct morphological appearances known as morphs: the flamboyant and dominant male “Independents” with dark neck ruffs and head tufts who fight vigorously for territories, the less dominant and less aggressive male “Satellites” with conspicuous white neck ruffs and head tufts who do not defend territories but who do mate when the dominant satellite male is preoccupied, and the male “Faeders” who look like females with their small size and lack of ornamental feathers, who neither defend territories nor perform courtship displays, but who do ‘sneak’ copulations whenever the opportunity arises (Figure 1A).
“Females visit leks to chose mates,” Professor Lank explained in email. “Satellites have fancy white plumages and cooperate with courtholders to attract females to a court. They are ‘wingmen’, but both are in fact trying to mate, so they both cooperate and compete. Since Satellites are only 15% of males, there are not enough to go around, so the Independents compete for them and try to recruit them. Both morphs display to visiting females, and she may chose to mate with either.”
Apparently, many females choose their mates as part of a contrasting duo. On the other hand, there are a few small, unassuming female mimics hanging around the leks too. They might be characterized as “nice guys.”
“Faeders — the female mimics — are small birds, just slightly larger than females and smaller than the other males, that lack fancy plumage and display behaviour. They get their share of matings by jumping in when females crouch, apparently for one of the plumed males,” Professor Lank explained in email. “However, females may actually choose to mate with them, even though they are not displaying.”
Each morph has distinct androgen levels
These three distinct morphs have distinct concentrations of androgens in their bodies. Androgens are a group of sex steroid hormones that have a variety of physiological and behavioral effects in both males and females, so they have pivotal roles in the formation and variation of sexual phenotypes. Although both sexes produce testosterone, males generally have higher circulating testosterone concentrations than females. In males, testosterone is synthesized primarily in the testes and typically peaks during the breeding season, but can vary considerably between individuals depending on genetics, life history, reproductive trade-offs, and alternative mating strategies.
Earlier studies of ruffs established that both Satellite and Faeder males have significantly lower testosterone levels throughout their bodies — except in the testes, where testosterone is essential to supporting the proper development of competitive sperm (ref, ref and ref). Not only do testosterone and other androgens regulate a range of male and female sexual characteristics, such as body size and ornamentation, but they also influence social behaviors, particularly those related to courtship and aggression.
Usually, dominant territorial males — Independent males in ruffs — have high levels of circulating testosterone whilst nonaggressive subdominant males — Satellite males — and males that employ alternative mating tactics, such as sneaking — these are the Faeder male ruffs — have low testosterone levels. In addition to high concentrations of circulating testosterone, Independent males have low levels of androstenedione, a much less potent androgen. The nonaggressive Satellite and Faeder males show the reverse pattern (Figure 1B).
A number of candidate genes are suspected to play a role in maintaining these disparate levels of testosterone and other androgens. However, a detailed characterization of the genetic variants linked to testosterone regulation, including a confirmation of their effects on testosterone production or metabolism, has so far remained elusive.
Androgen levels are regulated by enzymes encoded by a supergene
Prior studies found these three morphs were affiliated with an autosomal supergene region that encompasses roughly 90 tightly linked individual genes (ref and ref) that are inherited as a single unit, hence its name, “supergene.” (Note that autosomes are chromosomes that are not sex chromosomes. They carry the genetic codes that produce proteins that are essential for survival.)
This supergene has been maintained within the ruff population ever since it first arose, without any genetic reshuffling, as a balanced genetic polymorphism.
“The first inversion happened 20 million years ago, forming the female mimics,” Professor Lank explained in email. “Flipping this part of the chromosome disrupted the expression of fancy seasonal breeding plumage and behaviour. Much more recently, perhaps only 70,000 years ago, a complicated rearrangement happened between the original ancestral genes and the inversion to make a ‘hybrid inversion’ [’recombinant inversion’ in Figure 1] at that site. The birds with this are the current Satellites, which have fancy white plumages and display, but do not fight.”
This supergene also explains why Independent male morphs have either black or brown plumage.
“Another gene within the inversion is MC1R, which in 4 or 5 other species of birds controls the production of white versus black morphs,” Professor Lank explained in email, “and also shows expression differences among the morphs.”
Intrigued by the genetics underlying these shorebirds’ complex mating system, which is unique to them, an international collaboration of scientists examined the mutations in just one gene located within the supergene region. This particular gene encodes an enzyme that metabolizes testosterone to androstenedione. Interestingly, the team found that the inverted gene carries an accumulation of mutations that makes its enzyme faster than the ancestral enzyme at converting testosterone to androstenedione, thus rapidly lowering testosterone levels in the bloodstream, as seen in the two low-testosterone ruff morphs.
Testosterone apparently supports aggression but not courtship behavior
The study reports that differences in circulating androgens between the three male mating morphs in ruff sandpipers are linked to a single gene contained within the supergene that determines the morphs. This gene encodes the enzyme, 17-beta hydroxysteroid dehydrogenase 2 (HSD17B2).
“Independents have two copies of the ancestral HSD17B2 allele. In contrast, Satellites and Faeders each have a unique HSD17B2 inversion allele and one ancestral HSD17B2 allele,” the study’s co-lead author, neurobiologist Jasmine Loveland, told me in email. Dr Loveland was a postdoctoral fellow at both the Max Planck Institute for Biological Intelligence (MPIBI) and at the University of Vienna whilst conducting this research.
“It turns out that the male ruffs that are inversion carriers — Satellites and Faeders — have overall higher expression of HSD17B2 in the blood, with Satellites at an intermediate level and Faeders with the highest levels,” Dr Loveland said in email.
“Both in the brain and in some peripheral tissues, including the bloodstream, we showed that HSD17B2 transcripts are most abundant in Faeders, intermediate in Satellites and nearly absent in Independents,” said the study’s co-lead author, animal ethologist Alex Zemella, a PhD student in Behavioral Genetics and Evolutionary Ecology at both MPIBI and the University of Vienna.
These differences in HSD17B2 expression establish and maintain the corresponding differences in circulating concentrations of testosterone, which in turn, exert visible effects upon the birds’ morphology and behavior.
“Anecdotally, we know from behavioral observations, both in the wild and in our captive populations, that Faeders consistently show no aggressive behavior, and Satellites only very rarely do,” Mr Zemella reported in email. “This suggests that elevated circulating HSD17B2 expression in these two morphs is likely sufficient to suppress aggression almost entirely.”
If these low-testosterone morphs are not at all aggressive, do they still perform courtship displays?
“Intriguingly, while this gene seems to play a major role in modulating aggression, it does not appear to influence courtship displays, which are performed by both Satellites and Independents,” Mr Zemella replied in email. “HSD17B2 seems to be important only for certain aspects of their mating strategies but it does not completely explain them.”
Dr Loveland added in email that although male Satellite and Faeder ruffs have high levels of this enzyme in their blood, “which nearly eliminates all testosterone, this does not reduce their ‘sex drive’, because they are ready to mate whenever the opportunity arises.”
This observation is interesting because both testosterone and estradiol (another sex steroid hormone) in the brain are important for modulating courtship behavior in birds.
Derived versions of HSD17B2 enzymes are faster at metabolizing testosterone
In view of the fact that mutations tend to disrupt the functions of genes and their products, it is somewhat surprising that both the inverted and the recombined-inverted versions of HSD17B2 were found to convert testosterone to androstenedione faster than the ancestral version of the enzyme. Further, these derived versions of HSD17B2 were preferentially expressed in blood as well as in the brain of the low-testosterone morphs. So not only are there more HSD17B2 transcripts present in blood and brain of the low-testosterone morphs, but they metabolize testosterone to androstenedione faster than the ancestral isozyme.
“Two exciting twists […] are that 1) we saw that nearly all of the HSD17B2 expressed in the blood of Satellites and Faeders was actually coming from their respective inversion alleles and 2) the inversion HSD17B2 has accumulated mutations that make it even faster at its job!” Dr Loveland explained in email.
There is yet one more twist to this story, Professor Lank pointed out in email.
“One end of the inversion — the ‘breakpoint’ — [where the gene was] broken in half thus making it nonfunctional. This gene is essential to mitosis. This means that if you get the inversion from both your parents … you are dead,” Professor Lank said in email. “Homozygous lethal.”
As a result, all Faeders and Satellites are chromosomal heterozygotes for the inversion, which means that half of the sons (and daughters) of Faeders and Satellites inherit one of the two types of supergene inversions, and end up either as Faeders or Satellites themselves, whilst the other half do not, and end up as Independents.
But 85% of individual ruffs carry two ancestral copies of the supergene and thus are homozygous recessive Independents. This means that, in nature, matings between birds with either of the derived (inverted) supergene forms — where both parents carry them — are uncommon.
Besides the astounding complexity that underlies this mating system, what surprised you most about this study’s findings?
“This paper was full of exciting surprises for me,” Dr Loveland exclaimed in email. “The extremely high expression of the inversion HSD17B2 gene in the blood of Satellite and Faeder males was the biggest surprise. But also, the fact that Satellite and Faeder males had higher levels of testosterone in the testes compared to Independent males. Lastly, how in Satellites and Faeders the inversion allele for HSD17B2 appears to ‘take over’ the expression in some tissues, meaning that the ancestral allele is barely expressed at all.”
“What surprised me the most was how everything began to make sense once we combined the results from all the different analyses, including the initially puzzling findings from the hormonal analysis of the testes in Satellites and Faeders,” Mr Zemella replied in email. “They can be explained by the signal reaching the brain — specifically the hypothalamus — indicating a lack of circulating testosterone. In response, the testes compensate by producing more testosterone, which is inevitably converted to androstenedione in the bloodstream, restarting the cycle. While this mechanism may initially seem inefficient, it enables Satellite and Faeder males to continue producing testosterone in the testes, essential for sperm production, while simultaneously eliminating all circulating testosterone before it reaches the brain.”
What does these remarkable birds tell us about evolution?
“I think our findings are a wonderful example to demonstrate that evolution is not always proceeding straightforward and does not come up necessarily with the perfect solution right away,” replied the study’s senior author, evolutionary ethologist Clemens Küpper, the Group Leader at Max Planck Research, where he specializes in studying the evolution of social behavior, particularly those behaviors associated with mating and parental care.
“Rather [evolution] proceeds with a solution that is better suited for a current challenge that is somewhat better (or equally good) as existing ones,” Professor Küpper continued in email.
In the case of the ruff, the challenge has been that competition over mating between males is so high that very few males have the opportunity to mate whilst many miss out because they did not become dominant and defend a territory. So, there was a large potential for a ‘Plan B’ and ‘Plan C’ that takes the male-male competition to a different level and exploits the weakness of the dominant males.
The fighting and aggressive territorial (Independent) males are ill prepared to exclude sneakers (Faeder) males or cooperative (Satellite) males from the lek.
“Changes in the supergene seemed to help with the necessary physiological adaptations in this process,” Professor Küpper continued in email. “The supergene captured a single gene of steroid metabolism and, we knew from other species that relatively few changes to such steroid-related genes can result in dramatic changes to the steroid metabolism and then, presumably, also to behaviour, between the morphs.”
These findings reveal how evolutionary changes within a single gene’s structure, sequence, and regulation can drive significant diversity within a single species. And yet, this fascinating study leaves plenty of open questions for future exploration, such as how complex social behaviors are regulated in ruffs. Additionally, there are many promising avenues for research regarding how this supergene this affects female ruffs: for example, although they don’t show obvious physical differences as seen in the males, female ruffs also show distinct differences in circulating testosterone with the same pattern as males (ref).
“But we only have small sample sizes from females so far, and the effect is not as large, but it’s still there, which is pretty cool!” Dr Loveland exclaimed in email.
Could this discovery have medical applications for people?
“In combing through the literature to learn more about HSD17B2 in humans, there was a moment when I realized that ruffs and humans had something very special in common: one study had shown that a single amino acid change in the human HSD17B2 enzyme made it 5 times faster in its testosterone conversion rate,” Dr Loveland mentioned in email. “I noticed that Satellites and Faeders had also changed an amino acid at the exact same position! So I wondered, what if this mutation has also given ruffs a ‘hyper active’ HSD17B2?”
“I felt goosebumps thinking that this might be a huge breakthrough, so I contacted the researchers who had done the human study: did they think my hunch was crazy or might they be as enthusiastic as I was? Luckily, they were also in Munich (Helmholtz collaborators) so I pitched them the idea and they agreed that it was worth testing the conversion rates for the ‘ancestral’ and inversion HSD17B2s as well as seeing if this single amino acid change was enough to already make HSD17B2 work faster. Then of course, seeing these results for the first time and being able to confirm that initial hunch, was so satisfying and rewarding! I am so grateful for having such excellent collaborators!” Dr Loveland exclaimed in email.
Is it possible that this mutated HSD17B2 might help people?
“The potential clinical applications I imagine could be in terms of trying to slow down tumor growth,” Dr Loveland speculated in email. “For example, in cases where high levels of sex steroids, like testosterone, but also estradiol, promote tumor growth, and it is desirable to lower hormone levels.”
Source:
Jasmine L. Loveland, Alex Zemella, Vladimir M. Jovanović, Gabriele Möller, Christoph P. Sager, Bárbara Bastos, Kenneth A. Dyar, Leonida Fusani, Manfred Gahr, Lina M. Giraldo-Deck, Wolfgang Goymann, David B. Lank, Janina Tokarz, Katja Nowick, and Clemens Küpper (2025). A single gene orchestrates androgen variation underlying male mating morphs in ruffs, Science 387(6732):406-412 | doi:10.1126/science.adp5936
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