Female-biased sex ratios despite stable genetic sex determination across a climatic gradient in a marine fish

Despite a stable genetic XX/XY sex determination system and unbiased sex ratios in early life stages, Scandinavian populations of the two-spotted goby exhibit a consistent female-biased adult sex ratio across a climatic gradient, a pattern the authors attribute to sex-specific habitat distribution rather than environmental sex determination or differential mortality.

The Gist

Imagine a bustling underwater neighborhood where a tiny fish called the two-spotted goby lives. For years, scientists have noticed something strange about this neighborhood: whenever they count the grown-up fish, there are way more ladies than gents. It's like walking into a high school dance and finding that 75% of the people there are girls, even though the school was supposed to have an equal number of boys and girls.

This paper is a detective story trying to solve the mystery: Why are there so many more female gobies than males?

Here is the breakdown of the investigation, explained simply:

1. The Suspects: Three Possible Culprits

The scientists had three main theories about why the "dance floor" (the breeding area) was so full of females:

• Suspect A: The "Birth Factory" (Genetics). Maybe the fish are just born as girls more often. Perhaps their DNA is rigged to make more females.
• Suspect B: The "Early Exit" (Mortality). Maybe the boys are dying faster than the girls. Perhaps the stress of being a dad (since male gobies take care of the eggs) kills them off before they get old.
• Suspect C: The "Hiding Game" (Habitat). Maybe the boys and girls aren't actually missing; they just aren't in the same room. Maybe the boys are hiding in a different part of the ocean, or the girls are the only ones showing up to the party.

2. The Investigation: A High-Tech Detective Story

To solve this, the scientists went on a massive field trip. They swam along the coast of Scandinavia, from the warm south to the cold north, counting over 25,000 fish. They also used some serious high-tech tools:

• The "Genetic Fingerprint": They built a complete family tree (genome) for a male goby and compared it to females. They found that the fish have a standard XX/XY system (like humans), where females are XX and males are XY.
• The "Baby Check": They collected eggs and baby fish (larvae) and tested their DNA.
• The "Adult Check": They caught adult fish that were too young or shy to show their "party clothes" (secondary sexual characteristics) and tested their DNA to see who was actually a boy or a girl.

3. The Evidence: Ruling Out the Suspects

Ruling out Suspect A (The Birth Factory):
When they checked the eggs and babies, the ratio was perfect: 50/50.

• The Analogy: Imagine a factory that produces red and blue balls. If you check the conveyor belt right as the balls come off, you see an equal mix. So, the "birth factory" isn't the problem. Nature isn't making more girls at the start.

Ruling out Suspect B (The Early Exit):
They looked at the "teenage" fish (1-year-olds) and the adults. While the number of males did drop a bit as the breeding season went on (likely because being a dad is hard work and risky), the drop wasn't enough to explain why there were 75% females right at the start of the season.

• The Analogy: If the boys were dying off, you'd expect the "teenage" boys to be missing too. But they were there in equal numbers. The boys weren't dying before the party started; they just weren't at the party.

The Smoking Gun: Suspect C (The Hiding Game):
Since the babies are born equal, and the boys aren't dying off early, the only explanation left is that the boys and girls are hanging out in different places.

• The Analogy: Think of the breeding season as a big concert. The girls are all in the front row, dancing and singing. The boys are actually in the parking lot, or maybe in the VIP lounge, or maybe they just decided not to come because the crowd is too big.
• Why? The scientists think it's about competition.
  • One male goby can take care of eggs from many different females at once.
  • If there are too many other males around, the chance of a specific male getting to be a dad drops to zero.
  • So, smart male fish might decide, "Hey, this party is too crowded. I'll wait in the parking lot until it's quieter."
  • Females, on the other hand, need to find a male to lay their eggs with, so they have to show up to the party, even if it's crowded.

4. The Twist: The "Secret Gene"

While solving the mystery of the sex ratio, the scientists also found a cool genetic secret. They discovered a specific gene on the male fish's Y-chromosome called amhr2.

• The Analogy: It's like finding the "On/Off switch" for making a fish a boy. This gene is a duplicate of a gene found in other fish (like catfish), but in the goby, it got moved to a new spot on the chromosome and got surrounded by a lot of "junk DNA" (satellite DNA). It's a brand-new, slightly messy switch that nature just invented to make male gobies.

The Big Takeaway

The paper teaches us that nature is messy and complex.

We often think that if we know the genetics (the "blueprint"), we can predict exactly how many boys and girls there will be. But this study shows that ecology (how the fish live, where they hide, and how they compete) plays a huge role.

Even though the fish are born 50/50, the behavior of the fish (hiding when it's too crowded) creates a world where we mostly see females. It's a reminder that to understand nature, you can't just look at the DNA; you have to watch how the animals actually live their lives.

Technical Summary

1. Problem Statement

The Adult Sex Ratio (ASR) is a critical demographic parameter influencing population dynamics, sexual selection, and evolutionary trajectories. In the two-spotted goby (Pomatoschistus flavescens), historical data from Southern Sweden indicated a heavily female-biased ASR (approx. 75% females). However, the drivers of this bias were unclear. Potential causes included:

• Environmental Sex Determination (ESD): Climate gradients (temperature) potentially skewing sex ratios.
• Sex-biased Mortality: Differential survival rates between males and females due to reproductive costs (e.g., male-only parental care).
• Genetic Sex Determination (GSD) distortion: An inherent bias in the primary sex ratio.

The study aimed to disentangle these factors by investigating ASR across a steep climatic gradient (58°N to 68°N) in Scandinavia, combining large-scale demographic surveys with high-resolution genomic analysis to determine the sex-determination system and primary sex ratios.

2. Methodology

The research employed a multi-faceted approach combining field ecology, population genomics, and de novo genome assembly.

A. Field Sampling (Demographic Surveys)

• 2022 Survey: Conducted across 6 populations (30 transects) spanning 10° of latitude.
  • Census: Over 25,771 fish were counted via underwater snorkeling transects.
  • Capture: 8,374 fish were captured, phenotypically sexed (based on fin pigmentation and egg presence), and a subset (180 adults) was preserved for ddRAD sequencing.
  • Early Life Stages: Eggs and larvae were collected from artificial nests in 3 populations for genetic sexing.
  • Unsexed Adults: 100 adults from the northernmost population (Ringstad) lacking secondary sexual characteristics were collected for genetic sexing.
• Historical Data: Integrated data from 2007–2019 from Sweden, Norway, and Finland using snorkeling and beach seine methods to establish long-term trends.

B. Genomic Analysis

• De Novo Genome Assembly:
  • Generated a high-quality, long-read (PacBio HiFi) genome assembly from a single male (TH1).
  • Assembled using Hifiasm, resulting in a 871 Mb primary assembly with haplotype-resolved scaffolds.
  • Validated against a French reference genome (fGobFla1) and phylogenetically confirmed via cox1 barcoding.
• Sex Determination (SD) Locus Identification:
  • ddRAD Sequencing: Analyzed 180 adults (90 males, 90 females) to identify sex-associated SNPs.
  • K-mer Analysis: Merged Illumina reads from males and females to identify male-specific k-mers.
  • Candidate Gene Search: Annotated the SD region to identify potential master sex-determining (MSD) genes.
• Genotyping: Developed a targeted genotyping-by-sequencing (GBS) assay using 5 validated sex-linked markers to determine the genetic sex of eggs, larvae, and phenotypically unsexed adults.

C. Statistical Analysis

• Used Generalized Linear Mixed Models (GLMMs) to analyze ASR trajectories over time and latitude.
• Calculated heterozygosity and allele frequencies to assess sex-chromosome differentiation.

3. Key Contributions

• First Reference Genome: Produced the first high-quality, haplotype-resolved genome assembly for P. flavescens, enabling precise mapping of the sex-determination region.
• Identification of MSD Gene: Identified a male-specific duplicate of the amhr2 gene (anti-Müllerian hormone receptor type 2), designated amhr2y, located on the Y chromosome. This gene is part of the TGF-β signaling pathway, a common mechanism in teleost sex determination.
• Genomic Architecture: Characterized the SD region as a small (2.4 Mb) subtelomeric region on Chromosome 16 with low differentiation between X and Y haplotypes, interrupted by a massive expansion of satellite DNA (satDNA) within the introns of amhr2y.
• Decoupling ASR Drivers: Successfully separated the effects of primary sex determination from adult demographic processes across a wide latitudinal gradient.

4. Results

A. Genetic Sex Determination System

• P. flavescens possesses an XX/XY genetic sex determination system.
• The SD region is located on the distal end of Chromosome 16.
• The amhr2y candidate gene shows high amino acid identity (90.7%) with the ancestral amhr2 on Chromosome 10 but is separated by a ~350 Kb intronic region composed entirely of a specific satDNA family.
• There is low differentiation between the X and Y haplotypes, suggesting a young or recently evolved sex chromosome system.

B. Primary Sex Ratio (Eggs and Larvae)

• Genetic sexing of 585 eggs and larvae revealed a balanced sex ratio (SR ≈ 0.47–0.52) across all latitudes.
• There was no significant evidence of environmental sex determination (ESD) or sex-biased mortality in the first year of life.
• Immature 1-year-old fish in the northernmost population also showed a balanced sex ratio (SR = 0.54).

C. Adult Sex Ratio (ASR)

• Consistent Female Bias: Despite balanced primary ratios, the ASR in breeding habitats was consistently female-biased (~75% females) across the entire latitudinal gradient.
• Temporal Dynamics: The proportion of males generally decreased throughout the breeding season (May–July), likely due to male mortality associated with nest-guarding costs.
• Latitudinal Variation: The female bias was most pronounced in southern populations. In northern populations, the bias was less severe at the onset of the breeding season but still present later.

D. Ruling Out Alternative Hypotheses

• ESD: Ruled out as the primary driver because the primary sex ratio (eggs/larvae) remained balanced despite varying temperatures across the gradient.
• Mortality: While male mortality during breeding contributes to the decline of males over the season, it cannot explain the initial female bias observed at the start of the breeding season in southern populations, where most breeders are first-time spawners.

5. Significance and Conclusion

Primary Hypothesis: Sex-Specific Habitat Distribution
The authors propose that the female-biased ASR is driven by sex-specific habitat distribution rather than mortality or sex determination.

• Mechanism: Males and females may adopt different strategies regarding habitat use outside the breeding season or in response to local competition.
• Reproductive Strategy: Since one male can guard multiple clutches, the "prospect of breeding" for males decreases rapidly with increased male competition. Females, however, may have different thresholds for entering the breeding habitat.
• Ecological Context: Individuals may choose to remain in non-breeding habitats if the costs (predation, competition) outweigh the benefits of breeding. This decision is likely sex-specific and influenced by latitude (breeding season length) and local density.

Broader Implications

• Evolutionary Biology: The study highlights that ASR in natural populations cannot be predicted solely by GSD or mortality models; ecological context and behavioral plasticity are crucial.
• Genomic Evolution: The discovery of amhr2y with a massive satDNA expansion provides a novel case of convergent evolution in fish sex determination, distinct from the single-nucleotide substitution seen in pufferfishes and the truncation seen in catfishes.
• Climate Change: As a model system, P. flavescens demonstrates how demographic parameters like ASR are shaped by the interaction of genetics, ecology, and climate, offering insights into how marine populations may respond to ongoing environmental shifts.

In summary, the paper demonstrates that a stable, balanced genetic sex ratio can coexist with a highly skewed adult sex ratio, driven not by mortality or environmental cues, but by complex, sex-specific ecological behaviors and habitat choices.