Purging of inbreeding depression does not eliminate environmental variation in reproductive onset

Although enforced selfing in the flatworm *Macrostomum hystrix* successfully purged inbreeding depression and increased selfing propensity, it did not eliminate environmental variation in reproductive onset, as intermittent partner access still triggered accelerated reproduction despite the absence of inbreeding costs.

The Gist

The Big Picture: The "Plan B" Dilemma

Imagine you are at a party, and you are looking for a dance partner. Your goal is to find a new partner (outcrossing) because dancing with a stranger usually leads to more fun and better dance moves for your future kids. However, if you can't find anyone, you have a "Plan B": you can dance with yourself (selfing).

The problem with "Plan B" is that dancing with yourself is risky. It's like copying a document twice; eventually, you start copying the typos and errors. In biology, this is called inbreeding depression. The offspring might be weaker or less healthy because they inherited two copies of the same "bad instructions" (deleterious genes).

Because of this risk, most creatures have a "waiting period." They wait a long time hoping to find a partner before they give up and dance alone.

The Experiment: A Flatworm Family Tree

The scientists studied a tiny flatworm called Macrostomum hystrix. They wanted to test a specific theory: If a family is forced to "dance alone" for many generations, will they eventually stop worrying about the risks and just start dancing alone immediately?

They took a line of these worms (called the SR1 line) that had been forced to self-reproduce for about 100+ years. By this point, the "bad instructions" in their DNA should have been either deleted (purged) or fixed, meaning self-reproducing should be just as safe as finding a partner.

They set up three different "party scenarios" to see how the worms reacted:

1. The "Isolated" Group: These worms were locked in their own rooms. They had no choice but to dance alone.
2. The "Constant Party" Group: These worms were in a room with two friends 24/7. They could dance with a partner anytime they wanted.
3. The "Intermittent Party" Group: These worms were mostly alone, but every few days, they were brought together for a short 2-hour dance party, then sent back to their rooms. This was designed to simulate a world where partners are available, but the environment is a bit unstable or unpredictable.

The Surprising Results

The scientists measured two things:

1. How long did they wait to start reproducing? (The "Waiting Time")
2. Did the babies survive? (The "Inbreeding Depression")

Here is what happened:

• The "Bad Genes" are Gone: The scientists were right! The worms that had been self-reproducing for generations showed zero difference in baby survival between those who selfed and those who outcrossed. The "inbreeding depression" had been completely purged. The "Plan B" was now just as safe as "Plan A."
• The "Waiting Time" Disappeared: Because the risk was gone, the worms didn't wait around. Whether they were isolated or had constant partners, they started reproducing at roughly the same time. They had evolved to be ready to go immediately.

BUT... Here is the Twist:

The "Intermittent Party" group did something unexpected. Even though they had the same genetic safety as the others, they started reproducing much faster than the isolated worms or the constant party worms.

The Analogy: The "Unstable House" Theory

Why did the "Intermittent Party" worms rush to reproduce?

Think of it like this:

• Isolated Worms: They are in a quiet, boring house. They know they are alone, so they take their time getting ready.
• Constant Party Worms: They are in a stable, crowded house. They feel safe and secure, so they also take their time.
• Intermittent Worms: Imagine you are in a house where the lights flicker on and off, and the furniture is moved around every few days. You don't know if the house will be there tomorrow.

The scientists believe the "Intermittent" group sensed this instability. The constant shuffling of their housing (moving them from room to room for the short parties) acted like a signal that said, "The world is chaotic! You might not be here tomorrow, so have babies NOW!"

Even though their genes said, "It's safe to wait," their environment screamed, "Don't wait!"

The Takeaway

This paper teaches us two main things:

1. Evolution is fast: If you force a species to self-reproduce, they can quickly lose their fear of inbreeding and stop "waiting" for a partner.
2. Environment matters more than genes: Even when a species has evolved to be perfectly adapted to self-reproduce, chaos and instability (like moving furniture or unpredictable social groups) can still trigger them to reproduce immediately.

In short: You can train a worm to be okay with self-reproducing, but if you make its life feel unstable, it will panic and reproduce early anyway.

Technical Summary

1. Problem Statement and Research Context

The study addresses the evolutionary dynamics of mating systems in simultaneous hermaphrodites, specifically focusing on the trade-off between reproductive assurance (self-fertilization) and inbreeding depression.

• Theoretical Background: Organisms often employ "delayed selfing," where individuals wait for outcrossing opportunities before selfing to avoid the fitness costs of inbreeding. Theory predicts that if a population undergoes enforced selfing (e.g., during recolonization or bottlenecks), deleterious recessive alleles are purged by selection. This should eliminate inbreeding depression and subsequently reduce the "waiting time" (delay before reproduction), leading to a permanent shift toward high selfing propensity.
• Knowledge Gap: While previous studies (e.g., in Physa acuta) have shown that inbreeding depression can be reduced by enforced selfing, it remains unclear if this process completely eliminates inbreeding depression and waiting times, particularly after a period of relaxed selection. Furthermore, it is unknown whether environmental cues regarding social stability (grouping vs. isolation) continue to influence reproductive timing even after genetic purging.
• Specific Question: Does a highly inbred line of the flatworm Macrostomum hystrix exhibit a complete absence of inbreeding depression and waiting times, and how do different social environments (constant grouping vs. intermittent grouping vs. isolation) affect reproductive onset?

2. Methodology

Study Organism:

• Species: Macrostomum hystrix (a free-living, hermaphroditic flatworm).
• Strain: The SR1 line, derived from the 'PISA' population (known for delayed selfing).
• History: The line was subjected to 8 generations of enforced selfing and sib-sib mating, reaching an inbreeding coefficient ($F$) of ~0.998 by 2011. It was then maintained for ~15 years (>100 generations) in large populations (>1000 individuals) under relaxed selection, allowing for the potential accumulation of new mutations or the persistence of residual deleterious alleles.

Experimental Design:
The researchers tested three treatment groups using hatchlings from the SR1 line:

1. Isolated (I): Individuals housed alone, forced to self-fertilize.
2. Triplet (T): Three individuals housed together constantly, ensuring continuous outcrossing opportunities.
3. Intermittently Grouped (IG): Three individuals housed separately for most of the time but grouped together for 2 hours every 2nd or 3rd day in a neutral well to allow outcrossing. Crucially, housing wells were shuffled after every grouping event to prevent individuals from recognizing specific partners or locations.

Measurements:

• Reproductive Onset: Age (in days) at which the first offspring (hatchlings) were observed.
• Inbreeding Depression: Estimated via hatchling-to-adult survival rates. If inbreeding depression existed, offspring from selfed individuals (I) would show lower survival compared to outcrossed individuals (T/IG).
• Duration: The experiment ran for 137 days, with daily monitoring initially, transitioning to less frequent checks.

Statistical Analysis:

• Reproductive Onset: Analyzed using Linear Mixed Effects Models (LMM) with plate ID as a random effect.
• Survival: Analyzed using Linear Models (LM) on the proportion of surviving hatchlings per replicate.

3. Key Results

A. Purging of Inbreeding Depression

• Survival: There was no significant difference in hatchling-to-adult survival between the Isolated (selfing), Triplet (constant outcrossing), and Intermittently Grouped treatments ($p = 0.89$).
• Implication: This confirms that the SR1 line has successfully purged inbreeding depression. The fitness of selfed offspring is equivalent to outcrossed offspring, indicating that deleterious recessive alleles have been effectively eliminated or fixed.

B. Reproductive Onset (Waiting Time)

• Isolated vs. Triplet: As predicted by the purging of inbreeding depression, there was no significant difference in the age of reproductive onset between the Isolated (forced selfing) and Triplet (constant outcrossing) groups ($p = 0.49$). Both groups reproduced late (approx. 105–110 days).
• The Intermittent Grouping Anomaly: The Intermittently Grouped (IG) treatment reproduced significantly earlier (mean $\approx$ 80 days) than both the Isolated and Triplet groups ($p < 0.0001$).
• Interpretation: The accelerated reproduction in the IG group was not driven by the mating system itself (since I and T were similar) but by the specific environmental cue of intermittent grouping and shuffling.

4. Key Contributions

1. Confirmation of Complete Purging: The study provides robust experimental evidence that enforced selfing can lead to the complete elimination of inbreeding depression and the associated "waiting time" delay, even after a long period of relaxed selection. This supports theoretical models predicting rapid mating system shifts during colonization events.
2. Decoupling Social Stress from Mating System: By introducing the "Intermittently Grouped" treatment, the authors successfully distinguished between the effects of mating system (selfing vs. outcrossing) and social environment. They demonstrated that social instability (shuffling/grouping cues) is a potent driver of reproductive timing, independent of genetic inbreeding costs.
3. Plasticity in Reproductive Timing: The results highlight that even in a genetically "fixed" line where inbreeding depression is purged, reproductive onset remains highly plastic and responsive to environmental cues suggesting an unstable future (e.g., the "shuffling" in the IG treatment).

5. Significance and Implications

• Evolutionary Ecology: The findings suggest that while genetic purging can remove the costs of selfing, it does not necessarily erase the behavioral plasticity to respond to environmental cues. Organisms may retain the ability to accelerate reproduction when the environment signals instability, a trait likely crucial for colonizing new or fragmented habitats.
• Colonization Theory: The study supports the hypothesis that single-individual colonization events (founder effects) can rapidly select for high selfing propensity and early reproduction. However, the persistence of environmental sensitivity implies that reproductive strategies are not solely genetically determined but are a complex interplay of purged genetics and current environmental context.
• Experimental Design: The paper highlights the importance of controlling for "grouping stress" in mating system studies. Constant grouping (Triplet treatment) may induce stress or competition that masks the true baseline of reproductive timing, whereas intermittent grouping better mimics natural fluctuations and reveals hidden plasticity.

Conclusion:
While the SR1 line of M. hystrix has successfully purged inbreeding depression and eliminated the genetic need for a waiting period, environmental variation in reproductive onset persists. Specifically, cues of an unstable social environment (intermittent grouping) trigger a significant acceleration in reproductive timing, demonstrating that reproductive strategies are governed by both historical genetic purging and immediate environmental plasticity.