The cost of heat: How chronic thermal stress shapes the reproductive potential and fitness of a protogynous hermaphrodite killifish.

Organisms inhabiting temporary pools, such as annual killifishes, exist at the edge of environmental tolerance and are among the most vulnerable taxa to climate change. Understanding how heat stress affects these fragile ecosystems and the acclimation limits of their residents is critical for predicting biodiversity loss. We investigated the effects of a chronic thermal gradient on the somatic condition and reproductive traits of the endangered annual killifish Millerichthys robustus. Specifically, we evaluated reproductive potential in both sexes and explored whether trade-offs exist between somatic maintenance and reproductive investment under thermal stress. Our results reveal a progressive and synchronized decline in gamete production and quality across both sexes. However, we identified a marked sexual asymmetry in thermal tolerance: females exhibited a significantly lower threshold than males, likely due to the high bioenergetic demands of oogenesis. While embryo production plummeted at 32°C and failed completely at 34°C, males continued to produce viable gametes, although sperm motility-the primary trait affected by heat-was severely compromised. Contrary to life-history theory predictions, we found no evidence of compensatory energy reallocation; instead, deleterious effects on reproductive and somatic traits occurred synchronously, indicating a widespread physiological collapse rather than a strategic trade-off. These findings suggest that female physiology imposes a critical thermal bottleneck, limiting optimal reproduction to a narrow window between 26°C and 28°C. Such disruptions may stem from thermal interference with the hypothalamus-pituitary-gonadal axis and increased oxidative stress. Given the limited thermoregulatory refugia in shallow temporary ponds, these physiological constraints represent a severe threat to the fitness and population stability of M. robustus in a warming world.