Experimental treatment and procedure

To examine whether differences in habitat structure between ephemeral and non-ephemeral stream populations have affected the evolution of their growth strategies, we conducted a common garden experiment using the progenies of frogs from the mainland and Jeju Island under water temperature control (22 °C). First, we collected adult frogs from streams in the mainland (34.7°–34.9°N, 126.5°–126.7°E) and Jeju Island (33.45°N, 126.56°E) and brought them to the laboratory. Each collection location is represented by a single population. We placed three to five individual frogs from both sexes together in a semi-aquatic terrarium (40  × 23  × 23 cm) filled with dechlorinated water, rocks, and aquatic plants to facilitate mating. We provided calcium and vitamin D–powered juvenile crickets and mealworms ad libitum. We maintained seven mating chambers for each location until we collected enough eggs to fill all experimental cages (as described below). In total, 67 Jeju Island frogs and 48 mainland frogs (including both sexes; approximate sex ratio, 1:1) contributed to egg-laying. We controlled the room temperature using an air conditioner (22 °C) and provided natural light through transparent windows.

Egg clutches were found every 1–4 days in all chambers. On the day that new egg clutches were found in at least three mating chambers, we collected the eggs and relocated them to experimental cages (25  × 16  × 17 cm) filled with 3.5 L of dechlorinated water where the eggs were allowed to develop. To provide genetic variability within and among experimental cages, we filled each cage with eggs from at least three mating chambers (a similar number of eggs from each mating chamber was used to fill each cage). We never filled the same treatment on the same day. The order of egg filling was random among experimental treatments. Egg collection was completed within two weeks (from 1st to 13th July 2020). Because (1) females do not typically lay eggs in consecutive days, and (2) all experimental chambers were filled in two weeks, we consider that the eggs used for the experiment were not from a limited number of frogs but many different families. We released all adult frogs back to the collection sites after then.

We manipulated three treatments in a full factorial design: (1) the density of tadpoles (low vs. high), (2) the amount of food provided each day (scarce vs. abundant), and (3) the location where parental frogs were collected (ephemeral vs. non-ephemeral streams). For density treatment, we kept either five (low-density) or 30 (high-density) individuals in each cage. For food treatment, we provided either 0.01 (scarce) or 0.1 g (abundant) of fish food (TetraBits Complete; Tetra, Germany) every day. We proportionally reduced the amount of food provided to cages in which some tadpoles had died or became froglets. We replaced half of the water every 2–3 days and removed any remaining food daily. We replicated each treatment twice and tested 280 tadpoles in total.

We surveyed the survival of all tadpoles and whether any tadpoles became froglets (Gosner stage 46) (Gosner, 1960) each day. When a tadpole reached Gosner stage 46, we calculated the number of days that had passed from oviposition (larval period), measured the weight of the froglet (weight at metamorphosis), and then brought it out of the cage. We considered tadpoles to have been cannibalized when either tadpoles were missing or we directly observed cannibalistic behaviors. We note here that though the survival of tadpoles had been monitored a few times a day, we cannot distinguish between pre- and post-mortem cannibalism. We also noted the occurrence of non-cannibalistic deaths where tadpoles were found dead without any signs of attack. All froglets were released to the location where their parental frogs were collected at the end of the experiment. All protocols were approved by Mokpo National University Animal Care and Use Committee (approval no: MNU-IACUC-2020-001).