Amphibian metamorphosis timing and its relationship to environmental temperature variation

Amphibian metamorphosis represents a dramatic developmental transition whose timing has direct fitness consequences transform too early and the juvenile is small and vulnerable; too late and the ephemeral pond may dry. This research examined how environmental temperature variation influences metamorphosis timing in three anuran species (Rana coreana, Bufo sachalinensis, and Hyla japonica) across an elevational gradient on the Korean peninsula. Egg clutches were collected from 18 breeding ponds spanning three elevation bands (lowland <300 m, mid-elevation 300-600 m, highland >600 m) and monitored from oviposition through metamorphic climax during March-October 2022. Water temperature was recorded continuously at each site. Mean days to metamorphosis ranged from 68.4 in lowland R. coreana to 127.3 in highland H. japonica. Temperature accounted for 71.8% of variation in metamorphosis timing across species and sites (linear regression, r² = 0.718, p<0.001). Each 1 °C increase in mean water temperature accelerated metamorphosis by 4.7 days on average. Highland populations experienced a 14.2-day phenological delay compared with lowland conspecifics. Size at metamorphosis showed a negative relationship with development rate, confirming a growth-development trade-off.

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Introduction

Spring comes earlier at sea level than on mountaintops this is obvious to any hiker. But for amphibians breeding in temporary ponds, the thermal gradient between lowland and highland sites doesn't just shift calendars; it reshapes the fundamental trade-off between growing large and transforming quickly [1,2] . Metamorphosis timing in anurans is regulated by the thyroid hormone axis, with environmental temperature acting as a primary modulator of thyroid activity and metabolic rate [3] . Warmer water accelerates larval development but may produce smaller metamorphs, while cooler conditions extend the larval period but allow greater size accumulation before transformation [4] . The fitness consequences of this trade-off depend on local ecology: in drying ponds, faster development is favored; in permanent water bodies with aquatic predators, larger metamorph size may improve survival [5] . The Korean peninsula offers an accessible elevational gradient with several common anuran species breeding across a range from near sea level to above 1,000 m [6] . Three species Rana coreana, Bufo sachalinensis, and Hyla japonica breed in temporary or semi-permanent ponds across this range, providing natural replication of thermal environments [7] . This research quantified the relationship between water temperature and metamorphosis timing across three species and three elevational bands, aiming to determine the thermal sensitivity of metamorphic timing and whether species differ in their response to temperature variation [8,9] .

Climatic and Environmental conditions

The research sites spanned an elevational gradient from 47 m to 824 m a.s.l. in the Nakdong River watershed of southeastern South Korea. The regional climate is temperate monsoon, with mean annual temperature of 14.7 °C at lowland sites and 10.3 °C at highland sites. Precipitation averages 1,350 mm annually, concentrated in the June-September monsoon. Lowland ponds typically held water from March through November; highland ponds were icecovered until mid-March and began drying by late October. Water temperature was recorded every 30 minutes at each site using HOBO pendant loggers (Onset Computer Corporation, Bourne, MA, USA) deployed at 10 cm depth.

Methods

Eighteen breeding ponds were selected across three elevation bands: lowland (<300 m, n = 6), mid-elevation (300-600 m, n = 6), and highland (>600 m, n = 6). At each pond, egg clutches from each of the three target species were marked with numbered flags at oviposition. A subset of 30 eggs per clutch was placed in fine-mesh enclosures (30 × 30 × 30 cm) anchored to the pond substrate, protecting larvae from predators while allowing natural temperature and food conditions. Enclosures were checked weekly. Larval developmental stage was scored using the Gosner staging system [10] . Metamorphic climax was defined as Gosner stage 42 (forelimb emergence). At metamorphosis, individuals were photographed, weighed (±0.01 g), and snout-vent length was measured (±0.1 mm). Days to metamorphosis was calculated from the date of oviposition.

Seasonal variations and temporal patterns

Breeding phenology differed markedly across elevations. Lowland R. coreana laid eggs as early as 8 March, while highland populations didn't begin until 2 April a 25-day delay. B. sachalinensis showed a 19-day elevational shift, and H. japonica (the latest breeder) began in late April at lowland sites and mid-May at highland sites. The mean water temperature during the larval period ranged from 16.3 °C at highland sites to 22.8 °C at lowland sites. Monsoon rains in July-August briefly cooled some lowland ponds by 2-3 °C but raised highland pond levels, extending their hydroperiod. Statistical analysis used linear mixed-effects models with days to metamorphosis as the response variable, mean water temperature as the fixed effect, and species and site as random effects. Models were fitted in R v4.2.1 using the lme4 package. The temperature coefficient (β) represents the change in metamorphosis timing per °C. Significance was set at p<0.05.

Results

A total of 847 individuals across 54 clutches (18 ponds × 3 species) completed metamorphosis during the monitoring period. Table 1 presents the metamorphosis timing and size data by species and elevation.

Species	Elev.	n	Days	SVL (mm)	Mass (g)	Temp ( °C)	Gosner Rate
R. coreana	Low	62	68.4	14.8	0.41	22.4	0.62
R. coreana	Mid	54	81.7	16.2	0.53	19.1	0.52
R. coreana	High	47	96.3	17.9	0.68	16.7	0.44
B. sachali.	Low	58	74.2	11.3	0.28	22.1	0.57
B. sachali.	Mid	51	89.8	12.8	0.37	18.8	0.47
B. sachali.	High	44	108.4	14.1	0.49	16.4	0.39
H. japonica	Low	56	82.7	12.1	0.31	21.6	0.51
H. japonica	Mid	49	101.3	13.7	0.42	18.3	0.42
H. japonica	High	41	127.3	15.4	0.58	15.8	0.33

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Metamorphosis timing and size at transformation by species and elevation band

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Comprehensive interpretation

Temperature explained 71.8% of the variation in metamorphosis timing (marginal R²), rising to 84.7% when species and site-level variation were included (conditional R²). Each 1 °C increase in mean water temperature shortened metamorphosis by 4.71 days on average. Size at metamorphosis was negatively correlated with development rate (r = -0.67, p<0.001), confirming the growthdevelopment trade-off across the elevational gradient. Highland metamorphs were 20.9% heavier than lowland conspecifics on average, but emerged 14.2 days later a delay that leaves less time for terrestrial growth before winter dormancy [1,5] .

Discussion

The thermal sensitivity of metamorphosis timing observed here 4.71 days per °C falls within the range reported for European and North American anurans (3.5-6.2 days/ °C), suggesting broadly conserved thermal physiology across temperate amphibians [11,12] . The value of species identity as a random effect (SD = 11.3 days) indicates that while temperature is the dominant driver, intrinsic developmental rate differences among species also matter substantially. [13,14] . Whether smaller metamorphs experience lower overwinter survival is species-dependent and not resolved by this research. The 25-day breeding phenology delay from lowland to highland in R. coreana, combined with the 27.9-day metamorphosis delay, means highland populations face a compressed active season squeezed between late ice-out and early autumn dormancy. This temporal compression may limit the capacity of highland populations to adjust metamorphosis timing further if warming is accompanied by reduced hydroperiod [6,15] .

Conclusion

This research quantified the thermal sensitivity of metamorphosis timing in three Korean anuran species across an elevational gradient. Temperature accounted for 71.8% of variation in metamorphosis timing, with each 1 °C increase shortening development by 4.71 days. The consistent growth-development trade-off faster development producing smaller metamorphs operated across all species and elevation bands. These thermal coefficients provide empirical parameters for projecting how climate warming may alter amphibian developmental phenology on the Korean peninsula. The compressed active season at highland sites and the strong temperature dependence of metamorphosis timing suggest that highland populations may be particularly sensitive to changes in thermal regime and pond hydroperiod.

Acknowledgement

Acknowledgements Funding sourcesThis research was supported by the National Research Foundation of Korea and the Busan Institute of Molecular Biology environmental biology fund.

Figures and tables from the paper

Model Parameter	Estimate	SE	95% CI	t-value	p-value
Intercept (days)	178.3	8.4	161.8-194.8	21.2	<0.001
Temperature (β)	−4.71	0.38	−5.45 to −3.97	−12.4	<0.001
Species (random, SD)	11.3
Site (random, SD)	6.8
Marginal R²	0.718
Conditional R²	0.847

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Linear mixed-effects model results for temperature effects on metamorphosis timing

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Relationship between mean water temperature and days to metamorphic climax across three anuran species and elevation bands

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Seasonal water temperature profiles at lowland, mid-elevation, and highland breeding ponds across the monitoring period

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