| In this research, I collected data on reproductive biology, morphology, skin water exchange, behavioral performance, development and thermal physiology to explore the phenomenon and mechanism of thermal adaptation in Duttaphrynus (Bufo)melanosticus, a bufonid anuran that is widely distributed in warm-climate zones.Reproductive biologyTwo natural ponds at Lishui University (28°27'N,119°55?E) were selected as the observation sites to record the reproductive behavior and characteristics of D.melanosticus for three years from 2011 to 2013. This species often choose permanent ponds as the breeding sites, where courtship calling, amplectant pairs and spawning behavior, while mating ball means intense male-male competition. In Lishui, D.melanosticus is a spring breeder,with egg-laying occurring in March and April.Higher mean air temperature in March caused earlier annual first-breeding dates. The annual first spawning time happened 5 days and 21 days in advance, respectively,followed by 12.4, 13.2 and 15.6 ? in March from 2011 to 2013. Female D.melanosticus lay a single clutch per year and, depending on female size, clutch size ranges from 1313 to 9545 eggs. Holding female SVL constant with a partial correlation analysis, egg size (dry mass) was not correlated with clutch size. Mean snout-vent lengths (SVL) of males were greater in male-larger pairs than in female-larger ones, whereas mean SVL of females showed an inverse trend. Male SVL was positively correlated with female SVL in two types of amplectant pairs, so D. melanosticus belongs to size-assortative mating. One-way ANCOVA with female SVL as the covariate revealed that there were no difference in post-spawning body mass, clutch size, and egg dry mass between female-larger and male-larger pairs,whereas clutch dry mass were greater in male-larger pairs. Linear regression analysis revealed the ratio of male to female SVL was not a determinant of fertilization success. Size-assortative mating of D. melanosticus is due to male preference for larger females that were more fecund. Global warming would cause D. melanosticus to spawn in advance.Latitude variation in sexual dimorphismFemales were larger than males in SVL in all populations sampled. I therefore conclude that D. melanosticus is among anuran amphibians displaying female-biased sexual size dimorphism. In both sexes, SVL decreased with increase in latitude,namely the largest one was Qiongzhou population; the Lishui population was the smallest; Zhaoqing population had no significant difference from Shaoguan population. Latitude variation in SVL followed with a reverse Bergmann's rule in D.melanosticus, possibily due to the longer length of growth period at hiher latitudes. A principal component analysis on the 14 morphological variables other than SVL indicated that the first five axes explained 65.2% of total variation in original data.Head width, parotid gland length, hindlimb length, body width, abdominal cavity, eye diameter, forelimb width, and eardrum diameter had higher factor loadings in the first five axes, and the degree of latitude variation in these traits was very weak.Geographic variation in skin evaporation water loss and rehydrationAdult D. melanosticus from four populations (Lishui, Shaoguan, Zhaoqing, and Nanning) were used to examine geographic variation in skin evaporation water loss and rehydration. The evaporation water loss and rehydration of four populations were positively related to initial body weight, and the rate of rehydration was much greater than the rate of evaporative water loss (EWL). At any given body mass, EWL was lower in the Lishui population than in the other three populations, and EWLs were significantly higher in the Shaoguan and Nanning populations than in the other two populations. At any given body mass, rehydration of Lishui population was significantly less than other populations, followed by Shaoguan and Zhaoqing populations, Nanning population was the most. The skin resistance of D. melanosticus was weak, thus water was easily to evaporate and lose. This feature was treated as what the typical frogs had. In microhabitats, this species often took water-saving posture. The rate of rehydration was much faster than the rate of evaporative water loss, which showed that slow water loss and rapid water absorption were D.melanosticus' physiological adaptation to dehydration risk against terrestrial habitats.This adaptation helped to maintain suitable hydration balance. There was an obvious trend of "low in the north and high in the south" in the degree of evaporative water loss among four D. melanosticus populations.Habitat choice in summerIn comparison with B. gargarizans, field survey was carried out in July 2011 at Lishui university campus. I searched for shelter sites and feeding sites of D.melanosticus along four radiating sample-areas which centered on breeding water during 09:00-17:00 and 18:00-22:00. Meanwhile, we recorded vegetation type,canopy size, and the distance to tree trunk, road, the nearest water body and feeding site. Body mass, snout-vent length, body temperature, land surface temperature, and temperature difference between inside and outside shelter sites were measured. There were 22 shelter sites and 184 feeding sites found. In summer, D. melanosticus mainly chose the lawn, low temperature, wet, and far away from water body and roads as shelter sites during the day, while they chose the bare land and low canopy density of microhabitats as feeding sites. Meanwhile, foraging time mainly associated with the richness of nocturnal insects.The effects of temperature and density on growth and development of tadpolesI conducted a 4 (temperature treatments) × 3 (larval densities) factorial design experiemnt to assess the combined effects of temperature and density on the growth and development of D. melanosticus tadpoles. Of the four temperature treatments,three were controlled constant at 20, 25 and 30 ?, and one fluctuated naturally; the three larval densities were set at 30, 60, and 90 tadpoles per box, respectively. This design included 12 temperature × density combinations. Size at metamorphosis of D.melanosticus significantly became small and timing of metamorphosis significantly became short with natural fluctuation temperature, 25 and 30 ?. There was a significant interaction between temperature and density. D. melanosticus tadpoles could not complete the normal growth and development below 20 ?. The temperature of 25 ? was optimal for early growth. The percentage of metamorphosis was the highest at 30 ?. The rates of growth and development of D. melanosticus tadpoles at natural fluctuation temperature was close to its average temperature. There was significant density restriction effect during the growth and development of D.melanosticus tadpoles. Moreover, the plasticity in age and size at metamorphosis was high. Overall results indicated that temperature and density were both key ecological factors for the growth and development of D. melanosticus tadpoles. This study confirms that D. melanosticus tadpoles were fond of warm environment, which was consistent with actual south-biased distribution.Thermal preference and tolerance of tadpoles and adultsThis study examined selected body temperature (i.e. cloaca temperature, Tsel) of adults, preferred temperature (i.e. water temperature where they stay, Tpre) of tadpoles from Lishui and Shaoguan, and heat tolerance of D. melanosticus using self-made experimental equipments. Meanwhile, all of them were weighed and measured, and tadpoles were staged during the experiment following the table proposed by Gosner(1960). Tsel,critical thermal minimum (CTmin) and critical thermal maximum (CTmax)of D. melanosticus adults were significantly higher than B. gargarizans, on the contrary, thermal tolerance range (TTR) of the former was significantly lower than the later. Gosner stages of D. melanosticus tadpoles had significantly positive correlation with Tpre,CTmin and CTmax,while there were no significantly positive correlation between Gosner stages and TTR. Two-way ACNOVA with Gosner stage as a covariate indicated that different acclimation temperatures had no significant effect on the Tpre and TTR of D. melanosticus tadpoles from Lishui and Shaoguan, but the CTmin and CTmaxof Lishui population was significantly lower than Shaoguan population. CTmin, CTmax and TTR of tadpoles increased with acclimation temperatures. D. melanosticus tadpoles took obvious behavioral thermoregulation in the heterogeneous thermal environment. D. melanosticus tadpoles showed ontogenetic shifts in preferred temperature and thermal tolerance before metamorphosis.Acclimation temperatures had no significant effect on the Tpre of D. melanosticus tadpoles,while CTmin,CTmax, and TTR increased with acclimation temperatures. Tsel and heat tolerance of D. melanosticus adults and latitude variation in heat tolerance of tadpoles were closely related to geographical distribution and breeding season. |
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