Geographic variation in skin structure in male Andrew’s toad (Bufo andrewsi)

Variation in organ structure likely provides important clues on local adaptation and reflects the pressure target of natural selection. As one of the important organs, the skin plays a key role in adapting to complex environments by reducing water loss or increasing water absorption. Nevertheless, variation in the skin structure across different populations in a single species of anurans remains enigmatic. Here, we studied geographical variation in the skin structure of male Andrew’s toads (Bufo andrewsi) across ten populations using histological methods. We quantified thickness of the skin, the epidermis, the loose layer, the compact layer, and of the epidermis, area of granular glands (GGs) and of ordinary mucous glands (OMGs), width of the calcified layer, and number of capillary vessels. We found that the thickness of the skin, dermis and loose layer in dorsal skin increased with latitude whereas the area of granular glands decreased with altitude. Moreover, the width of the calcified layer in ventral skin decreased with latitude among populations. Our findings suggest that geographical variation in skin structure in male B. andrewsi is likely to reduce water loss or make water absorption occur faster in complex high-latitude environments, improving local adaptation.

Altitudinal implications in organ size in the Andrew’s toad (Bufo andrewsi)

Phenotypic flexibility of morphological and physiological traits within species is a common phenomenon across animal taxa. Hesse’s rule predicts that the size of an organ should exhibit an increase with increasing altitude along environmental gradients due to changes in oxygen supply and energy demands. Here, we test the prediction of Hesse’s rule by investigating geographical variation in the relative size of organs (i.e., heart, lungs, liver, and kidneys) across ten populations of Bufo andrewsi along an environmental gradient. We found that the relative size of these four specific organs did not increase with altitude or latitude across all populations. We also did not find that the relative size of the organs increased with increasing altitude among six populations located at a similar latitude and longitude, which is inconsistent with Hesse’s rule. Our findings suggest that oxygen supply and energy demands do not necessarily affect variation in organ size among populations.

Geographical variation in limb muscle mass of the Andrew’s toad (Bufo andrewsi)

Muscles are vital for the process of movement, mating and escape of predators in amphibians. During evolution, the morphological and genetic characteristics as well as the size of muscles in species will change to adapt different environments. Theory predicts that low male-male competition in high-altitude/latitude selects for small limb muscles. Here, we used the Andrew’s toad (Bufo andrewsi) as a model animal to test this prediction by analyzing geographical variation in the mass of limb muscles across nine populations from the Hengduan Mountains in China. Inconsistent with the prediction, we found that latitude and altitude did not affect the relative mass of total combined limb muscles and mass of combined hindlimb muscles among populations. Meanwhile, the relative mass of combined forelimb muscles, the two forelimb muscles (flexor carpi radialis and extensor carpi radialis) and the four hindlimb muscles (e.g. biceps femoris, semimebranous, semitendinosus and peroneus) was lowest in middle latitude and largest in low latitude whereas gracilis minor was largest in high latitudes. However, we did not find any correlations between the two forelimb muscles and the four hindlimb muscles and altitude. Our findings suggest that combined forelimb muscles, flexor carpi radialis, extensor carpi radialis, biceps femoris, semimebranous, semitendinosus and peroneus are largest in low latitudes due to pressures of mate competition.

Digestive tract adaptation associated with temperature and precipitation in male Bufo andrewsi

The digestive tract provides a functional relationship between energy intake and allocation. An understanding of effects of environmental factors on the evolution of digestive tract morphology is especially important. To investigate this, we studied the variation in digestive tract length across 10 populations of the Andrew’s toad (Bufo andrewsi) between 2012 and 2015 in Sichuan province, western China. These populations were collected in different habitats varying in temperature and precipitation. The results reveal an increase in the length of the digestive tract and gut with increasing temperature and decreasing precipitation, when controlling for the effect of body size. Our findings suggest that individuals of populations living in high-temperature and low-precipitation environments have longer digestive tracts, possibly because they consume less animal-based foods and more high-fiber foods.

Evolution of sexual dimorphism in the forelimb muscles of Andrew's toad (Bufo andrewsi) in response to putative sexual selection

Sexual differences in morphological traits are widespread among animals. Theory predicts that dimorphism in secondary sexual characters evolves as a consequence of sexual selection. We investigated the intersexual difference in mass of forelimb muscles of the Andrew's toad, Bufo andrewsi, an elongated species inhabiting montane regions in western China. Our results showed that average body size of females was significantly larger than males. However, when the influence of body size was removed, the forelimb muscle mass of males significantly exceeded that of females, and total mass of forelimb muscles of amplectant males was significantly larger than that of non-amplectant males. These results suggest that the robustness of the forelimbs can allow amplectant males to retain a firm grip on the female in amplexus, which may aid resistance to inference by conspecific males. Our findings are consistent with the prediction that sexual selection favors large forelimb muscle mass in males.

Proximate mechanisms leading to large male-mating advantage in the Andrew's toad, Bufo andrewsi

In animals, body size is a prominent trait that generally positively affects ecological and reproductive success. Through field observations and experiments, we investigated the effects of two mechanisms of sexual selection on large male mating advantage in the Andrew's toad, Bufo andrewsi, a species widely distributed in western China. We observed a large male mating advantage in the field. Field data were corroborated by experiments in which large males mated more frequently than smaller males. However, in female preference tests, in which females could choose freely between two males differing in body size, choosing females showed no size preference while many females did not exert mate choice at all. We suggest that the large male mating advantage observed in the field and laboratory is caused by competition among males rather than by female choice.