Biological sex identification in the endangered dusky gopher frog (Lithobates sevosa): a comparison of body size measurements, secondary sex characteristics, ultrasound imaging, and urinary hormone analysis methods.

BACKGROUND: Accurate sex identification techniques are important for wildlife demographic studies and for genetic management of captive breeding colonies. Various non-invasive methods for identification of biological sex in the weakly dimorphic endangered dusky gopher frog (DGF; Lithobates sevosa) were explored to support planned recovery efforts for this species including breeding and augmentation of wild populations. METHODS: Body size (snout-vent length and body weight) measurements, observation of nuptial pads, ultrasound imaging, and urinary hormone analysis for testosterone and estrone were performed on 27 male and 19 female DGFs. For each method, the mean and range of measurement values were determined for male and female DGFs housed in a captive breeding population. The ability of these methods to accurately predict the true biological sex of the individuals was assessed retrospectively. RESULTS: Body size measurements were of limited use for sex identification purposes, as males and females demonstrated overlapping body lengths and weights. Observation of the presence/absence of nuptial pads in males and females, respectively, proved to be accurate and easy to perform in most cases. Ultrasound imaging was useful for predicting the sex of female frogs, particularly when females were gravid. Commercial enzyme immunoassay kits were validated to measure urinary hormones in the DGF. Mean urinary testosterone (males: 2.22 ± 0.38 ng/ml; females: 0.92 ± 0.11 ng/ml) and estrone (males: 0.08 ± 0.01 ng/ml; females: 1.50 ± 0.39 ng/ml) concentrations were significantly (p < 0.05) different between the sexes. However, there was some overlap in hormone concentrations between the sexes. When a ratio of testosterone (T) to estrone (E) concentrations was calculated for each individual, males demonstrated significantly greater T/E ratios compared to females (p < 0.05). Use of this ratio showed greater accuracy in predicting the sex of the animal compared to using testosterone or estrone concentrations alone. CONCLUSIONS: Monitoring for presence/absence of nuptial pads and using urinary testosterone to estrone hormone ratios were the most accurate methods for identifying the biological sex of adult DGFs. Urinary hormone measurements for sex identification may be useful in other weakly dimorphic and monomorphic amphibian species in both ex situ and in situ settings.

Reintroduction and Post-Release Survival of a Living Fossil: The Chinese Giant Salamander.

Captive rearing and reintroduction / translocation are increasingly used as tools to supplement wild populations of threatened species. Reintroducing captive-reared Chinese giant salamanders may help to augment the declining wild populations and conserve this critically endangered amphibian. We released 31 captive-reared juvenile giant salamanders implanted with VHF radio transmitters at the Heihe River (n = 15) and the Donghe River (n = 16) in the Qinling Mountains of central China. Salamanders were monitored every day for survival from April 28th 2013 to September 3rd 2014. We attempted to recapture all living individuals by the end of the study, measured their body mass and total body length, and checked for abnormalities and presence of external parasites. Two salamanders at the Heihe River and 10 animals at the Donghe River survived through the project timeline. Nine salamanders were confirmed dead, while the status of the other 10 animals was undetermined. The annual survival rate of giant salamanders at the Donghe River (0.702) was 1.7-fold higher than that at the Heihe River (0.405). Survival increased as individuals were held longer following surgery, whereas body mass did not have a significant impact on survival rate. All salamanders recaptured from the Donghe River (n = 8) increased in mass (0.50 ± 0.13 kg) and length (5.5 ± 1.5 cm) after approximately 11 months in the wild, and they were only 7% lighter than wild animals of the same length (mean residual = -0.033 ± 0.025). Our results indicate that captive-reared Chinese giant salamanders can survive in the wild one year after release and adequate surgical recovery time is extremely important to post-release survival. Future projects may reintroduce older juveniles to achieve better survival and longer monitoring duration.