title: 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.
abstract: 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.
authors.affiliation: Department of Biochemistry, Molecular Biology, Entomology and Plant Pathology, Mississippi State University, Mississippi State, Starkville, MS, 39762, USA.
authors.forename: Katherine M
authors.initials: KM
authors.lastname: Graham
authors.affiliation: Department of Wildlife, Fisheries and Aquaculture, Mississippi State University, Mississippi State, Starkville, MS, 39762, USA. a.kouba@msstate.edu.
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Kouba
authors.affiliation: Department of Biochemistry, Molecular Biology, Entomology and Plant Pathology, Mississippi State University, Mississippi State, Starkville, MS, 39762, USA.
authors.forename: Cecilia J
authors.initials: CJ
authors.lastname: Langhorne
authors.affiliation: Department of Biochemistry, Molecular Biology, Entomology and Plant Pathology, Mississippi State University, Mississippi State, Starkville, MS, 39762, USA.
authors.forename: Ruth M
authors.initials: RM
authors.lastname: Marcec
authors.affiliation: Department of Biochemistry, Molecular Biology, Entomology and Plant Pathology, Mississippi State University, Mississippi State, Starkville, MS, 39762, USA.
authors.forename: Scott T
authors.initials: ST
authors.lastname: Willard
mesh_terms: D000818:Animals; D001001:Anura; D049628:Body Size / Q000502:physiology*; D001837:Body Weights and Measures / Q000379:methods; D056727:Endangered Species; D005260:Female; D005835:Genitalia / Q000000981:diagnostic imaging*; D012739:Gonadal Steroid Hormones / Q000652:urine*; D008297:Male; D012727:Sex Characteristics; D014463:Ultrasonography / Q000379:methods; D016482:Urinalysis / Q000379:methods
chemical_list: D012739:Gonadal Steroid Hormones
keywords: Body size; Dusky gopher frog; Lithobates sevosa; Mississippi gopher frog; Secondary sex characteristics; Sex identification; Ultrasound; Urinary hormone analysis