Urinary hormone metabolites identify sex and imply unexpected winter breeding in an endangered, subterranean-nesting frog.

Urinary hormone analysis has proved accurate for identifying sex and breeding periods in dimorphic amphibians with known reproductive cycles. We examined whether these techniques could provide this much needed information for a monomorphic anuran with an unconfirmed mating season in the wild. We analysed urinary estrone conjugate, testosterone, and progesterone metabolites to infer the time of breeding and to identify sex in the endangered Maud Island frog, Leiopelma pakeka. Testosterone metabolites in males and estrone and progesterone metabolites in females were at their peak during winter for both wild and captive frogs. These urinary metabolite patterns were consistent with the high proportion of females exhibiting enlarged ovarian follicles in winter months. Sex identification based on urinary estrone metabolite levels was 94% correct in this monomorphic species, in which the sexes overlap in snout-to-vent length (SVL) for over half of their adult size range and in which no other sexually dimorphic trait is known. The seasonal profiles imply unexpected winter or early spring breeding in L. pakeka. Overall, these results demonstrate use of urinary hormone metabolites for reproductive monitoring and sex identification in one of the world's most threatened and evolutionarily distinct amphibians.