From salvage to science: Overcoming initial challenges and developing an adaptively managed program for an endangered amphibian, the mountain-yellow legged frog (Rana muscosa)

Abstract

The mountain yellow-legged frog (Rana muscosa) recovery program at the San Diego Zoo Wildlife Alliance emerged from crisis-driven salvage rather than a planned conservation framework, resulting in a genetically limited founder base and a long-term need for adaptive management. Over more than a decade, the ex-situ population functioned as an experimental system in which husbandry, genetic pairing strategies, and assisted reproductive technologies (ARTs) were iteratively refined to overcome persistent reproductive challenges including mismatched timing to gamete release among males and females, low annual output in viable offspring, and a narrow breeding season. Understanding the factors that limit reproductive success is essential for sustaining ex situ populations of the endangered R. muscosa. We combined ten years of natural-breeding records with four years of assisted fertilization (AF) trials to identify where along the reproductive sequence line-specific constraints arise and to develop AF protocols to further evaluate whether AF and crossing of populations (admixture) can help overcome potential issues restricting natural breeding, such as behavior incompatibilities, and help elucidate factors reducing breeding success. Natural breeding revealed consistent differences at the earliest developmental stage: San Jacinto (SJ) pairs produced higher embryo-formation rates than San Bernardino (SB) pairs, whereas tadpole hatching success was similar once embryos cleaved. Embryo formation success increased over time with refinements to hormone timing, brumation, and environmental cueing, and shorter latency to oviposition was associated with improved outcomes. Despite these gains, SB-sired clutches remained consistently low in embryo formation. AF trials confirmed this pattern; SJ and early-generation admixed males produced the highest embryo formation, whereas SB males performed poorly across all combinations. SB females, however, reproduced successfully when paired with SJ males, indicating that reduced fertilization success in SB lines is primarily driven by male gamete traits rather than female physiology or behavioral mismatches. These findings support the strategic use of directional admixture to maintain SB genetic representation while improving overall reproductive output. Integrating natural-breeding data, AF outcomes, and cryobanking strategies will help sustain viable ex situ lines and support the recovery of R. muscosa in the wild. This case study demonstrates that incremental, data-guided improvements, rather than single breakthroughs, are essential for developing effective amphibian ARTs under genuine conservation conditions.