Limited Genetic Heterozygosity and Status of Two Populations of the Ramsey Canyon Leopard Frog: Rana subaquavocalis

—Using starch gel electrophoresis, we examined proteins specified by 41 presumptive loci representing two breeding populations of Rana subaquavocalis, a recently described species of leopard frog from Arizona. Individual and population levels of heterozygosity were low. Mean number of alleles detected (1.2) and mean levels of individual heterozygosity were higher at the Barchas Ranch location (0.042) compared with those from Ramsey Canyon (0.029). Nine loci among the 41 surveyed were polymorphic. Four of these were common to both populations. Each population was polymorphic for the remaining three loci, and each possessed one unique allele. Both populations went extinct by 1996. Rana subaquavocalis is a recently described species of leopard frog and member of the Rana pipiens complex from southeastern Arizona (Platz, 1993). In North America, there are currently seven additional species of leopard frogs, two widely distributed within the eastern half of the United States, including R. pipiens and Rana sphenocephala. Six species, R. pipiens, Rana berlandieri, Rana blairi, Rana chiricahuensis, Rana yavapaiensis, and Rana onca occur in the western half of the United States, and of these, the last five have more restricted distributions. Rana subaquavocalis was originally described (Platz, 1993) from a single locality in the Huachuca Mountains of southeastern Arizona. Subsequently, four additional populations were documented with breeding known to occur at two of these. No population estimate for any of the sites exceeds 50 adults, and the localities are geographically restricted to a 10 km radius within the Huachuca Mountains. As a result, the U.S. Fish and Wildlife Service (15 November 1994, Federal Register Notice) designated R. subaquavocalis as a category 1 candidate for federal listing as endangered or threatened. Collection and possession has been prohibited under Arizona Game and Fish Commission Order 41. Populations containing 50 or fewer individuals generally warrant concern regarding the long-term potential for loss of genetic variability (Shaffer, 1981). In this paper, we report the status of genetic variability in the two known breeding populations of R. subaquavocalis, identify factors that explain the low levels of heterozygosity and document the eventual fate of each population of this rare species. MATERIALS AND METHODS A sample of 15 large tadpoles from the concrete lined pond (type locality) in Ramsey Canyon (elevation 1622 m), 7 km southwest of Sierra Vista; 318269590N, 1108189130W, Cochise County, Arizona, were collected in July 1992 and flash frozen in liquid nitrogen, as were 17 newly metamorphosed juvenile leopard frogs from the Barchas Ranch stock tank (elevation 1528 m); 318289280N, 1108179530W, Cochise County, Arizona. Specimens were shipped on dry ice to the laboratory for tissue preparation. Muscle, liver, and heart tissues were dissected from juveniles and homogenized using a grinding buffer (0.1 M Tris, 1mM EDTA, 0.1mM NAD, 0.1mM NADP, 0.25% v/v B Mercaptoethanol, pH 7.0). Gut contents were removed from tadpoles and entire individuals homogenized in grinding buffer. Tissue homogenates were stored at 708C, thawed, and centrifuged at 13,000 rpm for 10 min prior to electrophoresis in 12% starch gels (60% Starch-Art starch, 40% U.S. Biochemicals starch). Two buffer systems were used: TAG 7 (Wright and White, 1992) and LiOH (Turner, 1983). The products of 41 presumptive protein-coding loci (Table 1) were resolved and genotype counts were analyzed using the BIOSYS1 program (Swofford and Selander, 1981). One of us (JEP) kept in close contact with personnel at the Ramsey Canyon Mile High Preserve since 1990 and resided on site in Ramsey Canyon from May 1 to 26 June of 1995 to monitor population composition and breeding activity at the type locality. Adult counts were made three to five nights per week. Counts made in 1990, 1991, and 1992 were total counts of transformed individuals. Those made after this time only included adults. Ramsey Creek was surveyed once per week during the 1995 season from the visitors center to 700 m upstream of the concrete lined pond.