Exposure to environmental contaminants contributes to the global decline of amphibian populations. The impacts of organic contaminants on amphibians are well documented. However, substantially less is known concerning the potential effects of metals on amphibian populations. Copper (Cu) is an essential element, but it can be toxic at concentrations only slightly higher than the normal physiological range. The present study examines the effects of chronic Cu exposure on embryos and larvae of southern leopard frogs, Lithobates (Rana) sphenocephalus. Groups of eggs from multiple clutches were collected from two wetlands and exposed to a range of Cu concentrations (0-150 µg/L) until they reached the free-swimming stage, and then individual larvae were reared to metamorphosis. Higher Cu concentrations significantly reduced embryo survival to the free-swimming stage but did not further reduce survival to metamorphosis. Larval period was affected by Cu treatment, but the clutch from which larvae originated (i.e., parentage) explained a higher proportion of the variation. Embryo survival to hatching varied significantly among clutches, ranging from 42.9 to 79.2%. Measurable levels of Cu were found in larvae with body burdens up to 595 µg Cu/g dry mass in the 100 µg/L treatment, and larval Cu body burdens were higher than in metamorphs. The present study also demonstrated that higher initial egg density ameliorated embryo mortality at higher Cu levels and should be accounted for in future studies.

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We isolated and characterized 10 microsatellite loci from the eastern spadefoot toad, Scaphiopus holbrookii. Loci were screened in 24 individuals from two schools of tadpoles in a single isolated wetland in South Carolina, USA. The number of alleles per locus ranged from 4 to 12, observed heterozygosity ranged from 0.200 to 0.875, and the probability of identity values ranged from 0.043 to 0.298. These new loci provide tools for examining the landscape genetics of a species facing continued destruction of its breeding habitat (small isolated wetlands) as well as fragmentation of upland life zones.Keywords Scaphiopus Á Spadefoot toad Á Microsatellite Á PCR primers Á SSR Á STR The eastern spadefoot toad (Scaphiopus holbrookii), a member of the family Pelobatidae, is found throughout much of the eastern US (Palis 2005). Adults are secretive, spending most of their time in underground burrows in sandy or loamy soils (Carr 1940;Pearson 1955). These pond-breeding amphibians typically use fish-free ephemeral wetlands, to which hundreds of adults may migrate during heavy rains in almost any month of the year (Hansen 1958;Neill 1957). Older tadpoles aggregate in schools of thousands of individuals (of unknown kinship) that forage voraciously throughout the pond (Scott 2008).A long-term mark-recapture metapopulation study (Greenberg and Tanner 2005) found limited inter-pond dispersal, but concluded that genetic analyses would be needed to understand metapopulation connectivity.We extracted total DNA from one S. holbrookii using the DNeasy tissue kit protocol (Qiagen, Valencia, CA) according to the manufacturer's instructions. We followed the enrichment procedure of Glenn and Schable (2005), with some exceptions. Briefly, DNA was digested with restriction enzyme RsaI (New England Biolabs), ligated to double-stranded linkers, denatured and hybridized to biotinylated microsatellite oligonucleotide mixes (mix 2 captured on magnetic streptavidin beads (Dynal). Unhybridized DNA was washed away and remaining DNA was eluted from the beads, amplified in polymerase chain reactions (PCR) using the forward SimpleX-6 as a primer. There were two primary changes to the Glenn and Schable (2005) protocol. First, a different linker was used (SimpleX-6 Forward 5 0 -AAAAG CACGAGCGGAACT and SimpleX-6 Reverse 5 0 -pAGT TCCGCTCGTGC). Second, the enriched libraries were sequenced on a 454 using titanium chemistry following standard Roche 454 library protocols (454 Life Sciences, a Roche company, Branford CT). Sequences were subjected to a 3 0 quality trim where only one base in the last 25 bases of the sequence contains a quality score less than 20 or alternatively contains one ambiguous base. CAP3 [33] was then used to assemble sequences at 98% sequence identity using a minimal overlap of 75 bp. Along with singlets, contigs of two or three sequences were searched for the presence of microsatellite DNA loci using the program MSATCOMMANDER version 0.8.1 (Faircloth 2008) and