Carlos Davidson

The role of natural history collections in documenting species declines

Efforts to document the decline of extant populations require a historical record of previous occurrences. Natural history museums contain such information for most regions of the world, at least at a coarse spatial scale. Museum collections have been successfully used to analyse declines in a wide range of plants and animals, at spatial scales ranging from single localities to large biotic and political regions. Natural history museum collections, when properly analysed, can be an invaluable tool in documenting changes in biodiversity during the past century.

DECLINES OF THE CALIFORNIA RED‐LEGGED FROG: CLIMATE, UV‐B, HABITAT, AND PESTICIDES HYPOTHESES

The federally threatened California red-legged frog (Rana aurora draytonii) has disappeared from much of its range for unknown reasons. We mapped 237 historic locations for the species and determined their current population status. Using a geographic information system (GIS), we determined latitude, elevation, and land use attributes for all sites and analyzed the spatial pattern of declines. We then compared the observed patterns of decline to those predicted by the climate change, UV-B radiation, pesticides, and habitat alteration hypotheses for amphibian decline. Declines were not consistent with the climate change hypothesis but showed a strong positive association with elevation, percentage upwind agricultural land use, and local urbanization. These results apply to patterns of decline across the entire range of R. a. draytonii in California, as well as within geographic subregions. The elevational gradient in declines is consistent with the UV-B hypothesis, although the UV-B hypothesis also predicts a north-to-south gradient in declines, which we did not observe. The association of declines with the amount of upwind agricultural land use strongly suggests that wind-borne agrochemicals may be an important factor in declines. This association was most pronounced within the Central Valley-Sierra region, where other studies have documented both transport and deposition of pesticides to the Sierra Nevada and the presence of pesticide residues in the bodies of congeneric (Rana muscosa) and more distantly related (Hyla regilla) frog species.

MULTIPLE STRESSORS AND AMPHIBIAN DECLINES: DUAL IMPACTS OF PESTICIDES AND FISH ON YELLOW-LEGGED FROGS

More than 40% of Earths 5700+ amphibian species have undergone recent declines. Despite the likely involvement of multiple factors in driving these declines, most studies continue to focus on single stressors. In California (USA), separate studies have implicated either introduced fish or pesticides as causal agents. To date, however, no study has simultaneously evaluated the respective roles of these two potential stressors nor attempted to assess their relative importance, information critical for the development of effective conservation efforts and environmental policies. We examined the role and relative effect of fish and pesticides on the mountain yellow-legged frog (Rana muscosa) using unusually detailed data sets for a large portion of R. muscosas historic range in Californias Sierra Nevada. Habitat characteristics and presence/absence of R. muscosa and fish were quantified at each of 6831 sites during field surveys. Pesticide use upwind of each site was calculated from pesticide application records and predominant wind directions. Using generalized additive models, we found that, after accounting for habitat effects, the probability of R. muscosa presence was significantly reduced by both fish and pesticides, with the landscape-scale effect of pesticides much stronger than that of fish. The degree to which a site was sheltered from the predominant wind (and associated pesticides) was also a significant predictor of R. muscosa presence. Taken together, these results represent the strongest evidence to date that windborne pesticides are contributing to amphibian declines in pristine locations. Our results suggest that amphibian declines may have complex multi-factorial causes, and caution that single-factor studies that demonstrate the importance of one factor should not be used as evidence against the importance of other factors.

Evidence from peptidomic analysis of skin secretions that the red-legged frogs, Rana aurora draytonii and Rana aurora aurora, are distinct species

The northern red-legged frog Rana aurora aurora and the California red-legged frog Rana aurora draytonii are traditionally classified together in the same species group. Ten peptides with antimicrobial activity were isolated from norepinephrine-stimulated skin secretions of R. aurora draytonii and purified to near homogeneity. The peptides were identified as belonging to the ranatuerin-2 family (two peptides), brevinin-1 family (four peptides), temporin family (three peptides), and a novel peptide, RV-23 (RIGVLLARLPKLFSLFKLMGKKV) that has limited structural similarity to the bee venom peptide, melittin. This distribution of peptides contrasts with that found previously in skin secretions from R. aurora aurora collected under the same conditions and at the same time of year (one ranatuerin-2 peptide, two brevinin-1 peptides, and one temporin peptide). The variation in amino acid sequences between corresponding R. aurora draytonii and R. aurora aurora peptides is comparable with the variation in sequences of orthologs from other members of the Amerana group of New World ranid frogs (Rana boylii, Rana muscosa, and Rana luteiventris). It is proposed, therefore, that the red-legged frogs should be regarded as separate species (R. aurora and R. draytonii) within the Amerana group rather than conspecific subspecies. The data emphasize that amino acid sequences of antimicrobial peptides in skin secretions may be used to infer taxonomic and phylogenetic relationships between species of ranid frogs.

Peptide defenses of the Cascades frog Rana cascadae: implications for the evolutionary history of frogs of the Amerana species group

The Cascades frog Rana cascadae belongs to the Amerana (or Rana boylii) group that includes six additional species from western North America (R. aurora, R. boylii, R. draytonii, R. luteiventris, R. muscosa, and R. pretiosa). R. cascadae is particularly susceptible to pathogenic microorganisms in the environment and populations have declined precipitously in parts of its range so that the protection afforded by dermal antimicrobial peptides may be crucial to survival of the species. Peptidomic analysis of norepinephrine-stimulated skin secretions led to the identification of six peptides with differential cytolytic activities that were present in high abundance. Structural characterization showed that they belonged to the ranatuerin-2 (one peptide), brevinin-1 (one peptide), and temporin (four peptides) families. Ranatuerin-2CSa (GILSSFKGVAKGVAKDLAGKLLETLKCKITGC) and brevinin-1CSa (FLPILAGLAAKIVPKLFCLATKKC) showed broad spectrum antibacterial activity (MIC</=32microM against Escherichia coli and Staphylococcus aureus) but only brevinin-1CSa was strongly hemolytic against human erythrocytes (LC(50)=5microM). The taxonomy of ranid frogs is currently in a considerable state of flux. The ranatuerin-2 gene is expressed in all members of the Amerana group studied to-date and cladistic analysis based upon a comparison of the amino acid sequences of this peptide indicates that R. cascadae, R. muscosa and R. aurora form a clade that is distinct from one containing R. draytonii, R. boylii, and R. luteiventris. This conclusion is consistent with previous analyses based upon comparisons of the nucleotide sequences of mitochondrial genes.

Contaminant residues and declines of the Cascades frog (Rana cascadae) in the California Cascades, USA

Populations of Cascades frogs (Rana cascadae) have declined precipitously in the Mount Lassen area, but remain abundant in the other half of their California range in the Klamath Mountains. To evaluate the role of contaminants in Cascade frog declines, we sampled sediment and frog tadpole tissue at 31 sites where Cascades frogs had disappeared and sites where Cascades frogs are still present across the Lassen and Klamath regions. Pacific chorus frogs (Pseudacris regilla) were tested and used as surrogates for residue concentrations in Cascades frogs. We analyzed a total of 79 tadpole samples for 73 semivolatile contaminants including pesticides, polychlorinated biphenyls (PCBs), and polycyclic aromatic hydrocarbons (PAHs). The most frequently detected residue was endosulfan sulfate, followed by dacthal, chlorpyrifos, PCB 187, endosulfan II, trans-chlordane, and trans-nonachlor. Chorus frogs had similar residue concentrations as Cascades frogs for most but not all chemicals, indicating that chorus frogs can serve as a reasonable proxy for chemical concentrations in Cascades frogs. None of the contaminants in tissue or sediment had significantly higher concentrations at sites where Cascades frogs have disappeared than at sites where Cascades frogs are still present. We found no evidence to support the hypothesis that the contaminants analyzed have contributed to the decline of Cascades frogs in northern California, although we were able to analyze only a handful of the over 300 pesticides currently used in the area.

Comparison of pressurized liquid extraction and matrix solid‐phase dispersion for the measurement of semivolatile organic compound accumulation in tadpoles

Analytical methods capable of trace measurement of semivolatile organic compounds (SOCs) are necessary to assess the exposure of tadpoles to contaminants as a result of long-range and regional atmospheric transport and deposition. The present study compares the results of two analytical methods, one using pressurized liquid extraction (PLE) and the other using matrix solid-phase dispersion (MSPD), for the trace measurement of more than 70 SOCs in tadpole tissue, including current-use pesticides. The MSPD method resulted in improved SOC recoveries and precision compared to the PLE method. The MSPD method also required less time, consumed less solvent, and resulted in the measurement of a greater number of SOCs than the PLE method.

The toxicity of glyphosate alone and glyphosate-surfactant mixtures to western toad (Anaxyrus boreas) tadpoles.

Pesticide choice based on toxicity to nontarget wildlife is reliant on available toxicity data. Despite a number of recent studies examining the effects of glyphosate on amphibians, very few have aimed to understand the toxicological effects of glyphosate in combination with surfactants as it is commonly applied in the field. Land managers interested in making pesticide choices based on minimizing impacts to nontarget wildlife are hindered by a lack of published toxicity data. Short-term acute toxicity trials were conducted for glyphosate in the form of isopropylamine salt (IPA) alone and mixed with 2 surfactants: Agri-dex and Competitor with western toad (Anaxyrus [Bufo] boreas) tadpoles. Glyphosate IPA mixed with Competitor was 6 times more toxic than glyphosate IPA mixed with Agri-dex, and both mixtures were more toxic than glyphosate IPA alone. The median lethal concentrations reported for 24-h and 48-h exposures were 8279 mg/L (24 h) and 6392 mg/L (48 h) for glyphosate IPA alone; 5092 mg/L (24 h) and 4254 mg/L (48 h) for glyphosate IPA mixed with Agri-dex; and 853 mg/L (24 h) and 711 mg/L (48 h) for glyphosate IPA mixed with Competitor. The present study indicates that the toxicity of a tank mix may be greatly increased by the addition of surfactants and may vary widely depending on the specific surfactant.