Effectiveness of antifungal treatments during chytridiomycosis epizootics in populations of an endangered frog.

The recently-emerged amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd) has had an unprecedented impact on global amphibian populations, and highlights the urgent need to develop effective mitigation strategies. We conducted in-situ antifungal treatment experiments in wild populations of the endangered mountain yellow-legged frog during or immediately after Bd-caused mass die-off events. The objective of treatments was to reduce Bd infection intensity ("load") and in doing so alter frog-Bd dynamics and increase the probability of frog population persistence despite ongoing Bd infection. Experiments included treatment of early life stages (tadpoles and subadults) with the antifungal drug itraconazole, treatment of adults with itraconazole, and augmentation of the skin microbiome of subadults with Janthinobacterium lividum, a commensal bacterium with antifungal properties. All itraconazole treatments caused immediate reductions in Bd load, and produced longer-term effects that differed between life stages. In experiments focused on early life stages, Bd load was reduced in the 2 months immediately following treatment and was associated with increased survival of subadults. However, Bd load and frog survival returned to pre-treatment levels in less than 1 year, and treatment had no effect on population persistence. In adults, treatment reduced Bd load and increased frog survival over the entire 3-year post-treatment period, consistent with frogs having developed an effective adaptive immune response against Bd. Despite this protracted period of reduced impacts of Bd on adults, recruitment into the adult population was limited and the population eventually declined to near-extirpation. In the microbiome augmentation experiment, exposure of subadults to a solution of J. lividum increased concentrations of this potentially protective bacterium on frogs. However, concentrations declined to baseline levels within 1 month and did not have a protective effect against Bd infection. Collectively, these results indicate that our mitigation efforts were ineffective in causing long-term changes in frog-Bd dynamics and increasing population persistence, due largely to the inability of early life stages to mount an effective immune response against Bd. This results in repeated recruitment failure and a low probability of population persistence in the face of ongoing Bd infection.

The amphibian microbiome exhibits poor resilience following pathogen-induced disturbance.

Infectious pathogens can disrupt the microbiome in addition to directly affecting the host. Impacts of disease may be dependent on the ability of the microbiome to recover from such disturbance, yet remarkably little is known about microbiome recovery after disease, particularly in nonhuman animals. We assessed the resilience of the amphibian skin microbial community after disturbance by the pathogen, Batrachochytrium dendrobatidis (Bd). Skin microbial communities of laboratory-reared mountain yellow-legged frogs were tracked through three experimental phases: prior to Bd infection, after Bd infection (disturbance), and after clearing Bd infection (recovery period). Bd infection disturbed microbiome composition and altered the relative abundances of several dominant bacterial taxa. After Bd infection, frogs were treated with an antifungal drug that cleared Bd infection, but this did not lead to recovery of microbiome composition (measured as Unifrac distance) or relative abundances of dominant bacterial groups. These results indicate that Bd infection can lead to an alternate stable state in the microbiome of sensitive amphibians, or that microbiome recovery is extremely slow-in either case resilience is low. Furthermore, antifungal treatment and clearance of Bd infection had the additional effect of reducing microbial community variability, which we hypothesize results from similarity across frogs in the taxa that colonize community vacancies resulting from the removal of Bd. Our results indicate that the skin microbiota of mountain yellow-legged frogs has low resilience following Bd-induced disturbance and is further altered by the process of clearing Bd infection, which may have implications for the conservation of this endangered amphibian.

Stepping into the past to conserve the future: Archived skin swabs from extant and extirpated populations inform genetic management of an endangered amphibian.

Moving animals on a landscape through translocations and reintroductions is an important management tool used in the recovery of endangered species, particularly for the maintenance of population genetic diversity and structure. Management of imperiled amphibian species rely heavily on translocations and reintroductions, especially for species that have been brought to the brink of extinction by habitat loss, introduced species, and disease. One striking example of amphibian declines and associated management efforts is in California's Sequoia and Kings Canyon National Parks with the mountain yellow-legged frog species complex (Rana sierrae/muscosa). Mountain yellow-legged frogs have been extirpated from more than 93% of their historic range, and limited knowledge of their population genetics has made long-term conservation planning difficult. To address this, we used 598 archived skin swabs from both extant and extirpated populations across 48 lake basins to generate a robust Illumina-based nuclear amplicon data set. We found that samples grouped into three main genetic clusters, concordant with watershed boundaries. We also found evidence for historical gene flow across watershed boundaries with a north-to-south axis of migration. Finally, our results indicate that genetic diversity is not significantly different between populations with different disease histories. Our study offers specific management recommendations for imperiled mountain yellow-legged frogs and, more broadly, provides a population genetic framework for leveraging minimally invasive samples for the conservation of threatened species.

Increased accuracy of species lists developed for alpine lakes using morphology and cytochrome oxidase I for identification of specimens.

The first step in many community ecology studies is to produce a species list from a sample of individuals. Community ecologists now have two viable ways of producing a species list: morphological and barcode identification. In this study, we compared the taxonomic resolution gained by a combined use of both methods and tested whether a change in taxonomic resolution significantly impacted richness estimates for benthic macroinvertebrates sampled from ten lakes in Sequoia National Park, USA. Across all lakes, 77 unique taxa were identified and 42% (32) were reliably identified to species using both barcode and morphological identification. Of the 32 identified to species, 63% (20) were identified solely by comparing the barcode sequence from cytochrome oxidase I to the Barcode of Life reference library. The increased resolution using a combined identification approach compared to identifications based solely on morphology resulted in a significant increase in estimated richness within a lake at the order, family, genus and species levels of taxonomy (P < 0.05). Additionally, young or damaged individuals that could not be identified using morphology were identified using their COI sequences to the genus or species level on average 75% of the time. Our results demonstrate that a combined identification approach improves accuracy of benthic macroinvertebrate species lists in alpine lakes and subsequent estimates of richness. We encourage the use of barcodes for identification purposes and specifically when morphology is insufficient, as in the case of damaged and early life stage specimens of benthic macroinvertebrates.

Removal of nonnative fish results in population expansion of a declining amphibian (mountain yellow-legged frog, Rana muscosa).

The mountain yellow-legged frog (Rana muscosa) was once a common inhabitant of the Sierra Nevada (California, USA), but has declined precipitously during the past century due in part to the introduction of nonnative fish into naturally fishless habitats. The objectives of the current study were to describe (1) the effect of fish removal from three lakes (located in two watersheds) on the small, remnant R. muscosa populations inhabiting those lakes, and (2) the initial development of metapopulation structure in each watershed as R. muscosa from expanding populations in fish-removal lakes dispersed to adjacent habitats. At all three fish-removal lakes, R. muscosa population densities increased significantly following the removal of predatory fish. The magnitude of these increases was significantly greater than that observed over the same time period in R. muscosa populations inhabiting control lakes that remained in their natural fishless condition. Following these population increases, R. muscosa dispersed to adjacent suitable (but unoccupied) sites, moving between 200 and 900 m along streams or across dry land. Together, these results suggest that large-scale removal of introduced fish could result in at least partial reversal of the decline of R. muscosa. Continued monitoring of R. muscosa at the fish-removal sites will be necessary to determine whether the positive effects of fish eradication are sustained over the long-term, especially in light of the increasingly important role played by an emerging infectious disease (chytridiomycosis, caused by Batrachochytrium dendrobatidis) in influencing R. muscosa populations.