Adam J. Sepulveda

Environmental DNA as a new method for early detection of New Zealand mudsnails (Potamopyrgus antipodarum)

Abstract.  Early detection of aquatic invasive species is a critical task for management of aquatic ecosystems. This task is hindered by the difficulty and cost of surveying aquatic systems thoroughly. The New Zealand mudsnail (Potamopyrgus antipodarum) is a small, invasive parthenogenic mollusk that can reach very high population densities and severely affects ecosystem functioning. To assist in the early detection of this invasive species, we developed and validated a highly sensitive environmental deoxyribonucleic acid (eDNA) assay. We used a dose–response laboratory experiment to investigate the relationship between New Zealand mudsnail density and eDNA detected through time. We documented that as few as 1 individual in 1.5 L of water for 2 d could be detected with this method, and that eDNA from this species may remain detectable for 21 to 44 d after mudsnail removal. We used the eDNA method to confirm the presence of New Zealand mudsnail eDNA at densities as low as 11 to 144 snails/m2 in a eutrophic 5th-order river. Combined, these results demonstrate the high potential for eDNA surveys to assist with early detection of a widely distributed invasive aquatic invertebrate.

Scale‐dependent genetic structure of the Idaho giant salamander (Dicamptodon aterrimus) in stream networks

The network architecture of streams and rivers constrains evolutionary, demographic and ecological processes of freshwater organisms. This consistent architecture also makes stream networks useful for testing general models of population genetic structure and the scaling of gene flow. We examined genetic structure and gene flow in the facultatively paedomorphic Idaho giant salamander, Dicamptodon aterrimus, in stream networks of Idaho and Montana, USA. We used microsatellite data to test population structure models by (i) examining hierarchical partitioning of genetic variation in stream networks; and (ii) testing for genetic isolation by distance along stream corridors vs. overland pathways. Replicated sampling of streams within catchments within three river basins revealed that hierarchical scale had strong effects on genetic structure and gene flow. amova identified significant structure at all hierarchical scales (among streams, among catchments, among basins), but divergence among catchments had the greatest structural influence. Isolation by distance was detected within catchments, and in‐stream distance was a strong predictor of genetic divergence. Patterns of genetic divergence suggest that differentiation among streams within catchments was driven by limited migration, consistent with a stream hierarchy model of population structure. However, there was no evidence of migration among catchments within basins, or among basins, indicating that gene flow only counters the effects of genetic drift at smaller scales (within rather than among catchments). These results show the strong influence of stream networks on population structure and genetic divergence of a salamander, with contrasting effects at different hierarchical scales.

Local and Landscape-Scale Influences on the Occurrence and Density of Dicamptodon aterrimus, the Idaho Giant Salamander

Abstract Species distribution and abundance depend on a balance between local and landscape-scale processes. To successfully manage populations in regions with anthropogenic disturbances and habitat fragmentation, an understanding of important processes at each of these spatial scales is important. We used a model selection approach to identify an effective spatial scale to manage the Idaho Giant Salamander, Dicamptodon aterrimus. We used data from field surveys to compare support for local and landscape-scale models that explain D. aterrimus occurrence and density in 40 streams distributed throughout the Lochsa River basin, Idaho. Local-scale models included covariates that reflect patch quality. Landscape-scale models included variables that reflect predictions from metapopulation theory about the importance of patch size, connectivity, and fragmentation. Our results suggest that landscape-scale processes are important controls on D. aterrimus occurrence and that this species has broad habitat requirements within streams. Specifically, we found that probability of D. aterrimus occurrence was highest in roadless drainages and lowest in spatially isolated streams and in drainages with high old-growth forest density. Surprisingly, we found that D. aterrimus density was greatest in streams with a high proportion of embedded substrate and fine sediment. The positive association with embedded substrate may reflect adaptation to a high frequency of natural disturbances, such as landslides, in our study area. We suggest that management and conservation efforts for this species focus on protecting roadless areas and restoring stream connectivity in human-impacted areas, rather than on only improving habitat quality within streams.

Using nitrogen stable isotopes to detect long- distance movement in a threatened cutthroat trout (Oncorhynchus clarkii utah)

Interior cutthroat trout occupy small fractions of their historic ranges and existing populations often are rele- gated to headwater habitats. Conservation requires balancing protection for isolated genetically pure populations with re- storation of migratory life histories by reconnecting corridors between headwater and mainstem habitats. Identification of alternative life history strategies within a population is critical to these efforts. We tested the application of nitrogen stable isotopes to discern fluvial from resident Bonneville cutthroat trout (BCT; Oncorhynchus clarkii utah) in a headwater stream. Fluvial BCT migrate from headwater streams with good water quality to mainstem habitats with impaired water quality. Resident BCT remain in headwater streams. We tested two predictions: (i) fluvial BCT have a higher d 15 N than residents, and (ii) fluvial BCT d 15 N reflects diet and d 15 N enrichment characteristics of mainstem habitats. We found that fluvial d 15 N was greater than resident d 15 N and that d 15 N was a better predictor of life history than fish size. Our data also showed that fluvial and resident BCT had high diet overlap in headwater sites and that d 15 N of lower trophic levels was greater in mainstem sites than in headwater sites. We conclude that the high d 15 N values of fluvial BCT were acquired in

Potential of Environmental DNA to Evaluate Northern Pike (Esox lucius) Eradication Efforts: An Experimental Test and Case Study.

Determining the success of invasive species eradication efforts is challenging because populations at very low abundance are difficult to detect. Environmental DNA (eDNA) sampling has recently emerged as a powerful tool for detecting rare aquatic animals; however, detectable fragments of DNA can persist over time despite absence of the targeted taxa and can therefore complicate eDNA sampling after an eradication event. This complication is a large concern for fish eradication efforts in lakes since killed fish can sink to the bottom and slowly decay. DNA released from these carcasses may remain detectable for long periods. Here, we evaluated the efficacy of eDNA sampling to detect invasive Northern pike (Esox lucius) following piscicide eradication efforts in southcentral Alaskan lakes. We used field observations and experiments to test the sensitivity of our Northern pike eDNA assay and to evaluate the persistence of detectable DNA emitted from Northern pike carcasses. We then used eDNA sampling and traditional sampling (i.e., gillnets) to test for presence of Northern pike in four lakes subjected to a piscicide-treatment designed to eradicate this species. We found that our assay could detect an abundant, free-roaming population of Northern pike and could also detect low-densities of Northern pike held in cages. For these caged Northern pike, probability of detection decreased with distance from the cage. We then stocked three lakes with Northern pike carcasses and collected eDNA samples 7, 35 and 70 days post-stocking. We detected DNA at 7 and 35 days, but not at 70 days. Finally, we collected eDNA samples ~ 230 days after four lakes were subjected to piscicide-treatments and detected Northern pike DNA in 3 of 179 samples, with a single detection at each of three lakes, though we did not catch any Northern pike in gillnets. Taken together, we found that eDNA can help to inform eradication efforts if used in conjunction with multiple lines of inquiry and sampling is delayed long enough to allow full degradation of DNA in the water.

Coexistence in streams: Do source-sink dynamics allow salamanders to persist with fish predators?

Theory suggests that source–sink dynamics can allow coexistence of intraguild predators and prey, but empirical evidence for this coexistence mechanism is limited. We used capture–mark–recapture, genetic methods, and stable isotopes to test whether source–sink dynamics promote coexistence between stream fishes, the intraguild predator, and stream salamanders (Dicamptodon aterrimus), the intraguild prey. Salamander populations from upstream reaches without fish were predicted to maintain or supplement sink populations in downstream reaches with fish. We found instead that downstream reaches with fish were not sinks even though fish consumed salamander larvae—apparent survival, recruitment, and population growth rate did not differ between upstream and downstream reaches. There was also no difference between upstream and downstream reaches in net emigration. We did find that D. aterrimus moved frequently along streams, but believe that this is a response to seasonal habitat changes rather than intraguild predation. Our study provides empirical evidence that local-scale mechanisms are more important than dispersal dynamics to coexistence of streams salamanders and fish. More broadly, it shows the value of empirical data on dispersal and gene flow for distinguishing between local and spatial mechanisms of coexistence.

The Shifting Climate Portfolio of the Greater Yellowstone Area.

Knowledge of climatic variability at small spatial extents (< 50 km) is needed to assess vulnerabilities of biological reserves to climate change. We used empirical and modeled weather station data to test if climate change has increased the synchrony of surface air temperatures among 50 sites within the Greater Yellowstone Area (GYA) of the interior western United States. This important biological reserve is the largest protected area in the Lower 48 states and provides critical habitat for some of the world’s most iconic wildlife. We focused our analyses on temporal shifts and shape changes in the annual distributions of seasonal minimum and maximum air temperatures among valley-bottom and higher elevation sites from 1948–2012. We documented consistent patterns of warming since 1948 at all 50 sites, with the most pronounced changes occurring during the Winter and Summer when minimum and maximum temperature distributions increased. These shifts indicate more hot temperatures and less cold temperatures would be expected across the GYA. Though the shifting statistical distributions indicate warming, little change in the shape of the temperature distributions across sites since 1948 suggest the GYA has maintained a diverse portfolio of temperatures within a year. Spatial heterogeneity in temperatures is likely maintained by the GYA’s physiographic complexity and its large size, which encompasses multiple climate zones that respond differently to synoptic drivers. Having a diverse portfolio of temperatures may help biological reserves spread the extinction risk posed by climate change.

Genetic reconstruction of a bullfrog invasion to elucidate vectors of introduction and secondary spread

Abstract Reconstructing historical colonization pathways of an invasive species is critical for uncovering factors that determine invasion success and for designing management strategies. The American bullfrog (Lithobates catesbeianus) is endemic to eastern North America, but now has a global distribution and is considered to be one of the worst invaders in the world. In Montana, several introduced populations have been reported, but little is known of their sources and vectors of introduction and secondary spread. We evaluated the genetic composition of introduced populations at local (Yellowstone River floodplain) and regional (Montana and Wyoming) scales in contrast to native range populations. Our objectives were to (1) estimate the number of introductions, (2) identify probable native sources, (3) evaluate genetic variation relative to sources, and (4) characterize properties of local‐ and regional‐scale spread. We sequenced 937 bp of the mitochondrial cytochrome b locus in 395 tadpoles collected along 100 km of the Yellowstone River, from three additional sites in MT and a proximate site in WY. Pairwise ΦST revealed high divergence among nonnative populations, suggesting at least four independent introductions into MT from diverse sources. Three cyt b haplotypes were identical to native haplotypes distributed across the Midwest and Great Lakes regions, and AMOVA confirmed the western native region as a likely source. While haplotype (H d = 0.69) and nucleotide diversity (π = 0.005) were low in introduced bullfrogs, the levels of diversity did not differ significantly from source populations. In the Yellowstone, two identified haplotypes implied few introduction vectors and a significant relationship between genetic and river distance was found. Evidence for multiple invasions and lack of subsequent regional spread emphasizes the importance of enforcing legislation prohibiting bullfrog importation and the need for continuing public education to prevent transport of bullfrogs in MT. More broadly, this study demonstrates how genetic approaches can reveal key properties of a biological invasion to inform management strategies.

Suppressing Bullfrog Larvae with Carbon Dioxide

Abstract Current management strategies for the control and suppression of the American Bullfrog (Lithobates catesbeianus = Rana catesbeiana Shaw) and other invasive amphibians have had minimal effect on their abundance and distribution. This study evaluates the effects of carbon dioxide (CO2) on pre- and prometamorphic Bullfrog larvae. Bullfrogs are a model organism for evaluating potential suppression agents because they are a successful invader worldwide. From experimental trials we estimated that the 24-h 50% and 99% lethal concentration (LC50 and LC99) values for Bullfrog larvae were 371 and 549 mg CO2/L, respectively. Overall, larvae that succumbed to experimental conditions had a lower body condition index than those that survived. We also documented sublethal changes in blood chemistry during prolonged exposure to elevated CO2. Specifically, blood pH decreased by more than 0.5 pH units after 9 h of exposure and both blood partial pressure of CO2 (pCO2) and blood glucose increased. These findings suggest that CO2 treatments can be lethal to Bullfrog larvae under controlled laboratory conditions. We believe this work represents the necessary foundation for further consideration of CO2 as a potential suppression agent for one of the most harmful invaders to freshwater ecosystems.

Evaluation of a Fine Sediment Removal Tool in Spring-fed and Snowmelt Driven Streams

The accumulation of fine-grained sediments impairs the structure and function of streams, so removing fine sediments may be required to achieve restoration objectives. There has been little work on methods of removing excess sediment or on the efficacy of the methods. We used a 4-year before-after-control-impact design in southeastern Idaho streams to test a fine sediment removal system (FSRS) manufactured by Streamside Environmental LLC. The FSRS agitates fine sediment in the substrate with clean pump water and then vacuums the sediment out of the stream with a second pump. Our objectives were: 1) to test if the FSRS can selectively remove fine sediment; 2) to monitor the bio-physical responses in FSRS treated and downstream waters; and 3) to compare the bio-physical responses to the FSRS in spring-fed and snowmelt driven stream reaches. The FSRS removed ~ 14 metric tons of sediment from the two treated reaches. More than 90% of this sediment was < 2 mm, indicating that the FSRS selected for fine sediment in both stream types. Sustained effects of removing this sediment were confined to substrate improvements in treated reaches. Embeddedness in the spring-fed reach decreased and subsurface grain size in spring-fed and snowmelt driven reaches increased. We did not detect any sustained invertebrate or fish responses in treated reaches or any detrimental bio-physical responses in downstream waters. These results indicate that the FSRS reduced fine sediment levels but sediment removal did not reverse the impacts of sediment accumulation to stream biota within our monitoring time frame.