Theses and Dissertations

Permanent URI for this collection


Recent Submissions

Now showing 1 - 5 of 362
  • Item
    Open Access
    Demystifying viruses: understanding the role of river viruses on microbial community structure and biogeochemical cycling through a multi-omic lens
    (Colorado State University. Libraries, 2023) Rodríguez-Ramos, Josué, author; Wrighton, Kelly, advisor; Hall, Edward, committee member; Metcalf, Jessica, committee member; Wilkins, Michael J., committee member
    Viruses are the most abundant entity on the planet, with estimates of up to 1031 viral particles dispersed across the globe in every ecosystem that can sustain life. Today, as the world responds to the COVID-19 pandemic, the word "virus" often evokes a negative response because of their impacts on human health and disease. Yet, most viruses that exist in the world can only infect bacteria and archaea. In fact, it has long been estimated that for every 1 bacterial or archaeal cell, there are 10 viruses that can infect it. While bacteria and archaea are long regarded as essential to overall ecosystem health and functionality, the roles of viruses in natural systems are much less understood and appreciated. Due to a scarcity of genome-resolved multi-omic studies, this lack of understanding is compounded in river ecosystems, which play critical roles modulating global carbon and nitrogen biogeochemistry. The overarching aims of this dissertation are to harness genome-resolved, multi-omic datasets to 1) decipher the impact that viruses can have on river microbial communities and biogeochemical cycling, and 2) to explain how viral ecology can enhance our understanding of river ecosystem function. To define the role that viral and microbial communities have on river function, I first set out to understand what is currently known of river viral ecology. In Chapter 1, I provided a background primer on viruses and their impacts on natural ecosystems. I then zoomed in on viral roles exclusively within rivers and described the current state of river viral ecology. I also highlighted some of the knowledge gaps addressed specifically by my thesis. My literature review revealed that while there are publicly available metagenomic datasets, there is a drastic underutilization of genome-resolved strategies which are critical for constraining microbial metabolism and viral impacts into informative units. Further, these datasets are largely unused because the data is collected in an un-coordinated manner, leading to the lack of similar sampling methods, and ultimately an inability to make results interoperable. Together, in this chapter I present compelling evidence for the need of genome-resolved, virus-host paired multi-omic analyses that are pivotal to our understanding of river ecosystems and lay the groundwork for the questions I will address throughout my dissertation. After identifying that there was a gap studies that leverage metagenome assembled genomes (MAGs) and viral metagenome assembled genomes (vMAGs), for Chapter 2 I focused on using a genome-resolved lens to uncover the microbial and viral metabolic underpinnings responsible for the biogeochemical cycling of carbon and nitrogen in the Columbia River system. This chapter used a dataset that was spatially resolved at the centimeter scale for three sediment cores across two transects of the Columbia River and included 33 samples, all of which had metagenomes that were paired to metaproteomes, biogeochemistry, and metabolites. Using this dataset, I created the first river microbial and viral database genome-resolved database called Hyporheic Uncultured MAG and vMAG (HUM-V). Leveraging metaproteomics paired to HUM-V database, I built a conceptual model outlining microbial and viral contributions to carbon and nitrogen biogeochemistry in these river sediments. With this metabolic reconstruction, I showed an intertwined carbon and nitrogen cycle that can likely contribute to the fluxes of nitrous oxide. Specifically, I demonstrated that well recognized river microbes like those of the phyla Nitrososphaeraceae as well as other less recognized phyla like Binatia encode and express genes for denitrification. I also showed that the clade II nosZ gene, which is responsible for nitrous oxide production, could possibly act as a nitrous oxide sink without contributing to its production. Linking viral members to microbial hosts demonstrated that viruses may be key modulators of carbon and nitrogen cycling. Specifically, I presented evidence that viruses can infect key nitrifying organisms (i.e., Nitrospiraceae) as well as key polymer degrading organisms (i.e., Actinobacteria). Highlighting their potential roles, linear regression analyses consistently identified viral organisms as key predictors of ecosystem biogeochemistry. Chapter 2 of my thesis yielded insights that uncovered some of the microbial contributions that were thought to occur but were poorly defined in river sediments (e.g., nitrogen mineralization), and presented a genome-resolved, virus-host paired strategy that I could then use to directly assess how viruses impacted host metabolism and ecosystem function. Ultimately, Chapter 2 highlights the power of genome-resolved database strategies to reduce existing predictive uncertainties in river corridor models. Having provided a genome-resolved view of metabolic processes in Chapter 2, for Chapter 3 I set out to expand upon our understanding of river viruses by providing insights into their temporal and spatial dynamics. For this, I worked with a finely tuned temporal dataset from an urban stream near Berlin, Germany called the Erpe River. The Erpe River dataset is a metagenomic timeseries where samples were collected every 3 hours for a total of 48 hours across both the surface water (SW) and pore water (PW) compartments. In addition to metagenomes, Fourier-transform ion cyclotron resonance mass spectrometry (FTICR-MS) and biogeochemistry were collected for each sample. Using this dataset, I created a database consisting of 1,230 vMAGs and 125 MAGs. Only 1% of our vMAGs clustered to known taxonomic representatives, highlighting the underrepresentation of river viruses in public databases. Due to this underrepresentation, I supplemented my viral taxonomic analyses with over 20,000 vMAGs spanning different publicly available studies that were relevant to rivers and wastewater treatment plants and showed that nearly half of the novel genera identified were cosmopolitan in aquatic ecosystems. I also characterized the spatial and temporal dynamics of the river microbiomes across the surface water (SW) and pore water (PW) compartments. Both the viral and microbial communities were distinct between the SW and PW samples and were both driven by the same chemical drivers. Given that these compartments had distinct communities, I set out to understand how they were changing over time. By employing multiple temporal statistical methods, I show that SW communities are more persistent and more stable relative to the PW communities, likely resulting from the homogeneous selection pressures of the SW, and the heterogeneity within the sediment. In addition to resolving these temporal dynamics, I highlight some specific virus and host genomes that influence biogeochemical cycling. In summary, my third chapter shows how river viral and microbial communities change across spatial and temporal gradients, and highlights how genome-resolved metagenomics enhances our interpretation of microbiome data. The final chapter of this dissertation (Chapter 4) summarizes the key findings of my thesis and provides future perspectives to inspire research in environmental river viral ecology. This section also showcases several publications that I have worked on throughout my doctoral degree that span multiple ecosystems like mouse guts, human guts, soils, and the development of the computational tool Distilled and Refined Annotation of Metabolism (DRAM). This final chapter also highlights a manuscript that I was involved in that showcases a new scientific framework: Interoperable, Open, Coordinated, and Networked (ICON). I further highlight this framework to address how these ICON strategies are beginning to be implemented in other fields and propose that in order to move the discipline of river microbial ecology forward, we need to implement ICON frameworks and the standardization and coordination of sampling collection. In summary, the aims of this dissertation were to summarize what is known in the field of river viral ecology (Chapter 1), to investigate viral roles that viruses play on river organic nitrogen and carbon processing (Chapter 2), to interrogate the temporal and spatial dynamics of viruses within rivers (Chapter 3), and to summarize how this dissertation has added to the understanding of river viral ecology, and what the next big questions for the field should be (Chapter 4). Ultimately, these works shine a spotlight on the viruses found in river ecosystems and shows that they likely play key roles in the regulation of microbial biogeochemical cycles.
  • Item
    Open Access
    The role of disturbance and local adaptation in altering tree regeneration responses to climate change
    (Colorado State University. Libraries, 2023) Nigro, Katherine M., author; Redmond, Miranda D., advisor; Battaglia, Michael A., committee member; Stevens-Rumann, Camille S., committee member; Rocca, Monique E., committee member
    Anthropogenic climate warming is predicted to reorganize the communities seen on the landscape today as species migrate to stay within their preferred climate niche. However, for long-lived trees, it is uncertain whether migrations will be able to keep pace with the rapidly changing climate. In addition, adaptations to local climate that have evolved within subpopulations of a species may become obsolete or maladaptive under climate change. Natural disturbances such as wildfire and insect outbreaks in forests may help to accelerate range shifts by reducing competition from other plant species and creating favorable microsites for establishment of novel species. For landscapes that are not recovering after disturbance events, human-assisted movement of populations within species (assisted gene flow) has been proposed to pre-adapt the forest to future climate conditions. In the first part of my dissertation, I analyze large-scale USDA Forest Service Forest Inventory and Analysis data from the interior western US and conduct a field study of trembling aspen in southern Colorado to understand how disturbances are interacting with climate change to alter species range shifts at broad and local scales. The first chapter of my dissertation shows that disturbances are catalyzing range shifts on a large-scale for the dominant tree species of the interior western US under both disturbed and undisturbed conditions, but mostly through reduced regeneration in hot and dry portions of species ranges rather than expansions into cooler and wetter areas. However, chapter 2 reveals that expansions are happening on more local scales for trembling aspen after wildfire, but not after beetle-kill mortality events. Therefore, this research shows that disturbance-facilitated species range expansions are limited to certain species and locations, which may not be enough to keep pace with climate warming. The second part of my dissertation investigates the potential for assisted gene flow in ponderosa pine by examining differences between seedlings from different populations and mother trees in both the greenhouse under drought and the field under a variety of microsite conditions. This research reveals that population climate and watering regime are important determinants of seedling growth in the greenhouse, but that ponderosa pine seedlings from hotter and drier populations do not possess adaptations typically associated with drought-tolerance and do not perform well when planted at the cooler range margin. In addition, mother tree sensitivity to climate was influential in seedling water use efficiency in the greenhouse and in the sensitivity of seedlings to microclimate conditions in the field, making the case that targeting seed collections from specific mother trees may be warranted. Overall, my dissertation research provides insight into how future disturbance events are likely to influence regeneration at species range margins and provides valuable information for land managers seeking to implement assisted gene flow at the upper elevational margin of ponderosa pine forests.
  • Item
    Open Access
    Ecological consequences of warming climes for cold-adapted species – evidence from mountain goats
    (Colorado State University. Libraries, 2023) Hayes, Forest Parker, author; Berger, Joel, advisor; Bailey, Larissa L., committee member; Wilson, Kenneth R., committee member; McGrath, Daniel, committee member
    Global climate change from human activity is changing ecological communities at unprecedented rates. Coupled with recent and extraordinary loss of biodiversity, assessing the consequences for vulnerable species – and effecting proactive conservation – will be fundamental to stymieing these losses. Among the areas most strongly impacted by these changes are montane regions, which are warming at rates 2–5x the global average. Within those, cold-adapted organisms are among the most strongly impacted as they may experience thermal stress at moderate temperatures. Past species- and taxa-level responses to warming environs includes numerous concentrated extirpations at the southern peripheries of distributional ranges during the late Pleistocene. Less clear are localized capacities of cold-adapted species to mitigate thermal challenges against warming temperatures, especially through proximate behavioral and physiological adjustments. In this dissertation, I address three key tendrils of the ecological consequences of warming climes for cold-adapted species. First, I evaluate the role of snow patches in mediating the physiological impacts of warming temperatures. Despite strong associations between many taxa and cold environs, great uncertainty remains about the biological benefits, if any, of using persistent snow during summer months. Contrary to the prevalent hypothesis that persistent snow provides thermal relief for cold-adapted species, I demonstrate that use of snow patches facilitates insect avoidance and not thermoregulatory gains. While the duration and spatial extent of snow declines globally as the climate warms, its diminishing availability is likely to have substantive impacts to populations given a general pattern of associations between insects and temperatures at high elevations and latitudes. Next, I evaluate changes in spatial use relative to high temperatures and the influence of snow on resource selection. While thermal challenges to persistence are well evidenced through historical extirpations, attention to near-term shifts in resource use and snow as a driver of summer resource selection remains under-investigated. I found strong shifts in resource selection during periods of high temperature, with increased selection for cooler habitats, little effect from snow, and evidence of ecological tradeoffs in avoidance of heat. These results point to possible demographic consequences of changing behavior and illustrate the importance of proactive consideration of changing patterns of use in management policies. Finally, I evaluate the importance of climate refugia and human translocation for the applied conservation of biodiversity. Despite a long history of relocating wildlife, translocations outside native ranges have rarely been conducted for conservation and populations introduced beyond their native ranges have seldom contributed to in situ species conservation within native ranges. Herein, I identify three cases of introductions to climate refugia and highlight the untapped conservation value of existing populations outside of native ranges. I report species in these refugia offer not only a reservoir for reintroductions but also a buffer against temperature-driven distributional change and may prove invaluable in stymieing current and future loss of biodiversity under continued climate challenges. In concert, this dissertation addresses physiological and geographical consequences of warming climes for cold-adapted species, identifies the need for proactive conservation, and highlights one avenue through which meaningful conservation benefits may be achieved.
  • Item
    A holistic evaluation of human-elephant interactions in multi-use landscapes
    (Colorado State University. Libraries, 2023) Carroll, Sarah Louise, author; Reid, Robin, advisor; Boone, Randy, committee member; Salerno, Jon, committee member; Wittemyer, George, committee member
    In East Africa, rangelands and savannas are complex social-ecological systems with a history of land sharing among pastoralists, their livestock and wildlife. Today, many are systems of global importance for biodiversity conservation. As in Earth's other biomes, East African rangelands and their inhabitants face growing challenges as the result of global change, namely ecological, climate, political, and socioeconomic changes that are threatening wildlife populations and straining human-wildlife relationships. African savanna elephants (Loxodonta africana) are key actors in these systems and the center of many of these challenges. Thus, sophisticated land-use planning that addresses the resource use and needs of both people and elephants that can be integrated into conservation policy at relevant scales of governance is a key need for elephant conservation. However, studies of African elephant spatial ecology rarely examine both ecological and human-elephant relationships in the ecosystems they inhabit, whereas existing studies from human dimensions disciplines, which focus on social aspects of human-elephant relationships, very rarely include landscape-specific analyses. Additionally, detailed, spatially explicit information describing human-elephant interactions is limited, particularly in regions where people interacting with elephants still herd livestock more than they grow crops. This dissertation addresses this research need by applying novel methods with a holistic approach to examine the spatial dynamics of human-elephant interactions in multi-use landscapes with a focus on elephant interactions with pastoral peoples and their livestock. Most research in this dissertation is specifically focused on the spatial ecology of human-elephant interactions in the Greater Mara Ecosystem of southwest Kenya and combines inference from images captured by remote camera traps, high-resolution GPS tracking data, and social survey data to: 1) Understand how livestock and land management influence spatiotemporal patterns of elephant occurrence, 2) Investigate elephant movement behaviors and understand how people and environmental variation influence elephant movement behaviors, 3) Map core elephant habitat and movement corridors to support conservation planning, 4) Understand how people in mixed-use savannas relate to elephants and how elephants and wildlife conservation impact their lived experiences, and 5) Map and quantify social willingness to coexist with elephants in mixed-use landscapes to support conservation planning that accounts for the needs of both people and elephants. I found that elephants shifted the quantity and timing of their activity in community conservancies where livestock are present relative to the neighboring protected area where livestock were absent. Elephants were also more likely to occur in the protected area than community conservancies even when controlling for habitat variation. Finally, I found that areas in community conservancies used with higher intensity by sheep and goats, and separately cattle, were somewhat less likely to be used by elephants during the daytime. However, this finding was not consistent across years and the estimated effects had low precision and additional alternative analyses may make this relationship clearer. I then apply network theory to analyze combined information on movement path properties, use intensity, and structural properties of movement networks calculated from GPS tracking data to delineate the functional landscape of movement for elephants in the wider Mara-Serengeti ecosystem. After identifying movement behaviors, I then investigate the environmental variables driving different movement behaviors with a focus on delineating the habitats that support high elephant use and elephant movement corridors. Finally, I contrast how movement behaviors and the environmental variables driving movement vary between elephants inhabiting the mesic, wet savannas of the wider Mara-Serengeti ecosystem with those previously published and observed in elephants inhabiting the xeric savannas of the Samburu-Laikipia ecosystem. Results showed that human presence strongly influenced elephant movement behavior in the Mara and specifically influenced the location of core areas, whereas in Samburu, water availability and vegetation productivity and predictability strongly were the most important variables explaining core area use for elephants. Although vegetation productivity also influenced elephant core area use in the Mara, predictability did not, and human presence and canopy cover strongly influenced core area use in the Mara more strongly than water availability. Overall, these findings indicate that elephants in the Mara are likely less constrained by water and forage availability than elephants in Samburu and have more flexibility to access these key resources while minimizing the risks posed by people. I apply cognitive hierarchy theory to understand how elephants impact people in the Greater Mara Ecosystem by investigating values and attitudes associated with African elephants and elephant conservation in communities sharing space with elephants. I use data collected from semi- structured interviewers at 177 households across a mixed-use, agropastoral landscape that also functions as an unprotected elephant corridor and analyzed responses using Bayesian hierarchical models to quantify positive attitudes towards elephants while accounting for self-reporting bias. I interpret quantitative model estimates in the context of qualitative attitude assessments and sociocultural values to gain a deeper understanding of what explains attitudes towards elephants in the region. We found that although a majority of people expressed positive attitudes about elephant conservation in general, most were not also positive about sharing space with elephants on community and private lands at a local scale. Model estimates showed that people who believed that elephants had sociocultural value were the most likely to be positive towards elephant conservation in general, but experiencing conflict with any wildlife lowered the probability of respondents to have a positive attitude towards sharing space with elephants at a local scale. Qualitative data revealed that safety and well-being concerns related to the perceived threats that elephants pose to human life, livestock, and crops, coupled with few social and economic incentives to support elephant conservation in community and private lands contribute to low local positive attitudes. Our results suggest that conservation approaches focused on sustaining existing sociocultural values and relationships with wildlife while also investing in human well-being and safety measures and could improve conservation outcomes in shared landscapes. Overall, this research develops contributions to the understanding of human-elephant interactions in East African savannas and provides practical applications for elephant conservation. Specifically, this dissertation through the creation of several map products can support conservation planning that accounts for both people and elephants in the Greater Mara Ecosystem. Some of the most important takeaways come from co-interpretation of results with agropastoral communities and can thus provide direct guidance to conservation practitioners on how to better address human well-being in community-based conservation efforts. Though this research was produced in collaboration with non-government organizations, community-based conservation leaders, and government wildlife officials in Kenya, I recommend that future work can improve the collaborative research process by more successfully including local communities as stakeholders at all stages of the collaborative research process.
  • Item
    Open Access
    Of toads and tolerance: intraspecific variation in host persistence when challenged by disease
    (Colorado State University. Libraries, 2023) Hardy, Bennett, author; Bailey, Larissa, advisor; Funk, W. Christopher, advisor; Huyvaert, Kathryn P., committee member; Muths, Erin, committee member; Hoke, Kimberly, committee member
    Infectious diseases are increasingly known to drive population declines and extinctions and ultimately contribute to the loss of global biodiversity. This phenomenon is none more apparent than in the extinctions and extirpations of over 500 amphibian species worldwide due to a disease caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd). This ongoing loss of amphibian diversity is concerning given the important roles amphibians serve for ecosystem function, ecosystem services, and pharmacology, among others. To mitigate these declines, scientists must understand the pathogen, environmental, and host factors that interact to affect disease outcomes. Despite over 25 years of research on amphibian declines and Bd, there is still much we can learn that can crucially inform our conservation actions. For instance, while Bd is undoubtedly a lethal pathogen of severe threat to many amphibians around the world, researchers have observed substantial variation in host responses to Bd infection or presence in the wild and in the laboratory. While many host populations are extirpated by Bd, some persist at lower abundances or rebound completely. Understanding variation in the mechanisms of host population persistence may provide vital hints for how to better conserve vulnerable amphibian populations. This leads me to the central question of my dissertation: "Why do some populations persist with Bd while others do not?". I address this question in three chapters by studying variation in boreal toad (Anaxyrus boreas boreas) responses to Bd at several locations across the toad's range. The boreal toad occurs from coastal southern Alaska down into Colorado. Boreal toads are susceptible to Bd and have experienced dramatic declines and extirpations at the southern edge of its range in New Mexico, Colorado, and southwestern Wyoming, which together are considered the southern Rocky Mountain population. Populations in the northern portion of its range, however, appear to be persisting despite the confirmed presence, and high prevalence, of Bd. Two major hypotheses for the difference in apparent persistence of boreal toad populations with Bd across regions include a) differences in demographic compensation and b) differences in host susceptibility. Therefore, I used observational and experimental studies to investigate these hypotheses to better understand the contexts in which boreal toads persist with Bd. In my first chapter, I leveraged a long-term mark-recapture dataset from multiple populations of boreal toads across a high-elevation gradient in Colorado to test for the existence of compensatory recruitment. Compensatory recruitment is a hypothesized mechanism of host persistence when challenged by disease where hosts increase their baseline recruitment, which compensates for decreased survival attributed to disease, and ultimately stabilizes or slows population declines. Limitations from prior investigations of this phenomenon in other amphibian-Bd systems include the lack of pre-Bd monitoring data, the lack of population replication, and studies that primarily examine low elevation populations. I found a life history trade-off between survival and recruitment across elevations, where high-elevation toads have high survival but low recruitment and vice versa at lower elevations. Once Bd arrived, however, recruitment was reduced across all populations and survival was reduced to zero. Estimates of population abundance and population growth rates were also variable prior to Bd arrival, but dramatically declined after. I did not find evidence for compensatory recruitment in these high elevation boreal toad populations. My findings highlight that demographic responses to disease may be environmentally context-dependent, and that high elevation amphibian populations are particularly vulnerable to the effects of Bd. In Chapter 2, I used a laboratory exposure experiment to identify an appropriate isolate of Bd to use in a future experiment designed to investigate differential susceptibility of boreal toad populations to Bd (Chapter 3). While researchers have known about the potential for laboratory-maintained Bd cultures to lose pathogenicity over time (i.e., pathogen attenuation), most exposure studies use isolates that are available to them, regardless of how long they have been maintained in the laboratory, or are unaware of their chosen isolate's culture history. I exposed wild-caught, captive-reared boreal toads to three different isolates of Bd that varied in the amount of time maintained in the lab (old vs. new) and the geographic origin of the isolate compared to the host (local vs. novel) to determine the best isolate for use in Chapter 3. I found that boreal toads exposed to the older isolates had higher weekly survival probabilities than those exposed to the new isolate, indicating pathogen attenuation for older isolates. This effect was also mediated by individual body mass, where larger toads had higher survival. My findings indicate that newer, local isolates are likely better choices when exposing amphibian hosts to Bd and that isolate age and host weight can dramatically affect our inferences from exposure studies. In my third chapter, I tested the hypothesis that boreal toads exhibit intraspecific variation in susceptibility to Bd. I expected that the host defense strategies of tolerance and resistance are stronger in boreal toads from Wyoming, and weaker in boreal toads from Colorado, and are primarily responsible for our observations of boreal toad population declines in Colorado, and relative population persistence in Wyoming. Previous studies investigating variation in amphibian host tolerance and resistance to Bd are predominantly focused on species-level comparisons, with fewer focusing on intraspecific variation. Most studies also lack replication among strata of interest (e.g., geography, disease prevalence, host genotypes), and none use a robust methodological framework that can reveal host and pathogen dynamics throughout experimental exposures. Therefore, I conducted a laboratory experimental exposure of boreal toads to Bd, informed by the results of Chapter 2. I included toads from two populations in Colorado and two populations in Wyoming, representing replicates from our strata of interest (i.e., differences in decline severity). Using a multistate modeling approach, I modeled the effects of static covariates (e.g., host population origin, treatment dose of Bd, etc.) and dynamic, individual, time-varying covariates (e.g., weekly individual Bd load, weekly change in individual body mass, etc.) on boreal toad weekly survival and state transition probabilities. State transitions included the weekly probability an individual would clear their infection, or whether a cleared individual would re-gain infection, providing insight into typically hidden infection dynamics. I found that boreal toads from Colorado populations had lower weekly survival probabilities than those from Wyoming when comparing identical Bd loads. This is evidence of increased tolerance to Bd in Wyoming toad populations. As in Chapter 2, individual mass was also important at predicting the effects of Bd on weekly survival probabilities of boreal toads. Boreal toads from Colorado had similar peak Bd loads and cleared Bd at the same probabilities as Wyoming. Colorado boreal toads, however, were on average quicker to reach their peak Bd infection loads and had increased probabilities of re-gaining their infections. These results provide some support for increased resistance among boreal toads in Wyoming compared to those in Colorado. My findings highlight that differential susceptibility to Bd among boreal toads from different regions may play a crucial role in generating the disparity in decline severity across the region. In conclusion, my dissertation provides evidence that intraspecific variation in persistence, when challenged by disease, is an important driver of host-pathogen dynamics. My research has filled vital research gaps for an imperiled amphibian species with the goal of helping wildlife managers make tough conservation decisions about host translocations, reintroductions, and captive breeding. I hope my work aids in the persistence of an iconic Rocky Mountain amphibian for years to come.