California Tiger Salamander Research Articles

A novel genetic strategy to enable rapid detection of rare non-native alleles

Established invasive species represent one of the most harmful and challenging threats to native biodiversity, necessitating methods for Early Detection and Rapid Response. Cryptic invasions are particularly challenging and often require expensive and time-consuming molecular surveys which limits their usefulness for management. We present a novel application of the Fluidigm SNP-Type Assay to identify rare non-native alleles that significantly reduces the cost and time to generate diagnostic results. We demonstrate the efficacy of this method using experimental Fluidigm pools (99% accuracy) and sequence data (96% accuracy). We apply our novel methodology to an endangered population of California tiger salamanders in Sonoma County where two individual non-native tiger salamander hybrids have previously been detected since 2008. We screened 5805 larvae in 387 sample-pools containing 15 larvae each. We did not detect any non-native hybrids in the population, a result that was verified with sequence data, though we strongly recommend additional years of sampling to confirm hybrid absence. Our success with a challenging, large-genome amphibian suggests this method may be applied to any system, and would be particularly useful when it is necessary for conservation practitioners to rapidly identify rare taxa or genes of interest.

Assessing hybrid vigour using the thermal sensitivity of physiological trade‐offs in tiger salamanders

Abstract Hybridization between species affects biodiversity and population sustainability in numerous ways, many of which depend on the fitness of the hybrid relative to the parental species. Hybrids can exhibit fitter phenotypes compared to the parental lineages, and this ‘hybrid vigour’ can then lead to the extinction of one or both parental lines. In this study, we analysed the relationship between water loss and gas exchange to compare physiological performance among three tiger salamander genotypes—the native California tiger salamander (CTS), the invasive barred tiger salamanders (BTS) and CTS × BTS hybrids across multiple temperatures (13.5°C, 20.5°C and 23.5°C). We developed a new index of performance, the water‐gas exchange ratio (WGER), which we define as the ratio of gas exchange to evaporative water loss (μL VO2/μL H2O). The ratio describes the ability of an organism to support energetically costly activities with high levels of gas exchange while simultaneously limiting water loss to lower desiccation risk. We used flow through respirometry to measure the thermal sensitivity of metabolic rate and resistance to water loss of each salamander genotype to compare indices of physiological performance. We found that temperature had a significant effect on metabolic rate and resistance to water loss, with both traits increasing as temperatures warmed. Across genotypes, we found that hybrids have a higher WGER than the native CTS, owing to a higher metabolic rate despite having a lower resistance to water loss. These results provide a greater insight into the physiological mechanisms driving hybrid vigour and offer a potential explanation for the rapid spread of salamander hybrids. More broadly, our introduction of the WGER may allow for species‐ or lineage‐wide comparisons of physiological performance across changing environmental conditions, highlighting the insight that can be gleaned from multitrait analysis of organism performance. Read the free Plain Language Summary for this article on the Journal blog.

Managing invasive hybrids with pond hydroperiod manipulation in an endangered salamander system.

When invasive and endangered native taxa hybridize, the resulting admixture introduces novel conservation challenges. Across a large region of central California, a hybrid swarm consisting of admixed endangered California tiger salamanders (CTS) (Ambystoma californiense) and introduced barred tiger salamanders (BTS) (Ambystoma mavortium) has replaced native populations, threatening the genetic integrity of CTS and the vernal pool systems they inhabit. We employed a large-scale, genomically informed field experiment to test whether shortening breeding pond hydroperiod would favor native CTS genotypes. We constructed 14 large, seminatural ponds to evaluate the effect of hydroperiod duration on larval survival and mass at metamorphosis. We tracked changes in non-native allele frequencies with a 5237-gene exon capture array and employed a combination of custom Bayesian and generalized linear models to quantify the effect of pond duration on salamander fitness. Earlier work on this system showed hybrid superiority under many conditions and suggested that hybrids are favored in human-modified ponds with artificially long hydroperiods. Consistent with these earlier studies, we found overwhelming evidence for hybrid superiority. Very short hydroperiods substantially reduced the mass (1.1-1.5 fold) and survival probability (10-13 fold) of both native and hybrid larvae, confirming that hydroperiod likely exerts a strong selective pressure in the wild. We identified 86 genes, representing 1.8% of 4723 screened loci, that significantly responded to this hydroperiod-driven selection. In contrast to earlier work, under our more natural experimental conditions, native CTS survival and size at metamorphosis were always less than hybrids, suggesting that hydroperiod management alone will not shift selection to favor native larval genotypes. However, shortening pond hydroperiod may limit productivity of hybrid ponds, complementing other strategies to remove hybrids while maintaining vernal pool ecosystems. This study confirms and expands on previous work that highlights the importance of hydroperiod management to control invasive aquatic species.

Habitat value of constructed breeding pools for the endangered Sonoma population of California tiger salamander

AbstractHabitat creation is a common element of efforts to recover populations threatened by altered habitat. Unfortunately, human‐created habitats do not necessarily support re‐establishment of target species. We investigated the value of constructed vernal pools as breeding habitat for the endangered Sonoma County population of California tiger salamanders (Ambystoma californiense) by conducting 19 years of larval surveys in 118 pools that were natural (n = 40), constructed (n = 70), or natural with substantial human alterations (human‐altered, n = 8). California tiger salamander larval densities in human‐altered and natural pools declined over the study period, while larval densities in constructed pools showed non‐negative trends. Larval declines in natural pools may be attributed to long‐term degradation of the surrounding terrestrial habitat. Mean larval densities over the course of the study were equivalent between constructed and natural pools, when corrected for pool morphometry. In contrast, human‐altered pools had lower larval densities. To assure consistent California tiger salamander breeding, pools needed to be ≥30 cm deep and 240 m2 in area, with deeper (50–70 cm) and larger (1,600–1,800 m2) pools providing increased larval productivity. Over the study, there was no evidence of decrease in the rate of colonization of new pools. We conclude that constructed pools can be important tools in the recovery of imperiled pool‐breeding amphibians if proper design elements for the target species are considered.

Applying stochastic and Bayesian integral projection modeling to amphibian population viability analysis.

Integral projection models (IPMs) can estimate the population dynamics of species for which both discrete life stages and continuous variables influence demographic rates. Stochastic IPMs for imperiled species, in turn, can facilitate population viability analyses (PVAs) to guide conservation decision-making. Biphasic amphibians are globally distributed, often highly imperiled, and ecologically well suited to the IPM approach. Herein, we present a stochastic size- and stage-structured IPM for a biphasic amphibian, the U.S. federally threatened California tiger salamander (CTS) (Ambystoma californiense). This Bayesian model reveals that CTS population dynamics show greatest elasticity to changes in juvenile and metamorph growth and that populations are likely to experience rapid growth at low density. We integrated this IPM with climatic drivers of CTS demography to develop a PVA and examined CTS extinction risk under the primary threats of habitat loss and climate change. The PVA indicated that long-term viability is possible with surprisingly high (20%-50%) terrestrial mortality but simultaneously identified likely minimum terrestrial buffer requirements of 600-1000 m while accounting for numerous parameter uncertainties through the Bayesian framework. These analyses underscore the value of stochastic and Bayesian IPMs for understanding both climate-dependent taxa and those with cryptic life histories (e.g., biphasic amphibians) in service of ecological discovery and biodiversity conservation. In addition to providing guidance for CTS recovery, the contributed IPM and PVA supply a framework for applying these tools to investigations of ecologically similar species.

The reference genome of the Vernal Pool Tadpole Shrimp, Lepidurus packardi.

In this paper, we report on the scaffold-level assembled genome for the federally endangered, California endemic crustacean Lepidurus packardi (the Vernal Pool Tadpole Shrimp). L. packardi is a key food source for other conserved California species including the California Tiger Salamander Ambystoma californiense. It faces significant habitat loss and fragmentation as vernal pools are threatened by urbanization, agricultural conversion, and climate change. This resource represents the first scaffold-level genome of any Lepidurus species. The assembled genome spans 108.6 Mbps, with 6 chromosome-length scaffolds comprising 71% of total genomic length and 444 total contigs. The BUSCO score for this genome is 97.3%, suggesting a high level of completeness. We produced a predicted gene set for this species trained on the Daphnia magna set of genes and predicted 17,650 genes. These tools can aid researchers in understanding the evolution and adaptive potential of alternative reproductive modes within this species.

Observations of extreme dehydration and rehydration leading to recovery of a threatened California tiger salamander

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Navigating the trade‐offs between environmental DNA and conventional field surveys for improved amphibian monitoring

AbstractThe need for efficient, accurate biodiversity monitoring is growing, especially for globally imperiled taxa, such as amphibians. Environmental DNA (eDNA) analysis holds enormous potential for enhancing monitoring programs, but as this tool is increasingly adopted, it is imperative for users to understand its potential benefits and shortcomings. We conducted a comparative study to evaluate the efficacy of two eDNA methodologies (quantitative (q)PCR and metabarcoding) and conventional field sampling approaches (seining, dipnetting, and visual encounter surveys) in a system of 20 ponds containing six different amphibian species. Using an occupancy modeling framework, we estimated differences in detection sensitivity across methods, with a focus on how eDNA survey design could be further optimized. Overall, both metabarcoding and qPCR were competitive with or improved upon conventional methods. Specifically, qPCR (species‐specific approach) was the most effective technique for detecting two rare species, the California tiger salamander (Ambystoma californiense) and California red‐legged frog (Rana draytonii), with a detection probability of >0.80 per survey. Metabarcoding (community approach) estimated amphibian diversity with comparable rates to field techniques on average, and detected an additional 41 vertebrate taxa. However, for two abundant species (western toads, Anaxyrus boreas, and Pacific chorus frogs, Pseudacris regilla), field techniques outperformed metabarcoding, especially as individuals metamorphosed. Our results indicate that eDNA approaches would be most effective when paired with visual encounter surveys to detect terrestrial life stages, and that more optimization, specifically primer choice and validation, is needed. By comparing methods across a diverse set of ponds and species, we provide guidance for future studies integrating eDNA approaches into amphibian monitoring.

Responses of migratory amphibians to barrier fencing inform the spacing of road underpasses: a case study with California tiger salamanders (Ambystoma californiense) in Stanford, CA, USA

Migratory amphibians are at high risk of negative impacts when roads intersect their upland and breeding habitats. Road mortality can reduce population abundance, survivorship, breeding, recruitment, and probability of long-term persistence. Increasingly, environmental planners recommend installation of under-road tunnels with barrier fencing to reduce mortality and direct amphibians towards the passages. Often, the permeability of these barrier and passage systems to amphibian population movements are unknown. We studied the movements of California tiger salamanders (CTS: Ambystoma californiense) in relation to solid and mesh barrier fencing attached to a 3-tunnel system between upland and breeding habitats in Stanford, California. We deployed active-trigger cameras along the fencing, used pattern recognition software to identify individuals by their unique spot patterns, and calculated individual salamander movement distances, speed, direction changes, and “success” at reaching the tunnel system. We found that migrating adult CTS moved an average of 40 m along barrier fencing before turning back into the habitat or “giving-up”. This short distance, in comparison to long migratory movements, may be explained by the orientation mechanisms salamanders use to reach their breeding sites. The probability CTS found a passage decreased rapidly with increasing distance from the tunnel system, particularly if individuals turned the “wrong” way after encountering the fence. Salamanders changed directions more often and spent more time along mesh fencing. Our results suggest that a maximum of 12.5 m between passages along CTS migration routes should allow approximately 90% of adult salamanders to encounter road crossings. Additionally, use of solid fencing or a visual barrier on mesh fencing may help to lead salamanders to passages most efficiently. These considerations can assist those seeking to design effective road mitigation for CTS and other migratory amphibians.

Acute mortality in California tiger salamander (Ambystoma californiense) and Santa Cruz long-toed salamander (Ambystoma macrodactylum croceum) caused by Ribeiroia ondatrae (Class: Trematoda).

In early September 2019, a morbidity and mortality event affecting California tiger salamanders (Ambystoma californiense) and Santa Cruz long-toed salamanders (Ambystoma macrodactylum croceum) in late stages of metamorphosis was reported at a National Wildlife Refuge in Santa Cruz County, California, U.S.A. During the postmortem disease investigation, severe integumentary metacercarial (Class: Trematoda) infection, associated with widespread skin lesions, was observed. Planorbid snails collected from the ponds of the refuge within seven days of the mortality event were infected with Ribeiroia ondatrae, a digenetic trematode that can cause malformation and death in some amphibians. We suggest sustained seasonal high-water levels due to active habitat management along with several years of increased rainfall led to increased bird visitation, increased over-wintering of snails, and prolonged salamander metamorphosis, resulting in a confluence of conditions and cascading of host-parasite dynamics to create a hyper-parasitized state.

Saving all the pieces: An inadequate conservation strategy for an endangered amphibian in an urbanizing area

Species conservation often focuses on preserving populations on remnant habitat patches, typically without evidence that this approach is sufficient for halting declines. We employed a 19-year dataset to examine the adequacy of this approach for recovering the Sonoma County distinct population segment of California tiger salamanders (SCTS; Ambystoma californiense), California, USA, which now exists almost entirely in remnant habitat patches designated as preserves across a rapidly urbanizing landscape. We estimated relative SCTS larval densities from standardized annual surveys from 2002 to 2020 across 118 vernal pools in eight preserves. We found that relative larval SCTS densities decreased by 48% over the study period, indicating that current efforts to conserve SCTS are inadequate for long-term viability. Increased densities were only observed at the single study preserve where SCTS were introduced. Temporal trends in larval density among preserves were best explained by the number of pools available to SCTS, and the ability of breeding pools to retain water throughout the larval period. Specifically, preserves with >1 breeding pool and ≥1 breeding pool that held water for at least two months following the breeding season (into late April) even in dry years had substantially lower rates of larval decline. Active conservation management of preserves, including provision of multiple breeding pools, at least some of which are resilient to variable future precipitation regimes, will be required to effectively conserve SCTS. We expect these findings to apply broadly to the conservation of many species of pool-breeding amphibians.

Use of atypical aquatic breeding habitat by the California tiger salamander

Use of atypical aquatic breeding habitat by the California tiger salamander

Coexistence within an endangered predator–prey community in California vernal pools

Abstract Globally endangered ecosystems, such as ephemeral wetlands, are often critical habitat for multiple interacting imperilled species. To conserve this biodiversity, managers must consider both species‐specific resource requirements and mechanisms for endangered species coexistence under variable habitat conditions. We examined communities native to California playa pools, ephemeral wetlands that have declined by >90% from their historic extent. Specifically, we describe the diet of a federally Threatened amphibian (Ambystoma californiense), and characterise interactions between this amphibian and two federally Endangered macrocrustaceans (Branchinecta conservatio and Lepidurus packardi) across three rain years to understand how these imperilled species coexist. We examined the dietary preferences of A. californiense larvae, metamorphs, juveniles, and breeding adults, and determined how prey electivity by larval A. californiense differs between natural playa pools and human‐modified stock ponds. Within playa pools, both breeding adult and larval A. californiense preyed extensively on L. packardi, whereas fully terrestrial juveniles had relatively empty stomachs. We provide evidence for size‐moderated optimal foraging by larval A. californiense, and show that larval prey differed dramatically between playa pools and stock ponds. Additionally, an ontogenetic progression from smaller to larger prey gave the relatively large, endangered macrocrustaceans an early‐season temporal refuge, during which they reached maturity in all three rain years. Consistent with complex life cycle theory, our results suggest that ephemeral wetland habitat offers abundant food resources for A. californiense relative to terrestrial habitat. Our findings also suggest that diet flexibility facilitates the persistence of this imperilled amphibian in human‐modified stock ponds. Temporal offsets in prey maturation rates and ontogenetic shifts in predator diets are likely to facilitate coexistence among the focal endangered species. We highlight the importance of accounting for spatial and temporal variation in interspecific interactions when predicting the effects of environmental change on biodiversity, particularly in highly threatened ecosystems.

Allele-specific expression and gene regulation help explain transgressive thermal tolerance in non-native hybrids of the endangered California tiger salamander (Ambystoma californiense).

Hybridization between native and non-native species is an ongoing global conservation threat. Hybrids that exhibit traits and tolerances that surpass parental values are of particular concern, given their potential to outperform native species. Effective management of hybrid populations requires an understanding of both physiological performance and the underlying mechanisms that drive transgressive hybrid traits. Here, we explore several aspects of the hybridization between the endangered California tiger salamander (Ambystoma californiense; CTS) and the introduced barred tiger salamander (Ambystoma mavortium; BTS). We assayed critical thermal maximum (CTMax) to compare the ability of CTS, BTS and F1 hybrids to tolerate acute thermal stress, and found that hybrids exhibit a wide range of CTMax values, with 33% (4/12) able to tolerate temperatures greater than either parent. We then quantified the genomic response, measured at the RNA transcript level, of each salamander, to explore the mechanisms underlying thermal tolerance strategies. We found that CTS and BTS have strikingly different values and tissue-specific patterns of overall gene expression, with hybrids expressing intermediate values. F1 hybrids display abundant and variable degrees of allele-specific expression (ASE), likely arising from extensive compensatory evolution in gene regulatory mechanisms between CTS and BTS. We found evidence that the proportion of genes with allelic imbalance in individual hybrids correlates with their CTMax, suggesting a link between ASE and expanded thermal tolerance that may contribute to the success of hybrid salamanders in California. Future climate change may further complicate management of CTS if hybrid salamanders are better equipped to deal with rising temperatures.

Using Environmental DNA to Monitor the Spatial Distribution of the California Tiger Salamander

AbstractGlobal efforts to conserve declining amphibian populations have necessitated the development of rapid, reliable, and targeted survey methods. Environmental DNA surveys offer alternative or complementary methods to traditional amphibian survey techniques. The California tiger salamander Ambystoma californiense (CTS) is endemic to California, where it breeds in vernal pools. In the past 25 y, CTS has faced a 21% loss of known occurrences, largely through habitat loss, and it is threatened by hybridization with an introduced congener. Protecting and managing remaining CTS populations rely on accurately monitoring changes in their spatial distribution. Current monitoring practices typically use dip-net surveys that are time-consuming and prone to false negative errors. To provide a new resource for monitoring and surveying larval CTS, we designed an assay and tested it on water samples collected from 29 vernal pools at two locations in California. We compared environmental DNA results to contemporaneous dip-net surveying results and found the assay agreed with positive dip-net results in 100% of cases. In several instances, we also detected the presence of CTS genetic material in the early spring before larvae hatched, potentially offering a new, earlier detection option for this imperiled species. This assay provides a valuable, noninvasive molecular tool for monitoring the spatial distribution of the CTS in vernal pools.

A refined ecological risk assessment for California red-legged frog, Delta smelt, and California tiger salamander exposed to malathion.

The California red-legged frog (CRLF), Delta smelt (DS), and California tiger salamander (CTS) are 3 species listed under the United States Federal Endangered Species Act (ESA), all of which inhabit aquatic ecosystems in California. The US Environmental Protection Agency (USEPA) has conducted deterministic screening-level risk assessments for these species potentially exposed to malathion, an organophosphorus insecticide and acaricide. Results from our screening-level analyses identified potential risk of direct effects to DS as well as indirect effects to all 3 species via reduction in prey. Accordingly, for those species and scenarios in which risk was identified at the screening level, we conducted a refined probabilistic risk assessment for CRLF, DS, and CTS. The refined ecological risk assessment (ERA) was conducted using best available data and approaches, as recommended by the 2013 National Research Council (NRC) report "Assessing Risks to Endangered and Threatened Species from Pesticides." Refined aquatic exposure models including the Pesticide Root Zone Model (PRZM), the Vegetative Filter Strip Modeling System (VFSMOD), the Variable Volume Water Model (VVWM), the Exposure Analysis Modeling System (EXAMS), and the Soil and Water Assessment Tool (SWAT) were used to generate estimated exposure concentrations (EECs) for malathion based on worst-case scenarios in California. Refined effects analyses involved developing concentration-response curves for fish and species sensitivity distributions (SSDs) for fish and aquatic invertebrates. Quantitative risk curves, field and mesocosm studies, surface-water monitoring data, and incident reports were considered in a weight-of-evidence approach. Currently, labeled uses of malathion are not expected to result in direct effects to CRLF, DS or CTS, or indirect effects due to effects on fish and invertebrate prey. Integr Environ Assess Manag 2018;14:224-239. © 2017 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals, Inc. on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

Population genetic and field-ecological analyses return similar estimates of dispersal over space and time in an endangered amphibian.

The explosive growth of empirical population genetics has seen a proliferation of analytical methods leading to a steady increase in our ability to accurately measure key population parameters, including genetic isolation, effective population size, and gene flow, in natural systems. Assuming they yield similar results, population genetic methods offer an attractive complement to, or replacement of, traditional field‐ecological studies. However, empirical assessments of the concordance between direct field‐ecological and indirect population genetic studies of the same populations are uncommon in the literature. In this study, we investigate genetic isolation, rates of dispersal, and population sizes for the endangered California tiger salamander, Ambystoma californiense, across multiple breeding seasons in an intact vernal pool network. We then compare our molecular results to a previously published study based on multiyear, mark–recapture data from the same breeding sites. We found that field and genetic estimates of population size were only weakly correlated, but dispersal rates were remarkably congruent across studies and methods. In fact, dispersal probability functions derived from genetic data and traditional field‐ecological data were a significant match, suggesting that either method can be used effectively to assess population connectivity. These results provide one of the first explicit tests of the correspondence between landscape genetic and field‐ecological approaches to measuring functional population connectivity and suggest that even single‐year genetic samples can return biologically meaningful estimates of natural dispersal and gene flow.

HEMATOLOGY AND PLASMA BIOCHEMISTRY INTERVALS FOR CAPTIVE-BORN CALIFORNIA TIGER SALAMANDERS (AMBYSTOMA CALIFORNIENSE).

Hematology and plasma biochemistry parameters were determined for 34 captive-born California tiger salamanders ( Ambystoma californiense ). The animals were manually restrained for general examination and venipuncture. This is the first comprehensive report of hematology and plasma biochemistry parameters in apparently healthy California tiger salamanders and may serve as a reference for clinical assessment and future study of this species.

Integrating occupancy models and structural equation models to understand species occurrence

Understanding the drivers of species occrrece s a fundamenal goal in basic and applied ecology. Occupancy models have emerged as a popular approach for inferring species occurrence because they account for problems associated with imperfect detection in field surveys. Current models, however, are limited because they assume covariates are independent (i.e., indirect effects do not occur). Here, we combined structural equation and occupancy models to investigate complex influences on species occurrence while accounting for imperfect detection. These two methods are inherently compatible because they both provide means to make inference on latent or unobserved quantities based on observed data. Our models evaluated the direct and indirect roles of cattle grazing, water chemistry, vegetation, nonnative fishes, and pond permanence on the occurrence of six pond-breeding amphibians, two of which are threatened: the California tiger salamander (Ambysloma californiense) and the California red-legged frog (Rana draytonil). While cattle had strong effects on pond vegetation and water chemistry, their overall effects on amphibian occurrence were small compared to the consistently negative effects of nonnative fish. Fish strongly reduced occurrence probabilities for four of five native amphibians, including both species of conservation concern. These results could help to identify drivers of amphibian declines and to prioritize strategies for amphibian conservation. More generally, this approach facilitates a more mechanistic representation of ideas about the causes of species distributions in space and time. As shown here, occupancy modeling and structural equation modeling are readily combined, and bring rich sets of techniques that may provide unique theoretical and applied insights into basic ecological questions.

Do Ecological Niche Models Accurately Identify Climatic Determinants of Species Ranges?

Defining species' niches is central to understanding their distributions and is thus fundamental to basic ecology and climate change projections. Ecological niche models (ENMs) are a key component of making accurate projections and include descriptions of the niche in terms of both response curves and rankings of variable importance. In this study, we evaluate Maxent's ranking of environmental variables based on their importance in delimiting species' range boundaries by asking whether these same variables also govern annual recruitment based on long-term demographic studies. We found that Maxent-based assessments of variable importance in setting range boundaries in the California tiger salamander (Ambystoma californiense; CTS) correlate very well with how important those variables are in governing ongoing recruitment of CTS at the population level. This strong correlation suggests that Maxent's ranking of variable importance captures biologically realistic assessments of factors governing population persistence. However, this result holds only when Maxent models are built using best-practice procedures and variables are ranked based on permutation importance. Our study highlights the need for building high-quality niche models and provides encouraging evidence that when such models are built, they can reflect important aspects of a species' ecology.