Roads networks may have profound impacts on the viability of wildlife populations. In particular, highways can be barriers to wildlife movement, leading to genetic diversity loss, inbreeding, and increased extinction risk for small, isolated populations on either side. The effects that highways have on wildlife movement can be variable, dependent on the unique dispersal behaviors of individual species. In this study, we test the hypothesis that highways will pose less of a barrier to coyotes, a species tolerant of human disturbance, than to gray fox, a species more sensitive to disturbance. We used landscape genetic tools to determine whether State Route 49, part of the California Essential Habitat Connectivity Project, was a barrier to coyote or gray fox movements. Genetic diversity among both coyotes and gray foxes was high and comparable to other studies. We found little evidence of contemporary genetic structure across State Route 49 for either species. All genetic differentiation that we observed was driven by family structure and relatives were found on both sides of the highway. Coyotes exhibited a significant signal of isolation by distance and a positive association between traffic volume and genetic distance. There are two possible explanations for these findings. State Route 49 may be permeable to coyote and gray fox movement due to successful road crossings. Alternatively, State Route 49 is a barrier to coyote and gray fox movement but there may be a time lag between initial reduction of gene flow and detection of population structure.

View the NCST Project Webpage

The application of genetic markers to investigate evolutionary and ecological questions about white sturgeon, Acipenser transmontanus, has been limited due to the species’ highly duplicated nuclear genome. Here, polysomic microsatellite markers were used to 1) examine the ancestral level of genome duplication in white sturgeon, 2) examine genetic diversity and patterns of population structure within and among drainages across the species’ range, and 3) provide genetic monitoring for a conservation aquaculture program sustaining an endangered white sturgeon population. In the first chapter, we followed the inheritance of eight microsatellite markers in 15 families of white sturgeon from a commercial caviar farm to determine whether white sturgeon (~250 chromosomes) should be classified as tetraploid or octoploid. The eight microsatellite loci were detected predominantly in four or eight copies, with one locus observed in >8 copies. Numbers of alleles per locus, patterns of allele transmission, and inference of gene copy number in parents suggested that white sturgeon should be considered ancient octoploids. The discovery of dodecaploid parents and their decaploid offspring in the farm population, confirmed by flow cytometry analysis, indicated that some aspect of sturgeon aquaculture was inducing spontaneous autopolyploidy in white sturgeon.

Next, microsatellite markers were applied to examine white sturgeon population structure across the species’ range. Population assignment testing was used to determine the origin of white sturgeon sampled in non-natal estuaries, or those not containing a spawning population, to evaluate marine dispersal behavior. The Sacramento-San Joaquin River system was found to contain a single white sturgeon population while the8Fraser River exhibited a hierarchical pattern of population structure. Strong levels of genetic divergence were detected above and below a natural barrier, Hells Gate, and fine-scale population substructure was identified above Hells Gate. Population structure in the Columbia River drainage (including the mainstem Columbia and Snake Rivers) was complex and suggested a pattern of isolation by distance. Net downstream gene flow also may have contributed to this pattern, with individuals migrating downstream through impoundments and over barriers with little upstream movement possible. There was no support for the current practice of managing each impounded reach on the Columbia or Snake Rivers as a separate population. Lack of population structure within historically continuous river habitat found across the species’ range suggested spawning site fidelity in white sturgeon may occur on a regional scale, with local gene flow among geographically proximate spawning sites. Population assignment of samples collected from non-natal estuaries indicated that all populations with ocean access make marine migrations, and individuals did not necessarily originate from the nearest spawning population.

Finally, microsatellites were used to conduct genetic monitoring of the Kootenai Tribe of Idaho’s conservation aquaculture program (CAP) for the endangered Kootenai River white sturgeon population. Continuous recruitment failure in this population has left it entirely dependent on the CAP for reproduction. A genetic profile database of wild broodstock used in the CAP was created to monitor hatchery-induced genetic changes in the Kootenai River population. Broodstock genotypes also were used to evaluate the accuracy of parentage assignment in the Kootenai River population, as hatchery managers soon will depend on this analysis to prevent inbreeding when most sexually mature adults available for captive breeding will be derived from hatchery production. Numbers of alleles and numbers of alleles per individual per locus were calculated to monitor the amount of wild type genetic diversity captured in broodstock utilized by the CAP. Parentage analysis with 18 microsatellite markers was validated in known hatchery families from the 2010 year class. Genetic diversity in the Kootenai River population was very low relative to other populations examined, likely due to founder effects and genetic drift after isolation from the mainstem Columbia c. 10,000 YBP. In less than one sturgeon generation, 96% of Kootenai River genetic diversity has been captured in broodstock that contributed offspring that survived to release in the Kootenai River and further propagation will likely preserve additional genetic variation. The 18 microsatellite panel improved parentage assignment accuracy and allowed a greater number of assignments relative to the previous panel used for parentage analysis, suggesting that this technique may become a useful tool in the management of this vulnerable population.

Freshwater fishes in California are challenged by anthropogenic activities, especially habitat fragmentation and the introduction of invasive species. Restriction Site-associated (RAD) Sequencing, a commonly applied Next Generation Sequencing technique in non-model organisms, can provide essential and actionable genomic information for managers to assess population statuses and for developing species conservation plans. In this dissertation, I explored the varied application of RAD sequencing to two native California species, Speckled Dace (Rhinichthys osculus) and Paiute cutthroat trout (Oncorhynchus clarkii seleniris). The Speckled Dace are widespread in western North America, and to this point, have been considered a single species. In Chapter One, the California Speckled dace is divided into three distinct evolutionary lineages based on the genetic differentiation detected by thousands of single nucleotide polymorphisms extracted using RAD sequencing. Paiute cutthroat trout are a subspecies of cutthroat trout threatened by competition and hybridization with introduced non-native trout. To protect and recover the subspecies, populations were previously translocated by conservation biologists to nine refuge populations thought to approximate historical habitats. Yet long-term genetic monitoring is required to monitor genetic diversity over space and time to assess the efficacy of these management actions. In Chapter Two, we selected 1,114 SNPs that generate comparable results in genetic diversity and genetic population structure to 6,187 SNPs in RAD sequencing after resolving technical questions related to the RAD sequencing approach. Results ultimately demonstrate the SNP panel is useful for genetic monitoring of population structure and heterozygosity, and thus helpful for conservation management. In Chapter Three, we applied 1,114 SNPs to estimate genetic diversity, and genetic population structure for all nine refuge populations. We also applied these SNPs to estimate parent-offspring relationships and the change of genetic diversity after the translocation between two refuge populations. We also applied these SNPs to estimate parent-offspring relationships and the change of genetic diversity after the translocation between two refuge populations.

Roads can have unintended effects on wildlife populations, such as causing direct mortality through animal collisions with cars, changing animal behaviors or distributions from traffic disturbance (e.g., noise, lighting), and fragmenting habitat. Roads may also act as barriers to wildlife movements, which prevents populations on either side from exchanging genes. Over time, wildlife populations isolated by barriers will lose genetic diversity, a process associated with an increased risk of extinction.

To better understand this dynamic in Northern California, a studywas completed examining whether State Route 49 (SR 49), a road initially constructed during the Gold Rush era, acts as a barrier to movements of two similar species with different tolerances to human activity, the coyote and gray fox. This policy brief summarizes findings from that study and discusses the policy implications.

View the NCST Project Webpage

Contemporary biodiversity loss is unprecedented and linked to decreased ecosystem function. Aquatic and wetland ecosystems are considered biodiversity hotspots and disproportionately at risk. In California, massive land-use changes continue to threaten freshwater, estuarine, and wetland ecosystems and species, but target species and species interactions are often difficult to study. Non-invasive genetic approaches are a suite of rapidly evolving technologies used to sample and study organisms non-invasively. These approaches facilitate better understanding of species, communities, and trophic interactions that are hard to glean using traditional methods. The research herein uses single- and multi-taxon methods in three local ecosystems. The theme that connects this work is the need to interpret a relatively new type of data in a manner that can be more easily operationalized for conservation and management. Chapter 1 examines and evaluates the use of single-taxon environmental DNA (eDNA) analysis for indirect detection of critically endangered delta smelt (Hypomesus transpacificus) from water samples in the San Francisco Estuary. Results indicate that turbidity and rarity of this small fish present challenges for eDNA detection. However, reliable and repeatable eDNA detection of rare species in turbid conditions is attainable with appropriate sampling and statistical methods. Chapter 2 applies multi-taxon eDNA metabarcoding to better understand fish community ecology in Putah Creek, a model system for stream restoration. Results show community differences across time and space, indicating that reordering is an important community dynamic. eDNA sampling was more sensitive for detection of rare species than conventional methods, facilitating a whole-ecosystem perspective. These findings complement long-term electrofishing survey data indicating that native fishes benefit from water management practices that replicate the natural flow regime. Finally, Chapter 3 applies non-invasive genetic sampling to study the diet of a widespread insectivorous predator in the Central Valley, a highly productive agricultural region. Results of guano analysis from the largest colony of Mexican free-tailed bats (Tadarida brasiliensis) in California show a diverse suite of prey characteristic of a generalist predator. Moths (Lepidoptera) and mosquitos (Diptera) comprised the highest relative abundance, including prey that are agricultural pests and disease vectors. Documenting this economically valuable ecosystem service may help increase support for bat conservation. Consumption of emergent taxa represents an aquatic-terrestrial food web linkage that has not been well-studied. Collectively, these studies demonstrate the value of emerging genetic technologies for conservation and provide new perspectives on ecologically relevant questions in imperiled, biodiverse ecosystems. Effective operationalization of non-invasive genetic approaches is part of the toolbox for conserving biodiversity and limiting the resulting socioeconomic and cultural losses.

To monitor the health of Chinook Salmon populations in California’s Central Valley, it’s necessary to measure and evaluate all stages of their life cycle. The outmigration journey of the pre-smolt juvenile Chinook is one of the least understood life stages, yet it’s also one of the most dangerous for the species. Rising water temperatures, drought, invasive predatory species and degradation of the marshland habitats in the San Francisco Bay-Delta (SFBD) created an unsustainable environment where most juvenile Chinook never make it to the Pacific Ocean. Although multiple marsh restoration projects are being developed to lessen the anthropogenic impact, the Chinook Salmon populations are still declining. An increased focus in monitoring the habitat use of pre-smolt Chinook salmon in the shallow-water marsh habitat of the upper SFBD can provide an invaluable insight into the best management strategies to ensure the survival of the species. Due to complications in monitoring the marsh habitats with conventional methods, I evaluated the use of environmental DNA (eDNA) as an alternative indicator of Chinook salmon presence. For the best use of this novel technology in the marsh conditions, we validated and optimized the performance of a Chinook salmon eDNA assay for use in an estuarine environment. Then we compared the effectiveness of eDNA detection to trawling, measuring the sensitivity of each method as well as identifying the biases, optimal working conditions, advantages, and disadvantages of these survey methods for mapping habitat use of the pre-smolt Chinook salmon.eDNA is a stable molecule that can persist in the environment for long periods. Since all living creatures release eDNA into the environment, almost any environmental sample contains eDNA. The eDNA can reflect current or historical distributions of the species living in the environment sampled, with environmental conditions such as eDNA transport, eDNA degradation, sedimentation and PCR inhibitors regulating the shape and duration of the eDNA plumes produced by our target species. Conducting a single species eDNA assay in an aquatic environment consists of four main steps: water filtration, DNA extraction, PCR inhibitor removal and qPCR amplification. In the filtration step a fixed amount of water, normally 1 L per sample, is filtered through a material which will capture the eDNA while the flowthrough is discarded. The filter pore size is the factor that determines the state of the eDNA that will be measured. Larger pores prioritize larger particles such as scales and eggs while smaller pores favor individual cells, subcellular structures and free eDNA. Then the extraction step isolates the eDNA from the filter, and the yield of this step largely influences the sensitivity of the assay. The PCR Inhibitor removal step works to remove any possible contaminants that might impact the amplification of eDNA; therefore, this step is essential to provide reliability of the assay across different environments. Last, the qPCR step amplifies the eDNA to a detectable amount of fluorescence. Marsh habitat is a challenging environment for eDNA surveys because it is associated with an elevated turbidity, which may clog filters and elevate presence of qPCR inhibitors. The first objective of my dissertation was to optimize a Chinook salmon qPCR assay to detect Chinook eDNA in the marshy and estuarine conditions of the SFBD. As the goal for the study was to implement the assay on a large scale, I not only focused on the sensitivity of the assay, but also accounted for the time and costs associated with sampling. I tested a total of 27 combinations of filters, extraction, and inhibitor removal methods. For the conditions of the SFBD we opted for a protocol using glass fiber filters, magnetic bead DNA extraction, and an extra step for PCR inhibitor removal, to best achieve our goals of a large scale eDNA monitoring system for juvenile Chinook salmon in the Upper SFBD. The second chapter of my dissertation determined how best to interpret the meaning of an eDNA positive or negative detection. To do so, I needed to evaluate the dispersion of the eDNA once it is released to better characterize the distribution of the juvenile Chinook salmon. In the literature, eDNA particles have initially been described as fine particulate organic matter (FPOM) particles and simulated as such with hydrological models. For all hydrological models for eDNA, three factors are essential: eDNA production, eDNA degradation and eDNA transport. In general, eDNA production is considered proportional to the mass of the target species, although more recent papers suggest that eDNA production is more correlated with surface area in the case of fishes. Stress, death, and environmental conditions may also affect the release of eDNA. Meanwhile eDNA degradation is mostly dependent on the environment with the bacterial activity dictating most of the eDNA destruction. eDNA can also become undetectable due to precipitation and binding to molecules that inhibit the amplification step of the assay. eDNA transport mostly is dictated by advection in the case of marine and lotic water bodies while in lentic systems turbulent diffusion is the main factor for eDNA dispersion. I built a simplified one-dimensional riverine eDNA transport model to estimate the detection radius of an individual eDNA source and evaluate the effects of the degradation rate, eDNA diffusion coefficient and river advection onto the distance that the eDNA particles can travel before degrading or settling in the bottom of the river. I identified that the main driver of eDNA dispersion is in most cases the advection of the system, which can greatly influence the transport of eDNA. Due to this effect the model can be used to estimate the effective part of the river that was measured for presence or absence of Chinook salmon with the eDNA assay. The modeling also suggests that transect sampling can be a valuable alternative to discrete, or point, sampling in order to increase the repeatability of detection and increase the sensitivity of the assay, with medium sized transects (250-500 m) providing most of the benefits of transect sampling. The model also uses a Bayesian Monte Carlo Markov chain to estimate degradation rates, production rates and diffusion coefficients for further cage studies. Between the years 2018 and 2021, I collaborated with the California Department of Water Resources on a study that performed pairwise sampling of eDNA and trawling to compare the detection rate of both methods to estimate their biases. Water conditions were also measured to describe the sampled environments. I observed that the eDNA survey had a higher detection rate than the trawling survey, with a 45% detection rate compared to 13% detection rate for trawling. eDNA detection was present in a broad set of environments while trawling detections were confined mostly to a narrow set of salinity and dissolved oxygen conditions. Trawling detections were correlated with shoals in the main corridor of Suisun Marsh while detection was minimal in the upper marshlands. eDNA positivity was similar throughout the sampled years while trawl detection occurred mostly in the wet year of 2019. Trawling seems more efficient when river discharge is high, possibly caused by a higher movement or by a higher number of juvenile Chinook throughout the system. My results suggest that trawling surveys may be hampered by dense vegetation and muddy substrates found in the upper marshlands, while eDNA is a promising alternative survey method for these conditions. With the increasing frequency of drought and reduced flows in the SFBD, we expect that eDNA surveys will be a valuable strategy to monitor the Chinook salmon population as well as measure the efficiency of conservation efforts in the region. Although the eDNA detection rate can be a better indicator of Chinook salmon habitat use, trawling and other fish capture survey methods can generate additional information about the health of populations with measures such as size, weight, diet, age, and origin. Therefore, eDNA and trawling provide a complementary view of the juvenile Chinook salmon population. We recommend that managers conduct eDNA surveys to estimate the habitat use and occupancy rates and use targeted trawling surveys to confirm Chinook presence and provide information that cannot be obtained from eDNA alone.

The principle of sustainable transportation suggests that impacts to environmental processes and patterns will be limited as much as possible during construction and operation of the transportation system. Wildlife populations are affected by transportation in various ways, including individual animal deaths due to collisions with vehicles, animal aversion to roads due to light and noise, habitat fragmentation, and human access for recreation and hunting. If individual animals of a species have limited movement in part or all of their range, then divided populations can become genetically different from each other, which can jeopardize survival of populations and even whole species. This population subdivision can happen even in the relatively short time since California highways were constructed.

This policy brief summarizes findings from a research that examined the potential population division effect of highways on coyote populations in the Bay Area and Sierra Nevada foothills.

View the NCST Project Webpage

Roads have the potential to fragment wildlife populations, leading to genetic diversity loss, inbreeding, and increased extinction risk for small, isolated populations. In this study, the authors used coyote as a model to investigate how four Northern California highways affect gene flow of ground-dwelling mammals. The authors collected coyote scat samples from opposite sides of a stretch of I-580 and I-680 in the Bay Area and I-80 and US 50 in the Sierra Nevada foothills. The authors extracted DNA and genotyped each coyote at 13 microsatellite loci. The authors estimated genetic diversity and determined how that diversity was partitioned across the landscape in each region. Genetic diversity levels in coyotes were high and comparable to other studies. The authors found significant genetic structure in both the Bay Area and Sierra Nevada foothills, although it didn’t always correspond to highway presence. In the Bay Area, two populations were identified and although some evidence suggested I-580 was a significant barrier to gene flow, the authors identified migrants across the highway. One of the two populations in the Bay Area contained many second order relatives, suggesting limited gene flow into that population. There was evidence of dispersal out of that population, however. In the Sierra Nevada foothills, the authors identified three populations. Individuals from one population were sampled across highway I-80 suggesting it was not a significant barrier to movement. The most genetically divergent population in the Sierra Nevada foothills was also the most geographically distant and therefore it was difficult to determine whether gene flow into that population was limited by highway presence or simply geographic distance from other populations. The conclusions drawn in their pilot study are limited by the small number of samples they were able to genotype completely in the timeframe of this project. The authors are going to continue analyzing samples that currently have only partial genotypes and add those to their regional datasets. Genetic analysis with these larger samples will allow them to better understand the role of highways in structuring coyote populations in the Bay Area and Sierra Nevada foothills.

View the NCST Project Webpage

Delta smelt, Hypomesus transpacificus (McAllister, 1963), is a federally threatened and California State endangered fish endemic to the San Francisco Estuary and Sacramento-San Joaquin Delta of North America (SFE). The species is a small, pelagic, mostly annual fish with freshwater resident, migratory, and semi-migratory life histories (Campbell et al., 2022; Hobbs et al., 2019). They have historically been considered an indicator species for water quality in the SFE. Over the last few decades, the species has undergone a population collapse associated with drought and anthropogenic disturbances, and it is now believed stochastic processes may push the species to extinction (Fisch et al., 2011; Moyle, Peter B., Brown, Larry R., Durand, John R., Hobbs, 2016). Meaningful conservation management of the species must encompass gaining a better understanding of the life history, ecology, demography, and physiology of the species so biological components contributing to success in the wild can be preserved. Because genetics, in combination with the environment, influence many aspects of individual and population level phenotypes, building a framework to better understand the species requires the development of genetic resources and monitoring of genetic diversity. Chapter one of this dissertation presents two chromosome-level genome assemblies -- one male and one female -- which are necessary resources for current and ongoing evolutionary and conservation genetics research concerning delta smelt and other declining and vulnerable species in the Osmeridae family, such as longfin smelt. Chapter two investigates three methods for identifying sex marker(s) within the assembled female and male delta smelt reference genomes. While ultimately no diagnostic sex-specific sequences were found in our RAD-sequencing dataset, abundance discrepancies in k-mers from female and male linked-read sequence data were identified. Chapter three is a first author paper I wrote titled "Association of the lactase persistence haplotype block with disease risk in populations of European descent" published in Frontiers in Genetics. This chapter switches organisms and investigates the potential for deleterious mutations to hitchhike in haplotype blocks which were heavily selected for in humans. This paper is a result of the work I completed in the first year and a half of my doctoral studies. Together this work contributes to the fields of evolutionary, comparative and conservation genomics. This work specifically contributes to delta smelt monitoring, management and research, and human disease risk studies. In summary my doctoral work has provided a novel delta smelt genome assembly which is the first chromosome-level and least fragmented publicly available male and female reference genomes within the Osmeridae (smelt) family; an examination of female and male delta smelt sequencing data showing a discrete difference between sexes and establishes a framework for further investigation; and results suggesting that despite the fact that the human lactase persistence haplotype block harbors increased deleterious mutations compared to the rest of the genome, they seem to have little effect on prostate cancer, cardiovascular disease, and bone mineral density disease phenotypes.