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San Fernando Valley spineflower (Chorizanthe parryi var. fernandina [S. Watson] Jeps.) (Polygonaceae) is an herbaceous annual plant, endemic to California, and until rediscovered in 1999 had been thought to be extinct for almost seven decades. Historically documented at 10 locations, it currently persists at 2, separated by approximately 27 km. State listed as endangered, a description of its genetic diversity and structure is of conservation interest. After determining a lack of variation in ploidy, we examined genetic variation from samples within both populations: a common garden study for potentially adaptive genetic variation in selected growth and phenological traits and analysis of single nucleotide polymorphisms identified through restriction-site associated DNA sequencing. Both measures indicated that this highly restricted taxon nevertheless harbors substantial levels of genetic diversity and has significant between- and within-population genetic structure. Combining approaches from population genomics and common garden studies provided more insight into the patterns and basis of genetic diversity than is typical for studies of non-model species. Although local adaptation was not specifically studied (i.e., via reciprocal transplant studies), the differences determined from these two independent lines of evidence indicate that mixing gene pools between populations is not recommended at this time. Further, with significant differences revealed among subpopulations, we caution against mixing genotypes across subpopulations for the most part, and without much more evidence that this would not pose a risk of outbreeding depression. The importance of supporting pollinator health and diversity is highlighted. With genetic diversity—particularly with an annual species—being dynamic, fluctuating with the usual processes and with contributions from the soil seedbank, we recommend periodic resampling to monitor genetic diversity and structure. Climate change is anticipated to contribute to this variability.

Environmental DNA (eDNA) methods complement traditional aquatic monitoring surveys and are especially advantageous for rare and listed species to detect spatial and temporal distribution patterns. However, improvements in ease of use and portability could increase the utility of eDNA methods, leading to more widespread application, including expanding its role in management decision-making processes. We describe the development of an eDNA detection assay for delta smelt (Hypomesus transpacificus), an endangered fish in the San Francisco Estuary, using SHERLOCK (Specific High-Sensitivity Enzymatic Reporter Unlocking). SHERLOCK is a clustered regularly interspaced short palindromic repeats (CRISPR)-based diagnostic tool with the ability to detect species-specific genetic variants, making it ideal for genetic-based taxonomic identification of any organism. Because of its high sensitivity and specificity, SHERLOCK is adaptable to eDNA detection in water samples. Here, we describe adaptation of a delta smelt SHERLOCK assay for use with estuarine water eDNA samples. This version of the assay exhibits increased sensitivity compared to the original delta smelt SHERLOCK protocol (new limit of detection approximately three copies per reaction compared to ~300 in original assay) and successfully detected delta smelt eDNA in both experimental and natural contexts. Overall, our results demonstrate that SHERLOCK eDNA detection offers managers an alternative, isothermal methodology, and highlights some challenges for detection of rare, endangered species at low abundance.

Accurate taxonomic identification is foundational for effective species monitoring and management. When visual identifications are infeasible or inaccurate, genetic approaches provide a reliable alternative. However, these approaches are sometimes less viable (e.g., need for near real-time results, remote locations, funding concerns, molecular inexperience). In these situations, CRISPR-based genetic tools can fill an unoccupied niche between real-time, inexpensive, but error-prone visual identification and more expensive or time-consuming, but accurate genetic identification for taxonomic units that are difficult or impossible to visually identify. Herein, we use genomic data to develop CRISPR-based SHERLOCK assays capable of rapidly (

After centuries of overexploitation and habitat loss, many of the world's sturgeon (Acipenseridae) populations are at the brink of extinction. Although significant resources are invested into the conservation and restoration of imperiled sturgeons, the burgeoning commercial culture industry poses an imminent threat to the persistence of many populations. In the past decade, the number and distribution of captive sturgeon facilities has grown exponentially and now encompasses diverse interest groups ranging from hobby aquarists to industrial-scale commercial facilities. Expansion of sturgeon captive culture has largely fallen outside the purview of existing regulatory frameworks, raising concerns that continued growth of this industry has real potential to jeopardize conservation of global sturgeon populations. Here, we highlight some of the most significant threats commercial culture poses to wild populations, with particular emphasis on how releases can accelerate wild population declines through mechanisms such as hybridization, introgression, competition, and disease transmission. We also note that in some circumstances, commercial captive culture has continued to motivate harvest of wild populations, potentially accelerating species' declines. Given the prevalence and trajectory of sturgeon captive culture programs, we comment on modifications to regulatory frameworks that could improve the ability of captive culture to support wild sturgeon conservation.

Accurate species identification is critical to monitoring programs because mis-identifications can lead to incorrect assessments of population status and trends. In the San Francisco Estuary, efforts to monitor the imperiled osmerid Delta Smelt (Hypomesus transpacificus) using morphology can be challenging because of the presence of the similar-looking non-native osmerid Wakasagi (Hypomesus nipponensis). In 2017, the US Fish and Wildlife Service’s field office in Lodi implemented a two-stage verification process for Wakasagi to help prevent Delta Smelt from being mis-identified as Wakasagi. Under this process, Wakasagi are initially identified in the field, independently identified a second time by an experienced staff member in the laboratory, then stored on-site where they can be made available for future studies. Using the recently developed Specific High-sensitivity Enzymatic Reporter un-LOCKing (SHERLOCK) assay for Wakasagi, we evaluated how well verification protocols performed by genetically identifying a subset of Wakasagi collected during routine sampling between 2017 and 2021. Through this study, we found that the protocols have served as an effective quality control measure for over 4 years and across multiple surveys. With the development of field-deployable genetics tools such as SHERLOCK, genetic identification will likely play an increasingly important role in ecological monitoring. We expect that hybrid approaches that combine morphological identifications by trained field crew with application of field-based genetic tools may offer an effective and efficient approach to ensuring data accuracy in the future.

White sturgeon (Acipenser transmontanus) are the largest freshwater fish in North America, with reproducing populations in the Sacramento-San Joaquin, Fraser, and Columbia River Basins. Of these, the Columbia River is the largest, but it is also highly fragmented by hydroelectric dams, and many segments are characterized by declining abundance and persistent recruitment failure. Efforts to conserve and supplement these fish requires an understanding of their spatial genetic structure. Here, we assembled a large set of samples from throughout the Columbia River Basin, along with representative collections from adjacent basins, and genotyped them using a panel of 325 single-nucleotide markers. Results from individual- and group-based analyses of these data indicate that white sturgeon in the uppermost Columbia River Basin, in the Kootenai and upper Snake Rivers, are the most distinct, while the remaining populations downstream in the basin can be described as a genetic gradient consistent with an isolation-by-distance effect. Notably, the population in the lowest reaches of the Columbia River is more distinct from the middle or upper reaches than from outside basins, and suggests historically a higher or more recent gene exchange through coastal routes than with populations in the interior Columbia Basin. Nonetheless, proximal reaches were generally only marginally or non-significantly divergent, suggesting that transplanting larvae or juveniles from nearby sources poses relatively little risk of outbreeding depression. Indeed, we inferred examples of dispersal between reaches via close-kin mark-recapture and genetic mark-recapture that indicate movement between nearby reaches is not unusual. Samples from the Kootenai and upper Snake Rivers exhibited notably lower genetic diversity than the remaining samples as a result of population bottlenecks, genetic drift, and/or historical divergence. Conservation actions, such as supplementation, are underway to maintain population viability and will require balanced efforts to increase demographic abundance while maintaining genetic diversity.

Environmental DNA (eDNA) detection methods can complement traditional biomonitoring to yield new ecological insights in aquatic systems. However, the conceptual and methodological frameworks for aquatic eDNA detection and interpretation were developed primarily in freshwater environments and have not been well established for estuaries and marine environments that are by nature dynamic, turbid, and hydrologically complex. Environmental context and species life history are critical for successful application of eDNA methods, and the challenges associated with eDNA detection in estuaries were the subject of a symposium held at the University of California Davis on January 29, 2020 (https://marinescience.ucdavis.edu/engagement/past-events/edna). Here, we elaborate upon topics addressed in the symposium to evaluate eDNA methods in the context of monitoring and biodiversity studies in estuaries. We first provide a concise overview of eDNA science and methods, and then examine the San Francisco Estuary (SFE) as a case study to illustrate how eDNA detection can complement traditional monitoring programs and provide regional guidance on future potential eDNA applications. Additionally, we offer recommendations for enhancing communication between eDNA scientists and natural resource managers, which is essential for integrating eDNA methods into existing monitoring programs. Our intent is to create a resource that is accessible to those outside the field of eDNA, especially managers, without oversimplifying the challenges or advantages of these methods.

With the increasing use of triploid fish for sport fisheries management, biologists, researchers, and managers working in field environments need practical methods to determine ploidy. Cytological methods (e.g., flow cytometry, Coulter counter) using erythrocytes are the most common for ploidy determination in fishes. However, collecting and storing erythrocytes can be logistically challenging during field work, and donor fish need to be alive or freshly killed. With rapid advances in molecular genetics, biologists, researchers, and managers may be unaware of molecular approaches for ploidy determination that could alleviate the difficulties associated with cytological methods and allow ploidy determination from archived samples (e.g., fin clips, scales, otoliths). In this study, we analyzed the agreement between molecular-based (using fin tissue) and Coulter counter-based (using blood) ploidy determinations for Walleyes Stizostedion vitreum—the first assessment of concordance between molecular and cytological methods for determining ploidy in the family Percidae. We found that agreement between these two methods was >98%. The high degree of agreement and greater ease of collecting and storing samples for molecular-based approaches relative to the traditional cytological ones support the utility of molecular methods for ploidy determination.

Using a system optimized for propagating human keratinocytes, culture of skin samples from white and green sturgeons generated epithelial cells capable of making cross-linked protein envelopes. Two distinct forms of TGM1-like mRNA were molecularly cloned from the cells of white sturgeon and detected in green sturgeon cells, accounting for their cellular envelope forming ability. The protein translated from each displayed a cluster of cysteine residues resembling the membrane anchorage region expressed in epidermal cells of teleosts and tetrapods. One of the two mRNA forms (called A) was present at considerably higher levels than the other (called B) in both species. Continuous lines of white sturgeon epidermal cells were established and characterized. Size measurements indicated that a substantial fraction of the cells became enlarged, appearing similar to squames in human epidermal keratinocyte cultures. The cultures also expressed CYP1A, a cytochrome P450 enzyme inducible by activation of aryl hydrocarbon receptor 2 in fish. The cells gradually improved in growth rate over a dozen passages while retaining envelope forming ability, TGM1 expression and CYP1A inducibility. These cell lines are thus potential models for studying evolution of fish epidermis leading to terrestrial adaptation and for testing sturgeon sensitivity to environmental stresses such as pollution.

Pathogen surveillance must be part of any population supplementation or reintroduction program for the conservation of threatened and endangered species. The unintended transmission of pathogens can have devastating effects on these already at-risk populations or the natural ecosystem at large. In the San Francisco Estuary (estuary), abundance of the endemic Delta Smelt (Hypomesus transpacificus) has declined to the point where regulatory managers are preparing to augment the wild population using fish propagated in a hatchery to prevent species extinction. Although disease is not an overt cause of population decline, comprehensive pathogen presence and prevalence data are lacking. Here, we performed a pilot study that applied molecular assays originally developed in salmonids to assess the presence of a wide variety of pathogens in the gill tissue of cultured and wild Delta Smelt—as well as cultured fish—deployed in enclosures in the estuary. We found the assays to be highly sensitive, and observed positive detections of a single pathogen, Ichthyophthirius multifiliis, in 13% of cultured Delta Smelt. We also detected ten other pathogens at very low levels in cultured, enclosure-deployed, and wild Delta Smelt that likely represent the ambient pathogen composition in the estuary (as opposed to actual infection). Our results corroborate previous work that cultured Delta Smelt do not appear to present a high risk for pathogen transmission during population supplementation or reintroduction. However, the molecular pathogen screening assays tested here have great utility as an early warning system indicator of when further diagnostic testing might be necessary to limit the extent and frequency of disease outbreaks; their utility will be further increased once they are customized for Delta Smelt.

Whole genome duplication is hypothesized to have played a critical role in the evolution of several major taxa, including vertebrates, and while many lineages have rediploidized, some retain polyploid genomes. Additionally, variation in ploidy can occur naturally or be artificially induced within select plant and animal species. Modern genetic techniques have not been widely applied to polyploid or ploidy-variable species, in part due to the difficulty of obtaining genotype data from polyploids. In this study, we demonstrate a strategy for developing an amplicon sequencing panel of single nucleotide polymorphisms for high-throughput genotyping of polyploid organisms. We then develop a method to infer ploidy of individuals from amplicon sequencing data that is generalized to apply to any ploidy and does not require prior identification of heterozygous genotypes. Combining these two techniques will allow researchers to both infer ploidy and generate ploidy-aware genotypes with the same amplicon sequencing panel. We demonstrate this approach with white sturgeon Acipenser transmontanus, a ploidy-variable (octoploid, decaploid and dodecaploid) imperiled species under conservation management in the Pacific Northwest and obtained a panel of 325 loci. These loci were validated by examining inheritance in known-cross families, and the ploidy inference method was validated with known ploidy samples. We provide scripts that adapt existing pipelines to genotype polyploids and an R package for application of the ploidy inference method. We expect that these techniques will empower studies of genetic variation and inheritance in polyploid organisms that vary in ploidy level, either naturally or as a result of artificial propagation practices.

Sturgeon and paddlefishes (Acipenseriformes) are cultured worldwide for commercial and conservation purposes. Over the past decade, we have learned many sturgeon culture programs experience spontaneous autopolyploidy, or the generation of triploidy without management intervention. Spontaneous autopolyploidy has been shown to negatively impact the physiology, reproductive development and fertility of sturgeon. Given these concerns, it is necessary to understand how it arises, how it affects production, and how it can be managed. The goal of this review is to describe the incidence of spontaneous autopolyploidy in sturgeon relative to other fishes, explain how spontaneous autopolyploids are formed, and report what is known about how triploidy and intermediate ploidy effect sturgeon reproductive development and physiology. We then describe four common methods for detecting spontaneous autopolyploids and their intermediate ploidy progeny and end with recommendations for reducing the incidence of spontaneous autopolyploidy based on the results of recent research. Although spontaneous autopolyploidy may never be completely eliminated from sturgeon culture, following these recommendations may significantly decrease its incidence.

Translocation has been used to conserve imperiled fishes and create new fisheries. One species for which translocation has played a significant role is the Sacramento Perch Archoplites interruptus. Extirpated from its native range, the Sacramento Perch has been introduced throughout California and Nevada through multiple translocation events, though historical records are incomplete. Recent assessments of eight previously uncharacterized Sacramento Perch populations have prompted reevaluation of range-wide population structure to inform a genetic management plan for long-term resiliency of this species. We examined Sacramento Perch genetic diversity and population structure across the current range of the species using 12 microsatellite markers. We analyzed samples from the eight uncharacterized populations and seven populations previously studied by Schwartz and May (2008). Bayesian clustering supported two distinct clusters of Sacramento Perch herein designated as A and B. Within these two clusters we detected hierarchical substructure, likely due to genetic drift after population founding. Genetic differentiation among populations within the same cluster was relatively low (FST = 0.023–0.176), while differentiation among populations from different clusters was higher (FST = 0.190–0.320). The existence of two strongly divergent genetic clusters in Sacramento Perch suggests two distinct translocation sources, and we recommend that these clusters be treated as genetic management units (GMUs). The B GMU populations had fairly low levels of genetic diversity relative to the A GMU populations. All populations showed evidence of past bottlenecks, and most had effective population sizes placing them at risk for inbreeding depression. Human-facilitated gene flow is recommended to prevent further genetic diversity loss. Due to uncertainty surrounding Sacramento Perch translocation history and strong levels of divergence between the two GMUs, translocations should be facilitated only between populations within the same GMU.

Previous studies have provided evidence of a reduced aerobic metabolic capacity, both at the cellular (metabolic enzyme activity) and the whole organism (aerobic scope) level in juvenile triploid white sturgeon, compared to diploid siblings. The downstream costs of this reduced metabolic capacity are still unclear, yet a lower aerobic scope suggests triploid white sturgeon likely have less energy to allocate to biological processes like growth and development. We conducted a 15-week growth trial to assess energy allocation to somatic growth in 2-month-old diploid and triploid white sturgeon. Spontaneous swimming activity, hepatosomatic index, condition factor, and deformities were also measured throughout the growth trial as indices of energy allocation to activity and fish condition. In general, our results indicate that triploid white sturgeon may have less energy available for processes beyond basal maintenance. This could be linked to a reduced overall performance as evidenced by lower weights and more deformities when compared with their diploid counterparts. However, many indices were still mostly unaffected by triploidy (condition factor, hepatosomatic index, and swimming activity). Whether this lower growth performance seen in juvenile triploid white sturgeon continues through sub-adulthood, puberty and final maturation requires further long-term studies.

One of the most fundamental aspects of ecological research and monitoring is accurate species identification, but cryptic speciation and observer error can confound phenotype-based identification. The CRISPR-Cas toolkit has facilitated remarkable advances in many scientific disciplines, but the fields of ecology and conservation biology have yet to fully embrace this powerful technology. The recently developed CRISPR-Cas13a platform SHERLOCK (Specific High-sensitivity Enzymatic Reporter unLOCKing) enables highly accurate taxonomic identification and has all the characteristics needed to transition to ecological and environmental disciplines. Here we conducted a series of "proof of principle" experiments to characterize SHERLOCK's ability to accurately, sensitively and rapidly distinguish three fish species of management interest co-occurring in the San Francisco Estuary that are easily misidentified in the field. We improved SHERLOCK's ease of field deployment by combining the previously demonstrated rapid isothermal amplification and CRISPR genetic identification with a minimally invasive and extraction-free DNA collection protocol, as well as the option of instrument-free lateral flow detection. This approach opens the door for redefining how, where and by whom genetic identifications occur in the future.

Triploid salmonids have been shown to underperform in suboptimal environments. It is thought this might be due to having larger cells to accommodate the increased number of chromosomes and therefore effects on aerobic metabolism from having smaller cellular surface area to volume ratios. The goal of this study was to examine the aerobic metabolism of diploid and triploid white sturgeon (Acipenser transmontanus) in ambient (18 °C) and elevated water temperatures (24 °C). Resting and maximum metabolic rates, recovery time from exhaustive exercise, and surface area to volume ratios of erythrocytes and their nuclei in diploid and triploid sturgeon were evaluated. Triploid sturgeon had a reduced aerobic scope and hematological response (hematocrit and hemoglobin) to exhaustive exercise. A reduced surface area to volume ratio of erythrocytes in triploid sturgeon provides evidence that cellular surface area could be one mechanism limiting aerobic metabolism in triploid fishes. A lower aerobic scope found in triploid sturgeon may impact reproductive and somatic growth, yet more research is needed to determine implications for management decisions on farms and hatcheries.

The Green Sturgeon is a long-lived anadromous fish known to spawn in only three locations. Southern Distinct Population Segment (DPS) Green Sturgeon spawn only in the Sacramento drainage, while the Northern DPS spawns in the Klamath and Rogue rivers. In 2011, a young of year (YOY) Green Sturgeon was captured in the Columbia River below Bonneville Dam, providing preliminary evidence of a fourth spawning location. In 2017, four additional Green Sturgeon YOY of similar size were captured in the Columbia River below Bonneville Dam. Genetic analysis of all YOY samples indicates that they belong to the Northern DPS, confirming that the Northern DPS actually spawns in at least three locations. The detection of Green Sturgeon spawning in the Columbia River only in years with relatively high spring flow (2011, 2017), combined with confirmed correlations between flow and spawning for other White and Green Sturgeon populations, suggests that appropriate spawning conditions may only exist above a specific flow threshold. The Columbia River has the potential to play an important role in Green Sturgeon climate change adaptation due to its location at the northernmost end of the species’ reproductive range. We recommend future research to better quantify Green Sturgeon recruitment magnitude, frequency and dynamics in the Columbia River.

The valley elderberry longhorn beetle (VELB), Desmocerus californicus dimorphus (Coleoptera: Cerambycidae), is a federally threatened subspecies endemic to the Central Valley of California. The VELB range partially overlaps with that of its morphologically similar sister taxon, the California elderberry longhorn beetle (CELB), Desmocerus californicus californicus (Coleoptera: Cerambycidae). Current surveying methods are limited to visual identification of larval exit holes in the VELB/CELB host plant, elderberry (Sambucus spp.), into which larvae bore and excavate feeding galleries. Unbiased genetic approaches could provide a much-needed complementary approach that has more precision than relying on visual inspection of exit holes. In this study we developed a DNA sequencing-based method for indirect detection of VELB/CELB from frass (insect fecal matter), which can be easily and non-invasively collected from exit holes. Frass samples were collected from 37 locations and the 12S and 16S mitochondrial genes were partially sequenced using nested PCR amplification. Three frass-derived sequences showed 100% sequence identity to VELB/CELB barcode references from museum specimens sequenced for this study. Database queries of frass-derived sequences also revealed high similarity to common occupants of old VELB feeding galleries, including earwigs, flies, and other beetles. Overall, this non-invasive approach is a first step towards a genetic assay that could augment existing VELB monitoring and accurately discriminate between VELB, CELB, and other insects. Furthermore, a phylogenetic analysis of 12S and 16S data from museum specimens revealed evidence for the existence of a previously unrecognized, genetically distinct CELB subpopulation in southern California.

Spontaneous autopolyploidy, a 1.5x increase in genome size resulting from second polar body retention after fertilization, has been documented in cultured sturgeons, with the proportion of spontaneous autopolyploid progeny ranging widely among maternal families. Sturgeon farms wish to reduce the number of spontaneous autopolyploids because their progeny, when crossed with a normal ploidy parent, exhibit intermediate ploidies, resulting in reproductive abnormalities. However, there is limited knowledge of the causes of the second polar body retention in sturgeon culture. In this study, we report the results of experiments performed from 2015 to 2019 aimed at identifying the sources of spontaneous autopolyploidy in white sturgeon (Acipenser transmontanus) culture. In collaboration with several sturgeon farms, we tested whether post-ovulatory ageing, mechanical shock during egg de-adhesion, and the combined effects of both factors increased spontaneous autopolyploidy. To test the effect of post-ovulatory ageing, eggs were collected from females and either fertilized at the industry normative time (2–5 h post-oviposition) or stored in ovarian fluid at 15 °C for 6–8 h before fertilization. To test the effect of mechanical shock, eggs were collected, fertilized 2–5 h post-oviposition and exposed to either gentle or vigorous mixing during the 60 min de-adhesion treatment. Results from this work reveal that post-ovulatory ageing does increase the incidence of spontaneous autopolyploidy in some females, but overall the proportions produced were low (range 0–15%). Proportions of spontaneous autopolyploids in eggs exposed to vigorous mixing were also variable (1–92%) but significantly higher in 75% of the females when compared to their respective controls or gentle mixing treatments, indicating that mechanical shock during egg de-adhesion is likely the primary cause of spontaneous autopolyploidy. To our knowledge this is the first study to document mechanical shock to eggs during de-adhesion as a cause of abnormal ploidy in cultured fishes. We observed high variability in egg quality among females and a significant relationship between embryo mortality and the incidence of spontaneous autopolyploidy when eggs were exposed to mechanical shock. Repeated spawning of a female that produced a high proportion of spontaneous autopolyploids provided preliminary evidence that genetic background may influence the likelihood that a female's eggs will be prone to second polar body retention when subjected to mechanical shock. Although spontaneous autopolyploidy in sturgeon culture will likely never be eliminated entirely, we provide practical recommendations to sturgeon producers to reduce its incidence in a production setting.

Environmental DNA (eDNA) analysis has gained traction as a precise and cost-effective method for species and waterways management. To date, publications on eDNA protocol optimization have focused primarily on DNA yield. Therefore, it has not been possible to evaluate the cost and speed of specific components of the eDNA protocol, such as water filtration and DNA extraction method when designing or choosing an eDNA protocol. At the same time, these two parameters are essential for the experimental design of a project. Here we evaluate and rank 27 different eDNA protocols in the context of Chinook salmon (Oncorhynchus tshawytscha) eDNA detection in an estuarine environment. We present a comprehensive evaluation of multiple eDNA protocol parameters, balancing time, cost and DNA yield. We collected samples composed of 500 mL estuarine water from Deverton Slough (38°11'16.7"N 121°58'34.5"W) and 500 mL from tank water containing 1.3 juvenile Chinook Salmon per liter. Then, we compared extraction methods, filter types, use of inhibitor removal kit for DNA yield, processing time, and protocol cost. Lastly, we used an MCMC algorithm together with machine learning to understand the DNA yield of each step of the protocol as well as the interactions between those steps. Glass fiber filtration was to be the most resilient to high turbidites, filtering the samples in 2.32 ± 0.08 min instead of 14.16 ± 1.86 min and 6.72 ± 1.99 min for nitrocellulose and paper filter N1, respectively. The filtration DNA yield percentages for paper filter N1, glass fiber, and nitrocellulose were 0.00045 ± 0.00013, 0.00107 ± 0.00013, 0.00172 ± 0.00013. The DNA extraction yield percentage for QIagen, dipstick, NaOH, magnetic beads, and direct dipstick ranged from 0.047 ± 0.0388 to 0.475 ± 0.0357. For estuarine waters, which are challenging for eDNA studies due to high turbidity, variable salinity, and the presence of PCR inhibitors, we found that a protocol combining glass filters, magnetic beads, and an extra step for PCR inhibitor removal, is the method that best balances time, cost, and yield. In addition, we provide a generalized decision tree for determining the optimal eDNA protocol for other studies in aquatic systems. Our findings should be applicable to most aquatic environments and provide a clear guide for determining which eDNA protocol should be used under different study constraints.

Biological invasion by non-native species has been identified as one of the major threats to native fish communities worldwide. The fish community of San Francisco Estuary is no exception, as the estuary has been recognized as one of the most invaded on the planet and the system has been impacted significantly by these invasions. Here, we summarize the introduction and probable establishment of a new species in the Sacramento–San Joaquin Delta, the Bluefin Killifish (Lucania goodei), as discovered by the US Fish and Wildlife Service Delta Juvenile Fish Monitoring Program (DJFMP). The DJFMP has conducted a large-scale beach seine survey since 1976, and it is the longest-running monitoring program in the San Francisco Estuary that extensively monitors the shallow-water nearshore habitat. Possibly introduced as discarded aquarium fish within the vicinity of the Delta Cross Channel, Bluefin Killifish is a close relative of the Rainwater Killifish (Lucania parva), another non-native fish species that has been present in the San Francisco Estuary system for decades. Studies in their native range suggest that Bluefin Killifish will fill a similar niche to Rainwater Killifish, albeit with a more freshwater distribution. The potential ecological impact of Bluefin Killifish remains unclear in the absence of additional studies. However, we have been able to track the spread of the species within the Sacramento–San Joaquin Delta through the existence of long-term monitoring programs. Our findings demonstrate the value of monitoring across various habitats for the early detection and proactive management of invasive species.

Prior to the Deepwater Horizon oil spill, we lacked a comprehensive baseline of oil contamination in the Gulf of Mexico's sediments, water column, and biota. Gaps in prespill knowledge limit our ability to determine the aftereffects of the Deepwater Horizon blowout or prepare to mitigate similar impacts during future oil spill disasters. We examined spatiotemporal differences in exposure to and metabolism of polycyclic aromatic hydrocarbons (PAHs) in 2 hake species (Urophycis spp.) to establish a current baseline for these ecologically important, abundant, and at-risk demersal fishes. Gulf hake (Urophycis cirrata) and southern hake (Urophycis floridana) were collected throughout the Gulf of Mexico during extensive longline surveys from 2012 to 2015. Analyses of biliary PAH metabolites and liver PAH concentrations provided evidence of exposures to di- and tricyclic compounds, with the highest concentrations measured in the northern Gulf of Mexico. Species-specific differences were not detected, but temporal trends observed in biliary PAHs suggest a decrease in acute exposures, whereas increasing liver PAHs suggest chronic exposures marked by greater assimilation than metabolism rates. To our knowledge, the present study provides the first multitissue contaminant analyses, as well as the most exhaustive biometric analyses, for both gulf and southern hakes. Though sources of exposure are complex because of multiple natural and anthropogenic PAH inputs, these results will facilitate the development of much needed health metrics for Gulf of Mexico benthos. Environ Toxicol Chem 2019;38:2740-2749. © 2019 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals, Inc. on behalf of SETAC.

The authors regret that the incorrect version of Figure 4 was included in the original paper.

Conservation-focused reintroduction or supplementation programs using wild-born individuals tend to be more successful than those based on release of captive-born individuals. Negative genetic consequences of captive breeding, such as genetic diversity loss, domestication selection, and inbreeding, may contribute to this lowered success. In this study, we compare genetic consequences of repatriation, or collection of wild-born juveniles for captive rearing and release, and broodstock-based breeding, where wild adults are bred in captivity, in a conservation program for white sturgeon, Acipenser transmontanus. Specifically, we use microsatellite markers to compare the genetic diversity and number of spawners, Nₛ, represented by each captive breeding strategy. We present a novel method to estimate Nₛ in which simulated known family configurations of varying complexity are used to identify appropriate inclusive and exclusive probability thresholds in the maximum likelihood-based pedigree reconstruction program Colony. These probability thresholds are then applied to reconstruct progeny arrays and estimate Nₛ in empirical datasets of two repatriation cohorts and a naturally produced year class. We find that a repatriation approach preserves more genetic diversity and represents more spawners than broodstock-based captive breeding. Furthermore, a repatriation approach represents levels of genetic diversity similar to those found in a wild cohort. These results are the first comparison of Nₛ between broodstock and repatriation-based methods with respect to a hatchery program, and provide evidence that when possible, repatriation can be a more effective strategy for conserving genetic diversity than broodstock-based breeding for supplementation and reintroduction.

Release of sturgeon with abnormal ploidy into the wild may result in reduced fitness due to lowered fertility in the F₂ and subsequent generations. Further, there is evidence that ploidy affects reproductive development and caviar yield. Therefore, the ability to accurately characterize the ploidy of white sturgeon (Acipenser transmontanus) is essential for both commercial and conservation aquaculture. This study compares nuclear volume and whole erythrocyte long-axis lengths obtained using Coulter counter and blood smears, respectively, from captive white sturgeon from populations originating in California and Idaho. We examine which method provides the most accurate, time efficient and cost-effective characterization of ploidy in this species. Results from Coulter counter and blood smears were compared to results from flow cytometry, the gold standard for genome size analysis. Previous work suggests that blood smears can distinguish between 8N (diploid) and 12N (triploid) sturgeon, but further analysis is required to see if this method can also be used to identify 10N fish and to provide robust evidence of its utility in 8N and 12N fish across populations. In this study, we demonstrated that the Coulter counter had 100% agreement with flow cytometry in ploidy assignment, while blood smears vary in their accuracy based on population. Blood smears showed a high degree of overlap in erythrocyte long-axis length between 8N and 10N individuals as well as some overlap between 10N and 12N individuals in the California fish, and a high degree of overlap between 8N and 12N individuals in the Idaho fish. Although blood smears are time-intensive and vary in their ploidy assignment accuracy, they are a low-cost technique and as such may have some utility for caviar farms attempting to identify 12N individuals in a small number of broodstock. By comparing the accuracy, efficiency and cost of these three methods, sturgeon farmers and conservation hatcheries will be able to choose the best method for their needs in determining the ploidy of their fish. We determined that Coulter counter is equally accurate to flow cytometry and is also the most time efficient method for ploidy determination in white sturgeon.

Previous studies have demonstrated reduced performance in triploid fish when reared under suboptimal conditions, which may be the result of a higher susceptibility to stressors when compared to diploids. The goal of this project was to investigate differences in the capacity of diploid (8 N) and triploid (12 N) white sturgeon, Acipenser transmontanus, to respond to both warm acclimation (6-weeks of acclimation to either 18 or 22 °C) and a subsequent acute stress (10-min low water stress). Following the 6-week acclimation, fish were sampled either before or following an acute low water stress. Bioindices of the primary and secondary stress response, hematology and cellular metabolic status were measured. We also sought to determine if time to peak cortisol levels were similar between diploid and triploid sturgeon after exposure to a severe acute stressor (netting stress). While both ploidies had similar primary and secondary responses to acute stress, both with and without warm acclimation, warm acclimation impacted the ability of diploid and triploid white sturgeon to mount a typical stress response to an acute stressor. In response to warm acclimation, triploids exhibited little change in branchial lactate dehydrogenase activity, while diploids increased activity. After exposure to an acute water reduction stress, diploids increased citrate synthase activity, yet triploids showed a decrease in activity. Differences in metabolic enzyme activity in response to warm acclimation and acute stress suggest triploid white sturgeon may have a reduced cellular metabolic capacity under chronic and acute stress, which may impact performance of triploid sturgeon in suboptimal conditions.

Previous studies suggest fish with additional copies of their genome (polyploids) underperform in suboptimal conditions and may be more susceptible to stress and disease. The objective of this study was to determine the role ploidy plays in the physiological response of white sturgeon to chronically elevated water temperatures. White sturgeon of two ploidies (8 N and 10 N) were acclimated to ambient (18 °C) and warm (22 °C) water. Bioindices of stress (plasma cortisol, glucose and lactate, total erythrocyte count, hematocrit, hemoglobin, mean erythrocyte volume, mean erythrocyte hemoglobin, and mean erythrocyte hemoglobin concentration), immunity (respiratory burst, plasma lysozyme, and total leukocyte count), and cellular metabolic capacity (lactate dehydrogenase and citrate synthase activity) were measured before and after a 6-week acclimation period. Both ploidies appear comparable in their constitutive immune and stress parameters and respond similarly to warming. Hematological indices suggest 8 N and 10 N sturgeon are similar in oxygen carrying capacity; however, differences in enzyme activity between ploidies indicate that 10 N sturgeon may have a lower cellular aerobic capacity. Our results have implications for the screening and management of ploidy on white sturgeon farms and hatcheries, as the differences between ploidies may affect 10 N sturgeon performance at elevated water temperatures. Further research is needed to elucidate the differences in inducible stress and immune responses and metabolism of white sturgeon of different ploidies.

A refugial population of the endangered delta smelt (Hypomesus transpacificus) has been maintained at the Fish Conservation and Culture Laboratory (FCCL) at UC Davis since 2008. Despite intense genetic management, fitness differences between wild and cultured fish have been observed at the FCCL. To investigate the molecular underpinnings of hatchery domestication, we used whole‐genome bisulfite sequencing to quantify epigenetic differences between wild and hatchery‐origin delta smelt. Differentially methylated regions (DMRs) were identified from 104 individuals by comparing the methylation patterns in different generations of hatchery fish (G1, G2, G3) with their wild parents (G0). We discovered a total of 132 significant DMRs (p <.05) between G0 and G1, 132 significant DMRs between G0 and G2, and 201 significant DMRs between G0 and G3. Our results demonstrate substantial differences in methylation patterns emerged between the wild and hatchery‐reared fish in the early generations in the hatchery, with a higher proportion of hypermethylated DMRs in hatchery‐reared fish. The rearing environment was found to be a stronger predictor of individual clustering based on methylation patterns than family, sex or generation. Our study indicates a reinforcement of the epigenetic status with successive generations in the hatchery environment, as evidenced by an increase in methylation in hypermethylated DMRs and a decrease in methylation in hypomethylated DMRs over time. Lastly, our results demonstrated heterogeneity in inherited methylation pattern in families across generations. These insights highlight the long‐term consequences of hatchery practices on the epigenetic landscape, potentially impacting wild fish populations. [ABSTRACT FROM AUTHOR]

Exotic species have been implicated as a major threat to native freshwater fish communities in the Unites States. The San Francisco Estuary watershed has been recognized as one of the most invaded systems where exotics often dominate the fish community. On October 6, 2014, members of the U.S. Fish and Wildlife Service detected a previously unknown exotic fish in a disconnected pool immediately upstream from the Chowchilla Bifurcation Structure in the San Joaquin River, a major tributary of the San Francisco Estuary. A member of the U.S. Fish and Wildlife Service initially identified the fish as an Oriental Weatherfish Misgurnus anguillicaudatus using external morphological characteristics. We conducted additional fish sampling near the Chowchilla Bifurcation Structure in November 2014 and collected a total of six additional specimens in disconnected pool habitats. Unexpectedly, genetic and meristic techniques revealed that these specimens were Large-Scale Loach Paramisgurnus dabryanus. To our knowledge this is the first confirmed occurrence of Large-Scale Loach in the United States and the suspected pathway of introduction is release from aquaria. Very little is known about the population in the San Joaquin River. We recommend further evaluation of the ecology, distribution, and abundance of Large-Scale Loach to better understand their potential impact on the fish communities of the San Joaquin River and the likelihood of establishment throughout the United States.

An extreme decline in Delta Smelt (Hypomesus transpacificus) abundance has led to a number of management actions to support this endangered species, including the development and refinement of culture techniques and the creation of a refuge population. The wild Delta Smelt population has diminished to the point that many in the scientific community believe population supplementation using cultured fish needs to be experimentally evaluated as a possible management tool. Concerns about supplementation include the effectiveness of this action, and its potential to divert attention and funding from other needed management actions such as habitat restoration. Here, we describe the outcomes of a 2-day workshop that described the current refuge population, and identified key issues for potential future use of cultured Delta Smelt for research and management. Expanded use of cultured Delta Smelt is controversial and requires consideration for complexities that include legal constraints and permitting requirements. Developing policies that allow for in situ experiments using cultured Delta Smelt appears to be a precursor for advancing policies that might allow supplementation actions. Releases of cultured fish, either experimentally or as a management action, clearly need to be conducted within an adaptive management program that is integrated with other strategies, including habitat restoration. We describe a general framework for evaluating the potential risks of supplementation and include suggestions for how to reduce risks and uncertainty. Overall, we conclude there is sufficient baseline information about Delta Smelt and the existing culture program to proceed with targeted field research that utilizes cultured fish. Finally, given the dire status of this species, we conclude that rapid progress toward the development of a viable and testable supplementation program must be a priority for Delta Smelt conservation.

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

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

Green sturgeon (Acipenser medirostris) is a highly migratory, marine oriented species that congregates in non-natal estuaries during summer and early fall. Individuals from the threatened Southern Distinct Population Segment (SDPS) and non-listed Northern Distinct Population Segment (NDPS) regularly co-occur in non-natal estuaries including the Columbia River estuary, Willapa Bay, and Grays Harbor in relative proportions not explained by abundance or distance from natal river. We used genetic markers to assign green sturgeon sampled in these estuaries from 2010 to 2012 to distinct population segments (DPS). We then examined interannual differences in DPS composition among estuaries. Fork length distributions were compared between SDPS and NDPS green sturgeon to determine whether size varied within and among DPSs and estuaries. The majority of green sturgeon sampled in the Columbia River estuary and Willapa Bay were assigned to the SDPS, while we assigned nearly even DPS proportions to our Grays Harbor samples. NDPS green sturgeon were significantly smaller than those originating from the SDPS within and among estuaries. We used these findings to develop several hypotheses about the mechanisms that may lead to specific patterns of non-natal estuary use. Genetic markers also assigned a single age-0 green sturgeon sampled in the Columbia River to the NDPS, although our analyses suggest that this individual’s parents may not have originated from known NDPS spawning populations. Because the Columbia River may serve as alternative spawning habitat for green sturgeon as climate change occurs, we recommend monitoring the Columbia River more closely for further evidence of green sturgeon spawning.

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

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

The goal of a day-long symposium on March 3, 2015, Sturgeon in the Sacramento–San Joaquin Watershed: New Insights to Support Conservation and Management, was to present new information about the physiology, behavior, and ecology of the green (Acipenser medirostris) and white sturgeon (Acipenser transmontanus) to help guide enhanced management and conservation efforts within the Sacramento–San Joaquin watershed. This symposium identified current unknowns and highlighted new electronic tracking technologies and physiological techniques to address these knowledge gaps. A number of presentations, each reviewing ongoing research on the two species, was followed by a round-table discussion, in which each of the participants was asked to share recom-mendations for future research on sturgeon in the watershed. This article presents an in-depth review of the scientific information presented at the sympo-sium with a summary of recommendations for future research.

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.