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Structural biology efforts using cryogenic electron microscopy are frequently stifled by specimens adopting preferred orientations on grids, leading to anisotropic map resolution and impeding structure determination. Tilting the specimen stage during data collection is a generalizable solution but has historically led to substantial resolution attenuation. Here, we develop updated data collection and image processing workflows and demonstrate, using multiple specimens, that resolution attenuation is negligible or significantly reduced across tilt angles. Reconstructions with and without the stage tilted as high as 60° are virtually indistinguishable. These strategies allowed the reconstruction to 3 Å resolution of a bacterial RNA polymerase with preferred orientation, containing an unnatural nucleotide for studying novel base pair recognition. Furthermore, we present a quantitative framework that allows cryo-EM practitioners to define an optimal tilt angle during data acquisition. These results reinforce the utility of employing stage tilt for data collection and provide quantitative metrics to obtain isotropic maps.

Wildfire events are becoming more frequent and severe on a global scale. Rising temperatures, prolonged drought, and the presence of pyrophytic invasive grasses are contributing to the degradation of native vegetation communities. Within the Great Basin region of the western U.S., increasing wildfire frequency is transforming the ecosystem toward a higher degree of homogeneity, one dominated by invasive annual grasses and declining landscape productivity. Greater sage-grouse (Centrocercus urophasianus; hereafter sage-grouse) are a species of conservation concern that rely on large tracts of structurally and functionally diverse sagebrush (Artemisia spp.) communities. Using a 12-year (2008-2019) telemetry dataset, we documented immediate impacts of wildfire on demographic rates of a population of sage-grouse that were exposed to two large wildfire events (Virginia Mountains Fire Complex-2016; Long Valley Fire-2017) near the border of California and Nevada. Spatiotemporal heterogeneity in demographic rates were accounted for using a Before-After Control-Impact Paired Series (BACIPS) study design. Results revealed a 40% reduction in adult survival and a 79% reduction in nest survival within areas impacted by wildfires. Our results indicate that wildfire has strong and immediate impacts to two key life stages of a sagebrush indicator species and underscores the importance of fire suppression and immediate restoration following wildfire events.

California's vernal pools are declining ecosystems that support valuable native plant and animal diversity. Vernal pool branchiopods are particularly at risk from vernal pool habitat loss and conservation efforts have targeted their long-term protection through the establishment of preserves and conservation banks. These conservation strategies require repeated, perpetual monitoring of preserved habitat, which is currently carried out through dip-net surveys and visual identification of specimens. Dip-netting may be destructive and frequently requires some sacrifice of protected species. Environmental DNA offers a new, modern method to monitor many protected freshwater organisms. We designed qPCR-based species-specific assays for four of California's vernal pool branchiopods: The Vernal Pool Fairy Shrimp Branchinecta lynchi (BRLY), the Midvalley Fairy Shrimp Branchinecta mesovallensis (BRME), and the Conservancy Fairy Shrimp Branchinecta conservatio (BRCO), and the Vernal Pool Tadpole Shrimp Lepidurus packardi (LEPA). We tested these assays using eDNA sampling protocols alongside traditional dip-net surveys to assess their viability as an alternative method to monitor vernal pool branchiopods. Based on occupancy modeling, each of our assays achieved a 95% or higher detection rate when using optimized sampling protocols.

BackgroundIn 2013, sarcoptic mange, caused by Sarcoptes scabiei mites, precipitated a catastrophic decline of the formerly stable urban population of endangered San Joaquin kit foxes (Vulpes macrotis mutica) in Bakersfield, California, USA. In 2019, a smaller sarcoptic mange outbreak affected kit foxes 58 km southwest of Bakersfield in the town of Taft, California. To determine whether the Taft outbreak could have occurred as spillover from the Bakersfield outbreak and whether epidemic control efforts must involve not only kit foxes but also sympatric dogs (Canis lupus familiaris), coyotes (Canis latrans), and red foxes (Vulpes vulpes), we evaluated genotypes and gene flow among mites collected from each host species.MethodsWe used 10 Sarcoptes microsatellite markers (SARM) to perform molecular typing of 445 S. scabiei mites collected from skin scrapings from twenty-two infested kit foxes, two dogs, five coyotes, and five red foxes from Bakersfield, Taft, and other nearby cities.ResultsWe identified 60 alleles across all SARM loci; kit fox- and red fox-derived mites were relatively monomorphic, while genetic variability was greatest in Bakersfield coyote- and dog-derived mites. AMOVA analysis documented distinct mite populations unique to hosts, with an overall FST of 0.467. The lowest FST (i.e. closest genetic relationship, FST = 0.038) was between Bakersfield and Taft kit fox-derived mites while the largest genetic difference was between Ventura coyote- and Taft kit fox-derived mites (FST = 0.843).ConclusionsThese results confirm the close relationship between the Taft and Bakersfield outbreaks. Although a spillover event likely initiated the kit fox mange outbreak, mite transmission is now primarily kit fox-to-kit fox. Therefore, any large-scale population level intervention should focus on treating kit foxes within the city.

Species' distributions are often discontinuous near the edge of the range where the environment may be more variable than the core of the range. Range discontinuity can reduce or cut off gene flow to small peripheral populations and lead to genetic drift and subsequent loss of genetic diversity. The southern extent of the Great Gray Owl (Strix nebulosa) range in North America is discontinuous, unlike their northern core range across the boreal forests. We sampled owls from five different locations on the periphery of the range across the western US (Wyoming, Idaho, California, northern Oregon, and southern Oregon) to investigate genetic population structure and genetic diversity. Using a reduced-representation genomic sequencing approach to genotype 123 individuals at 4817 single nucleotide polymorphic loci, we identified four genetically differentiated populations: California, southern Oregon, northern Oregon, and Wyoming and Idaho grouped together as a single Rocky Mountain population. The four genetically differentiated populations of Great Gray Owls identified in this study display high differentiation and low genetic variation, which is suggestive of long-term isolation and lack of connectivity, potentially caused by range discontinuity. The populations that lack habitat connectivity to the rest of the breeding range (i.e. those in California and Oregon) had lower genetic diversity than the Rocky Mountain population that is connected to the core of the range. These factors and other risks (such as disease and human-caused mortality) heighten susceptibility of these range-edge populations to future habitat and climate changes, genetic diversity erosion, and potential extinction vortex. For these reasons, protecting and monitoring this species on the southern edge of their range is vital.

The large-scale patterns of movement for the Sharp-shinned Hawk (Accipiter striatus), a small forest hawk found throughout western North America, are largely unknown. However, based on field observations we set out to test the hypothesis that juvenile migratory A. striatus caught along two distinct migration routes on opposite sides of the Sierra Nevada Mountains of North America (Pacific Coast and Intermountain Migratory Flyways) come from geographically different natal populations. We applied stable isotope analysis of hydrogen (H) and oxygen (O) of feathers, and large scale models of spatial isotopic variation (isoscapes) to formulate spatially explicit predictions of the origin of the migrant birds. Novel relationships were assessed between the measured hydrogen and oxygen isotope values of feathers from A. striatus museum specimens of known origin and the isoscape modeled hydrogen and oxygen isotope values of precipitation at those known locations. We used these relationships to predict the origin regions for birds migrating along the two flyways from the measured isotope values of migrant's feathers and the associated hydrogen and oxygen isotopic composition of precipitation where these feathers were formed. The birds from the two migration routes had overlap in their natal/breeding origins and did not differentiate into fully separate migratory populations, with birds from the Pacific Coast Migratory Flyway showing broader natal geographic origins than those from the Intermountain Flyway. The methodology based on oxygen isotopes had, in general, less predictive power than the one based on hydrogen. There was broad agreement between the two isotope approaches in the geographic assignment of the origins of birds migrating along the Pacific Coast Flyway, but not for those migrating along the Intermountain Migratory Flyway. These results are discussed in terms of their implications for conservation efforts of A. striatus in western North America, and the use of combined hydrogen and oxygen stable isotope analysis to track the movement of birds of prey on continental scales.

Complex coevolutionary relationships among competitors, predators, and prey have shaped taxa diversity, life history strategies, and even the avian migratory patterns we see today. Consequently, accurate documentation of prey selection is often critical for understanding these ecological and evolutionary processes. Conventional diet study methods lack the ability to document the diet of inconspicuous or difficult-to-study predators, such as those with large home ranges and those that move vast distances over short amounts of time, leaving gaps in our knowledge of trophic interactions in many systems. Migratory raptors represent one such group of predators where detailed diet studies have been logistically challenging. To address knowledge gaps in the foraging ecology of migrant raptors and provide a broadly applicable tool for the study of enigmatic predators, we developed a minimally invasive method to collect dietary information by swabbing beaks and talons of raptors to collect trace prey DNA. Using previously published COI primers, we were able to isolate and reference gene sequences in an open-access barcode database to identify prey to species. This method creates a novel avenue to use trace molecular evidence to study prey selection of migrating raptors and will ultimately lead to a better understanding of raptor migration ecology. In addition, this technique has broad applicability and can be used with any wildlife species where even trace amounts of prey debris remain on the exterior of the predator after feeding.

Single-particle cryogenic electron microscopy (cryo-EM) provides a powerful methodology for structural biologists, but the resolutions typically attained with experimentally determined structures have lagged behind microscope capabilities. Here, we exploit several technical advances to improve resolution, including per-particle contrast transfer function (CTF) refinement and correction for Ewald sphere curvature. The latter is demonstrated with several experimental samples and should become more standard as resolutions increase or at lower microscope accelerating voltages. The combined application of the described methods to micrographs recorded on a Titan Krios enables structure determination at ~1.86-Å resolution of an adeno-associated virus serotype 2 variant (AAV2), an important gene-delivery vehicle. The resulting structural details provide an improved model for understanding the biology of AAV that will guide future vector development for gene therapy.

Single-particle cryogenic electron microscopy (cryo-EM) provides a powerful methodology for structural biologists, but the resolutions typically attained with experimentally determined structures have lagged behind microscope capabilities. Here, we have exploited several technical solutions to improve resolution, including sub-Angstrom pixelation, per-particle CTF refinement, and most notably a correction for Ewald sphere curvature. The application of these methods on micrographs recorded on a base model Titan Krios enabled structure determination at ∼1.86-Å resolution of an adeno-associated virus serotype 2 variant (AAV2), an important gene-delivery vehicle.

An adult Buteo was found dead as a road-kill south of Sacramento, California, and was thought to represent the first state record of the eastern Red-shouldered Hawk (B. lineatus lineatus;). It is now a specimen in the Museum of Wildlife and Fisheries Biology (WFB 4816) at the University of California, Davis. We examined this specimen and found that many of its plumage characters differed from all other adult Red-shouldered Hawks examined, including nominate adults. Plumage markings and measurements were intermediate between Red-tailed Hawk (Buteo jamaicensis, ssp calurus) and Red-shouldered Hawk (ssp elegans), leading us to hypothesize that the bird was a hybrid. However, mtDNA sequences and nuDNA microsatellites proved definitively that the bird was a Red-shouldered Hawk, most likely of eastern origin. This case illustrates that apparent hybrids or apparent vagrants could be individuals with anomalous phenotypes caused by rare genetic variation or novel epigenetic effects.

Wetland habitats across the world are experiencing rapid modification and loss due to accelerating habitat conversion. Impacts to wetland habitats are particularly acute in California where up to 90% of wetland habitats have been modified or lost. Vernal pool ecosystems have therefore undergone a dramatic loss in habitat and along with them an entire endemic fauna is under threat of extinction. Recent efforts to conserve vernal pool habitat and associated species have involved restoration and creation of vernal pools as well as translocations of threatened species. The vernal pool fairy shrimp, Branchinecta lynchi, is one of several endemic and federally listed species being targeted for translocations. To guide reintroduction and conservation, detailed information on range-wide population structure and diversity is needed. We collected genetic data from two mitochondrial genes throughout the known extant range of B. lynchi to elucidate population structure and diversity of the species. We found support for phylogeographic structure throughout the range of B. lynch associated with isolated watersheds and vernal pool regions previously identified in the recovery plan for the species. The underlying mechanisms responsible for this broad pattern of genetic structure have yet to be identified. However, the evidence of only a few haplotypes being shared across the species range and patterns of isolation by distance within vernal pool regions suggests dispersal limitation may play a role. These results stress that conservation programs, at a minimum, should consider using individuals from regional populations as sources for reintroductions to maintain historical patterns of genetic differentiation. Additionally, because genetic structure is associated with vernal pool regions which are based on local hydrology and geology, translocations should proceed considering the distance between donor and recipient sites.

Management actions to protect endangered species and conserve ecosystem function may not always be in precise alignment. Efforts to recover the California Ridgway’s Rail (Rallus obsoletus obsoletus; hereafter, California rail), a federally and state-listed species, and restoration of tidal marsh ecosystems in the San Francisco Bay estuary provide a prime example of habitat restoration that has conflicted with species conservation. On the brink of extinction from habitat loss and degradation, and non-native predators in the 1990s, California rail populations responded positively to introduction of a non-native plant, Atlantic cordgrass (Spartina alterniflora). California rail populations were in substantial decline when the non-native Spartina was initially introduced as part of efforts to recover tidal marshes. Subsequent hybridization with the native Pacific cordgrass (Spartina foliosa) boosted California rail populations by providing greater cover and increased habitat area. The hybrid cordgrass (S. alterniflora × S. foliosa) readily invaded tidal mudflats and channels, and both crowded out native tidal marsh plants and increased sediment accretion in the marsh plain. This resulted in modification of tidal marsh geomorphology, hydrology, productivity, and species composition. Our results show that denser California rail populations occur in invasive Spartina than in native Spartina in San Francisco Bay. Herbicide treatment between 2005 and 2012 removed invasive Spartina from open intertidal mud and preserved foraging habitat for shorebirds. However, removal of invasive Spartina caused substantial decreases in California rail populations. Unknown facets of California rail ecology, undesirable interim stages of tidal marsh restoration, and competing management objectives among stakeholders resulted in management planning for endangered species or ecosystem restoration that favored one goal over the other. We have examined this perceived conflict and propose strategies for moderating harmful effects of restoration while meeting the needs of both endangered species and the imperiled native marsh ecosystem.

Abstract Background Genes at the major histocompatibility complex (MHC) are known for high levels of polymorphism maintained by balancing selection. In small or bottlenecked populations, however, genetic drift may be strong enough to overwhelm the effect of balancing selection, resulting in reduced MHC variability. In this study we investigated MHC evolution in two recently diverged bird species: the endemic Galápagos hawk (Buteo galapagoensis), which occurs in small, isolated island populations, and its widespread mainland relative, the Swainson's hawk (B. swainsoni). Results We amplified at least two MHC class II B gene copies in each species. We recovered only three different sequences from 32 Galápagos hawks, while we amplified 20 unique sequences in 20 Swainson's hawks. Most of the sequences clustered into two groups in a phylogenetic network, with one group likely representing pseudogenes or nonclassical loci. Neutral genetic diversity at 17 microsatellite loci was also reduced in the Galápagos hawk compared to the Swainson's hawk. Conclusions The corresponding loss in neutral diversity suggests that the reduced variability present at Galápagos hawk MHC class II B genes compared to the Swainson's hawk is primarily due to a founder event followed by ongoing genetic drift in small populations. However, purifying selection could also explain the low number of MHC alleles present. This lack of variation at genes involved in the adaptive immune response could be cause for concern should novel diseases reach the archipelago.

Abstract Background Phenotypic and molecular genetic data often provide conflicting patterns of intraspecific relationships confounding phylogenetic inference, particularly among birds where a variety of environmental factors may influence plumage characters. Among diurnal raptors, the taxonomic relationship of Buteo jamaicensis harlani to other B. jamaicensis subspecies has been long debated because of the polytypic nature of the plumage characteristics used in subspecies or species designations. Results To address the evolutionary relationships within this group, we used data from 17 nuclear microsatellite loci, 430 base pairs of the mitochondrial control region, and 829 base pairs of the melanocortin 1 receptor (Mc1r) to investigate molecular genetic differentiation among three B. jamaicensis subspecies (B. j. borealis, B. j. calurus, B. j. harlani). Bayesian clustering analyses of nuclear microsatellite loci showed no significant differences between B. j. harlani and B. j. borealis. Differences observed between B. j. harlani and B. j. borealis in mitochondrial and microsatellite data were equivalent to those found between morphologically similar subspecies, B. j. borealis and B. j. calurus, and estimates of migration rates among all three subspecies were high. No consistent differences were observed in Mc1r data between B. j. harlani and other B. jamaicensis subspecies or between light and dark color morphs within B. j. calurus, suggesting that Mc1r does not play a significant role in B. jamaicensis melanism. Conclusions These data suggest recent interbreeding and gene flow between B. j. harlani and the other B. jamaicensis subspecies examined, providing no support for the historical designation of B. j. harlani as a distinct species.

Artificial nest boxes have played an important role in American Kestrel (Falco sparverius) conservation worldwide, facilitating nesting and research efforts. However, these conservation efforts frequently unintentionally facilitate other cavity-nesting species including the European Starling (Sturnus vulgaris), a widespread invasive species in North America. Understanding how to deter European Starlings from occupying nest boxes is an essential responsibility of project design when deploying nest boxes. We examined the efficacy of five different abatement methods to mitigate the use of our nest boxes by European Starlings: nest removal, nest and egg removal, egg shaking, egg cracking, and egg oiling. We found spraying European Starling eggs with 100% food-grade corn oil and cracking eggs to be the most effective abatement methods. We recommend future American Kestrel nest box programs in this region use egg oiling or egg cracking on European Starling eggs. [ABSTRACT FROM AUTHOR]