Transboundary determinants of avian zoonotic infectious diseases: challenges for strengthening research capacity and connecting surveillance networks.

As the climate changes, global systems have become increasingly unstable and unpredictable. This is particularly true for many disease systems, including subtypes of highly pathogenic avian influenzas (HPAIs) that are circulating the world. Ecological patterns once thought stable are changing, bringing new populations and organisms into contact with one another. Wild birds continue to be hosts and reservoirs for numerous zoonotic pathogens, and strains of HPAI and other pathogens have been introduced into new regions via migrating birds and transboundary trade of wild birds. With these expanding environmental changes, it is even more crucial that regions or counties that previously did not have surveillance programs develop the appropriate skills to sample wild birds and add to the understanding of pathogens in migratory and breeding birds through research. For example, little is known about wild bird infectious diseases and migration along the Mediterranean and Black Sea Flyway (MBSF), which connects Europe, Asia, and Africa. Focusing on avian influenza and the microbiome in migratory wild birds along the MBSF, this project seeks to understand the determinants of transboundary disease propagation and coinfection in regions that are connected by this flyway. Through the creation of a threat reduction network for avian diseases (Avian Zoonotic Disease Network, AZDN) in three countries along the MBSF (Georgia, Ukraine, and Jordan), this project is strengthening capacities for disease diagnostics; microbiomes; ecoimmunology; field biosafety; proper wildlife capture and handling; experimental design; statistical analysis; and vector sampling and biology. Here, we cover what is required to build a wild bird infectious disease research and surveillance program, which includes learning skills in proper bird capture and handling; biosafety and biosecurity; permits; next generation sequencing; leading-edge bioinformatics and statistical analyses; and vector and environmental sampling. Creating connected networks for avian influenzas and other pathogen surveillance will increase coordination and strengthen biosurveillance globally in wild birds.

Ongoing Cooperative Engagement Facilitates Agile Pandemic and Outbreak Response: Lessons Learned Through Cooperative Engagement Between Uganda and the United States.

Pathogens threaten human lives and disrupt economies around the world. This has been clearly illustrated by the current COVID-19 pandemic and outbreaks in livestock and food crops. To manage pathogen emergence and spread, cooperative engagement programs develop and strengthen biosafety, biosecurity, and biosurveillance capabilities among local researchers to detect pathogens. In this case study, we describe the efforts of a collaboration between the Los Alamos National Laboratory and the Uganda Virus Research Institute, the primary viral diagnostic laboratory in Uganda, to implement and ensure the sustainability of sequencing for biosurveillance. We describe the process of establishing this capability along with the lessons learned from both sides of the partnership to inform future cooperative engagement efforts in low- and middle-income countries. We found that by strengthening sequencing capabilities at the Uganda Virus Research Institute before the COVID-19 pandemic, the institute was able to successfully sequence SARS-CoV-2 samples and provide data to the scientific community. We highlight the need to strengthen and sustain capabilities through in-country training, collaborative research projects, and trust.

Seroprevalence of Brucella spp. and Rift Valley fever virus among slaughterhouse workers in Isiolo County, northern Kenya.

Brucella spp. and Rift Valley fever virus (RVFV) are classified as priority zoonotic agents in Kenya, based on their public health and socioeconomic impact on the country. Data on the pathogen-specific and co-exposure levels is scarce due to limited active surveillance. This study investigated seroprevalence and co-exposure of Brucella spp. and RVFV and associated risk factors among slaughterhouse workers in Isiolo County, northern Kenya. A cross-sectional serosurvey was done in all 19 slaughterhouses in Isiolo County, enrolling 378 participants into the study. The overall seroprevalences for Brucella spp. and RVFV were 40.2% (95% CI: 35.2-45.4) and 18.3% (95% CI: 14.5-22.5), respectively while 10.3% (95% CI 7.4%-13.8%) of individuals were positive for antibodies against both Brucella spp. and RVFV. Virus neutralisation tests (VNT) confirmed anti-RVFV antibodies in 85% of ELISA-positive samples. Our seroprevalence results were comparable to community-level seroprevalences previously reported in the area. Since most of the study participants were not from livestock-keeping households, our findings attribute most of the detected infections to occupational exposure. The high exposure levels indicate slaughterhouse workers are the most at-risk population and there is need for infection, prevention, and control programs among this high-risk group. This is the first VNT confirmation of virus-neutralising antibodies among slaughterhouse workers in Isiolo County and corroborates reports of the area being a high-risk RVFV area as occasioned by previously reported outbreaks. This necessitates sensitization campaigns to enhance awareness of the risks involved and appropriate mitigation measures.

A scoping review of zoonotic parasites and pathogens associated with abattoirs in Eastern Africa and recommendations for abattoirs as disease surveillance sites.

Abattoirs are facilities where livestock are slaughtered and are an important aspect in the food production chain. There are several types of abattoirs, which differ in infrastructure and facilities, sanitation and PPE practices, and adherence to regulations. In each abattoir facility, worker exposure to animals and animal products increases their risk of infection from zoonotic pathogens. Backyard abattoirs and slaughter slabs have the highest risk of pathogen transmission because of substandard hygiene practices and minimal infrastructure. These abattoir conditions can often contribute to environmental contamination and may play a significant role in disease outbreaks within communities. To assess further the risk of disease, we conducted a scoping review of parasites and pathogens among livestock and human workers in abattoirs across 13 Eastern African countries, which are hotspots for zoonoses. Our search results (n = 104 articles) showed the presence of bacteria, viruses, fungi, and macroparasites (nematodes, cestodes, etc.) in cattle, goats, sheep, pigs, camels, and poultry. Most articles reported results from cattle, and the most frequent pathogen detected was Mycobacterium bovis, which causes bovine tuberculosis. Some articles included worker survey and questionnaires that suggested how the use of PPE along with proper worker training and safe animal handling practices could reduce disease risk. Based on these findings, we discuss ways to improve abattoir biosafety and increase biosurveillance for disease control and mitigation. Abattoirs are a 'catch all' for pathogens, and by surveying animals at abattoirs, health officials can determine which diseases are prevalent in different regions and which pathogens are most likely transmitted from wildlife to livestock. We suggest a regional approach to biosurveillance, which will improve testing and data gathering for enhanced disease risk mapping and forecasting. Next generation sequencing will be key in identifying a wide range of pathogens, rather than a targeted approach.

Scoping review of Culex mosquito life history trait heterogeneity in response to temperature.

BACKGROUND: Mosquitoes in the genus Culex are primary vectors in the US for West Nile virus (WNV) and other arboviruses. Climatic drivers such as temperature have differential effects on species-specific changes in mosquito range, distribution, and abundance, posing challenges for population modeling, disease forecasting, and subsequent public health decisions. Understanding these differences in underlying biological dynamics is crucial in the face of climate change. METHODS: We collected empirical data on thermal response for immature development rate, egg viability, oviposition, survival to adulthood, and adult lifespan for Culex pipiens, Cx. quinquefasciatus, Cx. tarsalis, and Cx. restuans from existing literature according to the PRISMA scoping review guidelines. RESULTS: We observed linear relationships with temperature for development rate and lifespan, and nonlinear relationships for survival and egg viability, with underlying variation between species. Optimal ranges and critical minima and maxima also appeared varied. To illustrate how model output can change with experimental input data from individual Culex species, we applied a modified equation for temperature-dependent mosquito type reproduction number for endemic spread of WNV among mosquitoes and observed different effects. CONCLUSIONS: Current models often input theoretical parameters estimated from a single vector species; we show the need to implement the real-world heterogeneity in thermal response between species and present a useful data resource for researchers working toward that goal.

Modeling the distribution of the endangered Jemez Mountains salamander (Plethodon neomexicanus) in relation to geology, topography, and climate.

The Jemez Mountains salamander (Plethodon neomexicanus; hereafter JMS) is an endangered salamander restricted to the Jemez Mountains in north-central New Mexico, United States. This strictly terrestrial and lungless species requires moist surface conditions for activities such as mating and foraging. Threats to its current habitat include fire suppression and ensuing severe fires, changes in forest composition, habitat fragmentation, and climate change. Forest composition changes resulting from reduced fire frequency and increased tree density suggest that its current aboveground habitat does not mirror its historically successful habitat regime. However, because of its limited habitat area and underground behavior, we hypothesized that geology and topography might play a significant role in the current distribution of the salamander. We modeled the distribution of the JMS using a machine learning algorithm to assess how geology, topography, and climate variables influence its distribution. The best habitat suitability model indicates that geology type and maximum winter temperature (November to March) were most important in predicting the distribution of the salamander (23.5% and 50.3% permutation importance, respectively). Minimum winter temperature was also an important variable (21.4%), suggesting this also plays a role in salamander habitat. Our habitat suitability map reveals low uncertainty in model predictions, and we found slight discrepancies between the designated critical habitat and the most suitable areas for the JMS. Because geological features are important to its distribution, we recommend that geological and topographical data are considered, both during survey design and in the description of localities of JMS records once detected.

Comparing western (Megascops kennicottii) and whiskered (M. trichopsis) screech-owl microbiomes in southern Arizona using a novel 16S rRNA sequencing method.

Microbiomes are essential to a host's physiology and health. Despite the overall importance of microbiomes to animal health, they remain understudied in wildlife. Microbiomes function as physical barriers to invading pathogens, and changes in the diversity or composition of microbes within a host may disrupt this barrier. In order to use microbiomes in wildlife ecology, knowledge of the natural variation within and among species is essential. We compare the diversity and composition of two avian species that share the same habitat and niche in our study area, the western screech-owl (Megascops kennicottii) and the whiskered screech-owl (M. trichopsis). We used a targeted 16S sequencing method to improve the taxonomic resolution of microbiomes. We found similar measures of alpha diversity between species and sample types (cloacal samples vs. fecal samples). However, there were significant differences in bacterial species richness among nestlings from different nest boxes, and the composition differed between the two bird species and among nestlings from different nest boxes. Western screech-owls had more variation in alpha diversity and composition and had fewer bacterial species in their core microbiome than whiskered screech-owls. Siblings are likely to yield similar findings for microbiomes; thus, sampling nestlings from different nests may be most informative for monitoring population-level changes.

Identification and distribution of pathogens coinfecting with Brucella spp., Coxiella burnetii and Rift Valley fever virus in humans, livestock and wildlife.

Zoonotic diseases, such as brucellosis, Q fever and Rift Valley fever (RVF) caused by Brucella spp., Coxiella burnetii and RVF virus, respectively, can have devastating effects on human, livestock, and wildlife health and cause economic hardship due to morbidity and mortality in livestock. Coinfection with multiple pathogens can lead to more severe disease outcomes and altered transmission dynamics. These three pathogens can alter host immune responses likely leading to increased morbidity, mortality and pathogen transmission during coinfection. Developing countries, such as those commonly afflicted by outbreaks of brucellosis, Q fever and RVF, have high disease burden and thus common coinfections. A literature survey provided information on case reports and studies investigating coinfections involving the three focal diseases. Fifty five studies were collected demonstrating coinfections of Brucella spp., C. burnetii or RVFV with 50 different pathogens, of which 64% were zoonotic. While the literature search criteria involved 'coinfection', only 24/55 studies showed coinfections with direct pathogen detection methods (microbiology, PCR and antigen test), while the rest only reported detection of antibodies against multiple pathogens, which only indicate a history of co-exposure, not concurrent infection. These studies lack the ability to test whether coinfection leads to changes in morbidity, mortality or transmission dynamics. We describe considerations and methods for identifying ongoing coinfections to address this critical blind spot in disease risk management.

Comparing variability in diagnosis of upper respiratory tract infections in patients using syndromic, next generation sequencing, and PCR-based methods.

Early and accurate diagnosis of respiratory pathogens and associated outbreaks can allow for the control of spread, epidemiological modeling, targeted treatment, and decision making-as is evident with the current COVID-19 pandemic. Many respiratory infections share common symptoms, making them difficult to diagnose using only syndromic presentation. Yet, with delays in getting reference laboratory tests and limited availability and poor sensitivity of point-of-care tests, syndromic diagnosis is the most-relied upon method in clinical practice today. Here, we examine the variability in diagnostic identification of respiratory infections during the annual infection cycle in northern New Mexico, by comparing syndromic diagnostics with polymerase chain reaction (PCR) and sequencing-based methods, with the goal of assessing gaps in our current ability to identify respiratory pathogens. Of 97 individuals that presented with symptoms of respiratory infection, only 23 were positive for at least one RNA virus, as confirmed by sequencing. Whereas influenza virus (n = 7) was expected during this infection cycle, we also observed coronavirus (n = 7), respiratory syncytial virus (n = 8), parainfluenza virus (n = 4), and human metapneumovirus (n = 1) in individuals with respiratory infection symptoms. Four patients were coinfected with two viruses. In 21 individuals that tested positive using PCR, RNA sequencing completely matched in only 12 (57%) of these individuals. Few individuals (37.1%) were diagnosed to have an upper respiratory tract infection or viral syndrome by syndromic diagnostics, and the type of virus could only be distinguished in one patient. Thus, current syndromic diagnostic approaches fail to accurately identify respiratory pathogens associated with infection and are not suited to capture emerging threats in an accurate fashion. We conclude there is a critical and urgent need for layered agnostic diagnostics to track known and unknown pathogens at the point of care to control future outbreaks.

Updated distribution maps of predominant Culex mosquitoes across the Americas.

BACKGROUND: Estimates of the geographical distribution of Culex mosquitoes in the Americas have been limited to state and provincial levels in the United States and Canada and based on data from the 1980s. Since these estimates were made, there have been many more documented observations of mosquitoes and new methods have been developed for species distribution modeling. Moreover, mosquito distributions are affected by environmental conditions, which have changed since the 1980s. This calls for updated estimates of these distributions to understand the risk of emerging and re-emerging mosquito-borne diseases. METHODS: We used contemporary mosquito data, environmental drivers, and a machine learning ecological niche model to create updated estimates of the geographical range of seven predominant Culex species across North America and South America: Culex erraticus, Culex nigripalpus, Culex pipiens, Culex quinquefasciatus, Culex restuans, Culex salinarius, and Culex tarsalis. RESULTS: We found that Culex mosquito species differ in their geographical range. Each Culex species is sensitive to both natural and human-influenced environmental factors, especially climate and land cover type. Some prefer urban environments instead of rural ones, and some are limited to tropical or humid areas. Many are found throughout the Central Plains of the USA. CONCLUSIONS: Our updated contemporary Culex distribution maps may be used to assess mosquito-borne disease risk. It is critical to understand the current geographical distributions of these important disease vectors and the key environmental predictors structuring their distributions not only to assess current risk, but also to understand how they will respond to climate change. Since the environmental predictors structuring the geographical distribution of mosquito species varied, we hypothesize that each species may have a different response to climate change.

Individual Nest Site Preferences Do Not Explain Upslope Population Shifts of a Secondary Cavity-Nesting Species.

Geographic ranges of plants and animals are shifting due to environmental change. While some species are shifting towards the poles and upslope in elevation, the processes leading to these patterns are not well known. We analyzed 22 years of western bluebird (Sialia mexicana) data from a large nest box network in northern New Mexico at elevations between 1860 m and 2750 m. This population has shifted to higher elevations over time, but whether this is due to changes in nesting behavior and preference for higher elevation within the population or driven by immigration is unclear. We banded adults and nestlings from nest boxes and examined nesting location and elevation for individual birds captured two or more times. Most recaptured birds nested at the same nest boxes in subsequent years, and the number of birds that moved upslope did not significantly differ from the number that moved downslope. Fledglings moved greater distances and elevations than adults, but these movements were not upslope specific. Female fledglings showed greater changes in elevation and distance compared to male fledglings, but again, movements were not consistently upslope. The upslope shift in this population may be due to birds immigrating into the population and not from changes in individual nesting behavior.

How Cooperative Engagement Programs Strengthen Sequencing Capabilities for Biosurveillance and Outbreak Response.

The threat of emerging and re-emerging infectious diseases continues to be a challenge to public and global health security. Cooperative biological engagement programs act to build partnerships and collaborations between scientists and health professionals to strengthen capabilities in biosurveillance. Biosurveillance is the systematic process of detecting, reporting, and responding to especially dangerous pathogens and pathogens of pandemic potential before they become outbreaks, epidemics, and pandemics. One important tool in biosurveillance is next generation sequencing. Expensive sequencing machines, reagents, and supplies make it difficult for countries to adopt this technology. Cooperative engagement programs help by providing funding for technical assistance to strengthen sequencing capabilities. Through workshops and training, countries are able to learn sequencing and bioinformatics, and implement these tools in their biosurveillance programs. Cooperative programs have an important role in building and sustaining collaborations among institutions and countries. One of the most important pieces in fostering these collaborations is trust. Trust provides the confidence that a successful collaboration will benefit all parties involved. With sequencing, this enables the sharing of pathogen samples and sequences. Obtaining global sequencing data helps to identify unknown etiological agents, track pathogen evolution and infer transmission networks throughout the duration of a pandemic. Having sequencing technology in place for biosurveillance generates the capacity to provide real-time data to understand and respond to pandemics. We highlight the need for these programs to continue to strengthen sequencing in biosurveillance. By working together to strengthen sequencing capabilities, trust can be formed, benefitting global health in the face of biological threats.

Sex ratio of Western Bluebirds Sialia mexicana is mediated by phenology and clutch size.

Mothers may produce more of one sex to maximize their fitness if there are differences in the cost of producing each sex or there are differences in their relative reproductive value. Breeding date and clutch size are known to influence offspring sex ratios in birds through sex differences in dispersal, social behaviours, differential mortality, and available food resources. We tested if breeding date, clutch size and drought conditions influenced offspring sex ratios in a sexually size-monomorphic species, the Western Bluebird, by interrogating a 21-year dataset. After controlling for differential mortality, we found that hatch dates late in the breeding season were associated with the production of more females, suggesting that the value of producing males declines as the breeding season progresses. When clutch size was taken into account, small clutches yielded significantly more females late in the breeding season compared to the early and middle parts of the breeding season that produced significantly more males. Large clutches early in the season tended to produce more females, although this was not significant. Drought severity was not correlated with sex ratio adjustment. We propose and discuss several explanations for these patterns, including male offspring, but not female offspring, acting as helpers, increased female nestling provisioning late in the breeding season, differences in food abundance, and egg-laying order. Future work will help to uncover the mechanisms leading to these patterns. Identifying patterns and mechanisms of sex ratio skew from long-term datasets is important for informing predictions regarding life-history trade-offs in wildlife populations.

Phoresy in animals: review and synthesis of a common but understudied mode of dispersal.

Phoresy is a type of interaction in which one species, the phoront, uses another species, the dispersal host, for transportation to new habitats or resources. Despite being a widespread behaviour, little is known about the ecology and evolution of phoresy. Our goal is to provide a comprehensive review of phoretic dispersal in animals and to bring renewed attention to this subject. We surveyed literature published between 1900 and 2020 to understand the extent of known higher-level taxonomic diversity (phyla, classes, and orders) and functional aspects of animals that use phoretic dispersal. Species dispersing phoretically have been observed in at least 13 animal phyla, 25 classes, and 60 orders. The majority of known phoronts are arthropods (Phylum Euarthropoda) in terrestrial habitats, but phoronts also occur in freshwater and marine environments. Marine phoronts may be severely under-represented in the literature due to the relative difficulty of studying these systems. Phoronts are generally small with low mobility and use habitats or resources that are ephemeral and/or widely dispersed. Many phoronts are also parasites. In general, animals that engage in phoresy use a wide variety of morphological and behavioural traits for locating, attaching to, and detaching from dispersal hosts, but the exact mechanisms behind these activities are largely unknown. In addition to diversity, we discuss the evolution of phoresy including the long-standing idea that it can be a precursor to parasitism and other forms of symbioses. Finally, we suggest several areas of future research to improve our understanding of phoresy and its ecological and evolutionary significance.

Microbial and Geochemical Dynamics of an Aquifer Stimulated for Microbial Induced Calcite Precipitation (MICP).

Microbially induced calcite precipitation (MICP) is an alternative to existing soil stabilization techniques for construction and erosion. As with any biologically induced process in soils or aquifers, it is important to track changes in the microbial communities that occur as a result of the treatment. Our research assessed how native microbial communities developed in response to injections of reactants (dilute molasses as a carbon source; urea as a source of nitrogen and alkalinity) that promoted MICP in a shallow aquifer. Microbial community composition (16S rRNA gene) and ureolytic potential (ureC gene copy numbers) were also measured in groundwater and artificial sediment. Aquifer geochemistry showed evidence of sulfate reduction, nitrification, denitrification, ureolysis, and iron reduction during the treatment. The observed changes in geochemistry corresponded to microbial community succession in the groundwater and this matched parallel geophysical and mineralogical evidence of calcite precipitation in the aquifer. We detected an increase in the number of ureC genes in the microbial communities at the end of the injection period, suggesting an increase in the abundance of microbes possessing this gene as needed to hydrolyze urea and stimulate MICP. We identify geochemical and biological markers that highlight the microbial community response that can be used along with geophysical and geotechnical evidence to assess progress of MICP.

Correction: California condor microbiomes: Bacterial variety and functional properties in captive-bred individuals.

[This corrects the article DOI: 10.1371/journal.pone.0225858.].

Ontology-Based Graphs of Research Communities: A Tool for Understanding Threat Reduction Networks.

Scientific research communities can be represented as heterogeneous or multidimensional networks encompassing multiple types of entities and relationships. These networks might include researchers, institutions, meetings, and publications, connected by relationships like authorship, employment, and attendance. We describe a method for efficiently and flexibly capturing, storing, and extracting information from multidimensional scientific networks using a graph database. The database structure is based on an ontology that captures allowable types of entities and relationships. This allows us to construct a variety of projections of the underlying multidimensional graph through database queries to answer specific research questions. We demonstrate this process through a study of the U.S. Biological Threat Reduction Program (BTRP), which seeks to develop Threat Reduction Networks to build and strengthen a sustainable international community of biosecurity, biosafety, and biosurveillance experts to address shared biological threat reduction challenges. Networks like these create connectional intelligence among researchers and institutions around the world, and are central to the concept of cooperative threat reduction. Our analysis focuses on a series of seven BTRP genome sequencing training workshops, showing how they created a growing network of participants and countries over time, which is also reflected in coauthorship relationships among attendees. By capturing concept and relationship hierarchies, our ontology-based approach allows us to pose general or specific questions about networks within the same framework. This approach can be applied to other research communities or multidimensional social networks to capture, analyze, and visualize different types of interactions and how they change over time.

California condor microbiomes: Bacterial variety and functional properties in captive-bred individuals.

Around the world, scavenging birds such as vultures and condors have been experiencing drastic population declines. Scavenging birds have a distinct digestive process to deal with higher amounts of bacteria in their primary diet of carcasses in varying levels of decay. These observations motivate us to present an analysis of captive and healthy California condor (Gymnogyps californianus) microbiomes to characterize a population raised together under similar conditions. Shotgun metagenomic DNA sequences were analyzed from fecal and cloacal samples of captive birds. Classification of shotgun DNA sequence data with peptide signatures using the Sequedex package provided both phylogenetic and functional profiles, as well as individually annotated reads for targeted confirmatory analysis. We observed bacterial species previously associated with birds and gut microbiomes, including both virulent and opportunistic pathogens such as Clostridium perfringens, Propionibacterium acnes, Shigella flexneri, and Fusobacterium mortiferum, common flora such as Lactobacillus johnsonii, Lactobacillus ruminus, and Bacteroides vulgatus, and mucosal microbes such as Delftia acidovorans, Stenotrophomonas maltophilia, and Corynebacterium falsnii. Classification using shotgun metagenomic reads from phylogenetic marker genes was consistent with, and more specific than, analysis based on 16S rDNA data. Classification of samples based on either phylogenetic or functional profiles of genomic fragments differentiated three types of samples: fecal, mature cloacal and immature cloacal, with immature birds having approximately 40% higher diversity of microbes.

Long-term phenology of two North American secondary cavity-nesters in response to changing climate conditions.

Wildlife populations can respond to changes in climate conditions by either adapting or moving to areas with preferred climate regimes. We studied nesting responses of two bird species, western bluebird (Sialia mexicana) and ash-throated flycatcher (Myiarchus cinerascens), to changing climate conditions (i.e., rising temperatures and increased drought stress) over 21 years in northern New Mexico. We used data from 1649 nests to assess whether the two species responded to changing climate conditions through phenological shifts in breeding time or shifts in nesting elevation. We also examined changes in reproductive output (i.e., clutch size). Our data show that western bluebirds significantly increased nesting elevation over a 19-year period by approximately 5 m per year. Mean spring temperature was the best predictor of western bluebird nesting elevation. Higher nesting elevations were not correlated with hatch dates or clutch sizes in western bluebirds, suggesting that nesting at higher elevations does not affect breeding time or reproductive output. We did not observe significant changes in nesting elevation or breeding dates in ash-throated flycatchers. Nesting higher in elevation may allow western bluebirds to cope with the increased temperatures and droughts. However, this climate niche conservatism may pose a risk for the conservation of the species if climate change and habitat loss continue to occur. The lack of significant changes detected in nesting elevation, breeding dates, and reproductive output in ash-throated flycatchers suggests a higher tolerance for changing environmental conditions in this species. This is consistent with the population increases reported for flycatchers in areas experiencing dramatic climate changes.