An understanding of habitat relationships is essential for managing hunted species, such as cougar, that are difficult to census. In the first phase of this study, I used aerial telemetry data to examine diurnal cougar resource selection at 2 scales, and compared results between 2 study sites, the Oquirrh Mountains and Monroe Mountain, Utah. In the second phase of this study, I used conditional logistic regression models and GPS collar data from the Oquirrh Mountains to determine whether cougar resource selection varied over 3 behaviors (prey caching, resting at a daybed site, and nocturnal activities) and 2 scales. Results from phase 1 indicated that in general, during diurnal hours cougars selected for woodland cover types, moderate to steep (20-70%) slopes, canyon and steep hillside landforms, and home ranges with a higher density of edge. However, selection for these resources was not consistent at both study sites, scales, or for every cougar. Small sample sizes and poor spatial accuracy of the aerial telemetry data likely precluded the ability to detect selection in every case. Results from phase 2 indicated that cougar resource selection varied by behavior, and selection of some resources was detected only at certain scales. Cougar cache sites were characterized by southern and eastern aspects; lower elevation; avoidance of edge; a greater diversity of land cover types; canyon landforms (ridges were avoided); riparian, deciduous, and coniferous woodland; and deciduous and coniferous forest cover types. Cougars selected daybed sites that avoided western aspects and edges, were further from roads, closer to streams, higher in elevation, on moderate to steep slopes, and in rocky, deciduous woodland, and riparian cover types. During nocturnal activities, cougars avoided northern aspects, and selected areas that were closer to streams and roads, on edges and in canyon landforms, with gentler slopes, and rock, riparian, and deciduous woodland cover types. Results from cross-validation procedures confirmed that the models were reliable and predictive of cougar resource selection. This study provides a comprehensive analysis of cougar resource selection over various scales and behaviors. Managers should use caution when using diurnal data to make conclusions about selection during other times of day or behaviors.

Cougars (Puma concolor) are difficult to census due to their large home ranges, low densities, and cryptic nature. The conventional “gold-standard” method for estimating cougar abundance entails the capture and radio-tagging of individuals in a study area in an attempt to acquire a direct enumeration of animals in the population. While this method provides an accurate abundance estimate, it is logistically challenging and prohibitively expensive. Noninvasive survey techniques may offer the ability to both accurately and inexpensively monitor cougar populations. While noninvasive techniques have been used on cougar populations, there remain questions on their accuracy and comparative efficacy. We estimated the density of a cougar population in Northwest Wyoming using direct enumeration, and used this estimate as a reference with which to evaluate the accuracy and cost-effectiveness of three types of noninvasive surveys performed between 2010 and 2014. The noninvasive methods included two annual mark- recapture sessions of: 1) remote camera trapping, 2) winter hair-collection transects, and 3) scat detection dog surveys. We GPS tracked 13 adult cougars (males = 5, females = 8) over 3 annual periods (Sep 2010 – Sep 2013). We used proportional home range overlap to determine density in a 1,570 km2 area. The average density was 0.82 cougars/100 km2 (± 0.10 SD; n = 3 years). The remote camera surveys produced a mean density of 0.60 cougars/100 km2 (n = 2 years; relative SD = 56.5%). The scat detection dog surveys produced an average density of 2.41 cougars/100 km2 (n = 2 years; relative SD = 12.6%). The winter transects failed to produce a sample size large enough for an abundance estimate. Due to the inclusion of non-adults in the scat sampling, and the fact that the reference estimate was essentially a minimum count of adults, we believe that the scat-based estimate was more accurate than the lower estimate produced by remote cameras. Additional analysis indicated that individual identification of cougars in photographs may not be reliable, challenging the validity of photo-based abundance estimates of cougars. On a cost-per-detection basis, scat detection dogs were the most cost effective method (scat detection dogs = $341; remote cameras = $3,241; winter transects = $7,627).

Cougar (Puma concolor) predation has been identified as being one of several factors contributing to the decline of mule deer (Odocoileus hemionus) throughout the Western United States. In order to better understand how these elusive felines utilize their surroundings and prey, I examined and analyzed cougar predation behavior in North-Central Utah, using global positioning systems (GPS) data from 2002-2010. Twenty-three cougars were fitted with GPS collars and monitored for prey caching behavior. In total 775 potential cache sites were visited and 546 prey remains found. Mule deer comprised the majority of prey at cougar cache sites, but 11 other species were also found. Collectively, adult female mule deer were killed more than any other demographic class. Proportionally there was no difference in the sex or age class of deer killed by cougars in three different population segments, but seasonal differences were found in the number of kills made between cougar groups. Female cougars with kittens had a higher predation rate than males or solitary females, and seasonally more kills were made in the winter vs. summer. Cougars spent an average of 3.3 days on deer kills, and 6.2 days on elk kills. Habitat analyses suggested that cougars preferentially used Gambel oak (Quercus gambelii) over other land cover types when caching prey, as well as selected unburned over burned areas for caching and foraging on prey. These results suggest that cougars utilize dense stands of vegetation cover when stalking and concealing their prey. Wildlife managers may want to consider the use of prescribed burns in areas of high cougar predation on mule deer. This habitat manipulation tool could simultaneously help mule deer populations by reducing the percent of stalking cover afforded to cougars when attempting to kill prey, along with increasing nutrient levels of newly burned foliage and allow for an increased diversity in forb and shrub species available to mule deer.

Background: The p53-up-regulated modulator of apoptosis (PUMA) protein is a pro-apoptotic, BH3-only member of the BCL2 family of effector proteins responsible for promoting organized cell death. PUMA is required for resolution of pneumococcal pneumonia in mice, as mice deficient of PUMA exhibit greater numbers of S. pneumoniae CFU within tissues and higher mortality rates than observed in Puma+/+ mice. Methods: Puma+/+ and Puma-/- mice were intranasally challenged with TIGR4 pneumococcus and sacrificed 24 h post-infection. Differences in cytokine levels from blood and whole lung tissue were detected by MILLIPLEX MAP Mouse Cytokine/Chemokine Magnetic Bead Panel. Lung transcriptomes from Puma+/+ and Puma-/- mice were prepared from total lung RNA using NEBNext Poly(A) mRNA Magnetic Isolation Module and NEBNext Ultra RNA Library Prep Kit for Illumina. Libraries were read by Illumina NovaSeq and transcript reads were referenced to Mus musculus. Results: Puma-/- mice exhibited significant differences in G-CSF, GM-CSF, IFN-gamma, IL-1-alpha and -beta, -6, -9, -10, -12 (p40 and p70), -13, and -17, IP-10, KC, MCP-1, MIP- iv 1alpha and -beta, MIP-2, RANTES, and TNF-alpha compared to Puma+/+ mice. Puma-/- lungs exhibited higher levels of IL-12, IFN-gamma, and IP-10. Loss of PUMA also resulted in expression of the pro-angiogenic genes Adam19 and Neurexin2. Additionally, Puma+/+ and Puma-/- mice displayed similar levels of colonization, but Puma-/- mice were more susceptible to subsequent dissemination to the lungs and blood. Conclusion: Polymorphonuclear cells (PMNs) were previously demonstrated to be one of the innate cell types responsible for Puma-dependent resolution of pneumococcal pneumonia in mice. Observations reported here suggest that this resolution is propelled by suppressing the inflammatory response via the inhibition of IL-12/IFN-gamma/IP-10 pro-inflammatory axis. Pulmonary tissue transcriptomic analysis also suggests PUMA-dependent positive regulation of homeostatic control of pulmonary vasculature, smooth muscle innervation, and maintenance of the interstitium. Gene ontological analysis further demonstrated Puma's modulatory role in Type I and II IFN signaling. For the first time, we report Puma's regulatory effects on pro-inflammatory cytokine signaling and gene expression during pneumococcal pneumonia.

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El aislamiento genético debido a la fragmentación del hábitat y a la consecuente pérdida de conectividad del paisaje es considerado como la mayor amenaza para la conservación de los grandes carnívoros, como es el caso del puma (Puma concolor). Debido a sus características biológicas, los mamíferos carnívoros son particularmente vulnerables a la pérdida y fragmentación del hábitat, como también a la persecución humana. Los ecosistemas argentinos han sido fuertemente modificados en las últimas décadas por la expansión de las actividades humanas y, aunque en el país el puma está protegido por la Ley Nacional N° 22.421, la especie se encuentra sometida a una alta presión de caza debido al creciente conflicto con la ganadería. Enfocado en la conectividad funcional a nivel de paisaje, este estudio representa la primera evaluación de la genética de poblaciones de puma en Argentina. A partir del uso combinando de muestras invasivas y no invasivas, se propuso analizar la estructura poblacional y la distribución espacial de la diversidad de este felino a través el genotipado de marcadores microsatélites específicos. Además, la inclusión de provincias con diferente tipo de manejo permitió evaluar el efecto de la presión de la caza legal sobre la estructura genética de las poblaciones. Se analizó un total de 401 muestras (180 cueros, 117 huesos, 61 heces y 43 músculos) en total, correspondientes a un período entre 1925-2018. La amplificación de 25 loci de microsatélites permitió la obtención de 199 genotipos individuales, siendo los cueros y músculos las tipologías de muestra con el éxito de genotipado mayor (70 y 67 %, respectivamente) y los huesos con el menor (30,8 %). Todos los marcadores resultaron polimórficos con un número promedio de alelos por locus de 8,2 (min: 4, máx: 14). Los análisis de estructuración poblacional sugieren la presencia de 4 grupos (clusters) genéticamente distintos. La diversidad genética general resultó ser moderada (heterocigosidad esperada He= 0,722) y heterogéneamente distribuida a lo largo del área de estudio, identificándose los mayores valores en el centro-norte y los menores en el sur y el este del país. El flujo de genes entre clusters resultó ser limitado y variable (0,6-16,2 % por generación), soportando la agrupación definida anteriormente. En general las poblaciones de puma de Argentina resultaron genéticamente empobrecidas, evidenciando indicios de cuello de botella. El tamaño poblacional efectivo resultó, en la mayoría de los casos, inferior al valor mínimo para prevenir la pérdida de diversidad genética por endogamia. En efecto, todas las poblaciones resultaron moderadamente endogámicas (Fis= 0,10- 0,39). Debido a que la mayoría de los sitios de muestreo están conectados por un paisaje permeable a los movimientos de los pumas, la existencia de una estructuración espacial marcada para P. concolor pareciera estar fuertemente asociada con la persecución humana, evidenciándose una tendencia decreciente de flujo génico, al aumentar la presión de caza. Trabajos futuros deberían explorar aspectos demográficos de estas poblaciones, e identificar los factores que limitan su conectividad para que, de esta forma, se puedan generar estrategias que garanticen su viabilidad y la conservación de la especie. / Genetic isolation due to habitat fragmentation and the consequent loss of landscape connectivity represents the main threat to the conservation of large carnivores, such as puma (Puma concolor). Due to their biological traits, carnivores are particularly vulnerable to habitat loss and fragmentation, as well as human persecution. During the last decades, Argentine ecosystems have been highly modified by the expansion of human activities. Although the National Law N° 22.421 protects pumas in Argentina, the species is under high hunting pressure due to the increasing conflict with livestock. Focused on functional connectivity at landscape level, this study represents the first evaluation of puma population genetics in Argentina. The study, based on the combined use of invasive and non-invasive samples, aims to analyze the population structure and spatial distribution of its diversity genotyping specific microsatellite markers. Moreover, the involvement of many provinces with different kind of management for the species enabled the assessment of legal hunting pressure effect on population genetics. A total of 401 samples (180 skins, 117 bones, 61 scats and 43 muscles) were analyzed, corresponding to the period between years 1925-2018. The amplification of 25 microsatellite loci resulted in 199 individual genotypes, where skin and muscle samples showed the highest genotyping success (70 and 67 %, respectively), and bone samples the lowest (30.8 %). All markers were polymorphic with an average number of alleles per locus of 8.2 (min: 4, max: 14). Population structure analyses suggest the presence of four genetically distinct groups (clusters). The overall genetic diversity is quite high (expected heterozygosity He= 0.722) and not evenly distributed across the study area, identifying the highest values in the center-north and the lowest in the south and east of the country. Gene flow estimations were low and variable between clusters (0.6-16.2 % per generation), supporting the grouping defined above. Puma populations in Argentina are genetically depauperated, expressing evidences of bottleneck events. The effective population size was in most of the cases less than the minimum value to prevent the loss of genetic diversity due to inbreeding. In fact, all populations were moderately inbred (Fis= 0.10-0.39). Since almost all sampling sites are connected through a landscape permeable to puma’s movements, the strong spatial structure of P. concolor in Argentina would appear to be highly related to human persecution, evidencing a gene flow pattern decreasing as hunting pressure increases. Future work should explore demographic aspects of these populations, and identify the factors that limit their connectivity, in order to generate plans that guarantee their viability and the conservation of the species. / TEXTO PARCIAL en período de teletrabajo

A lo largo de su distribución el puma, Puma concolor, protagoniza complejas interacciones con los humanos. En el Espinal del sudoeste bonaerense, donde los hábitats naturales han sido alterados por las actividades agro-ganaderas, este felino es el foco de intensos conflictos con los productores, quienes responden a la predación de ganado matando al puma. El primer objetivo de esta tesis fue el de determinar los patrones de actividad y uso del hábitat del puma en el SO bonaerense, explorando el efecto de los factores antrópicos. El segundo fue evaluar los conflictos con los humanos, mediante una revisión global del conflicto humano-puma y la caracterización de esta problemática en el Espinal bonaerense. A través de una revisión bibliográfica, se describió el conflicto entre ganadería y puma en las Américas. Los conflictos ocurrieron en el 78,1% de las publicaciones (n=69) y fueron más comunes en América del Sur y Central. El ovino fue la especie preferida por el puma y la presencia de conflictos fue mayor en ambientes con abundante cobertura de vegetación, lejos de las rutas y a distancias medias de ciudades. Las áreas protegidas no evitaron la presencia del conflicto. El análisis mediante Modelos Lineales Generalizados (MLG) indicó que el conflicto ocurriría en zonas con altas densidades de vacunos, cerca de ambientes con abundante cobertura vegetal y con menor riqueza de co-predadores. Aun así, se evidencia la escasez de informaciones sobre este tema. Se realizaron tres campañas de trampeo fotográfico en los partidos de Villarino y Patagones, Pcia. de Buenos Aires, y se registraron 105 eventos de puma. Los MLG mixtos mostraron que la proporción de pastizales con arbustos y monte, la riqueza de presas silvestres y la diversidad ambiental tuvieron un efecto positivo, y significativo, sobre la intensidad de uso del hábitat del puma, mientras que la fragmentación catastral la afectó negativamente. Este felino presentó una actividad crepuscularnocturna, con leves diferencias estacionales. Se realizaron 213 entrevistas semi-estructuradas en ambos partidos e inspecciones directas a sitios de predación de ganado en un área de Patagones. La predación se reportó en el 39,7% (n=75) de las propiedades y causó una pérdida promedio de 2134,1±6049,2 USD, afectando principalmente la producción de ovejas. Todos los eventos de predación ocurrieron de noche y preferentemente en cultivos y pastizales con arbustos. Las tasas de mortalidad fueron de 3,7 y 1,1-1,56 individuos/añox100km2 para ovinos y pumas, respectivamente. Los recintos nocturnos parecieron disminuir las pérdidas de ovejas pero los productores consideraron la caza de puma como la forma más efectiva de reducir la predación. La cobertura de vegetación, la presión antrópica y la disponibilidad de presas fueron factores recurrentes en este trabajo y parecen determinantes no sólo en la presencia del conflicto humano-puma globalmente y en el Espinal bonaerense, sino también en el uso del hábitat de este felino en esta región. Para la conservación del puma en áreas agrícolo-ganaderas son necesarias estrategias que combinen herramientas que favorezcan la tolerancia de los productores y la mitigación de las pérdidas por predación con la conservación de ambientes y especies presas claves del felino. / Throughout its distribution range, Puma concolor is involved in complex interactions with humans. In the Argentinean Espinal of the southwest of Buenos Aires province, where natural habitats have been altered by agricultural and livestock activities, this feline is the focus of intense conflicts with ranchers, who respond to predation on livestock by killing pumas. The first objective of this thesis was to determine the puma activity and habitat use patterns in southern Buenos Aires province, exploring the effect of human factors . The second was to carry out a review of human-puma conflicts through its distribution and characterize them in the southwest of the Espinal ecoregion. Human-puma conflict was described in the American continent through a bibliographical review. Conflicts occurred in 78,1% (n=69) of the literature and were more common in South and Central America. Sheep were the species preferred by pumas and the presence of conflicts was greater in areas with high vegetation coverage, far from main roads and at average distances of cities. Protected areas did not prevent the occurrence of conflict. Generalized Linear Models (GLM) identified that conflict would occur in areas with high cattle densities, in proximity of habitat with high vegetation coverage and with low co-predators richness. Lack of information on this subject is highlighted. 105 puma events were collected in three photographic trapping surveys carried out in Villarino and Patagones counties. The mixed GLMs showed that the proportion of grasslands with bushes and bushland, wild prey richness and habitat diversity had positive and significant effects on puma habitat use, while cadastral fragmentation affected it negatively. This feline presented a crepuscular-nocturnal activity with seasonal variations. Semi-structured interviews (n=213) were completed out in both counties and direct inspections on livestock kill site were carried out in an area of Patagones county. Predation was reported in 39,7% (n=75) of the ranches and caused an average loss of 2134,1±6049,2 USD, affecting mainly sheep production. All predation events occurred at night and preferably in cropland and grasslands with shrubs. Mortality rates were 3,7 and 1,1-1,56 individuals/year x 100km2 for sheep and pumas, respectively. Night enclosures appeared to reduce sheep losses, but puma hunting was considered by ranchers the most effective and implemented method of reducing predation. Vegetation coverage, anthropogenic pressure and prey availability were recurrent factors in this study and seem to be determinant not only in the presence of human-puma conflict globally and in the Espinal of Buenos Aires province, but also in the habitat use of this feline in this region. Puma conservation strategies in rangeland and agricultural- landscapes need to combine tools favoring tolerance by ranchers and mitigation of depredation losses with the conservation of key habitat and wild prey.

The elusive jaguar (Panthera onca) is extremely difficult to study due to its wide-ranging behavior, crepuscular activity peaks and its occurrence in low population densities in often dense forest habitats. Jaguars are also a species of concern, but our ability to provide for their survival is hampered by our inability to obtain reliable information on the status of their wild populations. This study combines innovative noninvasive research techniques such as scat detector dogs and molecular scatology to conduct the first genetic study on wild populations of Neotropical felids coexisting across fragmented forest habitats in Belize, Central America. Specifically, we analyzed multi-locus data in jaguars (Panthera onca), pumas (Puma concolor) and ocelots (Leopardus pardalis) collected from 1053 scat samples across their range in the country. First, we optimized 14 polymorphic microsatellite loci for jaguars (Panthera onca), pumas (Puma concolor), and ocelots (Leopardus pardalis), and assessed their utility for cross-species amplification. Additionally, we tested their reliability for species and individual identification using fecal DNA as he primary DNA source. All microsatellite loci examined successfully cross-amplified in the three target species, and were polymorphic. Second, to maximize PCR amplification success and genotyping accuracy rates, and to minimize genotyping error rates for fecal DNA samples, we evaluated the performance of two fecal DNA storage techniques (dimethyl sulfoxide saline solution/DET buffer, 95% EtOH) suitable for long-term preservation at remote tropical sites. Additionally, we tested fecal DNA samples collected from four different scat locations (top, side, bottom, inside). DET buffer was the superior fecal DNA preservation method and collecting fecal DNA from side and top locations of the scat resulted in the highest PCR success rates. For the main genetic study, we assessed the genetic conservation status of all three target species across the country of Belize. We examined levels of genetic diversity within different sites, (2) defined potential genetic clusters/populations, (3) and examined levels of gene flow and population structure for all three target species on a countrywide scale. Furthermore, we compared genetic diversity and gene flow levels among the three target species. Wild felids in Belize showed moderate levels of heterozygosity (HE = 0.60 - 0.70) with jaguars having the lowest genetic diversity with average expected heterozygosities of HE = 0.60 ± 0.05 and allelic richness (AR) of 4.94 ± 0.44 followed by pumas with HE = 0.65 ± 0.06 and AR of 7.52 ± 0.86 and ocelots with HE = 0.70 ± 0.05 and AR of 3.89 ± 0.23. We observed low to moderate levels of differentiation (FST = 0.00 - 0.15) and weak population structure using spatial Bayesian clustering techniques for all three target species. Although levels of genetic diversity and gene flow across the country are still fairly high, we did detect evidence of fragmentation indicating the risk of further habitat loss and fragmentation for wild felids. Felids were simultaneously monitored across all study sites by remote sensing camera traps, which allows for a comparison of density estimates obtained from two different noninvasive survey approaches. Furthermore, analytical methods for density estimation are advancing rapidly, making it difficult to choose the optimal technique. Thus, we compared a variety of density estimators including the conventional approach of estimating abundance ( ) in programs CAPTURE and MARK and dividing abundance by the effective trapping area (ETA), the recently developed spatially explicit capture-recapture (SECR) models, both the likelihood-based approach (ML-SECR) in program DENSITY and the Bayesian approach (B-SECR) in program SPACECAP, and finally the genetic-based mark-recapture one sampling occasion estimator in program CAPWIRE. Although different survey methods using various density estimators produced similar density estimates, confidence levels and coefficients of variation varied, with SECR methods resulting in the least precise estimates. Detection probabilities were generally higher for noninvasive genetic sampling than for camera trapping. Both techniques were shown to be reliable and highly efficient survey methods for density estimation of low-density Neotropical felids living in challenging environments such as the tropics. While less precise, SECR CMR models are probably a more realistic reflection of our uncertainty. They hold great promise for density estimation studies for wide-ranging and territorial carnivore species, especially if precision can be improved through study design or analysis advancements in the future. In conclusion, our results demonstrated that noninvasive sampling techniques such as molecular scatology and remote camera trapping are efficient research approaches to study multiple Neotropical felids in a multifaceted way and on a countrywide scale. We believe that the techniques and analyses developed in this study are widely applicable and relevant to the conservation and management of other elusive and difficult to study wild felids worldwide. / Ph. D.