Citellinema (Nematoda: Heligmosomidae) from North America with descriptions of 2 new species from the red squirrel Tamiasciurus hudsonicus and 1 from the Canadian woodchuck, Marmota monax.

Citellinema Hall, 1918 includes 6 valid species of gastrointestinal nematodes of sciurids. Two species occur in the Palearctic and 4 in the Nearctic, 3 of which occur minimally across Colorado, Idaho and Oregon and 1, Citellinema bifurcatum, has a wide distribution across North America. Members of the genus are didelphic, possess a cephalic vesicle, a terminal spine-like process in females and feature robust spicules, consisting of a proximal end fused and semicylindrical shaft connected to a lamina supported by 2 terminal filiform processes. Typically, the size of the spicules is used to differentiate species. As part of the Beringian Coevolution Project, specimens provisionally identified as C. bifurcatum were collected through intensive field sampling of mammals and associated parasites from across localities spanning the Holarctic. These specimens revealed considerable genetic variability at both mitochondrial and nuclear loci, supporting the identification of deeply divergent clades. Examination of these new specimens, along with the holotypes of C. bifurcatum and Citellinema quadrivittati indicates that Citellinema monacis (previously synonymized with C. bifurcatum) should be resurrected and 3 additional species described. We suggest that the apparent bifurcated nature of the spicule should be considered a generic diagnostic trait, while the proportional length of the lamina relative to that of the spicule is used as a specific character. We demonstrate the critical need for continued inventory of often poorly known assemblages of hosts and parasites, contributing to a growing baseline of archival specimens, collections and information that make explorations of faunal structure and diversity possible.

Maternal glucocorticoids have minimal effects on HPA axis activity and behavior of juvenile wild North American red squirrels.

As a response to environmental cues, maternal glucocorticoids (GCs) may trigger adaptive developmental plasticity in the physiology and behavior of offspring. In North American red squirrels (Tamiasciurus hudsonicus), mothers exhibit increased GCs when conspecific density is elevated, and selection favors more aggressive and perhaps more active mothers under these conditions. We tested the hypothesis that elevated maternal GCs cause shifts in offspring behavior that may prepare them for high-density conditions. We experimentally elevated maternal GCs during gestation or early lactation. We measured two behavioral traits (activity and aggression) in weaned offspring using standardized behavioral assays. Because maternal GCs may influence offspring hypothalamic-pituitary-adrenal (HPA) axis dynamics, which may in turn affect behavior, we also measured the impact of our treatments on offspring HPA axis dynamics (adrenal reactivity and negative feedback), and the association between offspring HPA axis dynamics and behavior. Increased maternal GCs during lactation, but not gestation, slightly elevated activity levels in offspring. Offspring aggression and adrenal reactivity did not differ between treatment groups. Male, but not female, offspring from mothers treated with GCs during pregnancy exhibited stronger negative feedback compared with those from control mothers, but there were no differences in negative feedback between lactation treatment groups. Offspring with higher adrenal reactivity from mothers treated during pregnancy (both controls and GC-treated) exhibited lower aggression and activity. These results suggest that maternal GCs during gestation or early lactation alone may not be a sufficient cue to produce substantial changes in behavioral and physiological stress responses in offspring in natural populations.

Optimisation of energetic and reproductive gains explains behavioural responses to environmental variation across seasons and years.

Animals switch between inactive and active states, simultaneously impacting their energy intake, energy expenditure and predation risk, and collectively defining how they engage with environmental variation and trophic interactions. We assess daily activity responses to long-term variation in temperature, resources and mating opportunities to examine whether individuals choose to be active or inactive according to an optimisation of the relative energetic and reproductive gains each state offers. We show that this simplified behavioural decision approach predicts most activity variation (R2  = 0.83) expressed by free-ranging red squirrels over 4 years, as quantified through accelerometer recordings (489 deployments; 5066 squirrel-days). Recognising activity as a determinant of energetic status, the predictability of activity variation aggregated at a daily scale, and the clear signal that behaviour is environmentally forced through optimisation of gain, provides an integrated approach to examine behavioural variation as an intermediary between environmental variation and energetic, life-history and ecological outcomes.

The new kid on the block: immigrant males win big whereas females pay fitness cost after dispersal.

Dispersal is nearly universal; yet, which sex tends to disperse more and their success thereafter depends on the fitness consequences of dispersal. We asked if lifetime fitness differed between residents and immigrants (successful between-population dispersers) and their offspring using 29 years of monitoring from North American red squirrels (Tamiasciurus hudsonicus) in Canada. Compared to residents, immigrant females had 23% lower lifetime breeding success (LBS), while immigrant males had 29% higher LBS. Male immigration and female residency were favoured. Offspring born to immigrants had 15-43% lower LBS than offspring born to residents. We conclude that immigration benefitted males, but not females, which appeared to be making the best of a bad lot. Our results are in line with male-biased dispersal being driven by local mate competition and local resource enhancement, while the intergenerational cost to immigration is a new complication in explaining the drivers of sex-biased dispersal.

Maternal glucocorticoids promote offspring growth without inducing oxidative stress or shortening telomeres in wild red squirrels.

Elevations in glucocorticoid (GC) levels in breeding females may induce adaptive shifts in offspring life histories. Offspring produced by mothers with elevated GCs may be better prepared to face harsh environments, where a faster pace of life is beneficial. We examined how experimentally elevated GCs in pregnant or lactating North American red squirrels (Tamiasciurus hudsonicus) affected offspring postnatal growth, structural size and oxidative stress levels (two antioxidants and oxidative protein damage) in three different tissues (blood, heart and liver) and liver telomere lengths. We predicted that offspring from mothers treated with GCs would grow faster but would also have higher levels of oxidative stress and shorter telomeres, which may predict reduced longevity. Offspring from mothers treated with GCs during pregnancy were 8.3% lighter around birth but grew (in body mass) 17.0% faster than those from controls, whereas offspring from mothers treated with GCs during lactation grew 34.8% slower than those from controls and did not differ in body mass around birth. Treating mothers with GCs during pregnancy or lactation did not alter the oxidative stress levels or telomere lengths of their offspring. Fast-growing offspring from any of the treatment groups did not have higher oxidative stress levels or shorter telomere lengths, indicating that offspring that grew faster early in life did not exhibit oxidative costs after this period of growth. Our results indicate that elevations in maternal GCs may induce plasticity in offspring growth without long-term oxidative costs to the offspring that might result in a shortened lifespan.

Territory acquisition mediates the influence of predators and climate on juvenile red squirrel survival.

Juvenile survival to first breeding is a key life-history stage for all taxa. Survival through this period can be particularly challenging when it coincides with harsh environmental conditions such as a winter climate or food scarcity, leading to highly variable cohort survival. However, the small size and dispersive nature of juveniles generally make studying their survival more difficult. In territorial species, a key life-history event is the acquisition of a territory. A territory is expected to enhance survival, but how it does so is not often identified. We tested how the timing of territory acquisition influenced the winter survival of juvenile North American red squirrels Tamiasciurus hudsonicus, hereafter red squirrels, and how the timing of this event mediated the sources of mortality. We hypothesized that securing a territory prior to when food resources become available would reduce juvenile susceptibility to predation and climatic factors overwinter. Using 27 years of data on the survival of individually marked juvenile red squirrels, we tested how the timing of territory acquisition influenced survival, whether the population density of red squirrel predators and mean temperature overwinter were related to individual survival probability, and if territory ownership mediated these effects. Juvenile red squirrel survival was lower in the years of high predator abundance and in colder winters. Autumn territory owners were less susceptible to lynx Lynx canadensis and possibly mustelid Mustela and Martes spp., predation. Autumn territory owners had lower survival in colder winters, but surprisingly non-owners had higher survival in cold winters. Our results show how the timing of a life-history event like territory acquisition can directly affect survival and also mediate the effects of biotic and abiotic factors later in life. This engenders a better understanding of the fitness consequences of the timing of key life-history events.

Indirect effects on fitness between individuals that have never met via an extended phenotype.

Interactions between organisms are ubiquitous and have important consequences for phenotypes and fitness. Individuals can even influence those they never meet, if they have extended phenotypes that alter the environments others experience. North American red squirrels (Tamiasciurus hudsonicus) guard food hoards, an extended phenotype that typically outlives the individual and is usually subsequently acquired by non-relatives. Hoarding by previous owners can, therefore, influence subsequent owners. We found that red squirrels breed earlier and had higher lifetime fitness if the previous hoard owner was a male. This was driven by hoarding behaviour, as males and mid-aged squirrels had the largest hoards, and these effects persisted across owners, such that if the previous owner was male or died in mid-age, subsequent occupants had larger hoards. Individuals can, therefore, influence each other's resource-dependent traits and fitness without ever meeting, such that the past can influence contemporary population dynamics through extended phenotypes.

Social effects of territorial neighbours on the timing of spring breeding in North American red squirrels.

Organisms can affect one another's phenotypes when they socially interact. Indirect genetic effects occur when an individual's phenotype is affected by genes expressed in another individual. These heritable effects can enhance or reduce adaptive potential, thereby accelerating or reversing evolutionary change. Quantifying these social effects is therefore crucial for our understanding of evolution, yet estimates of indirect genetic effects in wild animals are limited to dyadic interactions. We estimated indirect phenotypic and genetic effects, and their covariance with direct effects, for the date of spring breeding in North American red squirrels (Tamiasciurus hudsonicus) living in an array of territories of varying spatial proximity. Additionally, we estimated indirect effects and the strength of selection at low and high population densities. Social effects of neighbours on the date of spring breeding were different from zero at high population densities but not at low population densities. Indirect phenotypic effects accounted for a larger amount of variation in the date of breeding than differences attributable to the among-individual variance, suggesting social interactions are important for determining breeding dates. The genetic component to these indirect effects was however not statistically significant. We therefore showcase a powerful and flexible method that will allow researchers working in organisms with a range of social systems to estimate indirect phenotypic and genetic effects, and demonstrate the degree to which social interactions can influence phenotypes, even in a solitary species.

Is biasing offspring sex ratio adaptive? A test of Fisher's principle across multiple generations of a wild mammal in a fluctuating environment.

Fisher's principle explains that population sex ratio in sexually reproducing organisms is maintained at 1 : 1 owing to negative frequency-dependent selection, such that individuals of the rare sex realize greater reproductive opportunity than individuals of the more common sex until equilibrium is reached. If biasing offspring sex ratio towards the rare sex is adaptive, individuals that do so should have more grandoffspring. In a wild population of North American red squirrels (Tamiasciurus hudsonicus) that experiences fluctuations in resource abundance and population density, we show that overall across 26 years, the secondary sex ratio was 1 : 1; however, stretches of years during which adult sex ratio was biased did not yield offspring sex ratios biased towards the rare sex. Females that had litters biased towards the rare sex did not have more grandoffspring. Critically, the adult sex ratio was not temporally autocorrelated across years, thus the population sex ratio experienced by parents was independent of the population sex ratio experienced by their offspring at their primiparity. Expected fitness benefits of biasing offspring sex ratio may be masked or negated by fluctuating environments across years, which limit the predictive value of the current sex ratio.

Use of infrared thermography to detect reactions to stressful events: does animal personality matter?

The study of the relationship between animal stress and personality for free-ranging animals is limited and provides contrasting results. The perception of stressors by an individual may vary due to its personality, and certain personality traits may help individuals to better cope with them. Using non-invasive infrared thermography (IRT), we investigated the link between physiological and behavioral components expressed during an acute stress event by free-ranging Fremont's squirrels (Tamiasciurus fremonti). We expected that, during the acute stress event of being approached by the researcher, individuals that showed a fast pace-of-life syndrome (bolder, more active, and less social/more aggressive) based on an arena test would exhibit stronger sympathetic-adrenal-medullary system reactivity showing a more intense stress-induced hyperthermia (high core body temperature and low peripheral temperature) than individuals with a slow pace of life (shy, less active, and more social). We successfully employed IRT technology to images of Fremont's squirrels with identification of the individuals' body parts (eye, nose, ear, hind foot). However, we found no support for our hypothesis. Squirrels' body surface temperatures told us more about a squirrel's external environment and less about the thermal state of the body in that environment following a stressful event. Further studies need to assess how to make IRT effective and efficient in the field and improve its performance in studying the relationships between physiology and personality in wildlife.

High-resolution prediction of American red squirrel in Interior Alaska: a role model for conservation using open access data, machine learning, GIS and LIDAR.

American red squirrels (Tamiasciurus hudsonicus) are small mammals that are abundantly distributed throughout North America. Urbanization in the Anthropocene is now a global process, and squirrels live in affected landscapes. This leads to squirrels adjusting to human developments. Not much is known about the distribution of squirrels and squirrel middens near humans, especially not in the subarctic and sub-urbanized regions. Although this species is hunted, there are no real publicly available distribution and abundance estimates nor management plans and bag limits for squirrels in Alaska or in the United States known by us, except the endangered Mt. Graham squirrel. In general, insufficient squirrel conservation research is carried out; they are underrepresented in research and its literature. To further the science-based management for such species, this study aims to generate the first digital open access workflow as a generic research template for small mammal work including the latest machine learning of open source and high-resolution LIDAR data in an Open Source Geographic Information System (QGIS) and ArcGIS. Machine learning has proven to be less modeler biased and improve accuracy of the analysis outcome, therefore it is the preferred approach. This template is designed to be rapid, simple, robust, generic and effective for being used by a global audience. As a unique showcase, here a squirrel midden survey was carried out for two years (2016 and 2017). These squirrel middens were detected in a research area of 45,5 hectares (0,455 km2) in downtown Fairbanks, interior boreal forest of Alaska, U.S. Transect distances were geo-referenced with a GPS and adjusted to the visual conditions to count all squirrel middens within the survey area. Different layers of proximity to humans and habitat characteristics were assembled using aerial imagery and LIDAR data (3D data needed for an arboreal species like the red squirrels) consisting of a 3 × 3 m resolution. The layer data was used to train a predictive distribution model for red squirrel middens with machine learning. The model showed the relative index of occurrence (RIO) in a map and identified canopy height, distance to trails, canopy density and the distance to a lake, together, as the strongest predictors for squirrel midden distribution whereas open landscape and disturbed areas are avoided. It is concluded that squirrels select for high and dense forests for middens while avoiding human disturbance. This study is able to present a machine learning template to easily and rapidly produce an accurate abundance prediction which can be used for management implications.

Enhanced seed defenses potentially relax selection by seed predators against serotiny in lodgepole pine.

Serotiny, the retention of seeds in a canopy seed bank until high temperatures cause seeds to be released, is an important life history trait for many woody plants in fire-prone habitats. Serotiny provides a competitive advantage after fire but increases vulnerability to predispersal seed predation, due to the seeds being retained in clusters in predictable locations for extended periods. This creates opposing selection pressures. Serotiny is favored in areas of high fire frequency, but is selected against by predispersal seed predators. However, predation also selects for cone traits associated with seed defense that could reduce predation on serotinous cones and thereby relax selection against serotiny. This helps explain the elevated defenses in highly serotinous species. However, whether such interactions drive variation in seed defenses within variably serotinous populations has been studied rarely. We investigated the effects of phenotypic selection exerted by red squirrel (Tamiasciurus hudsonicus) predation on Rocky Mountain lodgepole pine (Pinus contorta latifolia) seeds. Squirrels preferentially harvested cones with more and larger seeds, indicating a preference for a higher food reward. We found evidence for stronger selection on trees with serotinous cones, which presumably accounts for the elevated defenses of and lower predation on serotinous compared to non-serotinous cones. Lower levels of predation on serotinous cones in turn lessen selection against serotiny by squirrels. This has important implications because the frequency of serotiny in lodgepole pine has profound consequences for post-fire communities and ecosystems widespread in the Rocky Mountains.

Isolation of the Lyme Disease Spirochete Borrelia mayonii From Naturally Infected Rodents in Minnesota.

Borrelia mayonii is a newly described member of the Borrelia burgdorferi sensu lato complex that is vectored by the black-legged tick (Ixodes scapularis Say) and a cause of Lyme disease in Minnesota and Wisconsin. Vertebrate reservoir hosts involved in the enzootic maintenance of B. mayonii have not yet been identified. Here, we describe the first isolation of B. mayonii from naturally infected white-footed mice (Peromyscus leucopus Rafinesque) and an American red squirrel (Tamiasciurus hudsonicus Erxleben) from Minnesota, thus implicating these species as potential reservoir hosts for this newly described spirochete.

Multilevel and sex-specific selection on competitive traits in North American red squirrels.

Individuals often interact more closely with some members of the population (e.g., offspring, siblings, or group members) than they do with other individuals. This structuring of interactions can lead to multilevel natural selection, where traits expressed at the group-level influence fitness alongside individual-level traits. Such multilevel selection can alter evolutionary trajectories, yet is rarely quantified in the wild, especially for species that do not interact in clearly demarcated groups. We quantified multilevel natural selection on two traits, postnatal growth rate and birth date, in a population of North American red squirrels (Tamiasciurus hudsonicus). The strongest level of selection was typically within-acoustic social neighborhoods (within 130 m of the nest), where growing faster and being born earlier than nearby litters was key, while selection on growth rate was also apparent both within-litters and within-study areas. Higher population densities increased the strength of selection for earlier breeding, but did not influence selection on growth rates. Females experienced especially strong selection on growth rate at the within-litter level, possibly linked to the biased bequeathal of the maternal territory to daughters. Our results demonstrate the importance of considering multilevel and sex-specific selection in wild species, including those that are territorial and sexually monomorphic.

Altered natal dispersal at the range periphery: The role of behavior, resources, and maternal condition.

Natal dispersal outcomes are an interplay between environmental conditions and individual phenotypes. Peripheral, isolated populations may experience altered environmental conditions and natal dispersal patterns that differ from populations in contiguous landscapes. We document nonphilopatric, sex-biased natal dispersal in an endangered small mammal, the Mt. Graham red squirrel (Tamiasciurus hudsonicus grahamensis), restricted to a single mountain. Other North American red squirrel populations are shown to have sex-unbiased, philopatric natal dispersal. We ask what environmental and intrinsic factors may be driving this atypical natal dispersal pattern. We test for the influence of proximate factors and ultimate drivers of natal dispersal: habitat fragmentation, local population density, individual behavior traits, inbreeding avoidance, competition for mates, and competition for resources, allowing us to better understand altered natal dispersal patterns at the periphery of a species' range. A juvenile squirrel's body condition and its mother's mass in spring (a reflection of her intrinsic quality and territory quality) contribute to individual behavioral tendencies for movement and exploration. Resources, behavior, and body condition have the strongest influence on natal dispersal distance, but affect males and females differently. Male natal dispersal distance is positively influenced by its mother's spring body mass and individual tendency for movement; female natal dispersal distance is negatively influenced by its mother's spring body mass and positively influenced by individual tendency for movement. An apparent feedback between environmental variables and subsequent juvenile behavioral state contributes to an altered natal dispersal pattern in a peripheral population, highlighting the importance of studying ecological processes at the both range center and periphery of species' distributions.

From the ground up: biotic and abiotic features that set the course from genes to ecosystems.

Spatial variation in cone serotiny in Rocky Mountain lodgepole pine (Pinus contorta ssp. latifolia) across Yellowstone National Park influences initial pine recruitment after stand-replacing fire with tremendous population, community, and ecosystem consequences. A previous study showed that much of the spatial variation in serotiny results from the balance of selection arising from high frequencies of fire favoring serotiny countered by opposing selection exerted by American red squirrels (Tamiasciurus hudsonicus) as seed predators. This earlier study, however, assumed stable local red squirrel densities over multiple generations of pines. Here, we examine environmental properties that might contribute to long-term stability in the densities of red squirrels among sites. We found that the amount of clay in the soil, an indicator of plant and fungal growth-the latter an important food resource for red squirrels-and the coefficient of variation (CV) in diameter at breast height (DBH) of forest trees together account for a substantial amount of variation in red squirrel density. Soil development occurs over very long time scales, and thus, intersite variation in the amount of clay is unlikely to shift across pine generations. However, CV of DBH and squirrel density increase with stand age, which acts to amplify selection against serotiny with increasing interfire interval. Regardless, much of the variation in the CV of DBH is accounted for by soil bulk density, mean annual temperature, and surface curvature, which are unlikely to vary in their relative differences among sites over time. Consequently, these soil and abiotic attributes could contribute to consistent spatial patterns of red squirrel densities from one pine generation to the next, resulting in consistent local and spatial variation in selection exerted by red squirrels against serotiny.

Patterns of ectoparasitism in North American red squirrels (Tamiasciurus hudsonicus): Sex-biases, seasonality, age, and effects on male body condition.

Within many species, males are often more heavily parasitised than females. Several hypotheses have been proposed to explain this phenomenon, including immunocompetence handicaps, sexual size dimorphism and behavioural differences. Here we set out to test the latter two hypotheses and make inferences about the former by assessing patterns of ectoparasitism across various life-history stages in a population of North American red squirrels (Tamiasciurus hudsonicus). We also conducted an ectoparasite removal experiment to investigate the effects of ectoparasites on male body condition. We found that males were more intensely parasitized than females, but only during the mating period. There was no difference in ectoparasite intensity between male and female juveniles at birth or at emergence, suggesting that ectoparasites do not exploit male red squirrels for longer-range natal dispersal. Male red squirrels in our population were slightly heavier than females, however we did not find any evidence that this dimorphism drives male-biased ectoparasitism. Finally, we could not detect an effect of ectoparasite removal on male body mass. Our results lend support to the hypothesis that ectoparasites exploit their male hosts for transmission and that male red squirrels are important for the transmission dynamics of ectoparasites in this population; however, the mechanisms (i.e., immunocompetence, testosterone) are not known.

Influence of Diversionary Food on Red Squirrel Populations and Damage to Crop Trees in Young Lodgepole Pine Forest.

This study was designed to test the hypothesis that large-scale provision of diversionary food (sunflower seeds) would reduce red squirrel (Tamiasciurus hudsonicus) feeding damage to lodgepole pine (Pinus contorta) crop trees. Study areas with managed lodgepole pine stands were located near Vernon and Quesnel in south-central British Columbia, Canada. Large-scale applications of sunflower seeds were conducted on a manual basis in 1989, and by manual and aerial means in 1990, and an operational level by aerial means in 1991. Feeding damage to crop trees was assessed in control and treatment blocks. Populations of the red squirrel, northwestern chipmunk (Eutamias amoenus), and Columbian ground squirrel (Spermophilus columbianus) were sampled intensively by live-trapping on control and treatment blocks in 1990. Manual application of seed (clumped distribution) significantly reduced damage in the treatment block (11.3% of trees damaged) compared with the control (57.5% of trees damaged). Aerial application of seed (uniform distribution) also significantly reduced damage in replicated treatment vs. control blocks. Provision of diversionary food resulted in a temporary increase in the overall number of red squirrels caught on the treatment areas followed by a return to control levels within 6 wk. This increase was primarily the result of an increased number of transients in the trapped sample. The population density of resident (transients excluded) red squirrels did not increase when diversionary food was added. Similarly, we could not detect differences in reproduction, body masses, or survival of squirrels between control and food-supplemented areas. Northwestern chipmunks and Columbian ground squirrels also showed a temporary increase in density when food was added. Application of sunflower seed on an operational basis significantly reduced damage by squirrels in replicated study areas covering three different forest ecological zones. Provision of diversionary food is an effective strategy to protect intensively managed stands of lodgepole pine from red squirrel feeding damage.

Diversification and gene flow in nascent lineages of island and mainland North American tree squirrels (Tamiasciurus).

Pleistocene climate cycles and glaciations had profound impacts on taxon diversification in the Boreal Forest Biome. Using population genetic analyses with multilocus data, we examined diversification, isolation, and hybridization in two sibling species of tree squirrels (Tamiasciurus douglasii and Tamiasciurus hudsonicus) with special attention to the geographically and genetically enigmatic population of T. hudsonicus on Vancouver Island, Canada. The two species differentiated only about 500,000 years ago, in the Late Pleistocene. The island population is phylogenetically nested within T. hudsonicus according to our nuclear analysis but within T. douglasii according to mitochondrial DNA. This conflict is more likely due to historical hybridization than to incomplete lineage sorting, and it appears that bidirectional gene flow occurred between the island population and both species on the mainland. This interpretation of our genetic analyses is consistent with our bioclimatic modeling, which demonstrates that both species were able to occupy this region throughout the Late Pleistocene. The divergence of the island population 40,000 years ago suggests that tree squirrels persisted in a refugium on Vancouver Island at the last glacial maximum, 20,000 years ago. Our observations demonstrate how Pleistocene climate change and habitat shifts have created incipient divergence in the presence of gene flow. Sequence data have been archived in GenBank­accession numbers: KF882736­KF885216.

THE IMPACT OF TREE SQUIRRELS (TAMIASCIURUS) ON LIMBER PINE SEED DISPERSAL ADAPTATIONS.

Tree squirrels (Tamiasciurus) are important selective agents on conifer reproductive strategies (Smith 1970, 1975). Although this is well established for wind-dispersed pines, the impact of tree squirrels on bird-dispersed pines has been largely ignored. I assessed the impact of tree squirrels on the allocation of reproductive energy in the bird-dispersed limber pine (Pinus flexilis) by comparing its cone and seed traits from three sites in the Rocky Mountains where tree squirrels (Tamiasciurus) are present to those from three mountain ranges in the Great Basin where tree squirrels are absent. As predicted, differences between the two regions in individual cone and seed traits are consistent with the hypothesis that tree squirrels are important selective agents on these traits. In the absence of tree squirrels, limber pine allocates more than twice as much energy to kernel compared with that invested in putative seed defenses (cone, resin, and seed coat) as does limber pine where tree squirrels are present. Such a large difference is particularly striking, because tree squirrels may have become extinct in the Great Basin in only the last 12,000 yr. Although many factors influence the allocation of energy to cones and seeds, no single factor other than the presence of tree squirrels is compatible with the large and consistent differences between limber pine in the Rocky Mountains and Great Basin. These results show that tree squirrels are an important constraint on the evolution of cone and seed traits that promote the dispersal of seeds by birds.