Population genomic structure of a widespread, urban-dwelling mammal: The eastern grey squirrel (Sciurus carolinensis).

Urbanization is a persistent and widespread driver of global environmental change, potentially shaping evolutionary processes due to genetic drift and reduced gene flow in cities induced by habitat fragmentation and small population sizes. We tested this prediction for the eastern grey squirrel (Sciurus carolinensis), a common and conspicuous forest-dwelling rodent, by obtaining 44K SNPs using reduced representation sequencing (ddRAD) for 403 individuals sampled across the species' native range in eastern North America. We observed moderate levels of genetic diversity, low levels of inbreeding, and only a modest signal of isolation-by-distance. Clustering and migration analyses show that estimated levels of migration and genetic connectivity were higher than expected across cities and forested areas, specifically within the eastern portion of the species' range dominated by urbanization, and genetic connectivity was less than expected within the western range where the landscape is fragmented by agriculture. Landscape genetic methods revealed greater gene flow among individual squirrels in forested regions, which likely provide abundant food and shelter for squirrels. Although gene flow appears to be higher in areas with more tree cover, only slight discontinuities in gene flow suggest eastern grey squirrels have maintained connected populations across urban areas in all but the most heavily fragmented agricultural landscapes. Our results suggest urbanization shapes biological evolution in wildlife species depending strongly on the composition and habitability of the landscape matrix surrounding urban areas.

The Expectations and Challenges of Wildlife Disease Research in the Era of Genomics: Forecasting with a Horizon Scan-like Exercise.

The outbreak and transmission of disease-causing pathogens are contributing to the unprecedented rate of biodiversity decline. Recent advances in genomics have coalesced into powerful tools to monitor, detect, and reconstruct the role of pathogens impacting wildlife populations. Wildlife researchers are thus uniquely positioned to merge ecological and evolutionary studies with genomic technologies to exploit unprecedented "Big Data" tools in disease research; however, many researchers lack the training and expertise required to use these computationally intensive methodologies. To address this disparity, the inaugural "Genomics of Disease in Wildlife" workshop assembled early to mid-career professionals with expertise across scientific disciplines (e.g., genomics, wildlife biology, veterinary sciences, and conservation management) for training in the application of genomic tools to wildlife disease research. A horizon scanning-like exercise, an activity to identify forthcoming trends and challenges, performed by the workshop participants identified and discussed 5 themes considered to be the most pressing to the application of genomics in wildlife disease research: 1) "Improving communication," 2) "Methodological and analytical advancements," 3) "Translation into practice," 4) "Integrating landscape ecology and genomics," and 5) "Emerging new questions." Wide-ranging solutions from the horizon scan were international in scope, itemized both deficiencies and strengths in wildlife genomic initiatives, promoted the use of genomic technologies to unite wildlife and human disease research, and advocated best practices for optimal use of genomic tools in wildlife disease projects. The results offer a glimpse of the potential revolution in human and wildlife disease research possible through multi-disciplinary collaborations at local, regional, and global scales.

Ectoparasites of Microtus californicus and Possible Emergence of an Exotic Ixodes Species Tick in California.

California voles (Microtus californicus Peale) harbor fleas and ticks, may be infected with vector-borne pathogens, and could themselves suffer from disease and serve as a source of infection for people and other animals. Here we summarize publications, museum archives, and recent records of ticks and fleas from California voles. There have been 18 flea species reported on California voles with geographic locations reported for 13. During recent statewide surveys, we found six flea species, with the highest species richness in Humboldt County. We found three of five previously reported tick species as well as a tick resembling the eastern North American tick Ixodes minor Neumann (which we here designate Ixodes "Mojave morphotype") on isolated Amargosa voles and Owens Valley voles (Microtus californicus vallicola Bailey) in Inyo County in 2012 and 2014. Additional incidental observations of this Mojave morphotype tick were on a western harvest mouse (Reithrodontomys megalotis Baird) at the Mojave site and a montane vole (Microtus montanus Peale) in the Owens Valley, both in March, 2014. We cannot rule out that this tick species has been present in remote areas of California but gone unrecognized, but these data are consistent with recent introduction of this tick, possibly from migrating birds. Changes in the ectoparasite fauna suggest changing ecologies of vectors and vector-borne pathogens that could influence animals and people as well.

Sources of bias in applying close-kin mark-recapture to terrestrial game species with different life histories.

Close-kin mark-recapture (CKMR) is a method analogous to traditional mark-recapture but without requiring recapture of individuals. Instead, multilocus genotypes (genetic marks) are used to identify related individuals in one or more sampling occasions, which enables the opportunistic use of samples from harvested wildlife. To apply the method accurately, it is important to build appropriate CKMR models that do not violate assumptions linked to the species' and population's biology and sampling methods. In this study, we evaluated the implications of fitting overly simplistic CKMR models to populations with complex reproductive success dynamics or selective sampling. We used forward-in-time, individual-based simulations to evaluate the accuracy and precision of CKMR abundance and survival estimates in species with different longevities, mating systems, and sampling strategies. Simulated populations approximated a range of life histories among game species of North America with lethal sampling to evaluate the potential of using harvested samples to estimate population size. Our simulations show that CKMR can yield nontrivial biases in both survival and abundance estimates, unless influential life history traits and selective sampling are explicitly accounted for in the modeling framework. The number of kin pairs observed in the sample, in combination with the type of kinship used in the model (parent-offspring pairs and/or half-sibling pairs), can affect the precision and/or accuracy of the estimates. CKMR is a promising method that will likely see an increasing number of applications in the field as costs of genetic analysis continue to decline. Our work highlights the importance of applying population-specific CKMR models that consider relevant demographic parameters, individual covariates, and the protocol through which individuals were sampled.

TOXOPLASMA GONDII PREVALENCE, PARTIAL GENOTYPES, AND SPATIAL VARIATION IN NORTH AMERICAN RIVER OTTERS (LONTRA CANADENSIS) IN THE UPPER PENINSULA OF MICHIGAN, USA.

Toxoplasma gondii is a ubiquitous parasitic protozoan that poses a health threat to wildlife and human health worldwide. Oocysts shed into the environment in felid host feces may persist for several years. Runoff from rainfall and snowmelt may carry the oocysts into waterways. Semiaquatic mammals such as the Northern American river otter (Lontra canadensis) are particularly at risk of exposure, as they may encounter infective stages in both terrestrial and aquatic environments. Despite this risk, only a small number of studies have examined the prevalence of T. gondii in US river otter populations. Tongue tissue was sampled from 124 otters from the Upper Peninsula of Michigan submitted by trappers to the Michigan Department of Natural Resources in the 2018-19 harvest season. Following DNA extraction, a portion of the B1 T. gondii gene was amplified with PCR. A subset of positive samples was genotyped for comparison with known T. gondii sequences. Of the 124 tongue samples, 35 (28%) were positive for T. gondii. Prevalence did not differ significantly between sexes or age classes across the entire study area. Most (53.8%) of the genotyped samples were type 4 (type 12), a genotype commonly found in North American wildlife. Genotypes 127 and 197 were also found. Three clusters of T. gondii prevalence were identified through SaTScan analysis, although they were not significant. When modeling prevalence of T. gondii with covariates at individual otter locations, the top three models included the presence of Sarcocystis, area of exotic plants, area of agriculture, and sex of the otter. Our results suggest that T. gondii is widespread in otter populations in the Upper Peninsula of Michigan.

FOOT INJURIES IN MICHIGAN, USA, GRAY WOLVES (CANIS LUPUS), 1992-2014.

The range of gray wolves (Canis lupus) in the contiguous US is expanding. Research and monitoring to support population recovery and management often involves capture via foothold traps. A population-level epidemiologic assessment of the effect of trap injuries on wolf survival remains needed to inform management. We describe the baseline rate, type, and severity of foot injuries of wolves born 1992-2013 in Michigan's Upper Peninsula, evaluate the reliability of field-scoring trap-related injuries, and the effect of injuries on wolf survival. We assessed foot injuries by physical and radiographic exam at postmortem and/or time of capture for 351 wolves using the International Organization for Standardization 10990-5 standard and the effects of injuries, sex, age, previous capture and body condition on survival using proportional hazards regression. We used ordinal regression to evaluate epidemiologic associations between sex, age, previous capture, body condition, cause of death and injury severity. Most wolves (53%) experienced no physically or radiographically discernable foot injuries over their lifetimes. Among those wolves that did experience injuries, 33% scored as mild. Foot injuries had little epidemiologically discernable effect on survival rates. Wolves with higher foot trauma scores did experience an increased risk of dying, but the magnitude of the increase was modest. Most limb injuries occurred below the carpus or tarsus, and scoring upper-limb injuries added little predictive information to population-level epidemiologic measures of survival and injury severity. There was little association between injury severity and cause of death. Based on necropsy exams, previous trap injuries likely contributed to death in only four wolves (1.1%). Our results suggest that injuries resulting from foothold traps are unlikely to be a limiting factor in recovery and ongoing survival of the Michigan gray wolf population.

Prion protein polymorphisms in Michigan white-tailed deer (Odocoileus virginianus).

Chronic Wasting Disease (CWD), a well-described transmissible spongiform encephalopathy of the Cervidae family, is associated with the aggregation of an abnormal isoform (PrPCWD) of the naturally occurring host prion protein (PrPC). Variations in the PrP gene (PRNP) have been associated with CWD rate of infection and disease progression. We analysed 568 free-ranging white-tailed deer (Odocoileus virginianus) from 9 CWD-positive Michigan counties for PRNP polymorphisms. Sampling included 185 CWD-positive, 332 CWD non-detected, and an additional 51 CWD non-detected paired to CWD-positives by sex, age, and harvest location. We found 12 polymorphic sites of which 5 were non-synonymous and resulted in a change in amino acid composition. Thirteen haplotypes were predicted, of which 11 have previously been described. Using logistic regression, consistent with other studies, we found haplotypes C (OR = 0.488, 95% CI = 0.321-0.730, P < 0.001) and F (OR = 0.122, 95% CI = 0.007-0.612, P < 0.05) and diplotype BC (OR = 0.340, 95% CI = 0.154-0.709, P < 0.01) were less likely to be found in deer infected with CWD. As has also been documented in other studies, the presence of a serine at amino acid 96 was less likely to be found in deer infected with CWD (P < 0.001, OR = 0.360 and 95% CI = 0.227-0.556). Identification of PRNP polymorphisms associated with reduced vulnerability to CWD in Michigan deer and their spatial distribution can help managers design surveillance programmesand identify and prioritize areas for CWD management.

HISTOPATHOLOGY AND RISK FACTORS ASSOCIATED WITH NEOTROMBICULA MICROTI INFESTATION IN THE ENDANGERED AMARGOSA VOLE (MICROTUS CALIFORNICUS SCIRPENSIS).

The Amargosa vole (Microtus californicus scirpensis) is a profoundly endangered rodent found only in the Central Mojave Desert, Inyo County, California, US. In 2010, severe cases of trombiculiasis, caused by larval Neotrombicula microti mites, were discovered among voles and sympatric small mammals. We evaluated Amargosa voles and sympatric rodents for infestation with N. microti December 2011-November 2012 and evaluated histopathology of ear tissue from 13 actively N. microti-infested Amargosa voles and 10 Amargosa voles with no gross evidence of current or past infestation. Rodents with current infestation had mites visible on tissue, typically ear pinnae, whereas mites were not seen on rodents with presumptive past infestation, but some of these animals had gross tissue scarring and loss consistent with healing from infestation. Ears from infested voles had severe granulocytic and necrotizing dermatitis, most associated with stylostome fragments, whereas few lesions were present in grossly uninfested voles. There was no association between body condition and infestation or severity of lesions. Significantly more voles were infested (37%) with N. microti than sympatric rodents (3%), suggesting that sympatric rodents do not serve as an important source of N. microti exposure to voles. Although this chigger infestation was common and induced severe localized pathology, we did not detect a fitness cost to infestation and recommend further evaluation of the disease to discern its significance in this conservation context.

An Ixodes minor and Borrelia carolinensis enzootic cycle involving a critically endangered Mojave Desert rodent.

Microtus californicus scirpensis is an endangered, isolated subspecies of California vole. It requires water pools and riparian bulrush (Schoenoplectus americanus) and occupies some of the rarest habitat of any North American mammal. The minimally vegetated, extremely arid desert surrounding the pools is essentially uninhabitable for Ixodes species ticks. We describe an enzootic cycle of Borrelia carolinensis in Ixodes minor ticks at a site 3500 km distant from the region in which I. minor is known to occur in Tecopa Host Springs, Inyo County, eastern Mojave Desert, California. Voles were live-trapped, and ticks and blood samples queried by PCR and DNA sequencing for identification and determination of the presence of Borrelia spp. Between 2011-2013, we found 21 Ixodes minor ticks (prevalence 4-8%) on Amargosa voles and Reithrodontomys megalotis. DNA sequencing of 16S rRNA from ticks yielded 99% identity to I. minor. There was 92% identity with I. minor in the calreticulin gene fragment. Three ticks (23.1%), 15 (24%) voles, three (27%) house mice, and one (7%) harvest mice were PCR positive for Borrelia spp. Sequencing of the 5S-23S intergenic spacer region and flagellin gene assigned Amargosa vole Borrelia strains to B. carolinensis. Ixodes minor, first described in 1902 from a single Guatemalan record, reportedly occurs only in the southeast American on small mammals and birds. The source of this tick in the Mojave Desert and time scale for introduction is not known but likely via migratory birds. Borrelia strains in the Amargosa ecosystem most closely resemble B. carolinensis. B. carolinensis occurs in a rodent-I. minor enzootic cycle in the southeast U.S. although its epidemiological significance for people or rodents is unknown. The presence of a tick and Borrelia spp. only known from southeast U.S. in this extremely isolated habitat on the other side of the continent is of serious concern because it suggests that the animals in the ecosystem could be vulnerable to further incursions of pathogens and parasites.

Pathogen infection and exposure, and ectoparasites of the federally endangered Amargosa vole (Microtus californicus scirpensis), California, USA.

Abstract We surveyed pathogens and ectoparasites among federally endangered Amargosa voles (Microtus californicus scirpensis) and sympatric rodents in Tecopa Hot Springs, Inyo County, California, December 2011-November 2012. We aimed to assess disease and detect possible spillover from or connectivity with other hosts within and outside the Amargosa ecosystem. We assessed 71 individual voles and 38 individual sympatric rodents for current infection with seven vector-borne zoonotic pathogens and past exposure to five pathogens. Thirteen percent of Amargosa voles were PCR positive for Toxoplasma gondii, a zoonotic protozoan that may alter host behavior or cause mortality. Additionally, we found antibodies against Borrelia burgdorferi sensu lato (SL) spp. in 21% of voles, against Anaplasma phagocytophilum in 2.6%, Rickettsia spp. in 13%, relapsing fever Borrelia (3.9%), and T. gondii (7.9%). Sympatric rodents also had active infections with Borrelia SL spp. (15%). Of the ectoparasites collected, the tick Ixodes minor is of particular interest because the study area is well outside of the species' reported range and because I. minor ticks infest migratory birds as well as rodents, showing a potential mechanism for pathogens to be imported from outside the Amargosa ecosystem.