Comparison of 454 pyrosequencing methods for characterizing the major histocompatibility complex of nonmodel species and the advantages of ultra deep coverage

Characterization and population genetic analysis of multilocus genes, such as those found in the major histocompatibility complex (MHC) is challenging in nonmodel vertebrates. The traditional method of extensive cloning and Sanger sequencing is costly and time‐intensive and indirect methods of assessment often underestimate total variation. Here, we explored the suitability of 454 pyrosequencing for characterizing multilocus genes for use in population genetic studies. We compared two sample tagging protocols and two bioinformatic procedures for 454 sequencing through characterization of a 185‐bp fragment of MHC DRB exon 2 in wolverines (Gulo gulo) and further compared the results with those from cloning and Sanger sequencing. We found 10 putative DRB alleles in the 88 individuals screened with between two and four alleles per individual, suggesting amplification of a duplicated DRB gene. In addition to the putative alleles, all individuals possessed an easily identifiable pseudogene. In our system, sequence variants with a frequency below 6% in an individual sample were usually artefacts. However, we found that sample preparation and data processing procedures can greatly affect variant frequencies in addition to the complexity of the multilocus system. Therefore, we recommend determining a per‐amplicon‐variant frequency threshold for each unique system. The extremely deep coverage obtained in our study (approximately 5000×) coupled with the semi‐quantitative nature of pyrosequencing enabled us to assign all putative alleles to the two DRB loci, which is generally not possible using traditional methods. Our method of obtaining locus‐specific MHC genotypes will enhance population genetic analyses and studies on disease susceptibility in nonmodel wildlife species.     

Modeling Wolverine Occurrence Using Aerial Surveys of Tracks in Snow

ABSTRACT We designed a novel approach to determining extent of distribution and area of occupancy for wolverines (Gulo gulo) by using aerial surveys of tracks in snow and hierarchical spatial modeling. In 2005 we used a small, fixed-wing aircraft with pilot and one observer to search 575 of 588 survey units for wolverine tracks in approximately 60,000 km² of boreal forest in northwestern Ontario, Canada. We used sinuous flight paths to scan open areas in the forest in the 100-km² survey units. We detected tracks in 138 (24%) of the 575 sampled units. There was strong evidence of occurrence (probability of occurrence >0.80) in 30% of the 588 survey units, weak evidence of occurrence (0.50–0.80) in 12%, weak evidence of absence (0.20–0.50) in 15%, and strong evidence of absence (< 0.20) in 43%. Wolverine range comprised 59% of the study area and area of occupancy was 33,400 km². With information on probability of occurrence and core areas of occupation for wolverines in our study area, resource managers and others can examine factors that influence wolverine distribution patterns and use this information to formulate best management practices that will maintain wolverines on the landscape in the face of increasing resource development. Comparing future survey results with those of our 2005 survey will provide an objective way to assess the efficacy of management practices.     

Spatial ecology of wolverines at the southern periphery of distribution

Wolverines (Gulo gulo) in the conterminous United States have experienced range contraction, are uncommon, and have been designated as warranted for protection under the United States Endangered Species Act. Data from the southern edge of the wolverine's circumpolar distribution is sparse, and development of effective conservation strategies would benefit from a more complete understanding of the species' ecology. We captured and radio-monitored 30 wolverines in the Greater Yellowstone Ecosystem (GYE), tested for seasonal habitat selection by elevation band, and examined a suite of spatial characteristics to clarify our understanding of the wolverine's niche. Wolverines in GYE selected for areas >2,600 m latitude-adjusted elevation (LAE; n = 2,257 wolverine locations [12 F, 6 M]). Wolverines avoided areas <2,150 m LAE, including during winter when the vast majority of ungulates are pushed to these elevations by deep snow. Wolverine home ranges were large relative to body size, averaging 303 km² for adult females and 797 km² for adult males (n = 13 [8 F, 5 M] and 33 wolverine-years). Resident adults fit with Global Positioning System (GPS) collars used an area >75% the size of their multi-year home range in an average of 32 days (n = 7 [5 F, 2 M]). Average movement rates of 1.3 km/2-hr indicated that both sexes move distances equivalent to the diameter of their home range every 2 days or the circumference of their home range in <1 week (n = 1,329 2-hr movements, n = 12 individuals [7 F, 5 M]). This capability for movement, the short time-frame over which home ranges were developed, and a lack of home range overlap by same sex adults ( , 90% CI = 0.0–4.8%, n = 22 pairs) suggested territoriality. We estimated wolverine density to be 3.5/1,000 km² of area >2,150 m LAE (95% CI = 2.8–9.6). Dispersal movements extended to at least 170 km for both sexes (n = 5 F, 2 M). At the southern edge of distribution, where suitable and unsuitable conditions exist in close proximity, wolverines selected high-elevation areas near alpine tree-line where a mix of forest, meadow, and boulder fields were present, deep snow-cover existed during winter, and low temperatures near freezing can occur throughout the year. Persistence in these areas where the growing season is brief requires large home ranges that are regularly patrolled, a social system that provides exclusive access to resources, and low densities. These characteristics, along with low reproductive rates, are prevalent throughout the species range, indicating that wolverines are specialists at exploiting a cold, unproductive niche where interspecific competition is limited. The vulnerability inherent in occupying this unproductive niche was likely influential in previous declines within the conterminous United States and will remain a factor as wolverines encounter modern human influences. Conserving wolverines in the conterminous United States will require collaborative management over a large geographic scale. © 2011 The Wildlife Society.     

Integrating motion-detection cameras and hair snags for wolverine identification

We developed an integrated system for photographing a wolverine's (Gulo gulo) ventral pattern while concurrently collecting hair for microsatellite DNA genotyping. Our objectives were to 1) test the system on a wild population of wolverines using an array of camera and hair-snag (C&H) stations in forested habitat where wolverines were known to occur, 2) validate our ability to determine identity (ID) and sex from photographs by comparing photographic data with that from DNA, and 3) encourage researchers and managers to test the system in different wolverine populations and habitats and improve the system design. Of the 18 individuals (10 M, 8 F) for which we obtained genotypes over the 2 years of our study, there was a 100% match between photographs and DNA for both ID and sex. The integrated system made it possible to reduce cost of DNA analysis by >74%. Integrating motion-detection cameras and hair snags provides a cost-effective technique for wildlife managers to monitor wolverine populations in remote habitats and obtain information on important population parameters such as density, survival, productivity, and effective population size. © 2011 The Wildlife Society.     

Modelling broad‐scale wolverine occupancy in a remote boreal region using multi‐year aerial survey data

Aim

We used data from aerial surveys of wolverine tracks collected in seven winters over a 10‐year period (2003–2012) within a 574,287 km² study area to evaluate the broad‐scale pattern of wolverine occurrence across a remote northern boreal forest region, identifying areas of high and low occupancy.

Location

Northern Ontario, Canada.

Taxon

Wolverine (Gulo gulo Linnaeus, 1758).

Methods

We collected wolverine tracks and observations in 100‐km² hexagonal survey units, making a total of 6,664 visits to 3,039 units, visiting each 1–9 times. We used hierarchical Bayesian occupancy modelling to model wolverine occurrence, and included covariates with the potential to affect detection and/or occupancy probability of wolverines.

Results

we detected wolverines on 946 visits, 14.2% of total visits. Probability of detecting a wolverine varied among years and between the two ecozones in the study area. Wolverine occupancy was negatively related to two important covariates, the geographical coordinate Easting and thawing degree‐days. A site occupancy probability map indicated that wolverine occupancy probabilities were highest, and standard error lowest, in the western and northern portions of the study area.

Main conclusions

The occupancy framework enabled us to use observation data from tracks of this elusive, wide‐ranging carnivore over a vast, remote area while explicitly considering detectability and spatial autocorrelation, yielding a map of probable wolverine distribution in northern Ontario that would not be possible using other methods of detection across a large region. With resource development pressures increasing in this globally significant region in the face of a changing climate, it is important to monitor changes in distribution of species like wolverines that have low population growth rates, large spatial requirements and sensitivity to human disturbance. This study demonstrates a relatively cost‐effective and non‐invasive alternative to monitoring based on wolverine harvest records, which have not been available since 2009 in Ontario due to changes in the provincial regulatory regime for this threatened species.     

Coarse-Scale Distribution Surveys and Occurrence Probability Modeling for Wolverine in Interior Alaska

ABSTRACT We determined wolverine (Gulo gulo) distribution and occurrence probabilities using aerial surveys and hierarchical spatial modeling in a 180,000-km² portion of Interior Alaska, USA. During 8 February-12 March 2006, we surveyed 149 of 180 1,000-km² sample units for wolverine tracks. We observed wolverine tracks in 99 (66.4%) sample units. Wolverine detection probability was ≥69% throughout the survey period. Posterior occurrence probabilities of whether a wolverine track occurred in a sample unit was dependent on survey timing, number of transects flown, number of neighboring sample units with detected tracks, percentage of the sample unit with elevation ≤305 m, and human influences. Our model indicated strong evidence of occurrence (>0.80) in 72% of the 180 survey units, strong evidence of absence (<0.20) in 12%, and weak evidence of occurrence or absence (0.20–0.80) in 16%. Wolverine area of occupancy made up 83% of the study area. Simulations illustrated that 2–4 survey routes were necessary for the survey technique to provide strong evidence of wolverine presence or absence in Interior Alaska if a track was not identified along the first route. The necessary number of survey routes depends on the occurrence probability in a sample unit. We provided managers with a map of wolverine distribution in Interior Alaska and an efficient and lower-cost method to detect coarse-scale changes in wolverine distribution. Our technique was effective in both Interior Alaska and Ontario, Canada, suggesting it would be effective throughout most of the boreal forest range of wolverines where tracks can be readily observed from the air. The technique requires a certain skill level in recognizing tracks; it is essential that tracks are identified correctly and training may be necessary depending on surveyor experience.     

Wolverine Occupancy,Spatial Distribution,and Monitoring Design

In the western United States, wolverines (Gulo gulo) typically occupy high-elevation habitats. Because wolverine populations occur in vast, remote areas across multiple states, biologists have an imperfect understanding of this species' current distribution and population status. The historical extirpation of the wolverine, a subsequent period of recovery, and the lack of a coordinated monitoring program in the western United States to determine their current distribution further complicate understanding of their population status. We sought to define the limits to the current distribution, identify potential gaps in distribution, and provide a baseline dataset for future monitoring and analysis of factors contributing to changes in distribution of wolverines across 4 western states. We used remotely triggered camera stations and hair snares to detect wolverines across randomly selected 15-km × 15-km cells in Idaho, Montana, Washington, and Wyoming, USA, during winters 2016 and 2017. We used spatial occupancy models to examine patterns in wolverine distribution. We also examined the influence of proportion of the cell containing predicted wolverine habitat, human-modified land, and green vegetation, and area of the cluster of contiguous sampling cells. We sampled 183 (28.9%) of 633 cells that comprised a suspected wolverine range in these 4 states and we detected wolverines in 59 (32.2%) of these 183 sampled cells. We estimated that 268 cells (42.3%; 95% CI = 182–347) of the 633 cells were used by wolverines. Proportion of the cell containing modeled wolverine habitat was weakly positively correlated with wolverine occupancy, but no other covariates examined were correlated with wolverine occupancy. Occupancy rates (ψ) were highest in the Northern Continental Divide Ecosystem (ψ range = 0.8–1), intermediate in the Cascades and Central Mountains of Idaho (ψ range = 0.4–0.6), and lower in the Greater Yellowstone Ecosystem (ψ range = 0.1–0.3). We provide baseline data for future surveys of wolverine along with a design and protocol to conduct those surveys. © 2020 The Authors. The Journal of Wildlife Management published by Wiley Periodicals, Inc. on behalf of The Wildlife Society.     

Wolverine Conservation and Management

ABSTRACT This Special Section includes 8 peer-reviewed papers on the wolverine (Gulo gulo) in southern North America. These papers provide new information on current and historical distribution, habitat relations at multiple spatial scales, and interactions with humans. In aggregate, these papers substantially increase our knowledge of wolverine ecology and population dynamics in North America, in many cases replacing previous speculations and informed judgments with empirical information. North American wolverines occur primarily in tundra, taiga, and subalpine environments. These environments become increasingly fragmented at southern latitudes, where wolverine populations occur at low densities and are potentially vulnerable to human-caused mortality. The combination of highly fragmented habitat, demographic sensitivity to adult mortality, and low population densities make local wolverine populations difficult to monitor and easy to overharvest. Where populations are fragmented, persistence is critically dependent on dispersal between habitat islands. Although dispersal dynamics are poorly understood, high levels of genetic structure observed in both current and historical populations indicate that dispersal between mountain ranges is limited. Wolverine biology remains poorly understood, and many fundamental issues need additional research.