Balancing sample accumulation and DNA degradation rates to optimize noninvasive genetic sampling of sympatric carnivores

Noninvasive genetic sampling, or noninvasive DNA sampling (NDS), can be an effective monitoring approach for elusive, wide‐ranging species at low densities. However, few studies have attempted to maximize sampling efficiency. We present a model for combining sample accumulation and DNA degradation to identify the most efficient (i.e. minimal cost per successful sample) NDS temporal design for capture–recapture analyses. We use scat accumulation and faecal DNA degradation rates for two sympatric carnivores, kit fox (Vulpes macrotis) and coyote (Canis latrans) across two seasons (summer and winter) in Utah, USA, to demonstrate implementation of this approach. We estimated scat accumulation rates by clearing and surveying transects for scats. We evaluated mitochondrial (mtDNA) and nuclear (nDNA) DNA amplification success for faecal DNA samples under natural field conditions for 20 fresh scats/species/season from <1–112 days. Mean accumulation rates were nearly three times greater for coyotes (0.076 scats/km/day) than foxes (0.029 scats/km/day) across seasons. Across species and seasons, mtDNA amplification success was ≥95% through day 21. Fox nDNA amplification success was ≥70% through day 21 across seasons. Coyote nDNA success was ≥70% through day 21 in winter, but declined to <50% by day 7 in summer. We identified a common temporal sampling frame of approximately 14 days that allowed species to be monitored simultaneously, further reducing time, survey effort and costs. Our results suggest that when conducting repeated surveys for capture–recapture analyses, overall cost‐efficiency for NDS may be improved with a temporal design that balances field and laboratory costs along with deposition and degradation rates.     

Landscape genetics of the nonnative red fox of California

Invasive mammalian carnivores contribute disproportionately to declines in global biodiversity. In California, nonnative red foxes (Vulpes vulpes) have significantly impacted endangered ground‐nesting birds and native canids. These foxes derive primarily from captive‐reared animals associated with the fur‐farming industry. Over the past five decades, the cumulative area occupied by nonnative red fox increased to cover much of central and southern California. We used a landscape‐genetic approach involving mitochondrial DNA (mtDNA) sequences and 13 microsatellites of 402 nonnative red foxes removed in predator control programs to investigate source populations, contemporary connectivity, and metapopulation dynamics. Both markers indicated high population structuring consistent with origins from multiple introductions and low subsequent gene flow. Landscape‐genetic modeling indicated that population connectivity was especially low among coastal sampling sites surrounded by mountainous wildlands but somewhat higher through topographically flat, urban and agricultural landscapes. The genetic composition of populations tended to be stable for multiple generations, indicating a degree of demographic resilience to predator removal programs. However, in two sites where intensive predator control reduced fox abundance, we observed increases in immigration, suggesting potential for recolonization to counter eradication attempts. These findings, along with continued genetic monitoring, can help guide localized management of foxes by identifying points of introductions and routes of spread and evaluating the relative importance of reproduction and immigration in maintaining populations. More generally, the study illustrates the utility of a landscape‐genetic approach for understanding invasion dynamics and metapopulation structure of one of the world's most destructive invasive mammals, the red fox.     

Size‐assortative choice and mate availability influences hybridization between red wolves (Canis rufus) and coyotes (Canis latrans)

Anthropogenic hybridization of historically isolated taxa has become a primary conservation challenge for many imperiled species. Indeed, hybridization between red wolves (Canis rufus) and coyotes (Canis latrans) poses a significant challenge to red wolf recovery. We considered seven hypotheses to assess factors influencing hybridization between red wolves and coyotes via pair‐bonding between the two species. Because long‐term monogamy and defense of all‐purpose territories are core characteristics of both species, mate choice has long‐term consequences. Therefore, red wolves may choose similar‐sized mates to acquire partners that behave similarly to themselves in the use of space and diet. We observed multiple factors influencing breeding pair formation by red wolves and found that most wolves paired with similar‐sized conspecifics and wolves that formed congeneric pairs with nonwolves (coyotes and hybrids) were mostly female wolves, the smaller of the two sexes. Additionally, we observed that lower red wolf abundance relative to nonwolves and the absence of helpers increased the probability that wolves consorted with nonwolves. However, successful pairings between red wolves and nonwolves were associated with wolves that maintained small home ranges. Behaviors associated with territoriality are energetically demanding and behaviors (e.g., aggressive interactions, foraging, and space use) involved in maintaining territories are influenced by body size. Consequently, we propose the hypothesis that size disparities between consorting red wolves and coyotes influence positive assortative mating and may represent a reproductive barrier between the two species. We offer that it may be possible to maintain wild populations of red wolves in the presence of coyotes if management strategies increase red wolf abundance on the landscape by mitigating key threats, such as human‐caused mortality and hybridization with coyotes. Increasing red wolf abundance would likely restore selection pressures that increase mean body and home‐range sizes of red wolves and decrease hybridization rates via reduced occurrence of congeneric pairs.     

Using the dog genome to find single nucleotide polymorphisms in red foxes and other distantly related members of the Canidae

Single nucleotide polymorphisms (SNP) are the ideal marker for characterizing genomic variation but can be difficult to find in nonmodel species. We explored the usefulness of the dog genome for finding SNPs in distantly related nonmodel canids and evaluated so-ascertained SNPs. Using 40 primer pairs designed from randomly selected bacterial artificial chromosome clones from the dog genome, we successfully sequenced 80-88% of loci in a coyote (Canis latrans), grey fox (Urocyon cinereoargenteus), and red fox (Vulpes vulpes), which compared favourably to a 60% success rate for each species using 10 primer pairs conserved across mammals. Loci were minimally heterogeneous with respect to SNP density, which was similar, overall, in a discovery panel of nine red foxes to that previously reported for a panel of eight wolves (Canis lupus). Additionally, individual heterozygosity was similar across the three canids in this study. However, the proportion of SNP sites shared with the dog decreased with phylogenetic divergence, with no SNPs shared between red foxes and dogs. Density of interspecific SNPs increased approximately linearly with divergence time between species. Using red foxes from three populations, we estimated F(ST) based on each of 42 SNPs and 14 microsatellites and simulated null distributions conditioned on each marker type. Relative to SNPs, microsatellites systematically underestimated F(ST) and produced biased null distributions, indicating that SNPs are superior markers for these functions. By reconstituting the frequency spectrum of SNPs discovered in nine red foxes, we discovered an estimated 77-89% of all SNPs (within the region screened) present in North American red foxes. In sum, these findings indicate that information from the dog genome enables easy ascertainment of random and gene-linked SNPs throughout the Canidae and illustrate the value of SNPs in ecological and evolutionary genetics.     

Individual dietary specialization in a generalist predator: A stable isotope analysis of urban and rural red foxes

Some carnivores are known to survive well in urban habitats, yet the underlying behavioral tactics are poorly understood. One likely explanation for the success in urban habitats might be that carnivores are generalist consumers. However, urban populations of carnivores could as well consist of specialist feeders. Here, we compared the isotopic specialization of red foxes in urban and rural environments, using both a population and an individual level perspective. We measured stable isotope ratios in increments of red fox whiskers and potential food sources. Our results reveal that red foxes have a broad isotopic dietary niche and a large variation in resource use. Despite this large variation, we found significant differences between the variance of the urban and rural population for δ¹³C as well as δ¹⁵N values, suggesting a habitat‐specific foraging behavior. Although urban regions are more heterogeneous regarding land cover (based on the Shannon index) than rural regions, the dietary range of urban foxes was smaller compared with that of rural conspecifics. Moreover, the higher δ¹³C values and lower δ¹⁵N values of urban foxes suggest a relatively high input of anthropogenic food sources. The diet of most individuals remained largely constant over a longer period. The low intraindividual variability of urban and rural red foxes suggests a relatively constant proportion of food items consumed by individuals. Urban and rural foxes utilized a small proportion of the potentially available isotopic dietary niche as indicated by the low within‐individual variation compared to the between‐individual variation. We conclude that generalist fox populations consist of individual food specialists in urban and rural populations at least over those periods covered by our study.     

The effectiveness of short‐term fox control in protecting a seasonally vulnerable species,the Eastern Long‐necked Turtle (Chelodina longicollis)

Reducing predation by introduced predators on seasonally vulnerable prey is of interest to biodiversity and game managers around the world. In Australia, the Red Fox (Vulpes vulpes) is a significant predator of freshwater turtle nests, destroying up to 93% of nests. We used a nonrandomized intervention study to assess the effectiveness of a short‐term (3‐week) but broad‐scale baiting operation in reducing the level of nest predation on artificial turtle nests around a complex lake system during a major flooding event in north‐western Victoria. Estimates of fox occupancy declined from 0.58 (0.44–0.70 95% CI) to 0.34 (0.21–0.46 95% CI) following fox control. Modelling of nest‐survival rates indicated there was no significant change in survival rates. Effective short‐term predator control to protect seasonally vulnerable prey is desirable and achievable. Knowledge of underlying predator density, predator–bait encounter and consumption rates, and the optimal duration of short‐term control is needed to reduce the risk to prey.     

Population structure of two rabies hosts relative to the known distribution of rabies virus variants in Alaska

For pathogens that infect multiple species, the distinction between reservoir hosts and spillover hosts is often difficult. In Alaska, three variants of the arctic rabies virus exist with distinct spatial distributions. We tested the hypothesis that rabies virus variant distribution corresponds to the population structure of the primary rabies hosts in Alaska, arctic foxes (Vulpes lagopus) and red foxes (Vulpes vulpes) to possibly distinguish reservoir and spillover hosts. We used mitochondrial DNA (mtDNA) sequence and nine microsatellites to assess population structure in those two species. mtDNA structure did not correspond to rabies virus variant structure in either species. Microsatellite analyses gave varying results. Bayesian clustering found two groups of arctic foxes in the coastal tundra region, but for red foxes it identified tundra and boreal types. Spatial Bayesian clustering and spatial principal components analysis identified 3 and 4 groups of arctic foxes, respectively, closely matching the distribution of rabies virus variants in the state. Red foxes, conversely, showed eight clusters comprising two regions (boreal and tundra) with much admixture. These results run contrary to previous beliefs that arctic fox show no fine‐scale spatial population structure. While we cannot rule out that the red fox is part of the maintenance host community for rabies in Alaska, the distribution of virus variants appears to be driven primarily by the arctic fox. Therefore, we show that host population genetics can be utilized to distinguish between maintenance and spillover hosts when used in conjunction with other approaches.     

Viral gut metagenomics of sympatric wild and domestic canids,and monitoring of viruses: Insights from an endangered wolf population

Animal host–microbe interactions are a relevant concern for wildlife conservation, particularly regarding generalist pathogens, where domestic host species can play a role in the transmission of infectious agents, such as viruses, to wild animals. Knowledge on viral circulation in wild host species is still scarce and can be improved by the recent advent of modern molecular approaches. We aimed to characterize the fecal virome and identify viruses of potential conservation relevance of diarrheic free‐ranging wolves and sympatric domestic dogs from Central Portugal, where a small and threatened wolf population persists in a highly anthropogenically modified landscape. Using viral metagenomics, we screened diarrheic stools collected from wolves (n = 8), feral dogs (n = 4), and pet dogs (n = 6), all collected within wolf range. We detected novel highly divergent viruses as well as known viral pathogens with established effects on population dynamics, including canine distemper virus, a novel bocavirus, and canine minute virus. Furthermore, we performed a 4‐year survey for the six wolf packs comprising this endangered wolf population, screening 93 fecal samples from 36 genetically identified wolves for canine distemper virus and the novel bocavirus, previously identified using our metagenomics approach. Our novel approach using metagenomics for viral screening in noninvasive samples of wolves and dogs has profound implications on the knowledge of both virology and wildlife diseases, establishing a complementary tool to traditional screening methods for the conservation of threatened species.     

Field Sedation of Coyotes,Red Foxes,and Raccoons With Medetomidine and Atipamezole

Abstract: We chemically restrained free-ranging coyotes (Canis latrans), red foxes (Vulpes vulpes), and raccoons (Procyon lotor) using medetomidine antagonized by atipamezole. All coyotes and 80% of red foxes were sedated with mean ± standard deviation doses of 0.12 ± 0.02 mg/kg and 0.14 ± 0.02 mg/kg medetomidine, respectively. Seventy-seven percent of raccoons were sedated with 0.21 ± 0.05 mg/kg medetomidine. In all species we observed occasional movement, muscle rigidity, and partial-arousal during sedation. Animals were alert within 4.3–8.6 ± 3.5–8.4 min following atipamezole at 0.4 mg/kg. Medetomidine and atipamezole provided safe handling in most animals and rapid recovery without use of a controlled substance. At these doses, biologists in the field should be prepared to administer a supplementary dose of medetomidine to some animals depending on ambient conditions and the objectives of the restraint event.     

Range‐wide multilocus phylogeography of the red fox reveals ancient continental divergence,minimal genomic exchange and distinct demographic histories

Widely distributed taxa provide an opportunity to compare biogeographic responses to climatic fluctuations on multiple continents and to investigate speciation. We conducted the most geographically and genomically comprehensive study to date of the red fox (Vulpes vulpes), the world's most widely distributed wild terrestrial carnivore. Analyses of 697 bp of mitochondrial sequence in ~1000 individuals suggested an ancient Middle Eastern origin for all extant red foxes and a 400 kya (SD = 139 kya) origin of the primary North American (Nearctic) clade. Demographic analyses indicated a major expansion in Eurasia during the last glaciation (~50 kya), coinciding with a previously described secondary transfer of a single matriline (Holarctic) to North America. In contrast, North American matrilines (including the transferred portion of Holarctic clade) exhibited no signatures of expansion until the end of the Pleistocene (~12 kya). Analyses of 11 autosomal loci from a subset of foxes supported the colonization time frame suggested by mtDNA (and the fossil record) but, in contrast, reflected no detectable secondary transfer, resulting in the most fundamental genomic division of red foxes at the Bering Strait. Endemic continental Y‐chromosome clades further supported this pattern. Thus, intercontinental genomic exchange was overall very limited, consistent with long‐term reproductive isolation since the initial colonization of North America. Based on continental divergence times in other carnivoran species pairs, our findings support a model of peripatric speciation and are consistent with the previous classification of the North American red fox as a distinct species, V. fulva.     

Dispatches from the neighborhood watch: Using citizen science and field survey data to document color morph frequency in space and time

Heritable color polymorphisms have a long history of study in evolutionary biology, though they are less frequently examined today than in the past. These systems, where multiple discrete, visually identifiable color phenotypes co‐occur in the same population, are valuable for tracking evolutionary change and ascertaining the relative importance of different evolutionary mechanisms. Here, we use a combination of citizen science data and field surveys in the Great Lakes region of North America to identify patterns of color morph frequencies in the eastern gray squirrel (Sciurus carolinensis). Using over 68,000 individual squirrel records from both large and small spatial scales, we identify the following patterns: (a) the melanistic (black) phenotype is often localized but nonetheless widespread throughout the Great Lakes region, occurring in all states and provinces sampled. (b) In Ohio, where intensive surveys were performed, there is a weak but significantly positive association between color morph frequency and geographic proximity of populations. Nonetheless, even nearby populations often had radically different frequencies of the melanistic morph, which ranged from 0% to 96%. These patterns were mosaic rather than clinal. (c) In the Wooster, Ohio population, which had over eight years of continuous data on color morph frequency representing nearly 40,000 records, we found that the frequency of the melanistic morph increased gradually over time on some survey routes but decreased or did not change over time on others. These differences were statistically significant and occurred at very small spatial scales (on the order of hundreds of meters). Together, these patterns are suggestive of genetic drift as an important mechanism of evolutionary change in this system. We argue that studies of color polymorphism are still quite valuable in advancing our understanding of fundamental evolutionary processes, especially when coupled with the growing availability of data from citizen science efforts.     

Biodiversity friend or foe: land use by a top predator,the dingo in contested landscapes of the Australian Wet Tropics

Dingoes (Canis dingo) in the coastal lowlands of Australia's Wet Tropics are perceived as a major threat to biodiversity and subjected to broad‐spectrum lethal control. However, evidence of their impacts is equivocal, and control programmes generally ignore the ecological benefits that dingoes might provide. Previous diet analysis has shown that dingoes in the Wet Tropics primarily prey on common, terrestrial mammals. However, little is known of dingo habitat use or prey acquisition in the region despite these activities having major implications for biodiversity conservation. We investigated land use by dingoes in the lowland Wet Tropics to enable predictions of potential prey types, relative prey use and modes of prey acquisition. Nine dingoes were tracked for 3–6 months. Home ranges and resting areas were estimated using multiple estimators, and habitat use was analysed using compositional analysis of habitat use and generalized additive models. Dingo ranging behaviour suggested that anthropogenic food subsidies were infrequently used. Each territory comprised several sclerophyll forest rest areas with adjacent sugarcane‐grassland high activity areas. Individuals used each rest‐activity area for extended durations before moving on to another. Sclerophyll and rainforests, which contain the fauna species of primary conservation concern, were generally used for rest/sleep, or movement between rest‐activity areas. Activity patterns were consistent with dingoes hunting in open sugarcane‐grassland habitats during daylight hours. Dingo activity was low in areas where fauna species of conservation concern occur, which suggests that dingoes do not pose a threat to their survival. Consequently, current broad‐spectrum lethal control may have minimal benefits or even incur costs for biodiversity. Maximizing the ecosystem services provided by dingoes while simultaneously minimizing their negative impacts requires a more targeted location‐specific management approach, one that assesses and mitigates impacts specifically where background circumstances suggest particular packs may be either a conservation or economic threat.     

Time‐dependent climate impact of a bioenergy system – methodology development and application to Swedish conditions

The area of dedicated energy crops is expected to increase in Sweden. This will result in direct land use changes, which may affect the carbon stocks in soil and biomass, as well as yield levels and the use of inputs. Carbon dioxide (CO₂) fluxes of biomass are often not considered when calculating the climate impact in life cycle assessments (LCA) assuming that the CO₂ released at combustion has recently been captured by the biomass in question. With the extended time lag between capture and release of CO₂ inherent in many perennial bioenergy systems, the relation between carbon neutrality and climate neutrality may be questioned. In this paper, previously published methodologies and models are combined in a methodological framework that can assist LCA practitioners in interpreting the time‐dependent climate impact of a bioenergy system. The treatment of carbon differs from conventional LCA practice in that no distinction is made between fossil and biogenic carbon. A time‐dependent indicator is used to enable a representation of the climate impact that is not dependent on the choice of a specific characterization time horizon or time of evaluation and that does not use characterization factors, such as global warming potential and global temperature potential. The indicator used to aid in the interpretation phase of this paper is global mean surface temperature change (ΔT_s(n)). A theoretical system producing willow for district heating was used to study land use change effects depending on previous land use and variations in the standing biomass carbon stocks. When replacing annual crops with willow this system presented a cooling contribution to ΔT_s(n). However, the first years after establishing the willow plantation it presented a warming contribution to ΔT_s(n). This behavior was due mainly to soil organic carbon (SOC) variation. A rapid initial increase in standing biomass counteracted the initial SOC loss.     

Landowners’ perspectives of black‐backed jackals (Canis mesomelas) on farmlands in KwaZulu‐Natal,South Africa

Despite continued efforts to eradicate black‐backed jackals (Canis mesomelas), they are considered an abundant mesopredator on agricultural land across South Africa, resulting in ongoing human–wildlife conflict and concern for farmers and wildlife managers. We conducted a questionnaire survey and semi‐formal interviews with farmers throughout KwaZulu‐Natal, examining farmers’ livestock husbandry, land‐use changes and perspectives towards jackals as a perceived threat to livestock. Many (75%) respondents acknowledged expanding agricultural activities on their farmlands since the onset of their farming careers. However, the perception was that these changes placed little pressure on mesopredators as farmers reported frequent daily (25%) and weekly (31%) sightings of jackal, and regular predation on livestock (72%). Some landowners (31%) reported between one and five livestock losses annually and suggest that mitigation strategies to prevent livestock losses are in place. Farmers suggested the increasing intensity in agricultural practices provided a greater food source for jackals allowing them to thrive in expanding agricultural conditions and, in some circumstances, farmers admitted to possibly being a cause through poor disposal techniques for dead animals. Feedback from farmers emphasized the importance of having collaboration between farmers to control jackal predation and reduce human–wildlife conflict.     

Hierarchical distance sampling to estimate population sizes of common lizards across a desert ecoregion

Multispecies wildlife monitoring across large geographical regions is important for effective conservation planning in response to expected impacts from climate change and land use. Unlike many species of birds, mammals, and amphibians which can be efficiently sampled using automated sensors including cameras and sound recorders, reptiles are often much more challenging to detect, in part because of their typically cryptic behavior and generally small body sizes. Although many lizard species are more active during the day which makes them easier to detect using visual encounter surveys, they may be unavailable for sampling during certain periods of the day or year due to their sensitivity to temperature. In recognition of these sampling challenges, we demonstrate application of a recent innovation in distance sampling that adjusts for temporary emigration between repeat survey visits. We used transect surveys to survey lizards at 229 sites throughout the Mojave Desert in California, USA, 2016. We estimated a total population size of 82 million (90% CI: 65–99 million) for the three most common species of lizards across this 66,830 km² ecoregion. We mapped how density at the 1‐km² scale was predicted to vary with vegetation cover and human development. We validated these results against independent surveys from the southern portion of our study area. Our methods and results demonstrate how multispecies monitoring programs spanning arid ecoregions can better incorporate information about reptiles.     

Comparative study of movement patterns of Mastomys natalensis in irrigated rice and fallow fields in eastern Tanzania

A 2‐year capture–mark–recapture study was conducted to estimate home ranges and weekly travel distance of Mastomys natalensis (Smith 1834) in an irrigated rice ecosystem and fallow fields. We found that adults have larger home ranges than subadults in fallow fields but not in rice fields, indicating that fallow fields are more suitable for breeding. Travel distances were larger in rice fields, especially in the transplanting stage, during which rice fields are flooded and provide less food, causing movements into neighbouring fallow fields that then temporarily experience higher population density. A decrease in travel distance was observed in rice fields during the maturity stage, which can be explained by higher food availability and a more suitable, nonflooded situation. Movement of M. natalensis in rice‐fallow mosaic landscapes thus seems to be driven by food availability and flooding status of the rice fields, which can be attributed to land use practices.     

Disentangling the effects of plant species invasion and urban development on arthropod community composition

Urban development and species invasion are two major global threats to biodiversity. These threats often co‐occur, as developed areas are more prone to species invasion. However, few empirical studies have tested if both factors affect biodiversity in similar ways. Here we study the individual and combined effects of urban development and plant invasion on the composition of arthropod communities. We assessed 36 paired invaded and non‐invaded sample plots, invaded by the plant Antigonon leptopus, with half of these pairs located in natural and the other half in developed land‐use types on the Caribbean island of St. Eustatius. We used several taxonomic and functional variables to describe community composition and diversity. Our results show that both urban development and A. leptopus invasion affected community composition, albeit in different ways. Development significantly increased species richness and exponential Shannon diversity, while invasion had no effect on these variables. However, invasion significantly increased arthropod abundance and caused biotic homogenization. Specifically, uninvaded arthropod communities were distinctly different in species composition between developed and natural sites, while they became undistinguishable after A. leptopus invasion. Moreover, functional variables were significantly affected by species invasion, but not by urban development. Invaded communities had higher community‐weighted mean body size and the feeding guild composition of invaded arthropod communities was characterized by the exceptional numbers of nectarivores, herbivores, and detritivores. With the exception of species richness and exponential Shannon diversity, invasion influenced four out of six response variables to a greater degree than urban development did. Hence, we can conclude that species invasion is not just a passenger of urban development but also a driver of change.     

The Helicobacter pylori adhesin protein HopQ exploits the dimer interface of human CEACAMs to facilitate translocation of the oncoprotein CagA

Helicobacter pylori infects half of the world's population, and strains that encode the cag type IV secretion system for injection of the oncoprotein CagA into host gastric epithelial cells are associated with elevated levels of cancer. CagA translocation into host cells is dependent on interactions between the H. pylori adhesin protein HopQ and human CEACAMs. Here, we present high‐resolution structures of several HopQ‐CEACAM complexes and CEACAMs in their monomeric and dimeric forms establishing that HopQ uses a coupled folding and binding mechanism to engage the canonical CEACAM dimerization interface for CEACAM recognition. By combining mutagenesis with biophysical and functional analyses, we show that the modes of CEACAM recognition by HopQ and CEACAMs themselves are starkly different. Our data describe precise molecular mechanisms by which microbes exploit host CEACAMs for infection and enable future development of novel oncoprotein translocation inhibitors and H. pylori‐specific antimicrobial agents.