Wolf feeding ecology in a multi-ungulate system – investigating the effect of individual predator traits and abundance of co-occurring species

Predation patterns by large carnivores are influenced by a combination of physical, behavioural, and environmental factors. With the recolonization of these apex predators reaching vast parts of their former ranges, there is a need to better understand carnivores’ ecology and effect on prey populations in human dominated landscapes. Data on individual traits of large carnivores are difficult to retrieve and even more to associate with individual feeding behaviour. Alongside cluster checks of GPS-collared wolves, the growing field of faecal DNA-based diet analysis in combination with individual genotyping has the potential to increase the feasibility of large-scale analyses of food use related to individual predator traits. However, the validation of prey DNA detection protocols is still lagging behind the methodological advances. In this thesis, the development and empirical validation of a molecular method for prey DNA detection was followed by the analysis of intrinsic and extrinsic factors affecting feeding ecology of wolves (Canis lupus). The method used nanofluidic array technology and a set of 80 multiple species-specific markers to detect DNA of 17 target prey from wolf scats. Through controlled feeding experiments with captive wolves, we estimated method sensitivity and accordingly calibrated the thresholds to reliably define a positive prey detection. The application of this methodology to the Scandinavian wolf population revealed variability at the landscape level in the use of the two main prey species, moose (Alces alces) and roe deer (Capreolus capreolus), indicating a dietary response of wolves to changes in wild ungulates relative abundance. In addition, GPS-data showed how scavenging constituted only a minor proportion of wolves’ feeding behaviour, related to season and with moderate support to bear and human density. By taking advantage of the long-term wolf monitoring, we showed that wolf feeding patterns (i.e. prey use and extent of scavenging) were affected by social status, sex, and level of inbreeding. These innovative patterns underline the relevance of considering predator individual traits when studying carnivore feeding ecology. The development and validation of our molecular method highlighted the overlooked relevance of assessing method sensitivity and including it in the evaluation of optimal thresholds for binary detection of prey species in predator scats. As the molecular method can be easily customized to different ecological settings, it may be further developed and applied to other areas and large carnivores. The knowledge gained in this study has the potential to help understanding the impact of recolonizing wolf populations on prey communities and inform the adaptive management of such predator and prey species living in a landscape highly managed by humans

Experimental feeding validates nanofluidic array technology for DNA detection of ungulate prey in wolf scats

The study of carnivores' diet is a key component to enhance knowledge on the ecology of predators and their effect on prey populations. Although molecular approaches to detect prey DNA in carnivore scats are improving, the validation of their accuracy, a prerequisite for reliable applications within ecological frameworks, is still lagging behind the methodological advances. Indeed, variation in detection probability among prey species can occur, representing a potentially insidious source of bias in food-habit studies of carnivores. Calibration of DNA-based methods involves the optimization of specificity and sensitivity and, whereas priority is usually given to the former to avoid false positives, sensitivity is rarely investigated so that false negatives may be largely overlooked. We conducted feeding trials with captive wolves (Canis lupus) to validate a nanofluidic array technology recently developed for the detection of multiple prey species in scats. Using 371 scat samples from 12 wolves fed with a single-prey diet, the sensitivity of our nanofluidic array method varied between 0.45 and 0.95 for the six main ungulate prey species. The method sensitivity was enhanced by using multiple markers per species and by a relatively low threshold of number of amplifying markers required to confirm a detection. Yet, at least two markers should be used to avoid false positives. By acknowledging sources of bias in sensitivity to reliably interpret the results of DNA-based dietary methods, our study highlights the relevance of feeding experiments to optimally calibrate the relative thresholds to define a positive detection and investigate the occurrence and extent of biases in sensitivity

Experimental feeding validates nanofluidic array technology for DNA detection of ungulate prey in wolf scats

The study of carnivores' diet is a key component to enhance knowledge on the ecology of predators and their effect on prey populations. Although molecular approaches to detect prey DNA in carnivore scats are improving, the validation of their accuracy, a prerequisite for reliable applications within ecological frameworks, is still lagging behind the methodological advances. Indeed, variation in detection probability among prey species can occur, representing a potentially insidious source of bias in food-habit studies of carnivores. Calibration of DNA-based methods involves the optimization of specificity and sensitivity and, whereas priority is usually given to the former to avoid false positives, sensitivity is rarely investigated so that false negatives may be largely overlooked. We conducted feeding trials with captive wolves (Canis lupus) to validate a nanofluidic array technology recently developed for the detection of multiple prey species in scats. Using 371 scat samples from 12 wolves fed with a single-prey diet, the sensitivity of our nanofluidic array method varied between 0.45 and 0.95 for the six main ungulate prey species. The method sensitivity was enhanced by using multiple markers per species and by a relatively low threshold of number of amplifying markers required to confirm a detection. Yet, at least two markers should be used to avoid false positives. By acknowledging sources of bias in sensitivity to reliably interpret the results of DNA-based dietary methods, our study highlights the relevance of feeding experiments to optimally calibrate the relative thresholds to define a positive detection and investigate the occurrence and extent of biases in sensitivity

Multiple species-specific molecular markers using nanofluidic array as a tool to detect prey DNA from carnivore scats

Large carnivore feeding ecology plays a crucial role for management and conservation for predators and their prey. One of the keys to this kind of research is to identify the species composition in the predator diet, for example, prey determination from scat content. DNA-based methods applied to detect prey in predators' scats are viable alternatives to traditional macroscopic approaches, showing an increased reliability and higher prey detection rate. Here, we developed a molecular method for prey species identification in wolf (Canis lupus) scats using multiple species-specific marker loci on the cytochrome b gene for 18 target species. The final panel consisted of 80 assays, with a minimum of four markers per target species, and that amplified specifically when using a high-throughput Nanofluidic array technology (Fluidigm Inc.). As a practical example, we applied the method to identify target prey species DNA in 80 wolf scats collected in Sweden. Depending on the number of amplifying markers required to obtain a positive species call in a scat, the success in determining at least one prey species from the scats ranged from 44% to 92%. Although we highlight the need to evaluate the optimal number of markers for sensitive target species detection, the developed method is a fast and cost-efficient tool for prey identification in wolf scats and it also has the potential to be further developed and applied to other areas and large carnivores as well

Scavenging patterns of an inbred wolf population in a landscape with a pulse of human-provided carrion

Scavenging is an important part of food acquisition for many carnivore species that switch between scavenging and predation. In landscapes with anthropogenic impact, humans provide food that scavenging species can utilize. We quantified the magnitude of killing versus scavenging by gray wolves (Canis lupus) in Scandinavia where humans impact the ecosystem through hunter harvest, land use practices, and infrastructure. We investigated the cause of death of different animals utilized by wolves, and examined how the proportion of their consumption time spent scavenging was influenced by season, wolf social affiliation, level of inbreeding, density of moose (Alces alces) as their main prey, density of brown bear (Ursus arctos) as an intraguild competitor, and human density. We used data from 39 GPS-collared wolves covering 3198 study days (2001-2019), including 14,205 feeding locations within space-time clusters, and 1362 carcasses utilized by wolves. Most carcasses were wolf-killed (80.5%) while a small part had died from other natural causes (1.9%). The remaining had either anthropogenic mortality causes (4.7%), or the cause of death was unknown (12.9%). Time spent scavenging was higher during winter than during summer and autumn. Solitary wolves spent more time scavenging than pack-living individuals, likely because individual hunting success is lower than pack success. Scavenging time increased with the mean inbreeding coefficient of the adult wolves, possibly indicating that more inbred individuals resort to scavenging, which requires less body strength. There was weak evidence for competition between wolves and brown bears as well as a positive relationship between human density and time spent scavenging. This study shows how both intrinsic and extrinsic factors drive wolf scavenging behavior, and that despite a high level of inbreeding and access to carrion of anthropogenic origin, wolves mainly utilized their own kills

Avskjutning av älg över tid och rum – effekter av rovdjur och skogsbruk

Älgjakt och skogsbruk är en viktig del av Inre Skandinaviens kulturarv där nyttjande av skog och älg som naturresurser är sammanlänkade. Älgpopulationen har ökat i antal på grund av ändringar i skogsbruket från plockhuggning till beståndsskogsbruk under mitten av 1900-talet vilket gett älgen ökad tillgång till foder i områden med ungskog. Detta har resulterat i mer jaktmöjligheter. Jakten på älg har både stort ekonomiskt och rekreativt värde medans älgens vinterbete på tall leder till stora förluster för markägare genom försämrad virkeskvalitet och lägre produktion av biomassa. Utöver människans påverkan via skogsbruk och jaktuttag så påverkan stora rovdjur dynamiken hos älgpopulationer. Förvaltning av lokala älgpopulationer till önskad täthet och/eller sammansättning påverkas därför av förekomst av varg och björn, och rovdjurens uttag påverkar i sin tur det möjliga jaktuttaget och dess sammansättning. Syftet med denna studie är att 1) ge en översikt på älgavskjutningen i Inre Skandinavien på regional och lokal nivå tvärs över riksgränsen, och 2) analysera vilka faktorer som påverkar variationen i avskjutning av älg med fokus på förekomst av rovdjur, effekter av skogsbruk, förekomst av rådjur (vargens näst vanligaste bytesdjur efter älg), och i en landskapsgradient. Vi använde avskjutningsstatistik från perioden 1995-2020, inventeringsdata från den årliga varginventeringen, antal skjutna björnar, andel jordbruksmark (index för rådjurstäthet), andel ungskog per år, och latitud för att förklara variationen i avskjutning över tid och mellan olika förvaltningsenheter i Sverige och Norge. Studien visar en variation i avskjutningen av älg både mellan olika älgförvaltningsområden i Sverige och Norge och inom länderna under de senaste decennierna. Sett över studieperioden för 1995-2017 var den totala avskjutningen i Sverige 50% lägre och i Norge 40% lägre i älgförvaltningsområden som helt överlappade med vargrevir jämfört med områden utan varg. Närvaro av varg och björn påverkade både den totala avskjutningen och andelen kor bland skjutna vuxna. I de flesta älgjaktområden ökade den totala avskjutningen med ökad andel ungskog vilket troligtvis är ett resultat av en ökad tillgång på foder som dels kan påverka älgtätheten men även fördelningen av älg i landskapet. Rådjurstätheten förklarade däremot inget av variationen i avskjutningen på älg vilket indikerar att rådjurstätheten i Hedmark, Värmland och Dalarna är för låga för att resultera i att vargarna dödar färre älgar. Den totala avskjutningen varierade inte med latitud men ålders- och könssammansättningen av jaktuttaget uppvisade viss variation i relation till latitud i Sverige men mindre i Norge. Ett viktigt resultat från denna studie var skillnaderna mellan Sverige och Norge för de olika älgförvaltningsområdena. En del av variationen kan bero på skillnader i den faktiska miljön som vi inte tagit hänsyn till i denna studie. En annan förklaring kan vara olikheter i förvaltningen av älg både historiskt sett och i nutid. Sammantaget visar denna studie en betydande påverkan av varg och björn på jaktuttaget samtidigt som det årliga jaktuttaget oftast är större (2.4-3.5 gånger) än det uttag som sker från varg

Scavenging patterns of an inbred wolf population in a landscape with a pulse of human-provided carrion

Scavenging is an important part of food acquisition for many carnivore species that switch between scavenging and predation. In landscapes with anthropogenic impact, humans provide food that scavenging species can utilize. We quantified the magnitude of killing versus scavenging by gray wolves (Canis lupus) in Scandinavia where humans impact the ecosystem through hunter harvest, land use practices, and infrastructure. We investigated the cause of death of different animals utilized by wolves, and examined how the proportion of their consumption time spent scavenging was influenced by season, wolf social affiliation, level of inbreeding, density of moose (Alces alces) as their main prey, density of brown bear (Ursus arctos) as an intraguild competitor, and human density. We used data from 39 GPS-collared wolves covering 3198 study days (2001–2019),including 14,205 feeding locations within space–time clusters, and 1362 carcasses utilized by wolves. Most carcasses were wolf-killed (80.5%) while a small part had died from other natural causes (1.9%). The remaining had either anthropogenic mortality causes (4.7%), or the cause of death was unknown (12.9%). Time spent scavenging was higher during winter than during summer and autumn. Solitary wolves spent more time scavenging than pack-living individuals, likely because individual hunting success is lower than pack success. Scavenging time increased with the mean inbreeding coefficient of the adult wolves, possibly indicating that more inbred individuals resort to scavenging, which requires less body strength. There was weak evidence for competition between wolves and brown bears as well as a positive relationship between human density and time spent scavenging. This study shows how both intrinsic and extrinsic factors drive wolf scavenging behavior, and that despite a high level of inbreeding and access to carrion of anthropogenic origin, wolves mainly utilized their own kills. Canis lupus, consumption time, human density, inbreeding, intraguild competition, prey density, social affiliationpublishedVersio

Fitness and fur colouration. Testing the camouflage and thermoregulation hypotheses in an Arctic mammal

Selection for crypsis has been recognized as an important ecological driver of animal colouration, whereas the relative importance of thermoregulation is more contentious with mixed empirical support. A potential thermal advantage of darker individuals has been observed in a wide range of animal species. Arctic animals that exhibit colour polymorphisms and undergo seasonal colour moults are interesting study subjects for testing the two alternative hypotheses: demographic performance of different colour morphs might be differentially affected by snow cover with a cryptic advantage for lighter morphs, or conversely by winter temperature with a thermal advantage for darker morphs. In this study, we explored whether camouflage and thermoregulation might explain differences in reproduction and survival between the white and blue colour morphs of the Arctic fox Vulpes lagopus under natural conditions. Juvenile and adult survival, breeding propensity and litter size were measured for 798 captive-bred and released or wild-born Arctic foxes monitored during an 11-year period (2007–2017) in two subpopulations in south-central Norway. We investigated the proportion of the two colour morphs and compared their demographic performance in relation to spatial variation in duration of snow cover, onset of snow season and winter temperatures. After population re-establishment, a higher proportion of blue individuals was observed among wild-born Arctic foxes compared to the proportion of blue foxes released from the captive population. Our field study provides the first evidence for an effect of colour morph on the reproductive performance of Arctic foxes under natural conditions, with a higher breeding propensity of the blue morph compared to the white one. Performance of the two colour morphs was not differentially affected by the climatic variables, except for juvenile survival. Blue morph juveniles showed a tendency for higher survival under colder winter temperatures but lower survival under warmer temperatures compared to white morph juveniles. Overall, our findings do not consistently support predictions of the camouflage or the thermoregulation hypotheses. The higher success of blue foxes suggests an advantage of the dark morph not directly related to disruptive selection by crypsis or thermoregulation. Our results rather point to physiological adaptations and behavioural traits not necessarily connected to thermoregulation, such as stress response, immune function, sexual behaviour and aggressiveness. Our findings highlight the need to explore the potential role of genetic linkage or pleiotropy in influencing the fitness of white and blue Arctic foxes as well as other species with colour polymorphisms

New molecular methods to assess biodiversity. Potentials and pitfalls of DNA metabarcoding: a workshop report

This report presents the outcome of the joint work of PhD students and senior researchers working with DNA-based biodiversity assessment approaches with the goal to facilitate others the access to definitions and explanations about novel DNA-based methods. The work was performed during a PhD course (SLU PNS0169) at the Swedish University of Agricultural Sciences (SLU) in Uppsala, Sweden. The course was co-organized by the EU COST research network DNAqua-Net and the SLU Research Schools Focus on Soils and Water (FoSW) and Ecology - basics and applications. DNAqua-Net (COST Action CA15219, 2016-2020) is a network connecting researchers, water managers, politicians and other stakeholders with the aim to develop new genetic tools for bioassessment of aquatic ecosystems in Europe and beyond. The PhD course offered a comprehensive overview of the paradigm shift from traditional morphology-based species identification to novel identification approaches based on molecular markers. We covered the use of molecular tools in both basic research and applied use with a focus on aquatic ecosystem assessment, from species collection to the use of diversity in environmental legislation. The focus of the course was on DNA (meta)barcoding and aquatic organisms. The knowledge gained was shared with the general public by creating Wikipedia pages and through this collaborative Open Access publication, co-authored by all course participants