Combining environmental DNA and remote sensing for efficient, fine-scale mapping of arthropod biodiversity.

Arthropods contribute importantly to ecosystem functioning but remain understudied. This undermines the validity of conservation decisions. Modern methods are now making arthropods easier to study, since arthropods can be mass-trapped, mass-identified, and semi-mass-quantified into 'many-row (observation), many-column (species)' datasets, with homogeneous error, high resolution, and copious environmental-covariate information. These 'novel community datasets' let us efficiently generate information on arthropod species distributions, conservation values, uncertainty, and the magnitude and direction of human impacts. We use a DNA-based method (barcode mapping) to produce an arthropod-community dataset from 121 Malaise-trap samples, and combine it with 29 remote-imagery layers using a deep neural net in a joint species distribution model. With this approach, we generate distribution maps for 76 arthropod species across a 225 km2 temperate-zone forested landscape. We combine the maps to visualize the fine-scale spatial distributions of species richness, community composition, and site irreplaceability. Old-growth forests show distinct community composition and higher species richness, and stream courses have the highest site-irreplaceability values. With this 'sideways biodiversity modelling' method, we demonstrate the feasibility of biodiversity mapping at sufficient spatial resolution to inform local management choices, while also being efficient enough to scale up to thousands of square kilometres. This article is part of the theme issue 'Towards a toolkit for global insect biodiversity monitoring'.

The origin, connectivity, and individual specialization of island wolves after deer extirpation.

Wolves are assumed to be ungulate obligates, however, a recently described pack on Pleasant Island, Alaska USA, is persisting on sea otters and other marine resources without ungulate prey, violating this long-held assumption. We address questions about these wolves regarding their origin and fate, degree of isolation, risk of inbreeding depression, and diet specialization by individual and sex. We applied DNA metabarcoding and genotyping by amplicon sequencing using 957 scats collected from 2016 to 2022, and reduced representation sequencing of tissue samples to establish a detailed understanding of Pleasant Island wolf ecology and compare them with adjacent mainland wolves. Dietary overlap was higher among individual wolves on Pleasant Island (Pianka's index mean 0.95 ± 0.03) compared to mainland wolves (0.70 ± 0.21). The individual diets of island wolves were dominated by sea otter, ranging from 40.6% to 63.2% weighted percent of occurrence (wPOO) (mean 55.5 ± 8.7). In contrast, individual mainland wolves primarily fed on ungulates (42.2 ± 21.3) or voles during a population outbreak (31.2 ± 23.2). We traced the origin of the Pleasant Island pack to a mainland pair that colonized around 2013 and produced several litters. After this breeding pair was killed, their female offspring and an immigrant male became the new breeders in 2019. We detected 20 individuals of which 8 (40%) were trapped and killed while two died of natural causes during the 6-year study. Except for the new breeding male, the pedigree analysis and genotype results showed no additional movement to or from the island, indicating limited dispersal but no evidence of inbreeding. Our findings suggest wolves exhibit more flexible foraging behavior than previously believed, and hunting strategies can substantially differ between individuals within or between packs. Nevertheless, anthropogenic and natural mortality combined with limited connectivity to the mainland may inhibit the continued persistence of Pleasant Island wolves.

Human disturbance in riparian areas disrupts predator-prey interactions between grizzly bears and salmon.

Wildlife must increasingly balance trade-offs between the need to access important foods and the mortality risks associated with human-dominated landscapes. Human disturbance can profoundly influence wildlife behavior, but managers know little about the relationship between disturbance-behavior dynamics and associated consequences for foraging. We address this gap by empirically investigating the consequences of human activity on a keystone predator-prey interaction in a region with limited but varied industrial disturbance. Using stable isotope data from 226 hair samples of grizzly bears (Ursus arctos horribilis) collected from 1995 to 2014 across 22 salmon-bearing watersheds (88,000 km2) in British Columbia, Canada, we examined how human activity influenced their consumption of spawning salmon (Oncorhynchus spp.), a fitness-related food. Accounting for the abundance of salmon and other foods, salmon consumption strongly decreased (up to 59% for females) with increasing human disturbance (as measured by the human footprint index) in riparian zones of salmon-bearing rivers. Declines in salmon consumption occurred with disturbance even in watersheds with low footprints. In a region currently among the least influenced by industrial activity, intensification of disturbance in river valleys is predicted to increasingly decouple bears from salmon, possibly driving associated reductions in population productivity and provisioning of salmon nutrients to terrestrial ecosystems. Accordingly, we draw on our results to make landscape-scale and access-related management recommendations beyond current streamside protection buffers. This work illustrates the interaction between habitat modification and food security for wildlife, highlighting the potential for unacknowledged interactions and cumulative effects in increasingly modified landscapes.

Creating, curating and evaluating a mitogenomic reference database to improve regional species identification using environmental DNA.

Species detection using eDNA is revolutionizing global capacity to monitor biodiversity. However, the lack of regional, vouchered, genomic sequence information-especially sequence information that includes intraspecific variation-creates a bottleneck for management agencies wanting to harness the complete power of eDNA to monitor taxa and implement eDNA analyses. eDNA studies depend upon regional databases of mitogenomic sequence information to evaluate the effectiveness of such data to detect and identify taxa. We created the Oregon Biodiversity Genome Project to create a database of complete, nearly error-free mitogenomic sequences for all of Oregon's fishes. We have successfully assembled the complete mitogenomes of 313 specimens of freshwater, anadromous and estuarine fishes representing 24 families, 55 genera and 129 species and lineages. Comparative analyses of these sequences illustrate that many regions of the mitogenome are taxonomically informative, that the short (~150 bp) mitochondrial 'barcode' regions typically used for eDNA assays do not consistently diagnose for species and that complete single or multiple genes of the mitogenome are preferable for identifying Oregon's fishes. This project provides a blueprint for other researchers to follow as they build regional databases, illustrates the taxonomic value and limits of complete mitogenomic sequences and offers clues as to how current eDNA assays and environmental genomics methods of the future can best leverage this information.

Humanity's diverse predatory niche and its ecological consequences.

Although humans have long been predators with enduring nutritive and cultural relationships with their prey, seldom have conservation ecologists considered the divergent predatory behavior of contemporary, industrialized humans. Recognizing that the number, strength and diversity of predator-prey relationships can profoundly influence biodiversity, here we analyze humanity's modern day predatory interactions with vertebrates and estimate their ecological consequences. Analysing IUCN 'use and trade' data for ~47,000 species, we show that fishers, hunters and other animal collectors prey on more than a third (~15,000 species) of Earth's vertebrates. Assessed over equivalent ranges, humans exploit up to 300 times more species than comparable non-human predators. Exploitation for the pet trade, medicine, and other uses now affects almost as many species as those targeted for food consumption, and almost 40% of exploited species are threatened by human use. Trait space analyses show that birds and mammals threatened by exploitation occupy a disproportionally large and unique region of ecological trait space, now at risk of loss. These patterns suggest far more species are subject to human-imposed ecological (e.g., landscapes of fear) and evolutionary (e.g., harvest selection) processes than previously considered. Moreover, continued overexploitation will likely bear profound consequences for biodiversity and ecosystem function.

Density-dependent changes in elk resource selection over successional time scales following forest disturbance.

There is an increasing need to understand how animals respond to modifications of their habitat following landscape-scale disturbances such as wildfire or timber harvest. Such disturbances can promote increased use by herbivores due to changes in plant community structure that improve forage conditions, but can also cause avoidance if other habitat functions provided by cover are substantially reduced or eliminated. Quantifying the total effects of these disturbances, however, is challenging because they may not fully be apparent unless observed at successional timescales. Further, the effects of disturbances that improve habitat quality may be density dependent, such that the benefits are (1) less valuable to high-density populations because the per-capita benefits are reduced when shared among more users or, alternatively, (2) more valuable to animals living in high densities because resources may be more depleted from the greater intraspecific competition. We used 30 years of telemetry data on elk occurring at two distinct population densities to quantify changes in space use at diel, monthly, and successional timescales following timber harvest. Elk selected logged areas at night only, with selection strongest during midsummer, and peak selection occurring 14 years post harvest, but persisting for 26-33 years. This pattern of increased selection at night following a reduction in overhead canopy cover is consistent with elk exploiting improved nutritional conditions for foraging. The magnitude of selection for logged areas was 73% higher for elk at low population density, consistent with predictions from the ideal free distribution. Yet elk avoided these same areas during daytime for up to 28 years post logging and instead selected untreated forest, suggesting a role for cover to meet other life history requirements. Our results demonstrate that while landscape-scale disturbances can lead to increased selection by large herbivores and suggest that the improvement in foraging conditions can persist over short-term successional timescales, the magnitude of the benefits may not be equal across population densities. Further, the enduring avoidance of logging treatments during the daytime indicates a need for structurally intact forests and suggests that a mosaic of forest patches of varying successional stages and structural completeness is likely to be the most beneficial to large herbivores.

Variation in adult and pup wolf diets at natal den sites is influenced by forest composition and configuration.

Although wolves are wide-ranging generalist carnivores throughout their life cycle, during the pup-rearing season wolf activity is focused on natal den sites where pup survival depends upon pack members provisioning food. Because prey availability is influenced by habitat quality within the home range, we investigated the relative importance of prey species for adults and pups and further examined the relationship between habitat characteristics, wolf diet, and litter size on Prince of Wales Island (POW) in Southeast Alaska. During 2012-2020, we detected 13 active den sites within the home ranges of nine wolf packs. We estimated minimum pup counts using motion-detecting cameras and individual genotypes from noninvasive samples (hair: n = 322; scat: n = 227) and quantified wolf diet composition using fecal DNA metabarcoding (n = 538). We assessed habitat composition, configuration, and connectivity within denning and annual home ranges estimated using wolf GPS-collar data. Contrary to expectations, wolves had a more constricted diet during denning season (April 15-July 31), and within this season pups had a narrower dietary niche (species richness [S] = 4) focused more on deer (relative frequency of occurrence [O/I] = 0.924) than adults (S = 15; deer O/I = 0.591). Litter size had a positive relationship with the relative frequency of deer in a wolf pack's diet. Wolf consumption of deer was positively associated with the proportion of young-growth forest (≤25 years old) within denning and annual home ranges. High levels of vegetation patch interspersion, and the density of closed logging roads were also important predictors, suggesting these habitat qualities were influential for increasing the availability of deer to wolves. Our results contrast with previous research indicating wolf pup diets included more alternate prey (i.e., beaver) than adults and emphasize the importance of deer to wolf viability on POW, especially during denning season.

Recovery of a marine keystone predator transforms terrestrial predator-prey dynamics.

Sea otters (Enhydra lutris) and wolves (Canis lupus) are two apex predators with strong and cascading effects on ecosystem structure and function. After decades of recovery from near extirpation, their ranges now overlap, allowing sea otters and wolves to interact for the first time in the scientific record. We intensively studied wolves during 2015 to 2021 in an island system colonized by sea otters in the 2000s and by wolves in 2013. After wolf colonization, we quantified shifts in foraging behavior with DNA metabarcoding of 689 wolf scats and stable isotope analyses, both revealing a dietary switch from Sitka black-tailed deer (Odocoileus hemionus), the terrestrial in situ primary prey, to sea otters. Here we show an unexpected result of the reintroduction and restoration of sea otters, which became an abundant marine subsidy for wolves following population recovery. The availability of sea otters allowed wolves to persist and continue to reproduce, subsequently nearly eliminating deer. Genotypes from 390 wolf scats and telemetry data from 13 wolves confirmed island fidelity constituting one of the highest known wolf population densities and upending standardly accepted wolf density predictions based on ungulate abundance. Whereas marine subsidies in other systems are generally derived from lower trophic levels, here an apex nearshore predator became a key prey species and linked nearshore and terrestrial food webs in a recently deglaciated and rapidly changing ecosystem. These results underscore that species restoration may serve as an unanticipated nutrient pathway for recipient ecosystems even resulting in cross-boundary subsidy cascades.

A seasonal pulse of ungulate neonates influences space use by carnivores in a multi-predator, multi-prey system.

The behavioral mechanisms by which predators encounter prey are poorly resolved. In particular, the extent to which predators engage in active search for prey versus incidentally encountering them has not been well studied in many systems and particularly not for neonate prey during the birth pulse. Parturition of many large herbivores occurs during a short and predictable temporal window in which young are highly vulnerable to predation. Our study aims to determine how a suite of carnivores responds to the seasonal pulse of newborn ungulates using contemporaneous global positioning system (GPS) locations of four species of predators and two species of prey. We used step-selection functions to assess whether coyotes, cougars, black bears, and bobcats encountered parturient adult female ungulates more often than expected by chance in a low-density population of mule deer and a high-density population of elk. We then assessed whether the carnivore species that encountered parturient prey more often than expected by chance did so by shifting their habitat use toward areas with a high probability of encountering neonates. None of the four carnivore species encountered GPS-collared parturient mule deer more often than expected by chance. By contrast, we determined that cougar and male bear movements positioned them in the proximity of GPS-collared parturient elk more often than expected by chance which may provide evidence of searching behavior. Although both male bears and cougars exhibited behavior consistent with active search for neonates, only male bears used elk parturition habitat in a way that dynamically tracked the phenology of the elk birth pulse suggesting that maximizing encounters with juvenile elk was a motivation when selecting resources. Our results suggest that there is high interspecific and intersexual variability in foraging strategies among large mammalian predators and their prey.

Diet analysis using generalized linear models derived from foraging processes using R package mvtweedie.

Diet analysis integrates a wide variety of visual, chemical, and biological identification of prey. Samples are often treated as compositional data, where each prey is analyzed as a continuous percentage of the total. However, analyzing compositional data results in analytical challenges, for example, highly parameterized models or prior transformation of data. Here, we present a novel approximation involving a Tweedie generalized linear model (GLM). We first review how this approximation emerges from considering predator foraging as a thinned and marked point process (with marks representing prey species and individual prey size). This derivation can motivate future theoretical and applied developments. We then provide a practical tutorial for the Tweedie GLM using new package mvtweedie that extends capabilities of widely used packages in R (mgcv and ggplot2) by transforming output to calculate prey compositions. We demonstrate this approach and software using two examples. Tufted Puffins (Fratercula cirrhata) provisioning their chicks on a colony in the northern Gulf of Alaska show decadal prey switching among sand lance and prowfish (1980-2000) and then Pacific herring and capelin (2000-2020), while wolves (Canis lupus ligoni) in southeast Alaska forage on mountain goats and marmots in northern uplands and marine mammals in seaward island coastlines.

Invertebrates for vertebrate biodiversity monitoring: Comparisons using three insect taxa as iDNA samplers.

Metabarcoding of environmental DNA (eDNA) is now widely used to build diversity profiles from DNA that has been shed by species into the environment. There is substantial interest in the expansion of eDNA approaches for improved detection of terrestrial vertebrates using invertebrate-derived DNA (iDNA) in which hematophagous, sarcophagous, and coprophagous invertebrates sample vertebrate blood, carrion, or faeces. Here, we used metabarcoding and multiple iDNA samplers (carrion flies, sandflies, and mosquitos) collected from 39 forested sites in the southern Amazon to profile gamma and alpha diversity. Our main objectives were to (1) compare diversity found with iDNA to camera trapping, which is the conventional method of vertebrate diversity surveillance; and (2) compare each of the iDNA samplers to assess the effectiveness, efficiency, and potential biases associated with each sampler. In total, we collected and analysed 1759 carrion flies, 48,686 sandflies, and 4776 mosquitos. Carrion flies revealed the greatest total vertebrate species richness at the landscape level, despite the least amount of sampling effort and the fewest number of individuals captured for metabarcoding, followed by sandflies. Camera traps had the highest median species richness at the site-level but showed strong bias towards carnivore and ungulate species and missed much of the diversity described by iDNA methods. Mosquitos showed a strong feeding preference for humans as did sandflies for armadillos, thus presenting potential utility to further study related to host-vector interactions.

Land-use effects on mosquito biodiversity and potential arbovirus emergence in the Southern Amazon, Brazil.

The Amazon rainforest is considered the largest reservoir of culicids and arboviruses in the world. It has been under intense human-driven alteration, especially in the so-called 'Arc of Deforestation', located in the eastern and southern regions. The emergence and transmission of infectious diseases are increasing, potentially due to land-use change. We used landscape-scale mosquito surveillance across a forest fragmentation gradient in the southern Amazon to evaluate the relationship between forest disturbance and the composition and structure of mosquito communities with a particular focus on the potential for arbovirus emergence in the region. Generalized linear models and logistic regression were used to associate the degree of landscape disturbance with arbovirus vectors' richness and abundance. A total of 1,960 culicids, belonging to 50 species, were collected from 2015 to 2016. Among these species, 20 have been associated with the transmission of arboviruses. Our results show an association of land use, more specifically small size of forest remnants with more irregular shape and higher edge density, with the increase of arbovirus vectors' richness and abundance. Six species of mosquito vectors exhibited a higher probability of occurrence in landscapes with medium or high degrees of disturbance. Our results indicate that land-use change influences mosquito communities with potential implications for the emergence of arboviruses.

Extensive aquatic subsidies lead to territorial breakdown and high density of an apex predator.

Energetic subsidies between terrestrial and aquatic ecosystems can strongly influence food webs and population dynamics. Our objective was to study how aquatic subsidies affected jaguar (Panthera onca) diet, sociality, and population density in a seasonally flooded protected area in the Brazilian Pantanal. The diet (n = 138 scats) was dominated by fish (46%) and aquatic reptiles (55%), representing the first jaguar population known to feed extensively on fish and to minimally consume mammals (11%). These aquatic subsidies supported the highest jaguar population density estimate to date (12.4 jaguars/100 km²) derived from camera traps (8,065 trap nights) and GPS collars (n = 13). Contrary to their mostly solitary behavior elsewhere, we documented social interactions previously unobserved between same-sex adults including cooperative fishing, co-traveling, and play. Our study demonstrates that aquatic subsidies, frequently described in omnivores, can also transform the ecology and behavior of obligate carnivores.

Predicted distribution of a rare and understudied forest carnivore: Humboldt marten (Martes caurina humboldtensis).

BACKGROUND: Many mammalian species have experienced range contractions. Following a reduction in distribution that has resulted in apparently small and disjunct populations, the Humboldt marten (Martes caurina humboldtensis) was recently designated as federally Threatened and state Endangered. This subspecies of Pacific marten occurring in coastal Oregon and northern California, also known as coastal martens, appear unlike martens that occur in snow-associated regions in that vegetation associations appear to differ widely between Humboldt marten populations. We expected current distributions represent realized niches, but estimating factors associated with long-term occurrence was challenging for this rare and little-known species. Here, we assessed the predicted contemporary distribution of Humboldt martens and interpret our findings as hypotheses correlated with the subspecies' niche to inform strategic conservation actions. METHODS: We modeled Humboldt marten distribution using a maximum entropy (Maxent) approach. We spatially-thinned 10,229 marten locations collected from 1996-2020 by applying a minimum distance of 500-m between locations, resulting in 384 locations used to assess correlations of marten occurrence with biotic and abiotic variables. We independently optimized the spatial scale of each variable and focused development of model variables on biotic associations (e.g., hypothesized relationships with forest conditions), given that abiotic factors such as precipitation are largely static and not alterable within a management context. RESULTS: Humboldt marten locations were positively associated with increased shrub cover (salal (Gautheria shallon)), mast producing trees (e.g., tanoak, Notholithocarpus densiflorus), increased pine (Pinus sp.) proportion of total basal area, annual precipitation at home-range spatial scales, low and high amounts of canopy cover and slope, and cooler August temperatures. Unlike other recent literature, we found little evidence that Humboldt martens were associated with old-growth structural indices. This case study provides an example of how limited information on rare or lesser-known species can lead to differing interpretations, emphasizing the need for study-level replication in ecology. Humboldt marten conservation would benefit from continued survey effort to clarify range extent, population sizes, and fine-scale habitat use.

Variable strategies to solve risk-reward tradeoffs in carnivore communities.

Mesopredator release theory suggests that dominant predators suppress subordinate carnivores and ultimately shape community dynamics, but the assumption that subordinate species are only negatively affected ignores the possibility of facilitation through scavenging. We examined the interplay within a carnivore community consisting of cougars, coyotes, black bears, and bobcats using contemporaneous Global Positioning System telemetry data from 51 individuals; diet analysis from 972 DNA-metabarcoded scats; and data from 128 physical investigations of cougar kill sites, 28 of which were monitored with remote cameras. Resource provisioning from competitively dominant cougars to coyotes through scavenging was so prolific as to be an overwhelming determinant of coyote behavior, space use, and resource acquisition. This was evident via the strong attraction of coyotes to cougar kill sites, frequent scavenging of cougar-killed prey, and coyote diets that nearly matched cougars in the magnitude of ungulate consumption. Yet coyotes were often killed by cougars and used space to minimize encounters, complicating the fitness benefits gained from scavenging. We estimated that 23% (95% CI: 8 to 55%) of the coyote population in our study area was killed by cougars annually, suggesting that coyote interactions with cougars are a complex behavioral game of risk and reward. In contrast, we found no indication that bobcat space use or diet was influenced by cougars. Black bears avoided cougars, but there was no evidence of attraction to cougar kill sites and much lower levels of ungulate consumption and carcass visitation than for coyotes. Interspecific interactions among carnivores are multifaceted, encompassing both suppression and facilitation.

Evaluating and integrating spatial capture-recapture models with data of variable individual identifiability.

Spatial capture-recapture (SCR) models have become the preferred tool for estimating densities of carnivores. Within this family of models are variants requiring identification of all individuals in each encounter (SCR), a subset of individuals only (generalized spatial mark-resight, gSMR), or no individual identification (spatial count or spatial presence-absence). Although each technique has been shown through simulation to yield unbiased results, the consistency and relative precision of estimates across methods in real-world settings are seldom considered. We tested a suite of models ranging from those only requiring detections of unmarked individuals to others that integrate remote camera, physical capture, genetic, and global positioning system (GPS) data into a hybrid model, to estimate population densities of black bears, bobcats, cougars, and coyotes. For each species, we genotyped fecal DNA collected with detection dogs during a 20-d period. A subset of individuals from each species was affixed with GPS collars bearing unique markings and resighted by remote cameras over 140 d contemporaneous with scat collection. Camera-based gSMR models produced density estimates that differed by <10% from genetic SCR for bears, cougars, and coyotes once important sources of variation (sex or behavioral status) were controlled for. For bobcats, SCR estimates were 33% higher than gSMR. The cause of the discrepancies in estimates was likely attributable to challenges designing a study compatible for species with disparate home range sizes and the difficulty of collecting sufficient data in a timeframe in which demographic closure could be assumed. Unmarked models estimated densities that varied greatly from SCR, but estimates became more consistent in models wherein more individuals were identifiable. Hybrid models containing all data sources exhibited the most precise estimates for all species. For studies in which only sparse data can be obtained and the strictest model assumptions are unlikely to be met, we suggest researchers use caution making inference from models lacking individual identity. For best results, we further recommend the use of methods requiring at least a subset of the population is marked and that multiple data sets are incorporated when possible.

Mesocarnivore landscape use along a gradient of urban, rural, and forest cover.

Mesocarnivores fill a vital role in ecosystems through effects on community health and structure. Anthropogenic-altered landscapes can benefit some species and adversely affect others. For some carnivores, prey availability increases with urbanization, but landscape use can be complicated by interactions among carnivores as well as differing human tolerance of some species. We used camera traps to survey along a gradient of urban, rural, and forest cover to quantify how carnivore landscape use varies among guild members and determine if a species was a human exploiter, adapter, or avoider. Our study was conducted in and around Corvallis, Oregon from April 2018 to February 2019 (11,914 trap nights) using 47 camera trap locations on a gradient from urban to rural. Our focal species were bobcat (Lynx rufus), coyote (Canis latrans), gray fox (Urocyon cinereoargenteus), opossum (Didelphis virginiana), raccoon (Procyon lotor), and striped skunk (Mephitis mephitis). Raccoon and opossum were human exploiters with low use of forest cover and positive association with urban and rural developed areas likely due to human-derived resources as well as some refugia from larger predators. Coyote and gray fox were human adapters with high use of natural habitats while the effects of urbanization ranged from weak to indiscernible. Bobcat and striped skunk appeared to be human avoiders with negative relationship with urban cover and higher landscape use of forest cover. We conducted a diel temporal activity analysis and found mostly nocturnal activity within the guild, but more diurnal activity by larger-bodied predators compared to the smaller species. Although these species coexist as a community in human-dominated landscapes throughout much of North America, the effects of urbanization were not equal across species. Our results, especially for gray fox and striped skunk, are counter to research in other regions, suggesting that mesopredator use of urbanized landscapes can vary depending on the environmental conditions of the study area and management actions are likely to be most effective when decisions are based on locally derived data.

A forest loss report card for the world's protected areas.

Protected areas are a key tool in the conservation of global biodiversity and carbon stores. We conducted a global test of the degree to which more than 18,000 terrestrial protected areas (totalling 5,293,217 km2) reduce deforestation in relation to unprotected areas. We also derived indices that quantify how well countries' forests are protected, both in terms of forested area protected and effectiveness of protected areas at reducing deforestation, in relation to vertebrate species richness, aboveground forest carbon biomass and background deforestation rates. Overall, protected areas did not eliminate deforestation, but reduced deforestation rates by 41%. Protected area deforestation rates were lowest in small reserves with low background deforestation rates. Critically, we found that after adjusting for effectiveness, only 6.5%-rather than 15.7%-of the world's forests are protected, well below the Aichi Convention on Biological Diversity's 2020 Target of 17%. We propose that global targets for protected areas should include quantitative goals for effectiveness in addition to spatial extent.

Pseudogymnoascus destructans growth in wood, soil and guano substrates.

Understanding how a pathogen can grow on different substrates and how this growth impacts its dispersal are critical to understanding the risks and control of emerging infectious diseases. Pseudogymnoascus destructans (Pd) causes white-nose syndrome (WNS) in many bat species and can persist in, and transmit from, the environment. We experimentally evaluated Pd growth on common substrates to better understand mechanisms of pathogen persistence, transmission and viability. We inoculated autoclaved guano, fresh guano, soil, and wood with live Pd fungus and evaluated (1) whether Pd grows or persists on each (2) if spores of the fungus remain viable 4 months after inoculation on each substrate, and (3) whether detection and quantitation of Pd on swabs is sensitive to the choice to two commonly used DNA extraction kits. After inoculating each substrate with 460,000 Pd spores, we collected ~ 0.20 g of guano and soil, and swabs from wood every 16 days for 64 days to quantify pathogen load through time using real-time qPCR. We detected Pd on all substrates over the course of the experiment. We observed a tenfold increase in pathogen loads on autoclaved guano and persistence but not growth in fresh guano. Pathogen loads increased marginally on wood but declined ~ 60-fold in soil. After four months, apparently viable spores were harvested from all substrates but germination did not occur from fresh guano. We additionally found that detection and quantitation of Pd from swabs of wood surfaces is sensitive to the DNA extraction method. The commonly used PrepMan Ultra Reagent protocol yielded substantially less DNA than did the QIAGEN DNeasy Blood and Tissue Kit. Notably the PrepMan Ultra Reagent failed to detect Pd in many wood swabs that were detected by QIAGEN and were subsequently found to contain substantial live conidia. Our results indicate that Pd can persist or even grow on common environmental substrates with results dependent on whether microbial competitors have been eliminated. Although we observed clear rapid declines in Pd on soil, viable spores were harvested four months after inoculation. These results suggest that environmental substrates and guano can in general serve as infectious environmental reservoirs due to long-term persistence, and even growth, of live Pd. This should inform management interventions to sanitize or modify structures to reduce transmission risk as well early detection rapid response (EDRR) planning.