Niche suitability and spatial distribution patterns of anurans in a unique Ecoregion mosaic of Northern Pakistan.

The lack of information regarding biodiversity status hampers designing and implementing conservation strategies and achieving future targets. Northern Pakistan consists of a unique ecoregion mosaic which supports a myriad of environmental niches for anuran diversity in comparison to the deserts and xeric shrublands throughout the rest of the country. In order to study the niche suitability, species overlap and distribution patterns in Pakistan, we collected observational data for nine anuran species across several distinct ecoregions by surveying 87 randomly selected locations from 2016 to 2018 in Rawalpindi District and Islamabad Capital Territory. Our model showed that the precipitation of the warmest and coldest quarter, distance to rivers and vegetation were the greatest drivers of anuran distribution, expectedly indicating that the presence of humid forests and proximity to waterways greatly influences the habitable range of anurans in Pakistan. Sympatric overlap between species occurred at significantly higher density in tropical and subtropical coniferous forests than in other ecoregion types. We found species such as Minervarya spp., Hoplobatrachus tigerinus and Euphlyctis spp. preferred the lowlands in proximal, central and southern parts of the study area proximal to urban settlements, with little vegetation and higher average temperatures. Duttaphrynus bengalensis and D. stomaticus had scattered distributions throughout the study area with no clear preference for elevation. Sphaerotheca pashchima was patchily distributed in the midwestern extent of the study area as well as the foothills to the north. Microhyla nilphamariensis was widely distributed throughout the study area with a preference for both lowlands and montane terrain. Endemic frogs (Nanorana vicina and Allopaa hazarensis) were observed only in locations with higher elevations, higher density of streams and lower average temperatures as compared to the other seven species sampled. It is recommended to provide legal protection to amphibians of Pakistan, especially endemic species, through revision in the existing wildlife laws. We suggest studying the effectiveness of existing amphibian tunnels and corridors or designing new ones tailored to the needs of our species to prevent their local extinction due to ongoing or proposed urban development which might affect their dispersal and colonization.

On the snow leopard Trails: Occupancy pattern and implications for management in the Pamir.

The snow leopard (Panthera uncia) inhabits one of the most challenging environments on Earth, referred to as the 'third pole'. Only a fraction of its vast range has been explored thus far, owing to myriad of barriers inflicted by the remote terrain and socio-ecological realities of the landscapes. Understanding distribution patterns of species is essential to devise practical management measures. This study aimed to understand the distribution pattern and factors influencing occupancy of snow leopard in the Pamir Mountain range through sign-based occupancy modelling. Our study confirmed that the Pamir range is a snow leopard stronghold, with occupancy estimated at 0.57 ± 0.02. The topographic features positively influenced the detection probability (p = 0.37 ± 0.005) of snow leopards. Occupancy was influenced by mean annual precipitation (ß = -6.12 ± 1.8), density of roads (ß = -1.61 ± 0.6) and water sources (ß = 0.74 ± 0.4). Our findings underpin that sign-based distribution surveys provide vigorous scientific knowledge about elusive species and merit replication being used for other species. We propose to redefine the protected area boundaries based on ecological knowledge and encourage transboundary cooperation to safeguard snow leopards at a landscape scale.

Expanding or shrinking? range shifts in wild ungulates under climate change in Pamir-Karakoram mountains, Pakistan.

Climate change is expected to impact a large number of organisms in many ecosystems, including several threatened mammals. A better understanding of climate impacts on species can make conservation efforts more effective. The Himalayan ibex (Capra ibex sibirica) and blue sheep (Pseudois nayaur) are economically important wild ungulates in northern Pakistan because they are sought-after hunting trophies. However, both species are threatened due to several human-induced factors, and these factors are expected to aggravate under changing climate in the High Himalayas. In this study, we investigated populations of ibex and blue sheep in the Pamir-Karakoram mountains in order to (i) update and validate their geographical distributions through empirical data; (ii) understand range shifts under climate change scenarios; and (iii) predict future habitats to aid long-term conservation planning. Presence records of target species were collected through camera trapping and sightings in the field. We constructed Maximum Entropy (MaxEnt) model on presence record and six key climatic variables to predict the current and future distributions of ibex and blue sheep. Two representative concentration pathways (4.5 and 8.5) and two-time projections (2050 and 2070) were used for future range predictions. Our results indicated that ca. 37% and 9% of the total study area (Gilgit-Baltistan) was suitable under current climatic conditions for Himalayan ibex and blue sheep, respectively. Annual mean precipitation was a key determinant of suitable habitat for both ungulate species. Under changing climate scenarios, both species will lose a significant part of their habitats, particularly in the Himalayan and Hindu Kush ranges. The Pamir-Karakoram ranges will serve as climate refugia for both species. This area shall remain focus of future conservation efforts to protect Pakistan's mountain ungulates.

Patterns of livestock depredation and Human-wildlife conflict in Misgar valley of Hunza, Pakistan.

Throughout the world, livestock predation by mammalian carnivores causes significant economic losses to poor farmers, and leads to human-wildlife conflicts. These conflicts result in a negative attitude towards carnivore conservation and often trigger retaliatory killing. In northern Pakistan, we investigated livestock depredation by large carnivores between 2014 and 2019, and subsequent Human-wildlife conflict, through questionnaire-based surveys (n = 100 households). We used a semi-structured questionnaire to collect data on livestock population, depredation patterns, predation count, and conservation approaches. We found a statistically significant increasing pattern of predation with influential factors such as age, gender, occupation, education of respondents, population of predators, threats index for predators and conservation efforts. Some 310 livestock heads with an average of 51 animals per year out of the total 9273 heads were killed by predators, and among them 168 (54%) were attributed to the wolf and 142 (45.8%) to snow leopard. Major threats to carnivores in the area included retaliatory killing, habitat destruction and climate change. Incentivization against depredation losses, guarded grazing and construction of predator-proof corral may reduce Human-wildlife conflict and both livelihood and predator can be safeguarded in the study area.

Large-scale whole-genome resequencing unravels the domestication history of Cannabis sativa.

Cannabis sativa has long been an important source of fiber extracted from hemp and both medicinal and recreational drugs based on cannabinoid compounds. Here, we investigated its poorly known domestication history using whole-genome resequencing of 110 accessions from worldwide origins. We show that C. sativa was first domesticated in early Neolithic times in East Asia and that all current hemp and drug cultivars diverged from an ancestral gene pool currently represented by feral plants and landraces in China. We identified candidate genes associated with traits differentiating hemp and drug cultivars, including branching pattern and cellulose/lignin biosynthesis. We also found evidence for loss of function of genes involved in the synthesis of the two major biochemically competing cannabinoids during selection for increased fiber production or psychoactive properties. Our results provide a unique global view of the domestication of C. sativa and offer valuable genomic resources for ongoing functional and molecular breeding research.

An empirical demonstration of the effect of study design on density estimations.

The simultaneous development of technology (e.g. camera traps) and statistical methods, particularly spatially capture-recapture (SCR), has improved monitoring of large mammals in recent years. SCR estimates are known to be sensitive to sampling design, yet existing recommendations about trap spacing and coverage are often not achieved, particularly for sampling wide-ranging and rare species in landscapes that allow for limited accessibility. Consequently, most camera trap studies on large wide-ranging carnivores relies on convenience or judgmental sampling, and often yields compromised results. This study attempts to highlight the importance of carefully considered sampling design for large carnivores that, because of low densities and elusive behavior, are challenging to monitor. As a motivating example, we use two years of snow leopard camera trapping data from the same areas in the high mountains of Pakistan but with vastly different camera configurations, to demonstrate that estimates of density and space use are indeed sensitive to the trapping array. A compact design, one in which cameras were placed much closer together than generally recommended and therefore have lower spatial coverage, resulted in fewer individuals observed, but more recaptures, and estimates of density and space use were inconsistent with expectations for the region. In contrast, a diffuse design, one with larger spacing and spatial coverage and more consistent with general recommendations, detected more individuals, had fewer recaptures, but generated estimates of density and space use that were in line with expectations. Researchers often opt for compact camera configurations while monitoring wide-ranging and rare species, in an attempt to maximize the encounter probabilities. We empirically demonstrate the potential for biases when sampling a small area approximately the size of a single home range-this arises from exposing fewer individuals than deemed sufficient for estimation. The smaller trapping array may also underestimate density by significantly inflating [Formula: see text]. On the other hand, larger trapping array with fewer detectors and poor design induces uncertainties in the estimates. We conclude that existing design recommendations have limited utility on practical grounds for devising feasible sampling designs for large ranging species, and more research on SCR designs is required that allows for integrating biological and habitat traits of large carnivores in sampling framework. We also suggest that caution should be exercised when there is a reliance on convenience sampling.

The response of culturally important plants to experimental warming and clipping in Pakistan Himalayas.

The relative effects of climate warming with grazing on medicinally important plants are not fully understood in Hindukush-Himalaya (HKH) region. Therefore, we combined the indigenous knowledge about culturally important therapeutic plants and climate change with experimental warming (open-top chambers) and manual clipping (simulated grazing effect) and compared the relative difference on aboveground biomass and percent cover of plant species at five alpine meadow sites on an elevation gradient (4696 m-3346 m) from 2016-2018. Experimental warming increased biomass and percent cover throughout the experiment. However, the interactive treatment effect (warming x clipping) was significant on biomass but not on percent cover. These responses were taxa specific. Warming induced an increase of 1 ± 0.6% in Bistorta officinalis percent cover while for Poa alpina it was 18.7 ± 4.9%. Contrastingly, clipping had a marginally significant effect in reducing the biomass and cover of all plant species. Clipping treatment reduced vegetation cover & biomass by 2.3% and 6.26%, respectively, but that was not significant due to the high variability among taxa response at different sites. It was found that clipping decreased the effects of warming in interactive plots. Thus, warming may increase the availability of therapeutic plants for indigenous people while overgrazing would have deteriorating effects locally. The findings of this research illustrate that vegetation sensitivity to warming and overgrazing is likely to affect man-environment relationships, and traditional knowledge on a regional scale.

Optimal sampling design for spatial capture-recapture.

Spatial capture-recapture (SCR) has emerged as the industry standard for estimating population density by leveraging information from spatial locations of repeat encounters of individuals. The precision of density estimates depends fundamentally on the number and spatial configuration of traps. Despite this knowledge, existing sampling design recommendations are heuristic and their performance remains untested for most practical applications. To address this issue, we propose a genetic algorithm that minimizes any sensible, criteria-based objective function to produce near-optimal sampling designs. To motivate the idea of optimality, we compare the performance of designs optimized using three model-based criteria related to the probability of capture. We use simulation to show that these designs outperform those based on existing recommendations in terms of bias, precision, and accuracy in the estimation of population size. Our approach, available as a function in the R package oSCR, allows conservation practitioners and researchers to generate customized and improved sampling designs for wildlife monitoring.

Identifying priority landscapes for conservation of snow leopards in Pakistan.

Pakistan's total estimated snow leopard habitat is about 80,000 km2 of which about half is considered prime habitat. However, this preliminary demarcation was not always in close agreement with the actual distribution-the discrepancy may be huge at the local and regional level. Recent technological developments like camera trapping and molecular genetics allow for collecting reliable presence records that could be used to construct realistic species distribution based on empirical data and advanced mathematical approaches like MaxEnt. The current study followed this approach to construct an accurate distribution of the species in Pakistan. Moreover, movement corridors, among different landscapes, were also identified through circuit theory. The probability of habitat suitability, generated from 98 presence points and 11 environmental variables, scored the snow leopard's assumed range in Pakistan, from 0 to 0.97. A large portion of the known range represented low-quality habitat, including areas in lower Chitral, Swat, Astore, and Kashmir. Conversely, Khunjerab, Misgar, Chapursan, Qurumber, Broghil, and Central Karakoram represented high-quality habitats. Variables with higher contributions in the MaxEnt model were precipitation during the driest month (34%), annual mean temperature (19.5%), mean diurnal range of temperature (9.8%), annual precipitation (9.4%), and river density (9.2). The model was validated through receiver operating characteristic (ROC) plots and defined thresholds. The average test AUC in Maxent for the replicate runs was 0.933 while the value of AUC by ROC curve calculated at 0.15 threshold was 1.00. These validation tests suggested a good model fit and strong predictive power. The connectivity analysis revealed that the population in the Hindukush landscape appears to be more connected with the population in Afghanistan as compared to other populations in Pakistan. Similarly, the Pamir-Karakoram population is better connected with China and Tajikistan, while the Himalayan population was connected with the population in India. Based on our findings we propose three model landscapes to be considered under the Global Snow Leopard Ecosystem Protection Program (GSLEP) agenda as regional priority areas, to safeguard the future of the snow leopard in Pakistan and the region. These landscapes fall within mountain ranges of the Himalaya, Hindu Kush and Karakoram-Pamir, respectively. We also identified gaps in the existing protected areas network and suggest new protected areas in Chitral and Gilgit-Baltistan to protect critical habitats of snow leopard in Pakistan.

Correction: Diet of the brown bear in Himalaya: Combining classical and molecular genetic techniques.

[This corrects the article DOI: 10.1371/journal.pone.0225698.].

Multiple observation processes in spatial capture-recapture models: How much do we gain?

Population monitoring data may originate from multiple methods and are often sparse and fraught with incomplete information due to practical and economic constraints. Models that can integrate multiple survey methods and are able to cope with incomplete data may help investigators exploit available information more thoroughly. Here, we developed an integrated spatial capture-recapture (SCR) model to incorporate multiple data sources with imperfect individual identification. We contrast inferences drawn from this model with alternate models incorporating only subsets of the data available. Using extensive simulations and an empirical example of multi-method brown bear (Ursus arctos) monitoring data from northern Pakistan, we quantified the benefits of including multiple sources of information in SCR models in terms of parameter precision and bias. Our multiple observation processes SCR model (MOP) yielded a more complete picture of the underlying processes, reduced bias, and led to more precise parameter estimates. Our results suggest that the greatest gains from integrated SCR models can be expected in situations where detection probability is low, a large proportion of detections is not attributable to individuals, and the degree of overlap between individual home ranges is low.

Correction to: Occurrence and seasonal variation of human Plasmodium infection in Punjab Province, Pakistan.

After publication of the original article [1], we were notified that an author's name has been erroneously spelled. Aamir Ali Khattak should be replaced with Aamer Ali Khattak.

Diet of the brown bear in Himalaya: Combining classical and molecular genetic techniques.

The ecological requirements of brown bears are poorly known in the Himalaya region, which complicates conservation efforts. We documented the diet of the Himalayan brown bear (Ursus arctos isabellinus) by combining classical scat analysis and a newly developed molecular genetic technique (the trnL approach), in Deosai National Park, Pakistan. Brown bears consumed over 50 plant species, invertebrates, ungulates, and several rodents. Eight plant families; Poaceae, Polygonaceae, Cyperaceae, Apiaceae, Asteraceae, Caryophyllaceae, Lamiaceae, and Rubiaceae were commonly eaten with graminoids comprising the bulk of the diet. Golden marmots comprised the major mammalian biomass in the park, and were also the main meat source for bears. Animal matter, making 36% of dietary content, contributed half of the digestible energy, due to its higher nutritious value. We did not find a significant temporal pattern in diet, perhaps because the availability of the major diet (graminoids) did not change over the foraging period. Male brown bears were more carnivorous than females, probably because of their larger size, which requires higher energy and also makes them more efficient in capturing marmots. Frequencies of three plant species were also significantly higher in male brown bears; Bistorta affinis, Carex diluta, and Carex sp. Diet of the brown bear differed significantly between the park and surrounding valleys. In valleys, diet consisted predominantly of graminoids and crops, whereas the park provided more nutritious and diverse foodThe estimated digestible energy available to brown bears in Deosai was the lowest documented among brown bear populations, due to the lack of fruits and a relatively lower meat content. The low nutritious diet and high cost of metabolism in a high-altitude environment, probably explains the very low reproductive potential of this population.

Occurrence and seasonal variation of human Plasmodium infection in Punjab Province, Pakistan.

BACKGROUND: Malaria is the fifth leading cause of death worldwide. Pakistan is considered as a moderate malaria-endemic country but still, 177 million individuals are at risk of malaria. Roughly 60% of Pakistan's population, live in malaria-endemic regions. The present study is based upon the survey of various health care centers in 10 major cities of Northern and Southern Punjab to find out the malarial infection patterns in 2015. The diagnosis, seasonal variations, age and gender-wise distribution of Plasmodium spp. circulating in the study area were also included in the objectives. METHODS: The malaria-suspected patients '16075' were enrolled for malaria diagnosis using microscopy, out of which 925 were malaria positive which were processed for molecular analysis using nested PCR. The 18S rRNA genes of Plasmodium species were amplified, sequenced, blast and the phylogenetic tree was constructed based on sequences using online integrated tool MEGA7. RESULTS: The 364 cases recruited from Northern Punjab with the highest incidence in Rawalpindi (25.5%) and lowest in Chakwal (15.9%). From Southern Punjab 561 cases were enlisted Rajanpur (21.4%) maximum and lowest from Multan and Rahim Yar Khan (18%). The slide positivity rate, annual parasite incidence, and annual blood examination rates were 5.7 per 1000 population, 0.1, and 0.2% respectively. The only P. vivax (66.7%), P. falciparum (23.7%) and mixed infection by these two species (9.6%) were diagnosed. The same trend (P. vivax > P. falciparum > mixed infection) in species identification %age was confirmed from molecular analysis. However, the occurrence of malaria was higher in Southern Punjab (5.5%) as compared to the Northern Punjab (4.0%). The overall malaria percentage occurrence of treatment-seeking patients in all recruited cities of Punjab was 4.9%. The age-group of 1-20 and males among genders were more affected by malaria. The comparison of different seasons showed that the malaria infection was at a peak in Summer and post-monsoon. CONCLUSION: The incidence of malaria was high in the flood infected rural areas of Southern Punjab, Summer, and post-monsoon. The age group (1-20) and gender-wise males were more affected by malaria.

Evolutionary history of enigmatic bears in the Tibetan Plateau-Himalaya region and the identity of the yeti.

Although anecdotally associated with local bears (Ursus arctos and U. thibetanus), the exact identity of 'hominid'-like creatures important to folklore and mythology in the Tibetan Plateau-Himalaya region is still surrounded by mystery. Recently, two purported yeti samples from the Himalayas showed genetic affinity with an ancient polar bear, suggesting they may be from previously unrecognized, possibly hybrid, bear species, but this preliminary finding has been under question. We conducted a comprehensive genetic survey of field-collected and museum specimens to explore their identity and ultimately infer the evolutionary history of bears in the region. Phylogenetic analyses of mitochondrial DNA sequences determined clade affinities of the purported yeti samples in this study, strongly supporting the biological basis of the yeti legend to be local, extant bears. Complete mitochondrial genomes were assembled for Himalayan brown bear (U. a. isabellinus) and black bear (U. t. laniger) for the first time. Our results demonstrate that the Himalayan brown bear is one of the first-branching clades within the brown bear lineage, while Tibetan brown bears diverged much later. The estimated times of divergence of the Tibetan Plateau and Himalayan bear lineages overlap with Middle to Late Pleistocene glaciation events, suggesting that extant bears in the region are likely descendants of populations that survived in local refugia during the Pleistocene glaciations.

Habitat suitability and movement corridors of grey wolf (Canis lupus) in Northern Pakistan.

Habitat suitability models are useful to understand species distribution and to guide management and conservation strategies. The grey wolf (Canis lupus) has been extirpated from most of its historic range in Pakistan primarily due to its impact on livestock and livelihoods. We used non-invasive survey data from camera traps and genetic sampling to develop a habitat suitability model for C. lupus in northern Pakistan and to explore the extent of connectivity among populations. We detected suitable habitat of grey wolf using a maximum entropy approach (Maxent ver. 3.4.0) and identified suitable movement corridors using the Circuitscape 4.0 tool. Our model showed high levels of predictive performances, as seen from the values of area under curve (0.971±0.002) and true skill statistics (0.886±0.021). The main predictors for habitat suitability for C. lupus were distances to road, mean temperature of the wettest quarter and distance to river. The model predicted ca. 23,129 km2 of suitable areas for wolf in Pakistan, with much of suitable habitat in remote and inaccessible areas that appeared to be well connected through vulnerable movement corridors. These movement corridors suggest that potentially the wolf range can expand in Pakistan's Northern Areas. However, managing protected areas with stringent restrictions is challenging in northern Pakistan, in part due to heavy dependence of people on natural resources. The habitat suitability map provided by this study can inform future management strategies by helping authorities to identify key conservation areas.

Trichophyton verrucosum infection in livestock in the Chitral district of Pakistan.

INTRODUCTION: Trichophyton verrucosum belongs to the dermatophyte fungi, closely related organisms that cause skin infections in animals and humans. T. verrucosum infection has been reported in livestock and people in different countries from all continents. Human cases have been reported in different areas of Pakistan, but there is little information about the animal source of the fungus. METHODOLOGY: Dermatological specimens collected in the Chitral district of Pakistan for a study on mange in livestock were retrospectively analyzed for the presence of T. verrucosum. In total, 5,873 animals (1,087 cows, 2,033 goats, and 2,753 sheep) were screened for evidence of dermatological lesions during two surveys performed in the summer and winter seasons. Skin scrapings collected from animals with lesions were analyzed by direct microscopic examination after digestion in sodium hydroxide and a real-time polymerase chain reaction (PCR) targeting pathogenic Trichophyton species. RESULTS: At microscopy, samples from 18 cows (1.6%), 3 sheep (0.1%), and 4 goats (0.2%) were positive for fungal elements consistent with T. verrucosum. PCR confirmed the microscopy results. The prevalence was lower than that reported in other countries in intensive breeding farms. Results agree with the literature regarding factors affecting T. verrucosum diffusion, i.e., infection was more prevalent in cattle, especially in younger animals during the winter season. CONCLUSIONS: This study reports, for the first time, the presence of T. verrucosum in livestock in Pakistan. A better knowledge of the animal role in the spread of this fungus may allow the adoption of more efficient control measures and prophylaxis.

The threatening but unpredictable Sarcoptes scabiei: first deadly outbreak in the Himalayan lynx, Lynx lynx isabellinus, from Pakistan.

Although neglected, the mite Sarcoptes scabiei is an unpredictable emerging parasite, threatening human and animal health globally. In this paper we report the first fatal outbreak of sarcoptic mange in the endangered Himalayan lynx (Lynx lynx isabellinus) from Pakistan. A 10-year-old male Himalayan lynx was found in a miserable condition with severe crusted lesions in Chitral District, and immediately died. Post-mortem examination determined high S. scabiei density (1309 mites/cm(2) skin). It is most probably a genuine emergence, resulting from a new incidence due to the host-taxon derived or prey-to-predator cross-infestation hypotheses, and less probable to be apparent emergence resulting from increased infection in the Himalayan lynx population. This is an alarming situation for the conservation of this already threatened population, which demands surveillance for early detection and eventually rescue and treatment of the affected Himalayan lynx.

New perspectives in diet analysis based on DNA barcoding and parallel pyrosequencing: the trnL approach.

The development of DNA barcoding (species identification using a standardized DNA sequence), and the availability of recent DNA sequencing techniques offer new possibilities in diet analysis. DNA fragments shorter than 100-150 bp remain in a much higher proportion in degraded DNA samples and can be recovered from faeces. As a consequence, by using universal primers that amplify a very short but informative DNA fragment, it is possible to reliably identify the plant taxon that has been eaten. According to our experience and using this identification system, about 50% of the taxa can be identified to species using the trnL approach, that is, using the P6 loop of the chloroplast trnL (UAA) intron. We demonstrated that this new method is fast, simple to implement, and very robust. It can be applied for diet analyses of a wide range of phytophagous species at large scales. We also demonstrated that our approach is efficient for mammals, birds, insects and molluscs. This method opens new perspectives in ecology, not only by allowing large-scale studies on diet, but also by enhancing studies on resource partitioning among competing species, and describing food webs in ecosystems.