An experimental design for obtaining DNA of a target species and its diet from a single non-invasive genetic protocol.

Next-generation sequencing technology has enabled accurate insights into the diet of wildlife species. The protocols for faecal sample collection and DNA extraction for diet analysis have differed from those focusing on target species, even in most studies combining questions on both aspects. We designed an experiment to evaluate two protocols using 11 parameters and select a single one that will generate both target species (Asiatic wild ass, Equus hemionus, in Israel) and diet DNA, as an effective strategy to minimise time, effort, and cost without hampering efficiency. In Protocol A, we swabbed the outer surface of faecal boluses and extracted DNA using a Stool Kit, while for Protocol B, we homogenised faecal matter from inside the bolus followed by extraction using a Powersoil Kit. Protocol A performed significantly better for four parameters, which included, for the target species, microsatellite amplification success and the quantity of the GAPDH gene; and for its diet, the number of exact sequence variants (ESVs) obtained at genus level and plant genus richness. However, there was no significant difference in the amplification success of sex-linked and plant markers, total reads at genus level, number of genera obtained and plant genus composition. Although we chose Protocol A, both protocols yielded results for the target species and its diet, demonstrating that one single protocol can be used for both purposes, although a pilot study is recommended to optimise the protocol for specific systems. This strategy may also be useful for studies combining target species and their gut microbiome and parasitic load.

Positive selection and heat-response transcriptomes reveal adaptive features of the Brassicaceae desert model, Anastatica hierochuntica.

Plant adaptation to a desert environment and its endemic heat stress is poorly understood at the molecular level. The naturally heat-tolerant Brassicaceae species Anastatica hierochuntica is an ideal extremophyte model to identify genetic adaptations that have evolved to allow plants to tolerate heat stress and thrive in deserts. We generated an A. hierochuntica reference transcriptome and identified extremophyte adaptations by comparing Arabidopsis thaliana and A. hierochuntica transcriptome responses to heat, and detecting positively selected genes in A. hierochuntica. The two species exhibit similar transcriptome adjustment in response to heat and the A. hierochuntica transcriptome does not exist in a constitutive heat 'stress-ready' state. Furthermore, the A. hierochuntica global transcriptome as well as heat-responsive orthologs, display a lower basal and higher heat-induced expression than in A. thaliana. Genes positively selected in multiple extremophytes are associated with stomatal opening, nutrient acquisition, and UV-B induced DNA repair while those unique to A. hierochuntica are consistent with its photoperiod-insensitive, early-flowering phenotype. We suggest that evolution of a flexible transcriptome confers the ability to quickly react to extreme diurnal temperature fluctuations characteristic of a desert environment while positive selection of genes involved in stress tolerance and early flowering could facilitate an opportunistic desert lifestyle.

Subspecies hybridization as a potential conservation tool in species reintroductions.

Reintroductions are a powerful tool for the recovery of endangered species. However, their long-term success is strongly influenced by the genetic diversity of the reintroduced population. The chances of population persistence can be improved by enhancing the population's adaptive ability through the mixing of individuals from different sources. However, where source populations are too diverse the reintroduced population could also suffer from outbreeding depression or unsuccessful admixture due to behavioural or genetic barriers. For the reintroduction of Asiatic wild ass Equus hemionus ssp. in Israel, a breeding core was created from individuals of two different subspecies (E. h. onager & E. h. kulan). Today the population comprises approximately 300 individuals and displays no signs of outbreeding depression. The aim of this study was a population genomic evaluation of this conservation reintroduction protocol. We used maximum likelihood methods and genetic clustering analyses to investigate subspecies admixture and test for spatial autocorrelation based on subspecies ancestry. Further, we analysed heterozygosity and effective population sizes in the breeding core prior to release and the current wild population. We discovered high levels of subspecies admixture in the breeding core and wild population, consistent with a significant heterozygote excess in the breeding core. Furthermore, we found no signs of spatial autocorrelation associated with subspecies ancestry in the wild population. Inbreeding and variance effective population size estimates were low. Our results indicate no genetic or behavioural barriers to admixture between the subspecies and suggest that their hybridization has led to greater genetic diversity in the reintroduced population. The study provides rare empirical evidence of the successful application of subspecies hybridization in a reintroduction. It supports use of intraspecific hybridization as a tool to increase genetic diversity in conservation translocations.

An appetite for pests: Synanthropic insectivorous bats exploit cotton pest irruptions and consume various deleterious arthropods.

Conservation biological control (CBC) seeks to minimize the deleterious effects of agricultural pests by enhancing the efficiency of natural enemies. Despite the documented potential of insectivorous bats to consume pests, many synanthropic bat species are still underappreciated as beneficial species. We investigated the diet of Kuhl's pipistrelle (Pipistrellus kuhlii), a common synanthropic insectivorous bat that forages in urban and agricultural areas, to determine whether it may function as a natural enemy in CBC. Faecal samples of P. kuhlii were collected throughout the cotton-growing season from five roost sites near cotton fields located in a Mediterranean agroecosystem, Israel, and analyzed using DNA metabarcoding. Additionally, data on estimated abundance of major cotton pests were collected. We found that the diet of P. kuhlii significantly varied according to sites and dates and comprised 27 species of agricultural pests that were found in 77.2% of the samples, including pests of key economic concern. The dominant prey was the widespread cotton pest, the pink bollworm, Pectinophora gossypiella, found in 31% of the samples and in all the roosts. Pink bollworm abundance was positively correlated with its occurrence in the bat diet. Furthermore, the bats' dietary breadth narrowed, while temporal dietary overlap increased, in relation to increasing frequencies of pink bollworms in the diet. This suggests that P. kuhlii exploits pink bollworm irruptions by opportunistic feeding. We suggest that synanthropic bats provide important pest suppression services, may function as CBC agents of cotton pests and potentially contribute to suppress additional deleterious arthropods found in their diet in high frequencies.

Detecting hierarchical levels of connectivity in a population of Acacia tortilis at the northern edge of the species' global distribution: Combining classical population genetics and network analyses.

Genetic diversity and structure of populations at the edge of the species' spatial distribution are important for potential adaptation to environmental changes and consequently, for the long-term survival of the species. Here, we combined classical population genetic methods with newly developed network analyses to gain complementary insights into the genetic structure and diversity of Acacia tortilis, a keystone desert tree, at the northern edge of its global distribution, where the population is under threat from climatic, ecological, and anthropogenic changes. We sampled A. tortilis from 14 sites along the Dead Sea region and the Arava Valley in Israel and in Jordan. In addition, we obtained samples from Egypt and Sudan, the hypothesized origin of the species. Samples from all sites were genotyped using six polymorphic microsatellite loci.Our results indicate a significant genetic structure in A. tortilis along the Arava Valley. This was detected at different hierarchical levels-from the basic unit of the subpopulation, corresponding to groups of trees within ephemeral rivers (wadis), to groups of subpopulations (communities) that are genetically more connected relative to others. The latter structure mostly corresponds to the partition of the major drainage basins in the area. Network analyses, combined with classical methods, allowed for the identification of key A. tortilis subpopulations in this region, characterized by their relatively high level of genetic diversity and centrality in maintaining gene flow in the population. Characterizing such key subpopulations may enable conservation managers to focus their efforts on certain subpopulations that might be particularly important for the population's long-term persistence, thus contributing to species conservation within its peripheral range.

Revealing life-history traits by contrasting genetic estimations with predictions of effective population size.

Effective population size, a central concept in conservation biology, is now routinely estimated from genetic surveys and can also be theoretically predicted from demographic, life-history, and mating-system data. By evaluating the consistency of theoretical predictions with empirically estimated effective size, insights can be gained regarding life-history characteristics and the relative impact of different life-history traits on genetic drift. These insights can be used to design and inform management strategies aimed at increasing effective population size. We demonstrated this approach by addressing the conservation of a reintroduced population of Asiatic wild ass (Equus hemionus). We estimated the variance effective size (Nev ) from genetic data (N ev =24.3) and formulated predictions for the impacts on Nev of demography, polygyny, female variance in lifetime reproductive success (RS), and heritability of female RS. By contrasting the genetic estimation with theoretical predictions, we found that polygyny was the strongest factor affecting genetic drift because only when accounting for polygyny were predictions consistent with the genetically measured Nev . The comparison of effective-size estimation and predictions indicated that 10.6% of the males mated per generation when heritability of female RS was unaccounted for (polygyny responsible for 81% decrease in Nev ) and 19.5% mated when female RS was accounted for (polygyny responsible for 67% decrease in Nev ). Heritability of female RS also affected Nev ; hf2=0.91 (heritability responsible for 41% decrease in Nev ). The low effective size is of concern, and we suggest that management actions focus on factors identified as strongly affecting Nev, namely, increasing the availability of artificial water sources to increase number of dominant males contributing to the gene pool. This approach, evaluating life-history hypotheses in light of their impact on effective population size, and contrasting predictions with genetic measurements, is a general, applicable strategy that can be used to inform conservation practice.

Taming the late Quaternary phylogeography of the Eurasiatic wild ass through ancient and modern DNA.

Taxonomic over-splitting of extinct or endangered taxa, due to an incomplete knowledge of both skeletal morphological variability and the geographical ranges of past populations, continues to confuse the link between isolated extant populations and their ancestors. This is particularly problematic with the genus Equus. To more reliably determine the evolution and phylogeographic history of the endangered Asiatic wild ass, we studied the genetic diversity and inter-relationships of both extinct and extant populations over the last 100,000 years, including samples throughout its previous range from Western Europe to Southwest and East Asia. Using 229 bp of the mitochondrial hypervariable region, an approach which allowed the inclusion of information from extremely poorly preserved ancient samples, we classify all non-African wild asses into eleven clades that show a clear phylogeographic structure revealing their phylogenetic history. This study places the extinct European wild ass, E. hydruntinus, the phylogeny of which has been debated since the end of the 19th century, into its phylogenetic context within the Asiatic wild asses and reveals recent mitochondrial introgression between populations currently regarded as separate species. The phylogeographic organization of clades resulting from these efforts can be used not only to improve future taxonomic determination of a poorly characterized group of equids, but also to identify historic ranges, interbreeding events between various populations, and the impact of ancient climatic changes. In addition, appropriately placing extant relict populations into a broader phylogeographic and genetic context can better inform ongoing conservation strategies for this highly-endangered species.

Inference and Analysis of Population Structure Using Genetic Data and Network Theory.

Clustering individuals to subpopulations based on genetic data has become commonplace in many genetic studies. Inference about population structure is most often done by applying model-based approaches, aided by visualization using distance-based approaches such as multidimensional scaling. While existing distance-based approaches suffer from a lack of statistical rigor, model-based approaches entail assumptions of prior conditions such as that the subpopulations are at Hardy-Weinberg equilibria. Here we present a distance-based approach for inference about population structure using genetic data by defining population structure using network theory terminology and methods. A network is constructed from a pairwise genetic-similarity matrix of all sampled individuals. The community partition, a partition of a network to dense subgraphs, is equated with population structure, a partition of the population to genetically related groups. Community-detection algorithms are used to partition the network into communities, interpreted as a partition of the population to subpopulations. The statistical significance of the structure can be estimated by using permutation tests to evaluate the significance of the partition's modularity, a network theory measure indicating the quality of community partitions. To further characterize population structure, a new measure of the strength of association (SA) for an individual to its assigned community is presented. The strength of association distribution (SAD) of the communities is analyzed to provide additional population structure characteristics, such as the relative amount of gene flow experienced by the different subpopulations and identification of hybrid individuals. Human genetic data and simulations are used to demonstrate the applicability of the analyses. The approach presented here provides a novel, computationally efficient model-free method for inference about population structure that does not entail assumption of prior conditions. The method is implemented in the software NetStruct (available at https://giligreenbaum.wordpress.com/software/).

Space-Use Patterns of the Asiatic Wild Ass (Equus hemionus): Complementary Insights from Displacement, Recursion Movement and Habitat Selection Analyses.

The way in which animals move and use the landscape is influenced by the spatial distribution of resources, and is of importance when considering species conservation. We aimed at exploring how landscape-related factors affect a large herbivore's space-use patterns by using a combined approach, integrating movement (displacement and recursions) and habitat selection analyses. We studied the endangered Asiatic wild ass (Equus hemionus) in the Negev Desert, Israel, using GPS monitoring and direct observation. We found that the main landscape-related factors affecting the species' space-use patterns, on a daily and seasonal basis, were vegetation cover, water sources and topography. Two main habitat types were selected: high-elevation sites during the day (specific microclimate: windy on warm summer days) and streambed surroundings during the night (coupled with high vegetation when the animals were active in summer). Distribution of recursion times (duration between visits) revealed a 24-hour periodicity, a pattern that could be widespread among large herbivores. Characterizing frequently revisited sites suggested that recursion movements were mainly driven by a few landscape features (water sources, vegetation patches, high-elevation points), but also by social factors, such as territoriality, which should be further explored. This study provided complementary insights into the space-use patterns of E. hemionus. Understanding of the species' space-use patterns, at both large and fine spatial scale, is required for developing appropriate conservation protocols. Our approach could be further applied for studying the space-use patterns of other species in heterogeneous landscapes.

Stochastic modelling of shifts in allele frequencies reveals a strongly polygynous mating system in the re-introduced Asiatic wild ass.

Small populations are prone to loss of genetic variation and hence to a reduction in their evolutionary potential. Therefore, studying the mating system of small populations and its potential effects on genetic drift and genetic diversity is of high importance for their viability assessments. The traditional method for studying genetic mating systems is paternity analysis. Yet, as small populations are often rare and elusive, the genetic data required for paternity analysis are frequently unavailable. The endangered Asiatic wild ass (Equus hemionus), like all equids, displays a behaviourally polygynous mating system; however, the level of polygyny has never been measured genetically in wild equids. Combining noninvasive genetic data with stochastic modelling of shifts in allele frequencies, we developed an alternative approach to paternity analysis for studying the genetic mating system of the re-introduced Asiatic wild ass in the Negev Desert, Israel. We compared the shifts in allele frequencies (as a measure of genetic drift) that have occurred in the wild ass population since re-introduction onset to simulated scenarios under different proportions of mating males. We revealed a strongly polygynous mating system in which less than 25% of all males participate in the mating process each generation. This strongly polygynous mating system and its potential effect on the re-introduced population's genetic diversity could have significant consequences for the long-term persistence of the population in the Negev. The stochastic modelling approach and the use of allele-frequency shifts can be further applied to systems that are affected by genetic drift and for which genetic data are limited.

Allelic richness following population founding events--a stochastic modeling framework incorporating gene flow and genetic drift.

Allelic richness (number of alleles) is a measure of genetic diversity indicative of a population's long-term potential for adaptability and persistence. It is used less commonly than heterozygosity as a genetic diversity measure, partially because it is more mathematically difficult to take into account the stochastic process of genetic drift for allelic richness. This paper presents a stochastic model for the allelic richness of a newly founded population experiencing genetic drift and gene flow. The model follows the dynamics of alleles lost during the founder event and simulates the effect of gene flow on maintenance and recovery of allelic richness. The probability of an allele's presence in the population was identified as the relevant statistical property for a meaningful interpretation of allelic richness. A method is discussed that combines the probability of allele presence with a population's allele frequency spectrum to provide predictions for allele recovery. The model's analysis provides insights into the dynamics of allelic richness following a founder event, taking into account gene flow and the allele frequency spectrum. Furthermore, the model indicates that the "One Migrant per Generation" rule, a commonly used conservation guideline related to heterozygosity, may be inadequate for addressing preservation of diversity at the allelic level. This highlights the importance of distinguishing between heterozygosity and allelic richness as measures of genetic diversity, since focusing merely on the preservation of heterozygosity might not be enough to adequately preserve allelic richness, which is crucial for species persistence and evolution.

A factorial design experiment as a pilot study for noninvasive genetic sampling.

Noninvasive genetic sampling has increasingly been used in ecological and conservation studies during the last decade. A major part of the noninvasive genetic literature is dedicated to the search for optimal protocols, by comparing different methods of collection, preservation and extraction of DNA from noninvasive materials. However, the lack of quantitative comparisons among these studies and the possibility that different methods are optimal for different systems make it difficult to decide which protocol to use. Moreover, most studies that have compared different methods focused on a single factor - collection, preservation or extraction - while there could be interactions between these factors. We designed a factorial experiment, as a pilot study, aimed at exploring the effect of several collection, preservation and extraction methods, and the interactions between them, on the quality and amplification success of DNA obtained from Asiatic wild ass (Equus hemionus) faeces in Israel. The amplification success rates of one mitochondrial DNA and four microsatellite markers differed substantially as a function of collection, preservation and extraction methods and their interactions. The most efficient combination for our system integrated the use of swabs as a collection method with preservation at -20 °C and with the Qiagen DNA Stool Kit with modifications as the DNA extraction method. The significant interaction found between the collection, preservation methods and the extraction methods reinforces the importance of conducting a factorial design experiment, rather than examining each factor separately, as a pilot study before initiating a full-scale noninvasive research project.

Effectiveness of multiple release sites in reintroduction of Persian fallow deer.

Releasing animals in more than one location may increase or decrease the probability of success of a reintroduction project, yet the question of how many release sites to use has received little attention. We used empirical data from the reintroduction program of the Persian fallow deer (Dama mesopotamica) (Galilee region in northern Israel) in an individual-based spatially explicit simulation model to assess the effects of releasing deer from multiple sites. We examined whether multiple release sites increase reintroduction success, and if so, whether the optimal number of sites for a given scenario can be determined and whether the outcome differs if animals are released alternately (i.e., the location of the release alternates yearly between sites) or consecutively (i.e., one release site is used for several years, then another is used, and so forth). We selected 8 potential release sites in addition to the original site and simulated the release of 180 individuals at a rate of 10 individuals per year in different combinations of the original site and 1-4 additional sites. In our model, releasing animals into the wild at multiple sites produced higher population growth and greater spatial expansion than releasing animals at only one site and a consecutive-release approach was superior to an alternate-release approach. We suggest that through the use of simulation modeling that is based on empirical data from previous releases, managers can make better-informed decisions regarding the use of multiple release sites and greatly improve the probability of reintroduction success.

Methods for assessing movement path recursion with application to African buffalo in South Africa.

Recent developments of automated methods for monitoring animal movement, e.g., global positioning systems (GPS) technology, yield high-resolution spatiotemporal data. To gain insights into the processes creating movement patterns, we present two new techniques for extracting information from these data on repeated visits to a particular site or patch ("recursions"). Identification of such patches and quantification of recursion pathways, when combined with patch-related ecological data, should contribute to our understanding of the habitat requirements of large herbivores, of factors governing their space-use patterns, and their interactions with the ecosystem. We begin by presenting output from a simple spatial model that simulates movements of large-herbivore groups based on minimal parameters: resource availability and rates of resource recovery after a local depletion. We then present the details of our new techniques of analyses (recursion analysis and circle analysis) and apply them to data generated by our model, as well as two sets of empirical data on movements of African buffalo (Syncerus caffer): the first collected in Klaserie Private Nature Reserve and the second in Kruger National Park, South Africa. Our recursion analyses of model outputs provide us with a basis for inferring aspects of the processes governing the production of buffalo recursion patterns, particularly the potential influence of resource recovery rate. Although the focus of our simulations was a comparison of movement patterns produced by different resource recovery rates, we conclude our paper with a comprehensive discussion of how recursion analyses can be used when appropriate ecological data are available to elucidate various factors influencing movement. Inter alia, these include the various limiting and preferred resources, parasites, and topographical and landscape factors.

Dynamics and management of infectious disease in colonizing populations.

The introduction of chronic, infectious diseases by colonizing populations (invasive or reintroduced) is a serious hazard in conservation biology, threatening the original host and other spillover species. Most research on spatial invasion of diseases has pertained to established host populations, either at steady state or fluctuating through time. Within a colonizing population, however, the spread of disease may be influenced by the expansion process of the population itself. Here we explore the simultaneous expansion of a colonizing population and a chronic, nonlethal disease introduced with it, describing basic patterns in homogeneous and structured landscapes and discussing implications for disease management. We describe expected outcomes of such introductions for three qualitatively distinct cases, depending on the relative velocities at which the population and epidemic expand. (1) If transmissibility is low the disease cannot be sustained, although it may first expand its range somewhat around the point of introduction. (2) If transmissibility is moderate but the wave-front velocity for the population, vp, is higher than that for the disease, vd, the disease wave front lags behind that of the population. (3) A highly transmissible disease, with vd > vp, will invade sufficiently rapidly to track the spread of the host. To test these elementary theoretical predictions, we simulated disease outbreaks in a spatially structured host population occupying a real landscape. We used a spatially explicit, individual-based model of Persian fallow deer (Dama mesopotamica) reintroduced in northern Israel, considering a hypothetical introduction of bovine tuberculosis. Basic patterns of disease expansion in this realistic setting were similar to our conceptual predictions for homogeneous landscapes. Landscape heterogeneity, however, induced the establishment of population activity centers and disease foci within them, leading to jagged wave fronts and causing local variation in the relative velocities at which the population and epidemic expanded. Based on predictions from simple theory and simulations of managed outbreaks, we suggest that the relative velocities at which the population and epidemic expand have important implications for the impact of different management strategies. Recognizing which of our three general cases best describes a particular outbreak will aid in planning an efficient strategy to contain the disease.