Sapindus emarginatus Vahl (Sapindaceae) also known as ‘Indian Soap nut’ is significantly important for saponin content in its fruits. However, its current population in India is heavily fragmented due to a lack of sustainable harvesting practices. Moreover, changing climatic regimes may further limit its distribution and possibly compromise the survival of the species in nature. The aim of the present study was to: predict the future distribution range of S. emarginatus; identify the bioclimatic variables limiting this distribution and to evaluate its adaptive fitness and genomic resilience towards these variables. To determine future species distribution range and identify limiting bioclimatic variables, we applied two different ecological niche models  on real occurrence data (n = 88 locations). The adaptive fitness of the species was evaluated by quantifying the genetic variability with AFLP markers and marker-environmental associations, using AFLP-associated Bayesian statistics. We found 77% overlap between the baseline (2030) and predicted (2100) species distribution ranges, which were primarily determined by maximum temperature (TMAX) and mean annual precipitation (MAP). The TMAX and MAP contributed 43.1% and 27.1%, respectively to ENM model prediction. Furthermore, AFLP loci significantly associated with bioclimatic variables, and TMAX and MAP represent the lowest proportion (6.15%), confirming to the severe response of the species genome towards these variables. Nevertheless, the very low Linkage disequilibrium (LD) in these loci (4.54%) suggests that the current sensitivity to TMAX and MAP is subject to change during recombination. Moreover, a combination of high heterozygosity (0.40–0.43) and high within-population variability (91.63 ± 0.31%) confirmed high adaptive fitness to maintain reproductive success. Therefore, the current populations of S. emarginatus have substantial genomic resilience towards future climate change, albeit significant conservation efforts (including mass multiplication) are warranted to avoid future deleterious impacts of inbreeding depression on the fragmented populations. Sapindus emarginatus Vahl (Sapindaceae) also known as ‘Indian ‘Soap nut’ is a tree species native to Indian sub-continent. In India, it is considered to be originated in the Western-Ghats and later extended up to the West- Central- North Indian biogeographical regions1. The species is of substantial importance for global trade in soap and perfumery industries due to the oil and saponin content obtained from its leaves, fruits and seeds. The global trade of saponin from all plant sources is expected to grow up to 970 million US$ in 2023, from 950 million US$ in 20172. India exports approximately 63,368 kg/year of saponin extract obtained from all plant sources to different countries3. The exact contribution of S. emarginatus to the global saponin market is unclear, but probably substantial as the species is widely used traditionally at Indian homes as detergent to wash precious clothes and ornaments and as shampoo to wash hair. Due to high commercial demand, the species has been enlisted under ‘priority species of economic importance’ in the country report of state of forest genetic resource in India4 and the available natural patches of the species in some places have been restored through reforestation4. Lack of sustainable harvesting practices has been causing problems in the natural regeneration, as the fruits are harvested without leaving seeds for its natural regeneration6, which led to fragmentation of the populations from wild and now the plants are found scattered in dry and moist deciduous forests and its periphery. Further, the distribution of the plants is subjected to significant impact due to changing climatic regimes. Therefore, there is a need to evaluate the available genetic resource of S. emarginatus and its adaptive fitness in projected bioclimatic regimes to ensure its sustainability.7. With the current occurrence data of a species, the ecological niche modeling (ENM) tools such as BioClim  and MaxEnt (maximum entropy), have been employed to predict the suitable niches for species population and their response toward different bioclimatic variables integrating the projected climatic regimes9. However, the actual climate tolerance of long-lived species is wider than the climatic envelope they currently occupy10. Specifically, the forest species are practiced to plant outside of its natural distribution for commercial, breeding and improvement purposes in monoculture. It has also been suggested for covering the commercial forestry trials, botanical gardens, and biodiversity database along with the natural distribution of a tree species to measure its climatic or environmental requirements through SDM/ENM11. Nevertheless, these model-based predictions are not practically successful until the species population itself is not adaptively fit12. A species needs to be resilient enough for local adaptation to maintain its survivability facing bottleneck selection pressures. In particular, the response of a species may vary toward dynamic extrinsic factors like changing climatic conditions. On the other hand, the existence of a high level of genetic heterogeneity within a population can lead to the increased adaptive fitness and evolutionary potential in longer-term16. An adaptive fit population is tend to transfer the same genetic characteristic to the future generations17. Ultimately, the response of species to the predicted changing climatic condition with high genetic heterogeneity leading to the adaptive fitness is of significant importance for the sustainability of population in limited distribution18.Species distribution modeling (SDM) based on fundamental ecological niche theory has become an integral tool to provide the biogeographic extent of a species distribution19, robust informativeness, higher multiplex ratio, wider genome coverage, higher heritability20 and fewer artifacts compared to ISSR and other markers21. In a previous report on S. emarginatus, the AFLP markers revealed comparatively higher polymorphism (P%) and polymorphic information content (PIC) values than the ISSR and DAMD markers22. Using ISSR markers the genetic diversity and differentiation among S. emarginatus populations from three biogeographical regions in India has been evaluated22. Assuming the population in Hardy-Weinberg Equilibrium (HWE) and without consideration of spatial and geographical factors, the study resulted into three distinct groups showing affinities towards their biogeographical regions22. Unlike the classical genetic algorithm, the Bayesian algorithm developed for AFLP markers25 is advantageous to estimate the genetic diversity and differentiation of populations without considering of HWE and inbreeding coefficient26. Moreover, it is also able to handle the biased estimates of genetic diversity measures due to unequal sample sizes.The DNA-based marker systems such as dominant markers i.e. amplified fragment length polymorphism (AFLP), inter-simple sequence repeats (ISSR), DAMD (directed amplification of minisatellite DNA) and co-dominant markers i.e. simple sequence repeats (SSR) have become an important tool to evaluate the distribution of heterogeneity and structure of the plant genetic resources. Among the dominant markers, AFLP exhibits higher reproducibilityS. emarginatus to respond to the projected climatic regime? What are the bioclimatic variables contributing as limiting factor in population distribution? In order to resolve these questions, ENM was performed with real occurrence data of the populations of S. emarginatus from three biogeographical regions in India  and projected (the year 2100) climatic regimes. The fitness of the populations was evaluated assessing the genetic variability using AFLP markers with Bayesian statistics. The signature of adaptation in genome of the species was evaluated using genome-wide association (GWAS) between marker and bioclimatic variables.The present investigation was performed to address the major questions: How the future climatic regime is going to affect the species population distribution? What is the state of the genomic resilience of different populations of dia Fig.  with mulp < 0.05, r = 0.85) was found between the predictions by both the models (BioClim and MaxEnt) for the species distribution on its range of occurrence in the baseline (the year 2030) and future (the year 2100) climatic regimes. Therefore, the species distribution prediction , and annual precipitation (MAP = 27.10%) with the highest contribution in the model predictions , temperature range  and increased MAP (<1000 mm).A significant correlation (the year 030 and fthe year 030 and ff (FIS) values were 0.18 and 0.98, respectively. The observed heterozygosity (Ho) ranged from 0.42 ± 0.0 to 0.44 ± 0.0 with an average of 0.43 ± 0.01  population revealed the highest number of common alleles < =50%, and no rare and common (<=25%) allele was found in the populations analyzed.The AFLP primers amplified 1957 loci (103 ± 39.37 loci/primer) with 94.79 ± 6.43% polymorphism (1859 polymorphic loci), 0.65 ± 0.02 major allele frequency (MAF) and 0.33 ± 0.01 PIC  to 32.39  between genetic distances and geographical distances matrix . The variation among locations was 6.46 ± 1.34% and within location among genotypes was 91.63 ± 0.31%.The Jaccard’s genetic similarity coefficient among the genotypes ranged from 0.24 to 0.61 with an average of 0.38 ± 0.05. The principal coordinate analysis (PCoA) differentiated the genotypes into three clusters  was found with the posterior odd (log10PO) > 0.5  was confirmed to avoid false-discovery in genome-wide association study (GWAS). Among all loci pairs, only 1.99% were found in significant LD (p < 0.01). Major LD decay was observed within a distance of 100 base pair (bp) and the length of LD block extended up to 1450 bp  association of 65 loci (3.59% out of 1810 polymorphic loci) from 15 AFLP markers with bioclimatic variables and altitude (Supplementary Table RANGE and the lowest proportion (6.15%) was with MAP and TMAX (Table p < 0.001) LD.In order to detect the outlier loci with a signature of adaptation, the only locus E-AGC/M-CTG-71 , and MAP distribution (CV = 22.51%) but with a narrow temperature gradient  which showed comparatively wider range of MAP (CV = 26.84%) and mean annual temperature (CV = 17.84%)27. The comparison of the CV and MAP data revealed that S. emarginatus might have been facing intrinsic incompetency to shift beyond the specified range of temperature gradient due to bionomic characteristics. Since the species distribution thrives on the fragmented populations in varied biogeographical regions, it may be significantly influenced by the extrinsic barriers like local conditions and selection pressures as well. Hence the adaptive fitness of the species led by the population genetic characteristics was determined to ensure the sustainability and genomic resilience of the population through dominant AFLP markers. The dominant markers have already been applied to confirm the adaptive fitness of trees29 and other plant species32. In the present study also, a high MAF (0.65 ± 0.02) of the AFLP markers and a high LD decay (1.99%) in loci-combinations supported their suitability for high-resolution GWAS33 confirming their frequent coverage of the species genome.The occurrence of es CV = 7.40%, and34. It can be inferred also from the results of ENM establishing the TMAX as one of the limiting factors to the species distribution with the highest (43.10%) relative contribution to the model prediction  from the Semi-arid biogeographic region Fig. , which hghest 43.0% relati35. Moreover, the sweet incense of its flowers attracts the pollinators, like Indian honeybees36 that may help to admix the population through pollination between the trees scattered away from each other35 maintaining within population variability. The Bayesian algorithm-based analysis of genetic structure has found the admixture model with independent allele frequency as the fittest and assumed four cryptic populations for the samples. Nevertheless, a weak or negligible genetic structure  exchanged among the sampled genotypes  for the measures of heterozygosity  supporting the sign of inbreeding depression on the species population. High heterozygosity with inbreeding depression in sub-divided populations represents the state of overdominance caused by heterozygote superiority37. The current status of the S. emarginatus populations in severe fragmentation along with the absence of natural regeneration through seeds, act as barrier for inter-population or long-distance out-crossing and seems to be the reason for inbreeding38. Inbreeding depression, causing loss of genetic variability in small or fragmented populations, may result in the reduced fitness of the offspring40. Despite inbreeding, the higher heterozygosity of the populations indicated that the population might not have crossed many generations with the inbreeding depression41. Therefore, being a long-lived forest tree, the reproductive success of the highly heterozygous population of S. emarginatus through mass multiplication leading to restoration may mask the deleterious impact of inbreeding42.The high heterozygosity of 43. This increment will have some influence on the climatic envelope of fundamental ecological niches leading their shifting to suitable conditions. Temperature and rainfall have been referred to as prime contributing factors in most of the projections for future climatic regimes44. In our investigation also, we found that TMAX and MAP with maximum contribution to the model prediction. The ENM establishes TMAX and MAP as extrinsic limiting factors of the species population distribution. Since the population distribution of the species has been observed within a narrow range of temperature gradient, it may have an influence of the predicted warm climate45. The GWAS approach found the most of the genomic proportion covered by the AFLP markers significantly associated with TRANGE and TMIN. It revealed the genomic resilience of the species to survive within varied TRANGE and TMIN. It is obvious, as the species is known to be frost hardy with occurrence in tropical deciduous forests in India46. The least proportion of the AFLP markers was found associated with TMAX and MAP establishing them as limiting factors for the genomic resilience of the species. In MLM, 3.45% of the species genome was found in significant association with the bioclimatic variables. The least proportion (6.15%) of the loci linked with TMAX and MAP has confirmed the severe response of the species genome toward these variables. Nevertheless, these associations are highly dependent on high LD to transfer to future generations. We observed only 4.54% LD among the loci-combinations found in significant association with the bioclimatic variables with high LD decay (within 100 bp). Since S. emarginatus maintains high heterozygosity preferring xenogamy, the current status of susceptibility of the species genome responding to TMAX and MAP is subject to change during recombination. In addition, the mating system preferred by S. emarginatus may help to avoid the deleterious effect of inbreeding on the species population. Therefore, it can be assumed that the S. emarginatus populations may have substantial genomic resilience toward the bioclimatic variables.A multi-model climate change projection for the Intergovernmental Panel on Climate Change  has predicted a 4% increase in warming and 14% increase in precipitation by the year 2080S. emarginatus populations using molecular marker technique with SDM/ENM approach. The ENM-based predictions have supported a stable distribution pattern of the species population towards the projected future climatic regime and confirmed maximum temperature and annual precipitation as the major limiting factors. However, the AFLP markers have detected the signature of adaptation on the genome of S. emarginatus, which supports that the species is resilient enough to survive on the threat of rising temperature and precipitation. The Deccan peninsular biogeographic region was found more suitable for the species, therefore the niches in the region can be preferred to conserve as strict nature reserves to maintain the existing genetic variability of the species populations. In light of the threat and vulnerability of ecological niches of Semi-arid region, sincere efforts can be made for screening of resilient genotypes of the species. Further, initiatives for the conservation and mass multiplication of the species are recommended to avoid the future deleterious impact of inbreeding depression on the fragmented populations. There is a scarcity of the genomic information related to S. emarginatus. Therefore, the 15 markers found significantly associated with the bioclimatic variables and altitudinal gradients, which can be further employed to find out their association with the functional adaptive traits.To the best of our knowledge, the present study seems to be a maiden attempt to study the adaptive fitness of https://www.gbif.org/occurrence/search?taxon_key=8086757), European herbaria; KEW (https://www.kew.org/search?textsearch=sapindus) and the herbarium records available at CSIR- National Botanical Research Institute, Lucknow (LWG), Central National Herbarium, Kolkata (CAL), Botanical Survey of India , Allahabad (BSA), Botanical Survey of India, Hyderabad (BSID) and Forest Research Institute, Dehradun (DD). The 157 GPS coordinates obtained were subjected to spatial filtration to avoid the sampling biases and equal richness based grid sampling applied through the program DIVA-GIS v7.547. Remaining GPS coordinates were selected for ground-truthing to obtain the real occurrence data of S. emarginatus populations. The locations were visited and finally 88 coordinates were recorded at the locations with verified abundance of the species in eight Indian states representing three biogeographical regions viz. Gangetic plain, Semi-arid and Deccan peninsula  and projected (the year 2070–2100) regimes was obtained from the website http://www.climond.org (Climond model CSIRO- MK 3.0) and extracted for the GPS coordinates of the locations using DIVA-GIS v7.547 (available at http://www.diva-gis.org/) in the resolution of 2.5 m.The data of 35 bioclimatic variables50. The BioClim algorithm was applied using the envelope method as implemented in program DIVA-GIS v7.547 for baseline and predicted bioclimatic variables. MaxEnt model was applied through program MaxEnt v3.4.152 to evaluate the response of niches to the bioclimatic variables for baseline and predicted climatic regimes. For training with threshold-depended sensitivity model (threshold = 0.5), default parameters in program setting were preferred with 50 replications and 500 iterations doing jackknife and to measure variable importance. The results obtained for two different models for two climatic regimes were correlated and the proportion of overlapping niches was determined through program ENMtools v1.853 applying relative rank statistics54. Based on the significant correlation among the bioclimatic variables obtained through program ENMtools v1.8, finally climatic variables viz. minimum temperature (TMIN), maximum temperature (TMAX), temperature range (TRANGE), isothermality (I), and mean annual precipitation (MAP) were tested to find out the limiting factor for the species distribution.The GPS coordinates were employed for ENM to predict the potential distribution of populations determining suitable niches for the species. For confirmation of the result both, BioClim and MaxEnt based models were applied as the assembling and comparing the results from different models can give robust projection22. The conventional AFLP protocol19 was used with minor modifications55 to develop an amplification profile for the 41 genotypes on 19 AFLP primer combinations . The program eliminates the band profiles with short sizer peaks (<50) to avoid the error on scoring. Further, the AFLP markers with <50 amplified loci were excluded for further analysis. The profile was analyzed applying both band-based and allele frequency-based approach26. The mean allele frequency (MAF), polymorphism percentage (P %) and polymorphic information content (PIC) of the markers were calculated by program POWERMARKER v3.2556. The genetic diversity of the sampled populations was estimated using Bayesian models. We preferred the θ-statistics  implemented in program HICKORY v1.124 that allows direct estimates of genetic differentiation measure (FST) from dominant markers without prior assumption of inbreeding and Hardy -Weinberg Equilibrium (HWE) within population even with small sample size. Location-wise panmictic heterozygosity (Hs) and total panmictic heterozygosity (Ht) were also estimated. All these estimations were performed with 50,000 steps of burn-in, 500,000 replicates, ‘thinning’ = 20 following the best-suited model for the data amongst all the four models  implemented in the program based on their corresponding DIC (deviance information criterion) value25. The inbreeding coefficient (f = FIS) resulted from the best-fitted model in HICKORY was incorporated in another Bayesian program AFLP-Surv v1.057 with the non-uniform prior distribution of allele frequencies at 10000 permutations to estimate the observed heterozygosity (Ho). Rare (private) and common alleles (<25%) among the populations were calculated through program GenAlEx v6.558. Jaccard’s genetic similarity coefficient among the genotypes was calculated by program DARWIN v5.059. A principal coordinate analysis (PCoA) was performed to cluster the genotypes in different axes applying program GenAlEx v6.5. To determine the genetic structure of the sampled population with an appropriate number of cryptic population (K) assigned by the genotypes, Bayesian statistics-based program STRUCTURE v2.3.160 was used. The project was run applying four prior-assumption with combinations of admixture/no-admixture models with correlated/independent allele frequencies on 100000 burn-in and 1000000 MCMC repeats with three-run of K from 1 to 9 for 2 ≤ K ≤ 8. The most suitable model and the best-suited ‘K’ for the data set were determined based on the highest Delta-K value resulted from an online program STRUCTURE HARVESTER61. The ancestry coefficient (Q) values of the genotypes were further used for mixed linear modeling (MLM) based association mapping. Pair-wise FST among populations were also calculated by program AFLP-Surv. The gene flow among populations was determined by calculating the number of migrants (Nm) among the populations62. To determine the effect of geographical distance and altitude on gene flow among locations, the Mantel’s tests were performed among pair-wise Nm, pair-wise geographical distance and pair-wise Euclidean distance based on altitude through program GenAlEx. Analysis of molecular variance (AMOVA) was also calculated.DNA profiling of 41 genotypes with 19 combinations of AFLP primers was developed based on scoring the presence (1) and absence (0) of the bands with help of program Genemapper63 was applied that identifies outlier loci with a Bayesian test based on a logistic regression model that decomposes FST values into a locus-specific component  and a population-specific component . Twenty pilot runs of 5,000 iterations were used to estimate the distribution of α-parameters, followed by 50,000 iterations for sampling. Outlier loci with substantial signature of adaptation were identified using a 5% false discovery rate with posterior odds of >10 (log10 (PO) > 0.5). Linkage disequilibrium among loci-combinations was performed by program TASSEL v3.064 to confirm the influence of demographic variables on the population genetic structure inferring LD decay. A genome-wide association study (GWAS) was performed between climatic variables , altitude and AFLP marker profile by program TASSEL v3.0 applying MLM to control the false-discovery due to the structured population66. The MLM considers the markers applied to the study as a fixed–effect factor and the population structure information of the sampled genotypes are considered as random effect factors. The required kinship (K) information was obtained from program TASSEL and the ancestry coefficient (Q) value was obtained from the program STRUCTURE output to implement the MLM + K + Q method. The loci significantly (p ≤ 0.01) associated with one or more climatic variables were taken into consideration. To find out the limiting factor, the proportions of AFLP markers linked with these climatic variables and altitude were calculated based on their number of loci in significant association with the climatic variables.To detect the signature of local adaptation on AFLP loci, program BAYESCAN v2.1Appendix S1.


Such high mitochondrial diversity could imply that Magdalenian people tamed and reared animals originating from different wolf lineages. We discuss our results in light of three ecological hypotheses and conclude that both domestication and the existence of a specialized wolf ecomorph are highly probable. However, due to their proximity to humans and a restricted diet, we propose domestication as the most likely scenario explaining the patterns observed herein.Dogs are known to be the oldest animals domesticated by humans. Although many studies have examined wolf domestication, the geographic and temporal origin of this process is still being debated. To address this issue, our study sheds new light on the early stages of wolf domestication during the Magdalenian period  in the Hegau Jura region (Southwestern Germany and Switzerland). By combining morphology, genetics, and isotopes, our multidisciplinary approach helps to evaluate alternate processes driving the early phases of domestication. The isotope analysis uncovered a restricted, low  This is exemplified by various studies that have investigated faunal adaptation to the environment before and after the Last Glacial Maximum (LGM), a period of climatic fluctuation and dramatic landscape changes2. One possibility to survive unfavorable conditions is to retreat into refugia, i.e. restricted and often isolated areas still harboring ecological and environmental features beneficial for the species in question4. Specifically, during the LGM, various species retreated to warmer areas such as the Iberian Peninsula5, subsequently preserving and shaping patterns of genetic variation prevalent during this epoch6.In line with several theories detailing biological diversity and evolution, the survival of species is often predicated on their ability to adapt and thrive within a changing environment. To do so, plant and animal species have developed several strategies, including the adaptation to existing, or the development of new ecological niches7. Ecomorphs are characterized by a genetic variety within a species or a variety of several species exposing the same phenotypic features, due to the adaptation to a local ecology. One prominent example of a species represented by many ecomorphs since prehistoric periods is the grey wolf (Canis lupus). The wolf is a mobile carnivore with an opportunistic diet8, and can occupy different trophic niches, best exemplified by Late Pleistocene wolves, which were characterized by two different dietary habits10.An alternative pathway for survival is to form so-called ecomorphs and thus adapt to environmental conditions and habitats14, they started to re-shape their local environment and thereby became a driving factor within the landscape providing artificial ecological and dietary (= trophic) niches17. The concept of humans as niche constructors is vividly debated and recently contributed to our understanding of domestication19. This debate includes the origin of modern dogs, which may have occurred intentionally by pet-keeping, a side-effect of goal-orientated domestication, or unintentionally (self-domestication), when humans created a niche for commensal scavengers with their food waste. The uniting element of these theories is the subsequent selection for tameness and reduced fear, resulting in decreased wariness and aggression, high tolerance of penning, and sexual precocity21.During the Late Pleistocene, as humans became more numerous and intrusive24. Once the process of domestication began, humans quickly gained control over the diet, reproduction, and health of their new companions and thus set the stage for a lasting human–dog bond25. For example, stable isotope analyses of canids found in Předmostí, a Gravettian site in the Czech Republic dated to 31,500 years ago, showed evidence of adaptation to two different dietary niches27; however, it remains unclear whether or not one of these two different canid groups are indeed early domesticated wolves28. While such older dates for potential onsets of wolf domestication have been challenged, it is widely accepted that 16,000 years ago is the approximate time for the first emergence of dogs10. From this time onwards, dog remains have been discovered at several archaeological sites throughout Europe, such as the famous dog from Bonn-Oberkassel excavated alongside two human burials30. Another important site with possible early dog remains is Kesslerloch32 (Canton Schaffhausen) located in the Hegau Jura and dating to the late Magdalenian period . Morphological examinations of these remains revealed a distinction between wolves and dogs33 and dietary reconstruction analyses16 demonstrated parallel occurrences of two distinct canid trophic niches at this site. However, it remains unclear whether Kesslerloch is a unique case or if wolf domestication was practiced in the entire region over a longer period of time. The Hegau Jura, including the Kesslerloch site and two other cave sites, Petersfels and Drexlerloch, represents a Magdalenian hotspot for human activity in the pre-Alpine region. In the heart of this setting lies the cave site Gnirshöhle, which also shows evidence of human occupation, with butchered animal bones, worked antlers, and bone needles34 (Supplementary Note 1), as well as provides a cohort of large canid remains, making this cave particularly valuable for investigating pre-Alpine canids.Although the when and where of wolf domestication are still disputed, it is now accepted that dogs are indeed the oldest domesticatesIn the present study, we applied a multidisciplinary approach to study canid remains from the Gnirshöhle and assessed canid population dynamics potentially shaped by environmental conditions and anthropogenic pressure during the Magdalenian in the Hegau Jura Fig. . By combCanis sp.) by archaeozoological classification. The mandible GN-999  were assigned to be large canid specimens  plotted against the length of the first molar (CLM1 = 27.3 mm) falls into the observed absolute ranges of recent Northern wolves and ‘Palaeolithic dog’ group35. Although, GN-999 is considerably shorter by one standard deviation than the lower bound of the range for modern and Pleistocene wolves, as well as the ‘Paleolithic dog’ group35. This specimen is also morphologically similar to a small wolf from Kesslerloch32 . Tooth crowding of the premolars is a typical trait for dogs and is related to shortening of the snout during the process of domestication37. However, crowding between P4 and M1 is also observed in wolves38. Additionally, a diastema is present between the P2 and the P3, which is rather typical for recent and Pleistocene wolves35. Thus, the morphological and metric assignments for GN-999 remain unresolved.The length of the tooth row of the mandible GN-999 39. Specimen HF-912 was removed from the isospace published by Baumann and colleagues16, as it was dated to the pre-LGM (Supplementary Note 3). The newly analyzed HF-1712 sample, contextually dated to pre-LGM, was also excluded. The slightly younger specimen, HF-1250.2 (Supplementary Note 3), was dated to 11,400 ± 30 BP , and therefore belonged to the Late Palaeolithic period. However, since both archaeological periods are temporarily close and the environmental conditions did not change dramatically, we decided to keep the isotopic values of this specimen in the reconstructed Magdalenian isospace. We calculated three canid niches: niche A, niche B, and niche C , additional mitochondrial genomes from canid remains of diverse temporal and geographic origin Fig.  were als25 and KSL-6141; additionally, the specimen HF-530 is placed with a third Swiss mitochondrial genome, KSL-62  was almost as high as that observed in a global assemblage of modern dogs  and slightly lower than the diversity determined for the Kesslerloch canids . These estimates of genetic diversity illustrate the high genetic variation in Magdalenian canids originated from caves located in the Hegau Jura region.As evidenced by the broad distribution of our samples throughout the phylogeny, the Magdalenian canids from southwestern Germany harbor a vast amount of genetic diversity. To assess the genetic diversity of the Magdalenian Gnirshöhle canids, we compared the nucleotide diversity (π) of those specimens with the observed nucleotide diversity of the Kesslerloch canids, in addition to modern dogs and wolves sampled worldwide Tables . Nucleot14C analyses were estimated. While the median ages for most samples do not differ substantially from their respective archaeological age .To evaluate the evolutionary timescale of the canid mitochondrial phylogeny, we performed BEAST analyses Figure . The resge Table , the age41, the coyote outgroup and the modern wolf samples from the Himalayas were excluded from this analysis. The TMRCA of all dogs and wolves was estimated to approximately 152 ka cal BP . It should be emphasized that the TMRCA is not equivalent to a population split44, nor does it represent the actual onset of domestication; it can, however, be used to assess an upper time limit of such events. We attribute slight discrepancies between the previously published TRMCAs and our new estimates to varying sampling regimes and parameter settings used in the different studies43. The most recent common ancestor of all closely contemporaneous samples from Gnirshöhle dates back to 135 ka BP (including GN-192). This is an intriguing finding for several reasons. First, Magdalenian mitochondrial genomes reconstructed from specimens originating from a single cave in southwestern Germany captured almost the entire breadth of genetic diversity of all contemporary and ancient dogs and most wolves. Second, the newly generated mitochondrial genomes from the Gnirshöhle canids introduce a yet unrecognized, ancient mitochondrial canid lineage that had survived into the Magdalenian. Several authors have now proposed that mitochondrial dog lineages in prehistoric Europe were replaced by expanding lineages arriving from the East46. This fate was possibly shared by the Gnirshöhle canids as an example of diversity that was replaced by the incoming lineages represented by today's dog clade A43. The age of the Gnirshöhle canids exceeds those of the samples analyzed by Frantz and colleagues24, and implies that with more samples, we may still discover divergent lineages representative of early dogs47, a prospect that helps to finally derive a more nuanced picture of modern wolf’s domestication history .In general, our TMRCA estimates are in line with previous findings placing a common ancestor of dogs and wolves in the late Pleistocene  the refugium hypothesis, (2) the ecomorph hypothesis, and 3) the domestication hypothesis . The larger an area, the more genetic variation could have accumulated consequently resulting in higher estimates of the genetic diversity of the species under consideration. If we accept the Hegau Jura region as a potential canid refugium, we should observe lower diversity in the mitochondrial genomes, as it is rather small compared to, for instance, the Iberian Peninsula refugium52. Furthermore, a retreat into glacial refugia would not necessarily require a shift in the trophic niche of the focal species, and hence under the refugium hypothesis, we would predict a diet composition similar to that of pre-LGM canids from southwestern Germany. Results of dietary reconstructions from the pre-LGM periods of the Swabian Jura17 have shown that all studied canids belonged exclusively to one group with high δ15N values that primarily fed on megafauna. While we observed the same pattern in the Magdalenian ‘wolf’ niche (niche A), canids from Gnirshöhle behaved differently, which led us to reject the refugium hypothesis.Previous studies of genetic diversity in 53 was sufficient to differentiate whether the canids of Gnirshöhle were dogs or wolves.With regard to the ecomorph and domestication hypotheses, we are not able to unequivocally differentiate between either solely based on our mitochondrial DNA data. Both hypotheses are equally supported by the high genetic diversity within the Magdalenian canids in addition to its dissociation from space and time. This demonstrates the limitation of genetic analysis of the maternally inherited mitochondrial genome to appropriately examine such complex hypotheses. From the archaeozoological perspective, neither the metrics nor tooth crowding9. All Gnirshöhle canids showed signals indicating they consumed a low δ15N protein diet (niche B), while those from the two nearby cave sites, Kesslerloch and Hohle Fels, showed a high δ15N protein dietary source like those consumed by other canids16. Thus it is unlikely that the Gnirshöhle canids represent a surviving wolf ecomorph with a similar high δ15N protein diet as the pre-LGM wolves17. However, it is possible that canids consumed reindeer and small game and may be defined by a new type of ecomorph, similar to the hypothesis proposed by Perri10 concerning canids from the Gravettian open-air site Předmostí (Czech Republic). Members of the Magdalenian niches A and B show similar reconstructed diets compared to those canid groups analyzed from Předmostí26. As Bocherens and colleagues26 showed, the ‘Pleistocene wolf’ group had free access to all dietary resources and consumed mostly megafauna, while the ‘Palaeolithic dog’ group consumed primarily reindeer27. We observed a dietary distribution in the Magdalenian isospace within niches A and B, similar to the Předmostí canid groups16. Ultimately, the ecomorph hypothesis becomes a reasonable explanation for the observed genetic and isotopic patterns, but it fails to answer the question of why the Gnirshöhle canids would form a separate ecomorph while a high δ15N protein diet source was likely available nearby.Considering the trophic niche observed among the Gnirshöhle canids being defined as a Late Pleistocene ecomorph and bearing in mind the properties of a surviving wolf ecomorph from the pre-LGM, we predict a restriction to a similar diet, as was observed for an Eastern Beringia Pleistocene ecomorph55. Direct dating of the Gnirshöhle samples implied that canids could have lived in close vicinity of Magdalenian people, occupying the Hegau Jura, and subsequently adapted to a restricted diet, possibly under human influence. Thus, we consider the Gnirshöhle canids to likely represent an early phase in wolf domestication—facilitated by humans actively providing a food resource for those early domesticates (niche B16). Moreover, the high mitochondrial genome diversity could be explained by the fact that Magdalenian people would have arbitrarily drawn individuals from a large pool of canid genetic variation in the region some 15,000 years ago. We suggest that the proximity Magdalenian hunter-gatherers is the most parsimonious explanation for the genetic, isotopic, and archaeozoological patterns observed in the Gnirshöhle canids.Besides environmental changes, humans can be a major driving factor to create ecological niches, and thus, domestication can lead to an ecomorph shaped by them46. While we cannot address the question of the domestication event’s singularity, our results support the hypothesis that the Hegau Jura was a potential center of early European wolf domestication. Such a scenario becomes plausible considering a close proximity of canids and humans thereby introducing a controlled, or at least a restrictive diet. This would foster their differentiation from wild conspecifics and may thus constitute a driving factor in the process of domesticating wolves. Lastly, we reiterated the importance of multidisciplinary approaches to investigate the origin of modern dogs, serving as a model for similar studies in the future.In conclusion, future investigations should include the analyses performed in our study, in addition to others for more detailed diet reconstructions , identification of the specimens’ geographic origin or range , as well as including nuclear genomes to further decipher details of the wolf’s domestication history. Intriguingly, a recent study focusing on the analysis of nuclear genomes of various ancient dogs suggested a single origin of modern dogs, but it failed to provide a geographic location for such an event58, and Drexlerloch59. All three sites contain faunal remains dominated by reindeer and horses; in addition, Gnirshöhle provides a considerable number of canids34 (Supplementary Note 1). These remains are well-suited to explore early interactions between humans and canids. To shed light on these scenarios, we combined diverse datasets generated from various disciplines of archaeological sciences .The Gnirshöhle (GN) is a small cave with two chambers (GN I and GN II) situated in the Bruder Valley close to Engen in the Hegau Jura of southwestern Germany. The new dates of the Gnirshöhle canid remains range from 15.5 to 15 ka cal BP  were obtained for six out of eight canid remains of Gnirshöhle, one red fox from Bockstein Cave (BS-21), and one pre-LGM wolf from Hohle Fels (HF-1712) in the Swabian Jura  of which only two specimens were measurable, namely a mandible  and the maximal length of the first molar (M1) . To enable a two-dimensional classification, we plotted the two measurements against each other and compared them with published metrical data from Germonpré and colleagues35 , and Pleistocene wolves , post-LGM dogs60, n = 4) in addition to metric data from modern wolves (n = 3) from the archaeozoological reference collection mentioned above , Hohle Fels  and Bockstein . Collagen extraction followed the protocol outlined by Bocherens and colleagues48 and is further detailed explained in Supplementary Note 4. The process was performed in the Biogeology working group laboratory at the University of Tübingen (Germany). Isotopic measurements of collagen were undertaken in duplicate at the Institute of Environmental Science and Technology of the Universitat Autònoma de Barcelona (ICTA-UAB) using a Thermo Flash 1112 (Thermo ScientificVC) elemental analyzer coupled to a Thermo Delta V Advantage mass spectrometer with a Conflo III interface. All details for elemental and isotopic analysis are provided in Supplementary Note 4.We performed new isotopic analyses from bone collagen for seven samples of canids from Gnirshöhle (δ (delta) value as follows:The isotopic ratios are expressed using the δ13C and δ15N values of six canids from Gnirshöhle, one newly analyzed red fox from Bockstein, and published isotopic values of 14 canids40, and applied a multivariate cluster analysis (using JMP 14) to the δ13C and δ15N isotopic values of all canids. We then used R63 and the R package SIBER to calibrate the core niches (standard ellipse area = SEA) out of the clusters64. The core niche depicts the center of a niche that is calculated by using a Bayesian most-likelihood estimation and explains roughly 40% of the expected variability64. This method is more reliable for analyzing small sample sizes and recommended for niche interpretations by Jackson and colleagues64. For dietary reconstructions, we utilized the same prey groups that were defined by Baumann and colleagues16: ‘Megaherbivores’, ‘Ungulates’, and ‘Small mammals’ that were constructed from a database of 91 carbon and nitrogen stable isotopic values from herbivores . The quality of the sequencing reads was estimated by FastQC tool69 and the adapters were trimmed by AdapterRemoval70 version 2.2.1a, both tools are integrated in the EAGER pipeline. MarkDuplicates v2.15.0 (Picard Tools) was applied to remove duplicates for genome reconstruction68. To demonstrate the authenticity of ancient DNA reads, the program MapDamage71 is utilized by the EAGER pipeline to estimate the distribution and frequency of any misincorporations at the 3′- and 5′-ends of the DNA reads. Potential contaminated DNA reads in sequencing data for the individuals GN-14 and GN-106 were identified by a low frequency of misincorporations estimated by MapDamage71. To overcome potentially false reconstruction of mitochondrial genomes due to contamination, we applied PMD-tools72 to separate endogenous ancient DNA reads from modern contaminant reads with a threshold PMD score of three. For these two specimens, filtered reads were then used for mitochondrial genome reconstruction.We used the Efficient Ancient Genome Reconstruction (EAGER) pipeline73 to reconstruct a comprehensive phylogeny. In total, a multiple sequence alignment database of 244 genomes was constructed (MAFFT74), including 221 mitochondrial genomes recruited for our comparative dataset73 and the reconstructed genomes of our study. Before the phylogenetic tree reconstruction, we defined the best-fit model for our data75, using IQ-TREE76, to obtain the highest statistically supported phylogenetic tree. Subsequently, a phylogenetic tree was generated employing IQ-TREE76 using a maximum likelihood approach with an estimation of 10,000 bootstrap replications.We used canid mitochondrial genome data published in previous studies77 2.1.1 into four partitions: (1) protein coding, gene position one , (2) position two , (3) position three with rRNA and tRNA , and (4) non-coding . Bayesian phylogenetic and timescale estimation was performed using BEAST78 1.10.4. Sample ages were used as tipdates for molecular clock calibration. Undated samples were assigned uniform age prior (0-100ky). Uncorrelated lognormal relaxed clock for each partition and Bayesian SkyGrid population model79 were used, as supported by the data. The MCMC chain was run for 200 M steps with sampling every 20,000th step. The first 20 M steps were removed as burn-in. Convergence and mixing were inspected  and the SkyGrid plot was generated in Tracer80 v 1.7.1. Maximum Clade Credibility trees were generated in TreeAnnotator (part of the BEAST package) and visualized in FigTree81 1.4.2.In order to estimate time-aware phylogeny in coalescent framework, we excluded the coyote mitogenomes, as well as three modern Tibetan/Mongolian wolves from China  and one ancient Siberian wolf (MK936996) that in preliminary runs revealed an unexpectedly old divergence time, which could be attributed either to genetic isolation or comparatively stronger selective forces. The alignment of 236 mitogenomes was partitioned using PartitionFinder73 assigned into four canid population groups: Kesslerloch (KSL), Gnirshöhle (GN), modern dogs, and modern wolves , sampling variance, and the standard deviation, defined by the square root of the variance82, according to well-established statistical algorithms83.To investigate the genetic diversity in more detail, the mitochondrial genomes from Kesslerloch and Gnirshöhle were compared to modern dog and wolf mitochondrial DNA sequenceses Table  and S8. Supplementary Information.Supplementary Table S1.Supplementary Table S9.


The GD varied from 0.235 to 0.911 across the 16 genotypes. Cluster analysis based on the GD estimated using SSRs classified the genotypes into three major groups and six sub-groups, almost consistent with the results of principal coordinate analysis. The combined data indicated that five hybrids showed 20% greater yield than mid-parent or better-parent. Two hybrids (P2 × P4) and (P2 × P5), which showed the highest performance of days to heading (DH), grain filling duration (GFD), and GY, and had large genetic diversity among themselves , were deemed as promising heat-tolerant hybrids. They showed the best mid-parent heterosis and better-parent heterosis (BPH) for DH , GFD , and GY . Correlation between GD and each of BPH and SCA effects based on SSR markers was significantly positive for GFD, hundred kernel weight, number of kernels per spike, harvest index, GY, and grain filling rate and was significantly negative for DH. These correlations indicate that the performance of wheat hybrids with high GY and earliness could be predicted by determining the GD of the parents by using SSR markers. Multivariate analysis (stepwise regression and path coefficient) suggested that GFD, hundred kernel weight, days to maturity, and number of kernels per spike had the highest influence on GY.Hybrid performance during wheat breeding can be improved by analyzing genetic distance (GD) among wheat genotypes and determining its correlation with heterosis. This study evaluated the GD between 16 wheat genotypes by using 60 simple sequence repeat (SSR) markers to classify them according to their relationships and select those with greater genetic diversity, evaluate the correlation of the SSR marker distance with heterotic performance and specific combining ability (SCA) for heat stress tolerance, and identify traits that most influence grain yield (GY). Eight parental genotypes with greater genetic diversity and their 28 F Triticum aestivum L.), one of the most prominent food crops worldwide, is one of the most essential sources of protein in humans. It represents 17% of the global crop area, feeding about 40% of world’s population and providing 20% of the total diet calories  showed the highest performance in DH, GFD, and GY, and both had large genetic diversity among themselves ; therefore, they were deemed as promising hybrids . The variances of SCA were lower than those of GCA for DH, GFD, and GY in S1, S2, and combined, indicating that additive genes primarily controlled the inheritance of these traits. Previous studies on wheat [The ANOVA showed significant variances for both GCA and SCA  of 0.722. Many investigators have used this model  are important tools for understanding the relationship between yield and its related traits . The tral e.g., –80). Bas. Basp < 1 hybrid varieties, which are essential for the occurrence of heterosis. Thus, we considered the two crosses (P2 × P5) and (P2 × P4) that showed the highest performance in DH, GFD, and GY and had large genetic diversity among themselves  as promising hybrids of heat stress tolerance.In conclusion, our results confirmed the importance of SSR markers as an effective tool for evaluating wheat GD, which varied from 0.235 to 0.911 between the 16 genotypes examined in this study. In general, the distance measure could be helpful for the detection of genetically similar/different genotypes. The eight parental genotypes selected in this study showed large genetic diversity among themselves, indicating increased potential for strong out-crossing and higher performance of FS1 Table(DOCX)Click here for additional data file.S2 TableMarkers in bold are specific SSR markers linked to earliness or yield and yield components in wheat.(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.


Fraxinus comprises about 40 species occupying several habitats in the Northern Hemisphere. With some species hybridizing and sharing genetic variants, questions remain on the species assignment of germplasm within the genus Fraxinus despite numerous species-specific assessments. A multidisciplinary approach was employed to provide a definitive insight into the genetics of an endangered Fraxinus “manna ash” collection, located in a rich plant biodiversity hotspot of the Madonie Mountains (Sicily). Although the collection size was small, genetic diversity, assessed by chloroplast (cpSSR) and nuclear (nSSR) microsatellites (SSR—Simple Sequence Repeats), allowed identifying three different chloroplast haplotypes, with one (H5) dominant, and several polymorphic loci, able to discriminate most of the local accessions studied. Molecular data were linked to cytofluorimetric and phenotypic evaluations and, contrary to popular belief that manna ash is Fraxinus ornus L., the germplasm currently used for manna production belongs to Fraxinus angustifolia Vahl. Interestingly, joint analysis of our genetic panel with a large European dataset of Fraxinus spp. suggested the presence of a possible glacial refuge in Sicily, confirming its importance as biodiversity source. Our results will be helpful for the design of long-term conservation programs for genetic resources, such as in situ and ex situ conservation, seed collection and tree reintroduction.The cosmopolitan genus  Fraxinus (Oleaceae) comprises 45–65 tree species and is represented across large areas of Europe, Asia and North America [Fraxinus species show considerable variability in their flowering biology, ecological requirements and distribution ranges as a result of dispersal and vicariance processes as well as adaptive evolution underlying diversification in the genus [The genus  America ,2. Fraxihe genus .Fraxinus excelsior L., the flowering ash, Fraxinus ornus L., and the narrow-leaved ash, Fraxinus angustifolia Vahl [F. excelsior, a polygamous species with male, female and hermaphrodite individuals, [F. ornus is androdiecious [F. angustifolia, a close relative to F. excelsior, shows hermaphrodite flowers and is very common around the Mediterranean basin and to the west of the Black Sea in the Danube basin. Due to the level of local adaptation [F. angustifolia. Three groups can be identified at subspecies level, structured by geographical regions [F. angustifolia ssp. oxycarpa (Bieb. ex Willd.) Franco and Rocha Afonso observed in the in East Central and Southeastern Europe, including the Balkans; F. angustifolia ssp. syriaca (Boiss.) Yalt. in the east of Europe, from Turkey to Pakistan; and F. angustifolia ssp. angustifolia in southwestern Europe. While F. excelsior grows on a wide range of soil types, preferring nutrient-rich substrates, F. angustifolia grows near surface and ground waters, and thus its habitat is more restricted [F. excelsior and F. angustifolia reflect the differential climatic and ecological affinities of both species, as well as the signature of ancient and contemporary hybridization [In Europe, three ash species are present: the common ash, lia Vahl . F. exceviduals, ,6, is fodiecious  and growdiecious ,8. Finalaptation , differe regions ,10: F. astricted ,12. Indestricted . The levdization ,14.Sicily boasts several botanical highlights. Both trees and endemic herbs survived through the last glacial maximum, highlighting the role of Sicily as a refuge area for thermophilous European plants during glacial times . In addiFraxinus in Sicily is restricted mainly to scattered groves and natural populations of F. angustifolia and F. ornus spread in the hilly area of the Madonie and Nebrodi Regional Natural Parks, at altitudes between 100 and 900 m a.s.l. In this area, the relevance of Fraxinus spp. is extremely high, from economical, botanical, naturalistic and historical points of view [F. ornus cultivation, brought to Sicily at the time of the Arab invasion (ninth-eleventh century) and known as manna ash, is grown for the production of manna, a crystal coagulate exuding after cutting 5–10 cm long slits with a billhook along and inside the whole thickness of the bark. From these slits a purplish and bitter liquid flows out. After contact with the air, this liquid turns white and becomes sweet, rapidly coagulating and forming crystal-like layers, called manna. Then manna is harvested and placed in specific trays for drying. Its production varies from 0.2 to 1.5 kg per tree per year, depending on ash cultivar [Fraxinus and to obtain the complete drying of the sap that exudes from cuttings. The most abundant constituent of manna is mannitol , a hexavalent alcohol known also as “manna sugar”; monosaccharide, oligosaccharide, mineral and volatile constituents are also well represented [Fraxinus groves has given rise to a traditional agroforestry landscape of unique value and relevant economic importance until the late 1950s. In the last 50–60 years, after the introduction of synthetic mannitol, the integrity and the extension of the manna ash landscapes have been dramatically reduced. Today, because of habitat fragmentation, naturally poor regeneration and land use change, the maintenance of the crop is threatened. The present landscape of Sicilian manna Fraxinus is limited to a fragmented and isolated area of few hundreds of hectares in the territory around Castelbuono and Pollina , in which natural populations, single and scattered trees are distributed. Large differences in several phenotypic traits, such as leaf morphology, inflorescence features and infructescence characteristics, are observed among the different plants. However, based on their distinctive morphological features, several cultivars (or varieties) were distinguished and identified with dialect names, since the nineteenth century [F. angustifolia and the remaining three putatively belonging to F. ornus [Fraxinus gene pool distributed in Sicily, to provide useful information aimed both to prevent biodiversity loss, safeguarding this endangered plant populations, and develop future breeding programs.The presence of the genus  of view ,18. In fresented . Due to resented . In Sici century ,20,21,22 century ,18. In lF. ornus . HoweverFraxinus spp. and clarify the hybridization process among ash species [Microsatellite  markers are codominant, highly polymorphic, reproducible, well distributed throughout the genome and suitable for automated analysis, and are; therefore, widely used markers for plant variety characterization ,26,27,28 species ,14,33,34Nuclear DNA content has an important role in systematics, and it is a useful tool in biodiversity evaluation ,36. FlowFraxinus in Sicily, belonging to a historical manna ash collection that, from previous studies [F. angustifolia. We also present the first assessment of evolutionary relationships between the F. angustifolia germplasm from Sicily and F. angustifolia germplasm from throughout Europe. Knowledge of the extent of genetic diversity and characterization of population genetic structure of native Fraxinus spp. in Sicily will have important implications for the conservation of this biodiversity, to provide useful knowledge for the management of the manna ash germplasm collection, in-situ/ex-situ conservation and to save this precious cultural heritage.In this study, we used morphological traits, genetic profiles (nuclear and plastid SSR markers) and genome size estimates (nuclear DNA content) to characterize the biodiversity within and among native accessions of  studies , seems tThis analysis is a first step towards selecting breeding material and establishing conservation strategies to sustain and potentially increase the overall production and productivity of manna ash and the continual use of this agroforestry tree resource through varietal improvement and suitable agronomic practices under southern Mediterranean conditions.Fraxinus spp.) germplasm located in Madonie National Park  were collected. Individual trees were selected according to specific criteria based on morphological features, historical notes and their use for manna production [F. ornus trees, collected in the same area, were also added in the study and used as reference. Young and fully expanded leaves were immediately frozen in liquid nitrogen and conserved at −80 °C until used for DNA extraction. This sample set, comprising 38 accessions (F. ornus (FOR) collected in Sicily, is named sample set #1 from here on.Leaf samples of native manna ash  was added to SSR profiles already available. To avoid redundancy in the results, this second sample set (#2) included only samples showing unique genetic profiles isolated from sample set #1, and SSR unique profiles of 475 individual ash trees described in Gérard et al. (2013) [F. angustifolia , F. excelsior  and hybrids between F. angustifolia and F. excelsior  collected in the natural, partially overlapping, distribution ranges of the two species across Europe  , belongis Europe . This seF. ornus trees, a set of morphological traits were evaluated  were recorded; three replicates for each trait were evaluated.For each accession from set #1, including four valuated . The mosF. ornus  as reference standard. Nuclei isolated from a single mature leaf were analyzed in three technical replicates for each accession of set #1. The analysis was carried out with a Partec PAS flow cytometer , equipped with a mercury lamp. Fully expanded leaves (1 cm2) were chopped in a glass Petri dish with 1 mL nuclei extraction buffer OTTO1 [®, Münster, Germany). The genome sizes of collected accessions  were ester OTTO1  and 3 drer OTTO1  supplemeGenomic DNA was extracted from 100 mg of powdered, frozen, young leaf tissue of each individual from sample set #1 using the QiagenDNeasy Plant Mini Kit . The purity and quantity of the DNA extracts were assessed with a NanoDrop 1000 spectrophotometer . Molecular investigations were carried out by amplifying 6 plastid (chloroplast) microsatellite  markers (cpSSR) , 3 plasPlastid DNA haplotypes were defined based on the concatenation of fragment length polymorphism at cpSSRs and amplified plastid DNA regions. To assign the species of origin of the sampled materials, the obtained haplotypes were compared visually with previously published cpSSR profiles from the three European ash species reported in Heuertz et al. (2006) .http://www.R-project.org). A dendrogram was computed from each distance matrix using the UPGMA (Unweighted Pair Group Method with Arithmetic Mean) implemented in the adegenet package [Genetic relationships between nSSR profiles of both sample sets  were estimated using Bruvo’s distance  in the p package  in R. BoF. angustifolia based on the listed analysis, we assessed the genetic differentiation between Sicilian manna ash and the collection of European F. angustifolia, F. excelsior and their hybrids using Wright’s fixation index (Fst) [F. angustifolia (FAN accessions belonging to set #2 was estimated using the observed (Ho) and expected (He) heterozygosities [I) and the inbreeding coefficient (Fis) for sampling locations in GenAlEx 6.5 [AR) within sampling locations was also evaluated though SPAGeDi software [Fst) values were then computed for only F. angustifolia accessions, grouped based on their sampling location or country and a neighbor-joining (NJ) tree using was developed using the adegenet package [After establishing that Sicilian manna ash is ex (Fst)  and Nei’ex (Fst)  computedex (Fst)  in R. Thgosities , ShannonAlEx 6.5 . Each loAlEx 6.5 , with thsoftware . Populat package .F. angustifolia distributed across Europe  was employed. Samples belonging to F. angustifolia were grouped based on their geographic area of origin  and DAPC analysis, implemented in the adegenet package [K-means algorithm “find.clusters” was used to independently verify the assignment of individuals to clusters.Finally, to evaluate the genetic structure of  package , was carF. ornus individual trees (set #1) was estimated based on the relative intensity of fluorescence using the known F. ornus genome size (1.98) as reference [n = 2 × = 46) [Ploidy level, DNA content and morphological trait variation of the manna ash collection DNA content of the 34 accessions belonging to the manna ash collection and of four eference . DNA con × = 46) . Flow cyF. ornus reference trees (F. ornus) and the colour of apical buds (red in manna ash vs. white in F. ornus) were clearly distinct for manna ash and F. ornus  out of nine plastid DNA markers were monomorphic in set #1, displaying fragment lengths of 97, 140, 117, 157 and 253 bp, respectively. The other loci showed low but significant levels of polymorphism : Three sF. ornus .F. ornus genotypes, the so called set #1, was investigated using 11 nSSR , with “Frassino monumentale” as the most distant sample among the manna ash genotypes. Except for varieties “Baciciu”, “Macigna” and “Nsiriddu”, all accessions were assigned to their expected local variety  [F. angustifolia and F. excelsior samples, respectively. As expected, samples belonging to F. ornus clustered as the most distant genotypes, behaving as outgroup, while the individuals morphologically classified as hybrids were distributed across the two main groups. Interestingly, F. angustifolia samples coming from Southern Italian regions from Gérard et al. (2013) [F. angustifolia germplasm in comparison with the range-wide collection of the species.In order to compare the genetic relationships and diversity between the manna ash collection (sample set #1) and the germplasm of . (2013)  into a s. (2013)  identifi. (2013)  data cluFst values with respect to the other FAN populations collected in Europe, whereas comparison to FEX populations resulted in higher values  and FEX  than to FAN    showed lower genetic distance  and lowr values . Hybrids= 0.536) .F. angustifolia sampling locations belonging to set #2 (Fis). However, except Hungary population (−0.607), Fis was close to zero therefore all groups could be considered in equilibrium  were also estimated for o set #2 . Hungaryo set #2 . Sixteenilibrium .AR) ranged from 1.173 (SSR431) to 3.746 (Fem12) for SSR431 and Fem12 markers, respectively (AR was higher in trees collected in Turkey (3.55) than the other populations. Macedonia, Montenegro, Portugal1, five out of seven French groups and the two population collected in Serbia showed similar AR values (ranging from 3.05 to 3.42), while population belonging to Hungary had the lowest value , belongs to F. angustifolia, the narrow-leaved ash. We also showed that this population contained high genetic diversity and, together with a population from Calabria, was strongly differentiated from F. angustifolia populations from other locations in Europe . The assessment of the genetic diversity is crucial to plan activities aimed to conservation of this important and endangered forest tree genetic resource.The genetics of endangered F. ornus. Specifically, in the local varieties the samara stalk length was greater (nearly three times) than in F. ornus, while the apical leaf length/width ratio and the number of samaras/raceme of manna ash samples were approximately two and a half and eight times lower, respectively, separating the two groups compared. Variety identification based on morphological data can be verified using molecular analysis, because the environmental effects, epistatic interactions and pleiotropic effects can interfere with morphological traits evaluation, as in many forest trees [Notwithstanding the limited number of individuals studied, plants belonging to the manna ash collection analyzed showed a wide variation in several phenotypic traits, such as leaf characteristics, inflorescence and infructescence features, plant vigor and morphology, showing a continuous and narrow range of values for individual traits. Leaf morphology is the strongest differentiating character within the local collection, although this trait is not successfully used to characterize the varieties, being strongly influenced by environmental factors. Indeed, it is well known that plants react to water stress, which is particularly strong in the Mediterranean area, by modifying their leaf traits. Considering that the trees collected come from a heterogeneous mountain environment in which neighboring individuals compete for the same ground and light resources, the observed phenotypic variations might be attributed to the impact of environmental factors on growth habit. Regarding fruit morphology, the shape and the length of the fruits of “manna” varieties were quite heterogeneous, as well as the colour or samaras changing from light green of most cultivars to veined brown of “Nsiriddu”. These traits; thus, showed a weak distinguishing power . Neverthst trees .F. angustifolia . The existence of intraspecific variation in genome size has been reported in several plant species ,65,66. H1.540 pg ) and are1.68 pg; ), and F.1.98 pg; ), showin1.98 pg; .F. ornus, specifically in trees from Italy and Corsica. In agreement with the increased botanical similarity of F. angustifolia and F. excelsior and a partially overlapping phenology [F. angustifolia and F. excelsior has been found previously but without common profiles between them and F. ornus [F. angustifolia and F. excelsior although in different geographic regions [F. angustifolia throughout central and southern Italy and in F. excelsior in a restricted area of the Eastern Alps; this particular distribution could be related to two possible different and independent glacial refuges of ash species [F. angustifolia trees collected in Portugal and Corsica [F. excelsior.Molecular analyses confirmed the differences highlighted through morphological and cytological approaches. In agreement with the low chloroplast DNA mutation rate detected in the Oleaceae , cpSSRs henology ,71,72, aF. ornus . Indeed, regions ,73. The  Corsica  and onlyF. angustifolia in Greece [Microsatellites are reported as very effective marker in terms of high information content and discrimination power owing to high allelic variation and allowing clear identification of populations or varieties in several plant species ,75,76,77n Greece . Among sn Greece , yieldinF. angustifolia and F. excelsior was reported [F. ornus clearly behaving like outgroup. Interestingly, a private branch grouping only F. angustifolia trees collected in the South of Italy, including the profiles recorded for manna ash collection, was highlighted. In agreement to cluster analysis, the pairwise fixation index (Fst) highlighted a clear distinctness for the populations from Southern Italy, close to Turkish and French populations. In addition both cluster and Fst analysis allowed to separate the two Italian populations highlight the feature belonging to manna ash collection. Our results are consistent with Fraxinus spp. distribution, indeed common ash (F. excelsior) is found mainly in the north regions throughout Europe [F. excelsior spp. siciliensis [F. angustifolia is mostly restricted to the Mediterranean region [F. angustifolia highlighted by both cp and nSSRs probably is related to the geographic isolation in which they have been found for about 10,000 years starting from the end of the last glaciation. This trend of speciation in Fraxinus, related to specific geographic zones, is in agreement to previous work [F. excelsior and F. angustifolia groups, reflecting the sharing of haplotypes between the two species [F. angustifolia trees collected in Balkan and Eastern Europe, in agreement to the species distribution [Although only a slight genetic differentiation between reported ,80, our t Europe ,81 and hiliensis ,83 is loiliensis  due to tn region ,85. The ous work , and wouous work  as reporous work . Phyloge species . More inribution , whereasF. angustifolia trees studied on the grounds of their geographic area, discriminant analysis based on nuclear microsatellites underlined a gradient from west to east in Y-axis. This evidence is consistent with the classification of F. angustifolia that can be grouped into three geographic subspecies: (i) F. angustifolia Vahl ssp. angustifolia , (ii) ssp. oxycarpa (M. Bieb. ex Willd.) Franco and Rocha Afonso (Northeast Spain to Turkey), and (iii) ssp. syriaca (Boiss.) Yalt (Turkey and Asia Minor) [F. angustifolia populations from South Italy, allow to hypothesize a possible glacial refuge of species in this area, according to a temperature increasing during the late glacial maximum  in the Mediterranean eastern compared to western one, as already reported for the Turkish populations [Finally, grouping the a Minor) ,7,8. Addulations ,88.F. angustifolia. This study provides useful information for germplasm management, finalized to improve the production and productivity of agroforestry species investigated. In addition, the evidences here reported suggest the presence of an additional glacial refuge for F. angustifolia in Italy, confirming the importance of Sicily as source of biodiversity. Furthermore, our study could represent an invitation for botanists to expand the historical knowledge of the collection. Due to the importance of ash germplasm studied, all local varieties were propagated in two repository fields, at the Professional Institute for Agriculture and the Environment (I.P.A.A.) “Luigi Failla Tedaldi”  and at the private Schicchi’s repository located in the Croce-Foresta district  (info at rosario.schicchi@unipa.it) making available this genetic resources for future breeding programs and to develop future national/international collaborations.In summary, using a multidisciplinary approach the present study was focused to characterize in depth a historical manna ash collection represented by local varieties collected in the Madonie area , in order to safeguard an important cultural heritage. Our finding definitively clarifies that the local varieties actually used for manna production belong to


Chlorocebus aethiops (Ch. aethiops) (vervet) and Papio anubis (baboon) are infected with S. mansoni, notably in Ethiopia, where they are expected to have potentially significant implications for transmission and control efforts.Schistosomiasis is a chronic parasitic disease, that affects over 207 million people and causes over 200,000 deaths annually, mainly in sub-Saharan Africa. Although many health measures have been carried out to limit parasite transmission, significant numbers of non-human primates such as S. mansoni isolates from human and non-human primates free-ranging in close proximity to villages in selected endemic areas of Ethiopia.The objective of this study was to assess and compare the genetic diversity and population structure of S. mansoni miracidia were directly isolated from fecal specimens of 104 hosts (i.e. 60 human hosts and 44 non-human primates). We performed DNA extraction and PCR amplification using fourteen microsatellite loci.A cross-sectional study was conducted in three transmission sites: Bochesa, Kime and Fincha. A total of 2,356 S. mansoni populations with cross-host infections.At population scale we showed strong genetic structure between the three sample sites. At the definitive host scale, we observed that host factors can shape the genetic composition of parasite infra-populations. First, in male patients, we observed a positive link between parasite genetic diversity and the age of the patients. Second, we observed a difference in genetic diversity which was high in human males, medium in human females and low in non-human primates (NHPs). Finally, whatever the transmission site no genetic structure was observed between human and non-human primates, however, there appears to be little barriers, if any, host specificity of the S. mansoni strains and inter- and intra-host genetic variations was observed. Substantial genetic diversity and gene flow across the S. mansoni population occurred at each site and non-human primates likely play a role in local transmission and maintenance of infection. Therefore, public health and wildlife professionals should work together to improve disease control and elimination strategies.Occurrence of infection of a single host with multiple  S. mansoni have provided insights into the variation of natural populations. Understanding S. mansoni genetic diversity and population structure of isolates from human and non-human primate hosts living in close proximity showed the occurrence of infection of a single host with multiple S. mansoni strains and inter- and intra-host genetic variations. In this article, the researchers assert the fact that genetic approach reveals that parasites from the three different sites are independent. Thus, we could consider the three sites as geographical replicates showing the influence of NHPs in parasitic transmission in Ethiopia. This study provides insights into the epidemiology, genetic diversity and population structure of S. mansoni in human and non-human primates in Ethiopia, all of which are crucial for the control of schistosomiasis.Schistosomiasis is a chronic disease caused by flukes (trematodes). The definitive host spectrum of schistosomes, whether human, non-human primates (NHPs) or other mammals, is highly dependent on the schistosome species concerned. Genetic diversity and population structure studies of  S. japonicum species is characterized by a broad host range and the host specificity seems to be a derived characteristic with some species such as S. intercalatum recorded to only infect humans [Schistosoma mansoni is present in South America and in Africa and it is responsible for the mesenteric form of the disease. Compared to S. japonicum, S. mansoni is less well recognized as a zoonotic parasite. We have identified evidence for 51 species being naturally infected with S. mansoni  and may have potentially significant implications for transmission and control efforts. Large-scale control programs are mainly based on morbidity control through a mass-drug chemotherapy program using a single drug treatment [S. japonicum [Schistosomiasis remains one of the most prevalent and debilitating helminthic infections in the world. Globally more than 207 million people are infected, and over 600 million people are at risk of infection . These p mansoni . It is ireatment . DespiteS. mansoni and may potentially exhibit similar clinical signs and symptoms [S. mansoni [S. mansoni infections have been evidenced in grivet monkeys (Chlorocebus aethiops) in Ethiopia [Erythrocebus patas) in Cameroon [Pan troglodytes) in Nigeria [Papio sp.) in Saudi Arabia [S. mansoni, other sympatric NHP species were described as free of the parasite. For example, in Kenya S. mansoni infection was identified in baboons but not in local vervet or sykes monkeys [S. mansoni infection in baboons in Ethiopia, but not in sympatric vervet monkeys. In several of these NHP schistosome infections, it has been suggested that forest fragmentation, increased proximity of humans to wild habitats and the emerging reliance of wild primates on human settlements for food, are at least partially responsible for increased exposure and risk of these animals contracting ‘human’ diseases [With close geographical and genetic proximity of many species of non-human primates (NHP) to humans, and given sufficient epidemiological opportunity, NHP can become infected with symptoms  and woulsymptoms , 7. Expesymptoms –10. Seve mansoni . NaturalEthiopia –15, KenyEthiopia , 17 and Ethiopia , in pataCameroon , in chim Nigeria  and Sene Nigeria , and in i Arabia , 22, Ethi Arabia , 15, 23,i Arabia –26, Kenyi Arabia –29 and Ni Arabia . Althoug monkeys . Similardiseases . This isS. japonicum where two distinct patterns have been identified according to the animal reservoir host implicated in the transmission: with parasites from rodent hosts emerging in the afternoon and those from cattle emerging in the early morning [S. japonicum where in villages of the Anhui province in China, different parasite lineages were identified between water buffalo or human hosts and goats, pigs, dogs or cats [S. mansoni or S. haematobium [S. mansoni has been reported in Ethiopia, the precise role in the transmission has never been inferred. In this study, we investigated the genetic diversity and population structure of S. mansoni from humans and NHPs in three selected endemic localities. The study will provide insights into the definitive host range and transmission dynamics of S. mansoni between humans and NHPs, and will deliver important information for schistosomiasis control in Ethiopia.A multi-host parasite system does not automatically imply a reservoir host for the human parasite. Parasite and/or host behavior may limit the encounter of animal and human infecting pathogens. For instance, the pattern of cercarial emergence, a genetically controlled behavior, can limit parasite exchanges between different host species. This has been demonstrated for  morning . Interes morning . Populat morning . This ha or cats . Similar or cats . Parasit or cats . In Ethimatobium . AlthougS. mansoni [S. mansoni infection in human and NHP were previously published and synthetized in A cross sectional study was conducted in Bochesa village, Kime area, and Fincha  located  mansoni , 37. Boc mansoni . Fincha  mansoni . There wS. mansoni positive egg specimens were kept in 0.85% saline solution and transported to the Medical Parasitology Laboratory of Aklilu Lemma Institute of Pathobiology, Addis Ababa University, to harvest miracidia. The age and the sex of each patient (i.e. host) were recorded [For humans, small plastic sheets were provided to study participants and a stool specimen was collected and examined microscopically using Kato-Katz method. recorded .S. mansoni positive samples were kept in 0.85% saline solution and transported to the same laboratory for miracidia hatching. Samples were collected once from baboons from Kime and twice from Fincha over two separate days. Samples were collected twice from vervets in Bochesa, at two different locations and at different times. During collection, much care was taken to select only distinct droppings so that each dropping would represent a single baboon or vervet monkey sample. This sampling method limit our investigations because contrary to human samples we cannot know precisely which sample collected from which individual NHP.For NHPs, fresh fecal droppings were collected early in the morning upon defecation and while the baboons were in the trees where they spent the night . MicroscS. mansoni miracidium isolation, samples from each infected host were prepared separately. The stool specimens and fecal droppings were suspended in dechlorinated water and filtered through tiered sieves of different mesh sizes  and kept for about 20 min in order to allow the eggs to settle in the bottom of the flask. The eggs were washed twice by filling the flask with distilled water and then allowing them to rest submerged for 30 minutes. Parasite eggs concentrated at the bottom of the flask were kept in the dark for 30 minutes and hatching was induced by exposure to bright light on a Petri dish. Miracidia were individually pipetted both for human and NHPs onto the Whatman FTA Elute card using a dissection microscope [For A 4.0mm disc was removed with micro-punch from the FTA Elute Card where the miracidia sample was loaded and put in a tube. 50μl Milli-Q water was added to each tube, incubated at room temperature for 10 minutes and then removed. 100μl of 5% (wt/vol) Chelex 100 solution was pipetted into each tube while the resin beads were evenly distributed (gentle mixing with a stir bar) in solution using a large bore pipette tip  followedFourteen previously published polymorphic microsatellite markers were chosen based on their consistent and reproducible amplification under standard conditions and used in two multiplexes (details of loci are provided in Multiplexed PCR products were genotyped by a subcontractor (Genoscreen) and scored with GeneMapper v. 2.2 software. All genotype calls were manually identified and rechecked by a second reader for consistency. Of the 2,476 genotyped individuals, 2,354 contained adequate information which could be included in further analyses. Here, we tested the divergence from Hardy–Weinberg expectations using procedure implemented in the FSTAT v.2.9.3.2  and baseFis) at each microsatellite locus and for each population using Fstat software 2.9.3.2. The most likely numbers of breeding males and females within an individual host were assessed using Colony software v. 2.0.5.8 assuming random-mating population [S. mansoni larvae were included in this sibship analysis. In each site, expected heterozygosity (He) and allelic richness (Ar) were compared between the parasite populations recovered in humans and NHP using pairwise t-tests using SPSS 20.0. The age of hosts, the number of breeders, the expected heterozygosity (He) and the allelic richness (Ar) were compared using ANOVA followed by LSD pos-hoct tests. Pearson correlation tests were performed to assess a possible link between the age of the human host and the infrapopulation genetic diversity indexes . Finally, in order to infer possible relationship between variables (age and sex) we built full factorial general linear models for each genetic diversity indexes with sex as an explanatory categorical variable and age as a continuous co-factor.Miracidia from all hosts (human or NHP) within a single site  were treated as a population while all miracidia from a single host were treated as an infrapopulation. The genetic diversity of the six schistosome populations and all infrapopulations were assessed by measuring the expected heterozygosity (He), the allelic richness (Ar) and the inbreeding coefficient , respectively. Non-human primates’ droppings sampling was initiated after explicit consent was obtained from local authorities.Ethical clearance was obtained from the Institutional Review Board (IRB) of Aklilu Lemma Institute of Pathobiology, Addis Ababa University (Ref. No.: ALIPB/994/2005/13) and the National Research Ethics Review Committee of Federal Republic of Ethiopia Ministry of Science and Technology (Ref. No.: 3.10/434/106). All ethical issues were strictly handled as per the International Ethical Guidelines for Biomedical Research. Informed verbal consent was obtained from all adults because the study participants provided only stool specimens and the procedure was not invasive presenting no more than minimal risk of harm to the study participants. Hence, the IRB approved the use of oral consent as we requested. For children younger than 18, informed verbal consent was obtained from their parents and the children also gave their assent. During specimen collection, oral consent for all those who agreed was documented as consented. All study subjects found positive for S. mansoni miracidia (+ 8.4) larvae per host were successfully genotyped and analyzed using 14 microsatellite loci (Database in One hundred and four different parasite infrapopulations (60 from human and 44 from NHP) with a total number of 2,354 iracidia , with anFis values showed significant departures from Hardy Weinberg expectations  with Fis values , revealing an excess of homozygosity (i.e. infrapopulations). We detected no deviation from Hardy Weinberg equilibrium at the infrapopulation scale  . At the population scale, 42 of 84 zygosity . These r = 0.02) . At the  = 0.02) . This alFis and number of breeders) for each infrapopulation are presented in 2,101 = 6.18, F2,101 = 7.87 and F2,87 = 4.36 for He, Ar and number of breeders respectively, p<0.05). Finally, we observed that the age of male patients was positively correlated to He , Ar , and the number of breeders  but not to the Fis  of the parasite infrapopulation they hosted. For female patients, no significant correlations were observed between genetic indices of the parasite infrapopulations they hosted whatever He , Ar , number of breeders  or Fis , and their ages (p>0.05). Full factorial generalized linear models . While this main genetic structure distinguished miracidia infrapopulations from the different transmission sites, results within study sites, did not separate humans and NHP infrapopulations, which were totally admixed. This genetic structure was corroborated by an individual-based approach implemented in Structure software which inferred a highest posterior probability for two genetic clusters (i.e. K = 2)  .However, the shoulder in the delta K curve at K = 3 See  suggestsSchistosoma mansoni parasite populations originating from humans and non-human primates (NHP). According to the geographical distances between study sites, this significant genetic structure was expected. Similar results were previously found in Ethiopia [S. haematobium [S. bovis [S. rodhaini in the parasite we collected. Because, the microsatellite markers we used have been specifically designed on S. mansoni, even if cross species amplification is possible, we could expect to have some inefficient markers and/or a difference in allele size on S. rodhaini parasites. We can also expect to observe a population differentiation of these parasites. These last points have not been observed in the present study. However, considering the growing importance of hybrid schistosomes, including S. mansoni x S. rodhaini hybrids, we preconize to consider S. rodhaini in future studies.This study was conducted at three study sites (30 to 500 km apart) and shows a very strong spatial genetic structure of Ethiopia , Kenya [Ethiopia , Brazil Ethiopia , Uganda Ethiopia  and SeneEthiopia . This pamatobium , 60 and S. bovis , where pS. mansoni parasite diversity has already been observed in Senegal [et al. 2014, because both studies sampled a broad range of ages, including children and adults, while most other studies focused exclusively on children. A noticeable difference between our study and that of Van den Broeck et al. (2014) is that we did not observe this link for female patients, and Van den Broeck et al. did not observe differences between genders. Indeed, the sex of the host can also shape the genetic diversity of the parasite. Even if no link between the sex of the host and the genetic diversity of the harbored parasite was highlighted in Senegal [S. mansoni genetic diversity has been observed in Rattus rattus males compared to females in the West indies [i.e. a difference in duration of cercariae recruitment or sex-biased dispersal and home range) or a difference in immunocompetence between sexes. The immunocompetence hypothesis has been experimentally tested and it has been shown that male mice are less susceptible to schistosome infection compared to females [S. haematobium genetic diversity has been observed in boys compared to girls in Zimbabwe [We also observed that host factors are able to shape the genetic composition of parasite populations. First, we observed a positive relationship between parasite genetic diversity and the age of the host in male patients, with older men being infected with a more diverse parasite population. Second, we observed differences in genetic diversity between host categories, with high diversity in human males, medium in human females and low in NHPs. Host behavior and/or physiology is expected to shape the genetic composition of the parasite infrapopulation that they host. It has been proposed that the high genetic diversity of parasites is the result of host behavior in sampling several parasites in space and/or in time, resulting in ‘genetic mixing bowl’ for parasite genes –64. A po Senegal , a subse Senegal . For inst indies . The aut females  and this females . These lZimbabwe . In thisZimbabwe .i.e. frequency of water contact), host immune defense mechanisms and parasite genetic diversity. The protective immunity hypothesis  states that the immune response stimulated by the adult parasite is more effective against new-coming larval schistosomes [Finally, the fact that NHPs harbor less parasite genetic diversity than humans could also result from behavioral differences including the frequency of water contact, which is much lower in NHPs than in humans. A general pattern can be proposed, establishing the link between host behavior (stosomes –73. Expestosomes . This gei.e. Fst) conducted between infrapopulations recovered in humans and NHP, revealed statistically significant genetic differentiations. However, these Fst values are less than 0.01 within the same study site and indicate a very low genetic differentiation between parasites harbored by humans and NHPs. This is consistent with NHP serving as reservoir hosts for human S. mansoni infections. Multi-host parasite systems have been well studied for Schistosoma japonicum because this parasite is recognized as a zoonotic parasite. In the marshland region in China, humans and bovines  share the same S. japonicum genotypes but not with other definitive hosts  [S. mansoni in Ethiopia. This is particularly relevant if we consider the high prevalence (21.6% to 51.7%) of NHP infections. Using barcodes, previous authors have also proposed that chimpanzees could act as reservoir host for S. mansoni [Even if our results reveal a primary genetic structure that is organized into three clusters which correspond to location (i.e. the 3 study sites), analyses (d goats) . Howeverd goats)  and in td goats) . Regardl mansoni . In many mansoni , the sam mansoni .S1 Table(DOCX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S1 Fig(TIFF)Click here for additional data file.